key: cord-0790965-uesjukpu authors: Ayasoufi, K; Wolf, DM; Namen, SL; Tritz, ZP; Jin, F; Pfaller, CK; Goddery, EN; Fain, CE; Gulbicki, LR; Khadka, RH; Yokanovich, LT; Hansen, MJ; Johnson, AJ title: Brain resident memory T cells rapidly expand and initiate neuroinflammatory responses following CNS injury and viral infection date: 2022-04-10 journal: bioRxiv DOI: 10.1101/2022.04.08.487707 sha: 9a1ba3a8214480fb4a8469c180bcc888d7a381c6 doc_id: 790965 cord_uid: uesjukpu The contribution of circulating verses tissue resident memory T cells (TRM) to clinical neuropathology is an enduring question due to a lack of mechanistic insights. The prevailing view is TRM cells are protective against pathogens in the brain. However, the extent antigen-specific TRM cells can induce neuropathology upon reactivation has not been determined. Using the described phenotype of TRMs, we found that brains of naïve mice harbor populations of CD69+ CD103− T cells. Notably, numbers of CD69+ CD103− TRM cells rapidly increase following neurological insults of physical, cancerous, or viral origins. This TRM expansion precedes infiltration of virus specific CD8 T cells and is due to proliferation of T cells within the brain. In contrast, the CD69+ CD103+ TRMs in the brain are generated after the initial expansion of CD69+ CD103− cells following injury and are antigen-specific. We next evaluated the capacity of antigen-specific TRMs in the brain to induce significant neuroinflammation post virus clearance, including infiltration of inflammatory monocytes, activation of T cells in the brain, and significant blood brain barrier disruption. These neuroinflammatory events were induced by TRMs, as depletion of peripheral T cells or blocking T cell trafficking using FTY720 did not change the neuroinflammatory course. Reactivation of antigen-specific TRMs in the brain also induced profound lymphopenia within the blood compartment. We have therefore determined that antigen-specific TRMs can induce significant neuroinflammation, neuropathology, and peripheral immune suppression. Importantly, understanding functions of brain TRMs is crucial in investigating their role in neurodegenerative disorders, CNS cancers, and long-term complications associated with viral infections including COVID-19. Graphical Abstract Healthy brain harbors populations of resident memory T cells (TRM). These TRM cells rapidly proliferate in response to CNS insults of various origins. Following clearance of the insult, populations of TRM cells in the brain decline, but an antigen-specific TRM subset remains within the brain. Antigen-specific reactivation of brain TRMs mediates neuroinflammatory sequalae involving activation and blasting of resident T cells, infiltration of inflammatory monocytes and blood brain barrier disruption. Severe neuroinflammation within the brain following antigen-specific TRM reactivation is concurrent with profound lymphopenia within the blood compartment. CD69 expression indicating resident memory T cells are present in the brain at baseline (Figure 289 1A, Figure S2A -C). While numbers and frequencies of CD69 + CD4 and CD8 T cells varied 290 between mice, all mice reproducibly had populations of CD69 + CD4 and CD69 + CD8 T cells in 291 their brains in the absence of any experimental stimuli ( Figure S2B -C). Further analysis of brain 292 resident CD4 and CD8 T cells demonstrated low expression levels of Thy1.2 iv label (iv-labeled) 293 on these T cells indicating they are not located within the vasculature ( Figure 1B ). Overall 294 frequencies and absolute numbers of iv-labeled T cells in the brain were lower when compared 295 to Thy1.2 -(iv -) T cells ( Figure 1C -D). This contrasts with the spleen where a high percentage of 296 T cells were iv-labeled even after perfusion ( Figure 1E -G). Interestingly, the proportion of T cells 297 that express CD69 was higher in the brain compared to spleen further demonstrating most 298 brain-resident T cells do in fact have a TRM phenotype ( Figure 1H ). Furthermore, amongst 299 CD69 + T cells, only a minority were iv-labeled in the brain as opposed to the spleen suggesting 300 brain resident T cells are not in/or close to the vasculature ( Figure 1I ). 301 Further phenotypic analysis of T cells in the brain, revealed high expression levels of CD44 and 302 CD69, and low expression levels of CD62L. Interestingly, a minor population of T cells 303 expressed CD103, and/or CD49a ( Figure 1K , and Figure S2A -G). Both CD103 + and CD103 -304 populations of CD69 + T cells were found in the brain albeit the CD103 + population remained a 305 minority in the naïve state ( Figure 1L -M and Figure S2A -C). In this study, however, we mainly 306 focused on the responses of the resident memory T cells within the brain. It is worth mentioning 307 that T cells are not the only infiltrating immune cell type found within the naïve brain 308 parenchyma ( Figure 1I ). Using high parameter flow cytometry coupled with UMAP analysis, we 309 determined that infiltrating immune cells in the naïve brain consist of CD4 and CD8 T cells, 310 TCRγδ + T cells, B220 + B cells, CD11b + populations of myeloid cells including those with Ly6C 311 and Ly6G expression, and populations of CD11b -TCRβ -B220immune cells ( Figure 1J ). We next sought to determine whether the variability in numbers of T cells in the brain of naïve 313 mice was due to sex differences. In five independent experiments, we quantified resident 314 memory T cells in the brain of male vs. female mice (results from one experiment is shown in 315 Figure S2H -K, and pooled results from four experiments are shown in Figure 1L -M). As 316 demonstrated in Figure 1L -M and Figure S2H -K, there are no significant differences between 317 frequencies and/or numbers of brain resident T cells between male and female mice. These 318 experiments further indicate that the variability in total numbers of resident memory T cells in the 319 brain is due to batch effects, isolation methods and Percoll gradients. This is a known and 320 reported caveat of T cell analysis from non-lymphoid tissues [46, 47] . To circumvent this problem 321 going forward, we included naïve controls in every experiments so that each batch of analysis is 322 comparable, can be reliably analyzed and compared to internal controls, and can then be 323 pooled with similar experiments. 324 In short, we demonstrated that brains of naïve mice, independent of sex, harbor populations of 325 T cells with phenotypic similarly to reported resident memory T cell subsets ( Figure 1 , and 326 Figure S2 ). Resident memory T cells are one subset amongst many infiltrating immune cells 327 found in the naïve brain at any given time ( Figure 1J ). Importantly, these T cells are not found 328 within the brain vasculature and are residing within the tissue parenchyma ( Figure 1B) . 329 We next sought to determine the extent the CD69 + CD103 + population of brain TRM cells, which 438 was generated at late times post PBS injection and GL261 tumor implantation, was also 439 detectable following TMEV infection. Remarkably, the CD8 + CD69 + CD103 + TRM cells were 440 identified in the brain beginning at day 15 post TMEV infection and remained detectable up to at 441 least 30 days post infection ( Figure 4H -I). We next sought to determine whether CD103 + CD8 442 TRM cells that appeared at late times during TMEV infection were D b : VP2121-130 epitope specific. 443 Our analysis determined that CD8 + CD69 + CD103 + populations of T cells that accumulate are 444 D b : VP2121-130 epitope specific ( Figure 4J -K and Figure S5E ). 445 Together, these data demonstrate that CD69 + CD103brain TRM cells rapidly expand 24-72 446 hours post CNS viral infections. This expansion of TRM cells between 24-72 hours post 447 infection is independent of virus antigens and precedes infiltration of antigen-specific CD8 T 448 cells. Interestingly, a second and distinct population of CD8 TRMs, which expresses both CD69 449 and CD103 surface markers, is identified in the brain between days 15-30 post TMEV infection. 450 The CD8 + CD69 + CD103 + TRM cells are virus antigen specific. In short, we demonstrated that 451 CNS viral infections generate two distinct TRM responses. First, existing CD69 + CD103 -TRM 452 cells expand in a virus antigen non-specific manner between 24-72 hours post infection. This 453 early expansion of TRM cells precedes infiltration of antigen-specific T cells which occurs 454 between days 5-7. At days 15-30 post infection, a second TRM population is detected that 455 expresses both CD69 and CD103 and has virus antigen specificity. 456 Brain injuries ranging from sterile injection of PBS, glioma growth, and a CNS viral infection all 458 result in a rapid increase in numbers of brain resident T cells. Hence, we next sought to 459 determine whether this expansion was due to infiltration of peripheral T cells into the brain 460 during injury or the result of in situ proliferation of T cells that already reside within the brain 461 parenchyma at the time of insult. In order to distinguish between these two possibilities, we 462 used the drug FTY720. FTY720 is phosphorylated by sphingosine kinase and has high affinity 463 for S1P1 and other S1P receptors and hence is a strong agonist of S1P1 receptor [51] . FTY720 464 treatment results in internalization of S1P receptors which in turn leads to sequestration of T 465 cells within the secondary lymphoid organs [51] [52] [53] . FTY720 leads to peripheral lymphopenia in 466 the blood as it sequesters T cells out of circulation. FTY720 treatment therefore blocks T cell 467 infiltration into the brain. Hence, if brain T cells still expand during TMEV infection, this 468 expansion is due to proliferation and not infiltration of new T cells into the brain following injury. 469 In this study, we initiated FTY720 or control (water) treatment one day before TMEV infection. 470 FTY720/water treatment was continued twice daily until euthanasia. AF488 conjugated anti 471 thy1.2 antibodies were also injected intravenously within 3 hours of TMEV infection to quantify 472 the proportion of brain infiltrate that passed through blood circulation in 18-24 hours. Mice also 473 received an intraperitoneal (IP) injection of Bromodeoxyuridine (BRDU) within 3 hours of TMEV 474 infection in order to quantify numbers of proliferating T cells in the brain. Mice were euthanized 475 one day after TMEV infection, perfused, and organs were processed for high parameter flow 476 cytometry (experimental design is shown in Figure 5A ). 477 First, we verified that FTY720 treatment depleted T cells from blood circulation. We found that 478 while total numbers of cells found in 100 µl of blood were comparable between all three groups, 479 frequencies, and numbers of TCRβ + cells, CD4 T cells, and CD8 T cells significantly declined in 480 frequencies and counts also decreased in TMEV+H2O treated groups when compared to naïve 485 controls ( Figure S6F ). This is potentially due to systemic immunosuppression as a result of 486 brain injury which we previously published [40] . Having established that FTY720 treatment efficiently depletes T cells out of blood circulation, we 488 next analyzed immune populations that infiltrate the brain within 24 hours of TMEV infection. 489 CD45 + populations from brain samples of each treatment group (naïve, TMEV+H2O, and 490 TMEV+FTY720) were concatenated and used for generation of UMAPs. As shown in Figure 5B , 491 our analysis is done on equal numbers of immune cells from each group and hence each 492 treatment group contributes exactly to 1/3 of total events. Unbiased UMAP analysis suggests 493 that while TMEV infected and naïve uninfected groups have obvious differences in their brain 494 immune cell populations, TMEV+H2O and TMEV+FTY720 groups appear identical ( Figure 5C ). 495 UMAP analysis was repeated with both male and female mice. Similar results were obtained 496 (data not shown). Intermediate vs. high levels of CD45 expression distinguishes infiltrating from 497 resident immune cells in the brain. The CD45 hi region consists of B cells, CD4 and CD8 T cells, 498 CD11b + Ly6C and Ly6G expressing monocytes, macrophages, and neutrophils, and B220 -499 TCRB -CD11b -Ly6C -Ly6Gpopulations, likely representing NK cells and other ILCs ( Figure 5D ). 500 Next, we sought to determine specific differences between TMEV infected FTY720 treated and 501 control mice. We demonstrate that CD8 T cells in brains of TMEV infected mice have higher 502 side scatter (SSC-A) indicating more activation and blasting compared to naïve controls ( Figure 503 5E right 8 samples compared to first 4 naïve samples-order is as follows from left to right: 1-4 504 naïve, 5-8 TMEV+H2O, and 9-12 TMEV+FTY720). Similarly, CD8 T cells in the TMEV infected 505 brains, regardless of treatment with FTY720, had increased CD69 expression compared to CD8 506 T cells from naïve brains ( Figure 5F left). Qualitatively, expression levels of AF488 Thy1.2 on 507 CD8 T cells decreased in intensity in FTY720 treated mice compared to controls (Figure 5F 508 right). Importantly, frequencies and numbers of BRDU + T cells were comparable between 509 TMEV+H2O and TMEV+FTY720 groups and both infected groups had higher BRDU + CD8 T 510 cells in their brains compared to naïve mice ( Figure 5G -J). As reported in Figure 4 , we found 511 higher numbers of T cells in the brain of TMEV infected mice one day post infection ( Figure 5K) . 512 Total T cell counts increased in both infected groups regardless of FTY720 treatment ( Figure 5K ). Interestingly, UMAP analysis indicated that while T cells are found in the brain, the 514 monocyte/myeloid infiltration at d1 post TMVE infection is the majority of the overall 515 neuroinflammatory response ( Figure 5D ). This was reflected on T cell percentages. If 516 percentages are represented of total CD45 hi cells, there is a slight decline in overall T cell 517 frequencies to account for the increase in monocytes ( Figure S7A ). However, if T cells are 518 represented as percentage of all live cells within the brain, then there is a significant increase in 519 TMEV infected mice compared to naïve controls ( Figure S7B ). Accordingly, absolute numbers 520 of CD4 and CD8 T cells in TMEV infected mice increased compared to naïve mice regardless of 521 FTY720 treatment ( Figure S7C -D). We next investigated TCRβ expression in infected vs. 522 uninfected controls. Consistent with the increased total T cell counts in the brain, side scatter 523 significantly increased and TCRβ expression levels decreased on T cells isolated from TMEV 524 infected brains when compared to naïve brain resident T cells ( Figure 5L -M). High side scatter 525 and concurrent downregulation of TCR expression indicates a state of T cell activation and 526 blasting. In short, there were no significant differences in numbers of BRDU + T cells between 527 FTY720 treated and control infected brains indicating this early expansion of T cells in the brain 528 one day post viral infection is due to in situ proliferation within the brain and does not depend on 529 infiltration of circulating T cells into the brain. 530 We next sough to determine whether the increase in TRM cells in the brain one day after 531 infection was due to increased numbers of T cells, or due to upregulation of CD69 on brain TRM 532 cells. Analysis of CD69 + CD4 T cells of live cells in the brain indicated an increase in CD69 533 expressing CD4 T cells. In contrast, the frequency of CD69 expressing cells calculated out of 534 total CD4 T cells did not change ( Figure 5N , and Figure S7E ). This was concurrent with an 535 increase in total absolute counts of CD69 + CD4 T cells in the brains of TMEV infected mice 536 compared to naïve controls regardless of FTY720 treatment ( Figure 5O ). This indicated that 537 during TMEV infection, overall numbers of brain resident CD69 + CD4 T cells increases. In 538 support of this observation, we found an overall increase in BRDU + CD69 + CD4 T cells in the 539 brains of infected mice compared to uninfected naïve controls indicating an increase in 540 proliferation of CD4 T cells within the brain ( Figure 5P ). 541 We next focused on CD8 T cells. Our analysis of the CD8 T cell compartment demonstrated 542 that the frequency of CD69 + CD8 T cells, CD69 + CD8 T cells of live cells, and absolute counts of 543 BRDU + CD69 + CD8 T cells all increased in the brains of infected mice compared to uninfected 544 controls, regardless of FTY720 treatment ( Figure 5Q -S, and Figure S7F ). These results indicate 545 that in TMEV infected brains, CD8 T cells both upregulate their CD69 expression, and 546 proliferate in response to TMEV infection. This early and rapid response of CD8 T cells in the 547 brain within 24 hours of TMEV infection is independent of infiltrating peripheral T cells. 548 We next sought to quantify numbers of iv-labeled T cells that infiltrate the brain within 24 hours 549 of TMEV infection. When frequency of Thy1.2 + T cells were calculated out of CD69 + CD4 or 550 CD69 + CD8 T cells in the brain, no significant changes were detected between naïve and 551 infected groups ( Figure S7G -H). Similarly, total numbers of Thy1.2 + TCRβ + , or Thy1.2 + CD69 + 552 CD4 + , or Thy1.2 + CD69 + CD8 + T cells were comparable between groups ( Figure S7G -I). 553 Interestingly, both TMEV infected groups had a lower frequency of Thy1.2 + cells, when 554 calculated out of total T cells, in comparison to the naïve group ( Figure S7I ). 555 All together, these data indicate that infiltration of peripheral T cells into the brain one day post 556 TMEV infection does not contribute to the overall expansion of brain resident T cells. Instead, 557 Virus antigen specific T cells reside within the brain following TMEV clearance, enabling 559 We have thus far demonstrated that brains of naïve mice have populations of TRM cells that 561 rapidly proliferate during acute neurological insults in an antigen non-specific manner. In 562 parallel, we demonstrated that TMEV infection results in accumulation and retention of antigen-563 specific TRMs within the brain 3-4 weeks after infection ( Figure 4G -K). We next sought to determine the extent antigen-specific memory T cells induce neuropathology. To determine the 565 function of accumulated antigen-specific T cells in the brain, we first intracranially infected 566 C57BL/6 mice with TMEV and allowed 60 days for viral clearance and memory T cell formation. 567 TMEV infection is cleared in C57BL/6 mice between days 30-45 post infection due to a strong 568 CD8 T cell response against the immunodominant epitope VP2121-130 which can be tracked using 569 D b : VP2121-130 specific tetramers [36, 38, 50, 54] . We therefore reactivated antigen-specific 570 memory T cells by intravenously injecting the VP2121-130 peptide (or control TMEV-irrelevant E7 571 peptide) 60 days post infection, a timepoint when measurable virus has been cleared from the 572 CNS [38, [54] [55] [56] . In these experiments, mice additionally received an intravenous injection of anti 573 thy1.2 antibodies 24 hours prior to euthanasia to identify T cells infiltrating the brain from 574 circulation ( Figure 6A ). 575 residency is distinct between an unmanipulated naïve and a quiescent antigen-577 experienced brain 578 Before proceeding with TRM reactivation studies, we first investigated dynamics of TRM cells in 579 brains of naïve vs. antigen-experienced mice that have cleared TMEV infection. 580 Interestingly, when comparing all naïve and control TMEV infected mice (TMEV+E7), 581 frequencies of trafficking T cells in an antigen-experienced brain was significantly lower than 582 that of a naïve brain ( Figure 6B ). While frequencies of iv-labeled T cells in the brain of antigen-583 experienced mice decreased, their overall numbers increased when comparing TMEV+E7 to 584 naïve mice indicating that the overall T cell counts in the brain is higher in the antigen-585 experienced brain despite lack of an ongoing neurological insult ( Figure 6B -C). Together, these 586 data combined with data presented in our parabiosis studies ( Figure 2 ) indicate that T cells from 587 circulation constantly infiltrate the brain parenchyma and become tissue resident in the naïve 588 state. The rate of this re-circulation is significantly decreased in an antigen-experienced brain. While injection of the VP2121-130 peptide acutely during the effector phase of TMEV response 592 (d7) induces extreme neuroinflammation leading to CD8 T cell mediated blood-brain-barrier 593 disruption and death [50, 57] , neuropathological consequences of VP2121-130 injection during the 594 memory phase remain unknown. We hence sought to determine the extent reactivation of 595 antigen-specific T cells induces neuroinflammation. We determined that following VP2121-130 596 injection, total frequencies, and numbers of infiltrating CD45 hi immune cells in the brain 597 significantly increased in comparison to E7, or naïve controls ( Figure 6D -E). This 598 neuroinflammatory response in VP2121-130 injected mice was concurrent with significant blood 599 brain barrier permeability as measured by gadolinium leakage in MRI images ( Figure 6F -G). In 600 short, injection of the immunodominant peptide from TMEV reactives antigen-specific memory 601 CD8 T cells including brain TRMs. This reactivation is associated with rapid and enhanced 602 infiltration of immune cells into the brain and detectable blood brain permeability ( Figure 6D -G). 603 We have previously demonstrated that acute neurological insults including TMEV infection 605 induce severe peripheral immunosuppression [40] . Here, we sought to determine whether 606 reactivation of brain TRMs, in the absence of an external physical hit to the brain, induces 607 similar peripheral lymphopenia. Importantly, VP2121-130 injection significantly reduced 608 frequencies and overall counts of CD4 and CD8 T cells in peripheral blood in comparison to 609 naïve and E7 control groups ( Figure 6I -J). In fact, blood brain barrier permeability, as measured 610 by MRI, inversely correlated with CD8 T cell counts in the blood indicating that lymphopenia in 611 the blood is a direct correlate of brain injury and inflammation ( Figure 6K ). It is important to 612 mention, however, that the total number of T cells between E7 and VP2 groups did not change in the brain while these counts significantly declined in the blood ( Figure 6H brain vs. Figure 6I -J 614 blood). This argues against lymphopenia being simply due to migration of T cells from the blood 615 into the brain. Together, these data indicate that reactivation of antigen-specific memory T cells 616 in the brain is a neurologic insult associated with blood brain barrier permeability and severe 617 neuroinflammation in the brain and is concurrent with severe peripheral lymphopenia. 618 TRMs whose responses can be distinguished from that of peripheral T cells 620 To investigate the extent brain resident TRMs induce neuropathology following antigen-specific 621 reactivation, we used two parallel strategies to deplete peripheral T cells. First, groups of mice 622 received two intraperitoneal injections of low dose anti CD8 (aCD8LD) depleting antibodies on 623 prior to peptide injection. This dose of depleting antibody efficiently depletes CD8 T cells in 624 blood and spleen, while sparing TRM populations in the brain ( Figure 6L -O, Figure S9G , I, and 625 K, Figure S10A -D, comparing groups E7 and E7+aCD8LD). On day -2, mice either received an 626 intravenous injection of the TMEV immunodominant peptide VP2121-130 to reactivate TMEV 627 specific memory T cells, or an irrelevant peptide E7 from the human papilloma virus, both of 628 which are presented in the context of the H-2D b class I molecule. Additionally, on d-1, mice 629 received an intravenous injection of AF488 conjugated anti Thy1.2 antibodies to label T cells 630 within circulation (Experimental design is shown in Figure 6L -Top). In summary, this strategy will 631 distinguish peripheral T cell responses from those of the resident memory T cells in the brain 632 during antigen-specific TRM reactivation. 633 In parallel, a group of mice received either FTY720 or sham injection beginning on day -4 in 634 order to block infiltration of T cells into the brain without depletion. These mice received peptide 635 and antibody injections as described in the aCD8LD group above ( Figure 6L -Bottom). Like the 636 aCD8LD treated groups, FTY720 treatment depleted T cells in circulation, but did not affect total 637 spleen count, or numbers of T cells in the brain ( Figure 6M -O, Figure S9A -F, Figure S11A -D 638 comparing E7 control to E7+FTY720). Frequencies of CD4 T cells in blood or spleen were not 639 altered with aCD8LD treatment, while FTY720 treatment resulted in decreased CD4 T cell levels 640 in the blood ( Figure S8A -B). This is while CD4 T cell counts in the brain were unaffected in all 641 E7 injected groups regrades of aCD8LD or FTY720 treatment ( Figure S8C ). These strategies 642 together will mechanistically dissect the role of peripheral T cells (outside) and brain resident 643 memory T cells (inside) during antigen-specific reactivation and the consequential 644 neuroinflammation. 645 To this point, we demonstrated increased infiltration of immune cells into the brain during 648 antigen-specific T cell reactivation in mice infected with and recovered from TMEV infection. We 649 next sought to measure the magnitude of neuroinflammation within the brains of mice following 650 antigen-specific T cell reactivation when peripheral T cells are eliminated with aCD8LD or 651 FTY720 treatment. We first evaluated the state of neuroinflammation in mice with following 652 antigen-specific T cell reactivation with and without low dose (aCD8LD) CD8 depleting 653 antibodies. We hypothesized that the TRM response generated from within the brain upon 654 peptide delivery is responsible for the resultant neuroinflammation and hence aCD8LD 655 treatment should not alter the neuroinflammatory sequalae in the brain. 656 To identify global changes between inflamed brains and quiescent brains following antigen-657 specific TRM reactivation, we performed unbiased UMAP clustering of high parameter flow 658 cytometry data obtained from total immune infiltrates (CD45 + ) of perfused brains from 659 TMEV+E7, TMEV+VP2, TMEV+E7+aCD8LD, and TMEV+VP2+aCD8LD groups ( Figure 7A ). 660 UMAP analysis determined that E7 and E7+aCD8LD groups clustered similarly, while VP2, and 661 VP2+aCD8LD groups clustered together ( Figure 7A ). We demonstrated that the total CD45 + 662 cells in the brain are clustered as two separate infiltrating (CD45 hi ), and resident (CD45 mid ) 663 populations ( Figure 7B ). Antigen-specific reactivation of memory T cells induced changes in the 664 microglia that include slight increase in CD45 expression, similar CD11b expression, and 665 enhanced MHCII expression as these clusters are completely remodeled in VP2121-130 injected 666 mice compared to E7 controls ( Figure 7A -C). The infiltrating immune cell (CD45 hi ) cluster is 667 mostly comprised of T cells, B cells, and CD11b + (B220 -TCRβ -) myeloid, and CD11b -(B220 -668 TCRβ -) populations ( Figure 7D ). T cell clusters are further divided into CD4 T cells, and D b : 669 VP2121-130 + and D b : VP2121-130 -CD8 T cells ( Figure 7E ). This is while the CD11b + cluster can be 670 further divided into MHCII hi and MHCII low monocytes, and Ly6G hi neutrophils ( Figure 7F ). 671 Importantly, manual gating confirmed that the majority of infiltrating myeloid cells in the VP2 672 groups are CD11b + MHCII hi cells when compared to E7 groups ( Figure 7G ). Finally, we 673 analyzed the CD8 cluster in the UMAPs for expression of T cell lineage and residency markers 674 ( Figure 7H ). CD69 was highly expressed on majority of brain resident T cells, while CD103 was 675 detectible on a subset of CD8 T cells ( Figure 7H ). Majority of T cells in the brain were virus-676 specific as they stained positive for D b : VP2121-130 tetramer ( Figure 7H ). CD44 expression 677 remained high while CD62L was detectible on a small subset, likely the infiltrating iv-labeled T 678 cells ( Figure 7H ). Additionally, subsets of CD8 T cells demonstrated upregulation of activation 679 markers Ly6C and CD11b ( Figure 7H ). 680 Following unbiased UMAP analysis, we performed manual gating and quantification to further 681 analyze the neuroinflammatory response in the brain following TRM reactivation. We first 682 evaluated total immune cells within the brain. We found that VP2121-130 injected groups, 683 regardless of aCD8LD treatment status, demonstrated an increase in immune infiltrate as 684 measured by total numbers of CD45 hi cells when compared to naïve or E7 controls ( Figure 7I ). 685 One major immune cell type that increased during antigen-specific TRM reactivation following 686 and MHCII ( Figure 7J -K left and middle histograms). Interestingly, the increase in CD11b + S10H). Neuroinflammation as measured by an increase in total immune cell infiltrates and 690 specific increases in inflammatory monocytes occurred to a similar extent in VP2, and 691 VP2+aCD8LD groups. These findings indicate that myeloid cell infiltration and 692 neuroinflammation during reactivation of antigen-specific T cells in the brain is mediated by 693 brain resident T cells and is independent of peripheral CD8 T cell responses ( Figure 7I -J). While 694 infiltration of inflammatory monocytes into the brain is a major hallmark of neuroinflammation, 695 CD8 T cell activation and blasting is crucial in this inflammatory process. CD8 T cells isolated 696 from brains of both VP2 and VP2+aCD8LD groups had larger side scatter (SSC-A) indicating 697 blasting and activation when compared to E7 controls ( Figure 7K right histogram, and L). 698 We then further analyzed T cells found in the brain of mice following antigen-specific TRM 699 reactivation. While most groups had a higher frequency of CD4 T cells compared to naïve mice, 700 no major differences between E7 and VP2 groups were found, regardless of treatment with 701 aCD8LD ( Figure 7M ) to deplete peripheral CD8 T cells. However, total numbers of CD4 T cells 702 in the brains of VP2 121-130 injected mice increased when compared to E7 controls ( Figure 7M ). 703 This increase in total CD4 T cells during VP2121-130 administration occurred to the same extent in 704 the VP2 and VP2+aCD8LD groups as depletion of CD8 T cells specifically leaves CD4 T cells 705 untouched ( Figure 7M ). Total frequencies of CD8 T cells in E7 and VP2 groups remained stable 706 but appeared higher than naïve groups ( Figure 7N ). VP2+aCD8LD groups of mice had 707 significantly decreased frequencies of CD8 T cells in the brain compared to E7+aCD8LD group. 708 Total numbers of CD8 T cells were also comparable between E7, and VP2 controls, while 709 VP2+aCD8LD groups demonstrated a reduction compared to E7+aCD8LD control ( Figure 7N ). 710 We next evaluated numbers of antigen-specific CD8 T cells in the brain. The majority of CD8 T 711 cells in brains of E7, VP2, E7+FTY720, and VP2+FTY20 treated mice stained positive for 712 tetramer and were antigen-specific ( Figure 7O ). Frequencies of D b : VP2121-130 CD8 T cells were comparable between E7, E7+aCD8LD, and VP2+aCD8LD groups while the VP2 control group 714 had slightly lower frequencies of tetramer positive CD8 T cells in the brain ( Figure 7O bottom) . 715 Correspondingly, absolute counts of tetramer positive CD8 T cells in the brain were comparable 716 between E7, VP2, and E7+aCD8LD while they were found to be slightly lower in VP2+aCD8LD 717 group ( Figure 7O Top) . This data indicates that majority of the neuroinflammatory response 718 following antigen-specific T cell reactivation is functionally due to actions of brain resident T 719 Given that a proportion of CD8 T cells following VP2121-130 injection likely originate from the 721 periphery, we next quantified frequencies and numbers of iv-labeled T cells accumulated in the 722 brain within 24 hours. Frequencies and numbers of iv-labeled CD4 T cells between E7 and VP2 723 and between E7+aCD8LD and VP2+aCD8LD groups increased ( Figure 7P -Q). Additionally, 724 compared to E7 control, VP2121-130 injected mice had an accumulation of iv-labeled CD8 T cells 725 measured by both increased frequencies and numbers ( Figure 6R -S, comparing E7 to VP2). In 726 comparison to VP2 groups, VP2+aCD8LD treated mice had significantly lower proportions of iv-727 labeled CD8 T cells in the brain which is due to depletion of peripheral CD8 T cell sources 728 ( Figure 6R -S, comparing VP2 to VP2+aCD8LD). However, while significant, the frequency of 729 brain infiltrating CD8 T cells that stemmed from circulating sources ranged between 7-31% of 730 the overall pool. This demonstrates the predominant T cell response is resident to the brain and 731 is not originating from circulation. 732 Finally, we determined that frequencies and numbers of iv-labeled CD4 and CD8 T cells 733 increased in the brain, whereas their counterparts in the spleen did not. Reactivation of brain TRMs induces significant neuroinflammation within the brain 743 We have demonstrated that following antigen-specific TRM activation, depletion of peripheral 745 CD8 T cells did not change hallmark features of neuroinflammation within the brain. Included in 746 this analysis was assessment of T cell blasting and inflammatory monocyte infiltration. We next 747 aimed to determine the extent FTY720 treatment alters the course of neuroinflammation 748 following TRM reactivation. Similar to VP2+aCD8LD treatment group, VP2 and VP2+FTY720 749 treated mice demonstrated comparable increases in total CD45 hi infiltrating immune cells, 750 inflammatory CD11b + MHCII hi cells, and CD8 T cell blasting as measured by increased side 751 scatter when compared to E7 and E7+FTY720 controls ( Figure 8A -C and E). This increase in 752 brain CD11b + MHCII hi cells in VP2 groups did not occur in the spleen ( Figure S11H ). Moreover, 753 frequencies and numbers of total CD8 T cells were comparable amongst E7, VP2, E7+FTY720, 754 and VP2+FTY720 groups ( Figure 8D ). Additionally, frequencies of CD4 T cells in the brain 755 remained consistent between E7 and VP2 and E7+FTY720 and VP2+FTY720 treated groups 756 ( Figure 8F left) . Total numbers of CD4 T cells increased between E7 and VP2 groups but was 757 significantly lower when VP2 and VP2+FTY720 groups were compared ( Figure 8F right) . This 758 indicates that during VP2121-130 injection, a minor population of peripheral CD4 T cells enters the 759 brain. Upon FTY720 treatment, circulating CD4 T cells are reduced and hence CD4 T cells do 760 not increase in the brains of VP2+FTY720 group compared to E7+FTY720 control. When 761 analyzing antigen-specific CD8 T cells, we determined comparable numbers of tetramer + CD8 T 762 cells between E7, VP2, E7+FTY720, and VP2+FTY720 groups indicating these cells were not affected by FTY720 treatment ( Figure 8G ). Together, these data indicate that responses of the 764 brain resident T cells prevail over those in the periphery following antigen-specific memory T cell 765 reactivation. 766 We next further analyzed iv-labeled T cells amongst groups. In this experiment, no statistically 768 significant difference was detected between E7 vs. VP2 and E7+FTY720 vs. VP2+FTY720 769 groups in frequencies of iv-labeled CD4 T cells (Figure S11I left). Yet when absolute counts of 770 iv-labeled CD4 T cells were quantified, VP2 treated mice had significantly more iv-labeled CD4 771 T cells compared to E7 controls. FTY720 treatment diminished this increase due to depletion of 772 peripheral CD4 T cell sources ( Figure 8H left). Frequencies of iv-labeled CD8 T cells increased 773 between both E7 vs VP2 and E7+FTY720 vs. VP2+FTY720 groups (Figure S11I Right). 774 Differences between absolute counts of iv-labeled CD8 T cells in the brain of VP2+FTY720 775 treated mice did not reach statistical significance when compared to E7+FTY720 group; but 776 there were no differences between the two VP2 groups indicating similar responses occurred in 777 the brain following VP2 121-130 injection regardless of FTY720 treatment ( Figure 8H right) . 778 Together, these data indicate that the neuroinflammatory response during antigen-specific 779 memory T cell reactivation does not rely on responses of T cells outside the brain. 780 The neuroinflammatory response following antigen-specific memory T cell reactivation is 781 While neuroinflammation within the brain occurred following TRM reactivation to the same 783 extent between VP2 and VP2+FTY720 treated groups, FTY720 treatment altered peripheral 784 responses in the spleen. For example, VP2+FTY720 and E7+FTY720 groups demonstrated a 785 significant reduction in iv-labeled CD4 T cells in the spleen when compared to E7 control likely 786 stemming from lack of circulation from the blood and the lymphatics system ( Figure S11E ). We 787 did not find significant differences in iv-labeled CD8 T cells in the spleen of FTY720 treated mice compared to controls ( Figure S11F ). Importantly, despite comparable frequencies of tetramer + 789 CD8 T cells in the brain, VP2+FTY720, and VP2+aCD8LD treated groups exhibited significantly 790 lower numbers of antigen-specific CD8 T cells in their spleens in comparison to VP2 groups 791 indicating that despite lower peripheral responses, brain responses were robust and fully 792 functional ( Figure S11H and Figure 8I ). This is in accordance with our above findings and re-793 enforces that brain resident T cells mediate neuroinflammatory responses independently of 794 peripheral T cell responses following antigen-specific reactivation. 795 Both strategies using low dose anti CD8 depletion and FTY720 during memory T cell 797 reactivation led to diminished antigen-specific responses in the spleen ( Figure 8I ). Yet, neither 798 of these strategies altered the course or hallmark features of neuroinflammation in the brain 799 (Figure 7-8) . We then sought to determine the extent blood brain barrier permeability occurs in 800 FTY720 and aCD8LD treated mice following VP2121-130 injection. Using T1-weighted MRI 801 following gadolinium injection, we determined that neither FTY720 treatment nor aCD8LD 802 treatment blocked permeability due to neuroinflammation following memory T cell reactivation 803 ( Figure 8J -L). This demonstrates that reactivation of antigen-specific memory T cells in the brain 804 results in neuroinflammation characterized by: 1) increased total immune cell infiltration into the 805 brain, 2) increased inflammatory monocytes within the brain, 3) blasting and activation of brain 806 resident CD8 T cells, and 4) disruption of the blood brain barrier. Treatment with FTY720 and 807 aCD8LD efficiently eliminated peripheral T cell responses, but the neuroinflammatory response 808 remained intact. Hence, antigen-specific brain resident T cells upon reactivation mediate 809 neuroinflammation and blood brain barrier disruption independently of peripheral T cells. 810 While the existence and responses of resident memory T cells in barrier tissues have been well-812 described, their characterization and function in the brain remains poorly understood. 813 Specifically, the characterization of brain TRM cells in naïve mice and their subsequent 814 responses during neurological insults remains unclear. In this study, we characterized two 815 subsets of brain resident T cells: one TRM subset was CD69 + CD103and the other was CD69 + 816 CD103 + . We found that naïve brains harbor CD69 + CD103population of CD4 and CD8 TRM 817 cells at baseline (Figure 1) . We further demonstrated that CD8 T cells both with a CD103and 818 CD103 + phenotype accumulate in the brain as a function of age. Meanwhile, CD4 TRM cells do 819 not accumulate in the brain of aging mice ( Figure 2 ). Additionally, we found that the CD69 + 820 CD103population of brain resident memory T cells expand acutely during neurological insults 821 of various origins (Figure 3-5) . This rapid expansion of brain TRMs occurs during sterile injury, 822 CNS viral infections, and CNS cancer progression (Figure 3-5) . In a glioma model, TRM cells 823 persist in the brain and do not fade or take up an exhausted phenotype indicating they can be 824 targeted, and their powers harvested against the tumor (Figure 3) . The rapid expansion of 825 CD69 + CD103 -CD8 TRM cells following CNS viral infection was not dependent on T cell 826 responses outside of the brain and preceded entry of viral antigen-specific T cells. 827 In the memory time point, however, reactivation of brain TRMs induced severe 828 neuroinflammation in the brain that was accompanied by activation and blasting of CD8 T cells, 829 infiltration of inflammatory monocytes, and concurrent blood brain barrier permeability ( Figure 6 -830 8). These neuroinflammatory events occur inside the brain upon antigen-specific memory T cell 831 reactivation even in the absence of CD8 T cells outside of the brain. Hence, we determined that 832 responses of the brain resident memory T cells occur independently of peripheral T cell 833 responses and that the neuroinflammatory cascade is initiated from within the brain. 834 These data together suggest that the CD69 + CD103population of resident memory T cells is 835 found in the brain of healthy mice and is capable of promptly reacting towards various 836 neurological insults through proliferation within the brain. The early antigen-independent TRM 837 response is followed by generation of a CD69 + CD103 + TRM population that is antigen-specific. 838 Re-activation of the antigen-specific TRM population leads to severe neuroinflammation in the 839 brain which is concurrent with profound lymphopenia in the blood compartment. Our study helps 840 shed light on distinct populations of resident memory T cells in the brain: one that responds 841 quickly to various insults independently of antigens and then subsides, and one that is 842 generated after the expansion of the first TRM population and is antigen-specific. The antigen-843 specific population responds powerfully upon reactivation, does not require help from outside T 844 cells, induces neuroinflammation concurrent with peripheral lymphopenia, and remains in the 845 brain long after induction of the initial neurological insult. 846 CD8, but not CD4, TRMs accumulate in the naïve brain during the process of aging 847 Our data demonstrates CD8 TRM cells accumulate within the brain during aging (Figure H-I) . 848 This is in contrast to CD4 TRM cells. CD8 T cells were previously found to be increased in 849 brains of 18-22-month-old mice; yet no sequential analysis with aging was performed [28, 32, 850 58] . Our data demonstrates accumulation of total CD8 T cells as well as CD8 T cells with a TRM 851 phenotype beginning at 6 months of age which continues to 1 year. Furthermore, we provide 852 evidence that CD103 + TRM cells only accumulate in naïve old mice, while brains of young 853 unchallenged mice mainly harbor CD69 + CD103 -TRM populations. Given the correlation 854 between neuroinflammation and aging[59], it is conceivable to hypothesize that this correlation 855 between age and accumulation of CD8 T cells is inflammation dependent. Interestingly, the 856 process of aging alone did not lead to accumulation of CD4 TRM cells in the brain compared to 857 young mice, nor did it affect upregulation of CD103 on CD4 TRMs. The specific signals leading 858 to CD8 vs. CD4 TRM cell accumulation within the aging brain, and how this T cell accumulation 859 interplays with age-associated cognitive decline are amongst future intriguing questions that 860 remain to be answered. Both CD103and CD103 + populations of resident memory T cells have been described during 864 infections of several tissues including the brain [20, 23, 25, 26, 34 ]. Yet, to date, no systematic 865 study has distinguished differences between the requirements for generation and maintenance 866 of these two distinct TRM populations in the brain. Our study systematically showed that CD69 + 867 CD103memory T cells are found in brains of all naïve specific pathogen free (SPF) C57BL/6 868 mice, and they expand acutely during neurological insults. This acute expansion of TRM cells in 869 both the CD4 and CD8 T cell compartments occurred with similar kinetics during sterile physical 870 injury, viral infection, and in a model of CNS cancer (Figure 3-5) . Additionally, at 24 hours post 871 TMEV infection, the expansion of TRM cells was not antigen-specific and was due to 872 proliferation within the brain rather than infiltration of circulating T cells ( Figure 5 ). The observed 873 kinetics of the response by the CD69 + CD103populations was distinct from that of the CD69 + 874 CD103 + TRM cells. This is best illustrated by the detection of CD69 + CD103 + TRM populations 875 long after the initial expansion of the existing CD69 + CD103populations post neurological 876 injury. For example, the CD69 + CD103 + TRM populations were exclusively detectable after 877 induction of neurological insults in mice under 6 months of age. During sterile injury, the CD103 + 878 TRM cell expansion of both CD4 and CD8 T cells occurred at day 28 post injury. Similarly, 879 during TMEV infection, CD103 + CD8 + TRM cells appeared at day15 and persisted to 60 days 880 post infection. Although this CD69 + CD103 + population is small compared to its CD103 -881 counterpart, it is detectible, and its expansion occurs after the detected responses of the CD69 + 882 CD103population. Due to the distinct responses generated by CD103and CD103 + TRM 883 populations in the brain, future studies are needed to investigate the role of each population in 884 the context of neurological diseases. Interestingly, aging alone is sufficient to induce generation 885 of a CD69 + CD103 + CD8 + TRM population within the brain that is durably maintained. Yet, CD4 + 886 CD69 + CD103 + TRM cells within the brain were only found following induced neurological 887 The CD103 + CD8 + TRMs generated during TMEV infection were virus antigen specific 889 suggesting this TRM population may confer antigen-specific protection locally while the CD103 -890 counter part is capable of cross-reactive protection against a multitude of acute insults. This Our data suggests that two types of TRM cells exist in the brain: 1) TRMs that can react against 897 insults independent of specific antigens, and 2) antigen-specific TRMs that accumulates upon 898 pathogen encounter. While the existence of both CD103 + and CD103 -CD8 T cells in the brain 899 was previously described, the kinetics of their responses and their existence in naïve mice was 900 not directly studied [26] . Importantly, we demonstrate that the rapid expansion and proliferation 901 of the CD69 + CD103 -TRM cells in the brain precedes infiltration of antigen-specific T cells into 902 the brain. Together, both TRM populations may be required for optimal responses during 903 neurological insults. 904 TRM cells are thought to not re-circulate upon entry into non-lymphoid tissues [4] . This point has 907 been best shown using parabiosis studies [4, 11, 20] . This work was mainly performed following 908 tissue specific immunizations or infections [4, 11, 21, 25] . However, these studies do not test or 909 rule out the possibility that the circulation harbors a TRM precursor population that upon entry to the tissues will become tissue resident, especially in naïve mice. It was recently shown that 911 brain resident T cells do not expand ex vivo upon re-stimulation and it was hypothesized that 912 the brain environment supports maintenance of the TRM cells long after infection [25] . Our data 913 with naïve-naïve parabiosis indicates that, even in naïve mice, brain allows infiltration of 914 peripheral T cells into the parenchyma (Figure 2A-G) . These T cells in turn become 915 phenotypically identical to brain resident memory T cells and express CD69. Identification of 916 TRM precursor cells from blood will be useful in several applications. For example, these cells 917 may be enriched and used to produce CAR T cells against glioblastoma as these cells may 918 have enhanced brain homing potential. Such CNS localization could allow CAR transduced 919 TRMs to rapidly become activated upon tumor encounter and proliferate within the brain, and 920 ultimately confer long term anti-tumor immunity. 921 to the antigen-experienced brain. 923 We determined that the rate of recirculation and turnover in a naïve un-manipulated mouse 924 brain is significantly higher than a quiescent antigen-experienced brain ( Figure 6B -C). This 925 increased frequency of circulating T cells in the naïve brain is contrasted with significantly fewer 926 overall T cell counts compared to the quiescent antigen-experienced brain. This argues that as 927 the brain experiences insults and accumulates antigen-specific T cells, the capacity to allow 928 entry to newly arriving T cells is reduced. This is likely due to space restrictions within the tissue 929 parenchyma. One can then visualize the human brain as a collection of many antigen-specific T 930 cells with strong potential for reactivation upon delivery of cognate antigens. This has important 931 implications in the GBM field. We have shown that TRMs can persist in the brain of a glioma-932 bearing mouse even as the tumor progresses (Figure 3 ). Other groups have also shown antigen 933 specific TRMs that accumulated in the brain following viral infections prior to the tumor 934 implantation. Activation of viral specific TRMs in the brain has been shown to improve outcomes 935 in tumor-bearing mice [52, 60] . Interestingly, some peripheral viral infections have recently been 936 shown to also induce robust TRM populations in the brain[61] indicating the human brain TRM 937 pool is likely a collection of expanded T cells against previous neurological insults and some 938 against peripheral stimuli. 939 We demonstrated that frequencies, and to an extent, counts of iv-labeled T cells in the brain 942 increased during antigen-specific reactivation of memory T cells following injection of the 943 immunodominant viral peptide in previously infected mice. Peptide delivery induced reactivation 944 of brain resident memory T cells and the resulting neuroinflammation also induced blood brain 945 barrier (BBB) disruption, which is an event that can lead to upregulation of FC receptors [62] [63] [64] [65] . 946 Upregulation of FC receptors together with enhanced permeability might lead to increased 947 delivery of antibodies to the brain and lead to increased labeling of resident T cells. This is 948 possible as frequencies of iv-labeled T cells in brains of both VP2+aCD8LD and VP2+FTY720 949 groups increased significantly when compared to E7 controls (Figure 7 -8 and Figure S11I ). This 950 increase occurred despite significant depletion of CD8 T cells in the blood of mice treated with 951 aCD8LD or FTY720 (Figure 7P-S and Figure 8I and Figure S11I ). It is also possible that the 952 increase in iv-labeled T cells in the brain during TRM reactivation stems from infiltration from not 953 blood, but unusual reservoirs of T cells near the brain including meninges and brain lymphatics. 954 While the relevance of these sources to brain health, behavior, and recently in GBM and 955 Alzheimer's disease has been explored [66] [67] [68] [69] [70] [71] ; several concepts regarding exact mechanism of 956 exit for T cells from meninges and brain lymphatic into the brain during TRM reactivation remain 957 unclear. For example, we do not currently know the extent aCD8LD strategy depletes T cells in 958 the meninges. Additionally, the S1P receptor requirement for exit out of brain lymphatics and 959 meningis, and whether FTY720 affects these processes remain unknown. We definitively showed that reactivation of brain resident memory T cells is concurrent with 963 profound lymphopenia in the blood compartment ( Figure 6I-K) . In fact, the extent of BBB 964 disruption inversely correlated with the magnitude of this lymphopenia ( Figure 6K ). We have 965 previously showed that neurological insults varying in origin induced peripheral 966 immunosuppression [40] . This peripheral immunosuppression in GBM and stroke patients has 967 been reported before [72] [73] [74] [75] [76] [77] . The study here further indicates that reactivation of memory T 968 immunosuppression. This is important if TRM reactivation as a treatment for GBM is to be 970 tested. GBM is a neurological insult that already induces profound peripheral 971 immunosuppression. Activation of brain resident memory T cells further will likely enhance the 972 immunosuppression. However, our data also indicates that brain TRMs induce severe 973 neuroinflammation in the absence of help from outside T cells and in the presence of 974 lymphopenia (Figure 6-8) . Therefore, using TRMs as therapy might be possible and its efficacy 975 may depend on a delicate balance between neuroinflammation and controlling the peripheral 976 brain-insult induced lymphopenia. Future studies are required to determine ideal strategies to 977 activate brain TRMs while avoiding profound peripheral lymphopenia. 978 Patients with active COVID infection and those who have recovered often present with lingering 980 neurological deficiencies and neuroinflammation of unknown etiology [78] [79] [80] [81] [82] [83] [84] . Two important 981 neuropathologies in human COVID patients have been specifically reported which are blood 982 brain barrier disruption and brain atrophy [78, [85] [86] [87] . We hypothesize that SARS-COV2 infection 983 induces strong immune responses both in the periphery and in the brain. While direct infection 984 of the brain with SARS-COV2 remains controversial [83, [87] [88] [89] [90] [91] . Meanwhile, CD8 T cell 985 infiltration into the brain, TRM reactivation and subsequent neuroinflammation can occur 986 following strong peripheral immune responses that induce blood brain barrier disruption or 987 endothelial cell dysfunction for which there is recent evidence in COVID patients and mouse 988 models [83, 84, [86] [87] [88] [89] [90] [91] . Whether brain was directly infected by the virus, or insulted through 989 within the brain is likely. Our working hypothesis which is supported by data presented in this 991 paper presents the following mechanism for neuropathologies in COVID patients. COVID 992 infection induces strong CD8 T cell activation and infiltration into the brain. Acute responses of 993 CD8 T cells in the brain can lead to neuronal loss and eventual brain atrophy. Following 994 recovery from infection, a population of TRMs reside within the brain. Upon secondary 995 reencounter or strong peripheral inflammatory stimuli, brain TRMs are reactivated, and 996 neuroinflammation and BBB disruption is induced, which can then lead to symptoms including 997 brain fog and memory loss. Brain TRM re-activation due to lingering viral antigens or antigen 998 reencounter can lead to substantial neuroinflammation and hence neuropathology. This 999 neuroinflammation might also partially explain the lymphopenia reported in some COVID 1000 patients [80, 82, 92] . 1001 Together, the analysis of brain TRM cells during homeostasis and following neurological injuries 1002 can help us better understand the balance between pathogenic and protective role of brain 1003 resident memory T cells in health and in disease. Understanding the intricate balance between 1004 neuroprotection and neuropathology during TRM responses will be crucial in designing better 1005 therapeutics to control brain inflammation in autoimmune settings, while enhancing local 1006 immunity to combat CNS tumors. This study further helps move the field forward by recognizing 1007 the unique nature of brain TRM cells through detailed identification of distinct TRM populations as well as determining kinetics of their responses at baseline, acutely, and during the memory 1009 phase. intravenously injected with 6 µg of AF488 conjugated anti Thy1.2 antibodies to in vivo label 2 circulating T cells. Following 3 minutes, mice were euthanized and intracardiac perfusion was 3 performed. Perfused brains and spleens were then harvested and processed and recovered 4 single cells were stained with a panel of antibodies and analyzed on a Cytek Aurora spectral 5 flow cytometer. A) Gating strategy for brain T cells analysis. B) Representative Thy1.2+ brain 6 TCRβ + cells are shown. Majority of brain resident T cells do not stain with the iv-label indicating 7 that brain resident T cells reside within the tissue parenchyma. C) Frequencies of iv-labeled 8 (circulating) T cells in the brain are quantified and found to be very low. Thy1.2 + frequencies are 9 shown out of TCRβ + cells, CD4 T cells, and CD8 T cells. D). Absolute counts of iv + (circulating) 10 and iv -(resident) T cells in the brain are shown. E) Representative Thy1.2 expression (iv-label) 11 levels on spleen TCRβ + cells are shown. Substantial proportion of splenic T cells stain with the 12 iv-label indicating the percentage of splenic T cells within the vasculature. F) Frequencies of iv + 13 T cells in the spleen are quantified. G). Absolute counts of iv + and iv -T cells in the spleen are 14 shown. H) When comparing the brain to the spleen, majority of CD4 and CD8 T cells in the 15 brain express CD69, while a minor percentage in the spleen expresses CD69. I) After gating on 16 CD69 + TCRβ + cells, only a minority in the brain have the iv-label whereas a higher percentage 17 are found within circulation in the spleen. J) Total CD45 + populations from the naïve brain were 18 concatenated and a UMAP was build using FlowJo software. UMAP analysis (using Euclidean 19 methods with default 15 nearest neighbors, minimum distance of 0.5 and 2 set as the number of 20 components) was performed. UMAPs were built based on SSC-A and the following 21 compensated parameters: CD103, CD8, CD44, Ly6G, CD4, Thy1.2 iv, Ly6C, CD69, CD45, 22 CD11b, TCRβ, TCRγδ, B220, and CD62L. Left UMAP shows that total CD45 + cells in the brain 23 are divided between resident and infiltrating cells distinguishable by the level of CD45 24 expression as infiltrating cells express high levels and resident cells express mid-levels of 25 CD45. Right UMAP shows different sub-populations of immune cells within the gated CD45 hi 26 expressing infiltrating immune cells. K) Heat maps further detail levels of expression of 27 residency, memory, and the iv-label on T cell sub populations. L) Absolute numbers of CD69 + 28 CD103 -populations of CD4 and CD8 T cells in the brain is compared between male and female 29 mice. M) Absolute numbers of CD69 + CD103 + populations of CD4 and CD8 T cells in the brain 30 is compared between male and female mice. No significant sex-specific changes in brain TRMs 31 were found. Data are shown as individual mice and mean. Error bars represent standard 32 deviation. Data were tested for normal distribution using Shapiro-Wilk test first. If data was 33 normally distributed, an unpaired t test was used to compare between two groups. If data was 34 not normally distributed, non-parametric test of Mann-Whitney was performed. Designation of 35 symbols is as follows: ns for P > 0.05, * for P ≤ 0.05, ** for P ≤ 0.01, *** for P ≤ 0.001, **** for P 36 ≤ 0.0001. Experiments in A-I were repeated 6 times and results from one experiment is shown. 37 Data from 4 naïve mice is pooled together for the UMAPs analysis in J. Data in L-M is pooled 38 from 4 independent experiments, n=5-6 per group. shown. A GFP-WT parabiosis was performed and mice were euthanized and perfused 60 days 54 later. Brains were harvested, single cell suspensions were made and stained with a panel of 55 antibodies and analyzed on a BD LSRII flow cytometer. B) Frequencies of GFP + and GFP -56 CD4 + CD69 + T cells in the GFP host are shown indicating circulating sources contribute to the 57 brain resident T cell pool. C) Frequency of GFP + and GFP -CD4 + CD69 + in the WT host is shown 58 confirming circulating sources contribute to the brain resident pool. D) Frequencies of GFP + and 59 GFP -CD8 + CD69 + T cells in the GFP host are shown. E) Frequencies of GFP + and GFP -CD8 + 60 CD69 + in the WT host is shown. F-G) Absolute counts of GFP and WT CD4 and CD8 CD69 + UMAP_1 UMAP_2 Error bars represent standard deviation. Data were tested for normal distribution using Shapiro-188 Wilk test first. If data was normally distributed, a one-way anova test was used to compare three 189 or more groups and an Unpaired t test was used to compare between two groups. If data was Experimental design is shown in A) Mice were treated with FT720 twice a day starting one day 218 before TMEV infection. 1 mg of FTY720 was dissolved in 6.6 ml of water and 100 µl of this 219 solution was injected intraperitoneally (IP) into each mouse to achieve at least a 0.5 mg/kg 220 dosage. 100 µl of water was used as control. Naïve mice remained untreated. both TMEV infected groups regardless of treatment when compared to the naïve control. I) 247 Absolute counts of BRDU + proliferating T cells are increased in both TMEV infected groups 248 regardless of treatment when compared to the naïve control. J) Representative flow plot for 249 BRDU expression on gated T cell population is shown. Both TMEV+H 2 O and TMEV+FTY720 250 have higher BRDU incorporating T cells compared to the naïve control. K) Total counts of T 251 cells in the brain of TMEV infected mice are higher than the uninfected control. This increase in 252 T cell counts during infection is independent of FTY720 treatment. L) Geometric mean 253 fluorescence intensity (gMFI) of TCRβ expression on T cells isolated from TMEV infected brains 254 is lower than naïve controls indicating T cells from infected brain, regardless of treatment, are 255 more activated when compared to naïve resident T cells. M) gMFI of side scatter (SSC-A) is 256 higher on T cells from TMEV infected groups compared to controls indicating T cells isolated 257 from TMEV infected brains are more activated, and blasting compared to naïve brain resident T 258 cells. Mice were intracranially infected with TMEV and allowed 8 weeks for resolution of the infection 320 and TRM generation. Mice then received either the TMEV immunodominant peptide VP2 121-130 , 321 or irrelevant peptide E7 on day -2 with respect to euthanasia. Injection of VP2 121-130 peptide will 322 reactivate TMEV specific T cells including TRMs in the brain. On day -1, mice received an 323 intravenous injection AF488 conjugated anti Thy1.2 antibodies to label T cells within the blood 324 circulation at this time point. 24 hours post injection of antibodies, mice were euthanized, 325 perfused and analyzed. A) experimental design is shown. B) Proportions of iv-labeled CD4 and 326 CD8 T cells (T cells that infiltrate brain parenchyma from circulation within 24 hours) decreases 327 in a quiescent antigen-experienced brain compared to the unmanipulated naïve brain. C) 328 Absolute counts of iv-labeled CD4 and CD8 T cells (T cells that infiltrate brain parenchyma from 329 circulation within 24 hours) is significantly increased in a quiescent antigen-experienced brain 330 compared to the unmanipulated naïve brain. Data in B-C are pooled from naïve and E7 controls 331 presented in Figure 7P -S and Figure 8H -I and re-graphed. D-E) Memory T cell reactivation 332 following VP2 121-130 injection induces severe neuroinflammation associated with increased 333 frequencies and numbers of infiltrating immune cells into the brain. E shows representative flow 334 plots. Data in D are pooled from experiments presented in Figure 7I and Figure 8A . F-G) 335 Neuroinflammation following VP2 121-130 injection is also associated with increased blood brain 336 barrier permeability as measured by MRI analysis. T1 weighted MRIs are analyzed for 337 gadolinium leakage into the tissue. H) Total numbers of T cell in the brain of VP2 121-130 injected 338 mice do not change when compared to E7 controls. I) Frequencies of CD4 and CD8 T cells are 339 significantly decreased in the blood of mice following reactivation of brain resident memory T 340 cells. J) Absolute numbers of CD4 and CD8 T cells are significantly decreased in the blood of 341 mice following reactivation of brain resident memory T cells. Data shown in I-J is pooled from all 342 replicates individually presented in Figure S9 . K) The magnitude of blood brain barrier 343 permeability negatively correlates with CD8 T cell counts in the blood. Because total T cells in 344 the brain (H) did not increase, this correlation indicates that severity of damage in the brain is 345 directly associated with the extent of lymphopenia in the blood compartment. L) To determine 346 the extent brain resident antigen-specific memory T cells mediate neuroinflammation upon 347 reactivation, we depleted peripheral T cells but left brain TRMs intact using the following 348 experimental design. Mice were intracranially infected with TMEV. 8 weeks post infection, 349 groups of mice either received low dose anti CD8 depleting antibodies (aCD8LD) or FTY720 350 intraperitoneally. Depleting antibodies were given to mice for two consecutive days while 351 FTY720 was given twice daily beginning on day 4 prior to euthanasia. On day -2, mice received 352 an intravenous injection of either TMEV immunodominant peptide VP2 121-130 , or irrelevant 353 peptide E7. On d-1, mice received an intravenous injection of AF488 conjugated anti Thy1.2 354 antibodies to label T cells in circulation. One day after antibody injection, mice were euthanized 355 and perfused, and analyzed using high parameter flow cytometry. M) CD8 T cells in the blood 356 are depleted efficiently with both aCD8LD and FTY720 strategies. N) CD8 T cells are depleted 357 from spleens of mice in aCD8LD treated group, but not in FTY720 group as FTY720 only blocks 358 exit of T cells into the blood. O) Both aCD8LD and FTY720 strategies leave the brain resident 359 memory T cells intact and hence only affect peripheral T cells (naïve, Comparing E7 vs. 360 E7+aCD8LD vs. E7+FTY720 groups). 361 Grey shaded box represents data from brain, while the red shaded box represents data from 362 blood. In B-D, H, and I-J, data represented is pooled from 4 independent experiments each. F, 363 G, and K represent data from one experiment. used to compared between selected groups. When certain groups were selected for statistical 376 analysis, all comparisons are shown on the graph ns for P > 0.05, * for P ≤ 0.05, ** for P ≤ 0.01, 377 *** for P ≤ 0.001, **** for P ≤ 0.0001. Brains of mice described in Figure 6 in the cohort with FTY720 treatment were analyzed further. A) Reactivation of memory T cells in the brain leads to increased infiltration of CD45 hi immune 486 cells in the brain despite FTY720 treatment as both VP2 and VP2+FTY720 groups had 487 comparable increases in overall immune infiltrate. B) CD11b + MHCII hi (B220 -TCRβ -) myeloid 488 cells infiltrate the brain upon memory T cell reactivation and FTY720 does not impact their 489 infiltration as VP2 and VP2+FTY720 groups had comparable increases in this population. C) 490 Memory T cell reactivation through VP2 121-130 peptide injection induced CD8 T cell activation and 491 blasting in the brain as measured by increased side scatter in the VP2 groups despite FTY720 492 treatment. D) Frequencies of CD8 T cells (left) are comparable amongst infected groups but 493 appear higher than naïve controls. Total absolute counts of CD8 T cells (right) in the brains of 494 mice with previous TMEV infection, regardless of FTY720 treatment, is comparable indicating 495 resident memory T cells in the brain were not affected by FTY720 treatment. All previously 496 infected groups have higher overall counts of CD8 T cells in the brain when compared to naïve 497 unmanipulated controls. E) Representative histograms indicate high expression of MHCII and 498 Ly6C on myeloid cells and increased side scatter on gated CD8 T cells in both VP2 and 499 VP2+FTY720 treated groups compared to respective E7 controls. F) Frequencies of CD4 T 500 cells (left) in E7, VP2, and E7+FTY720 groups are higher than naïve groups but appear 501 comparable amongst groups. Absolute counts of CD4 T cells (right) in the brains of mice with T 502 cell reactivation is higher than control (VP2 vs. E7). However, FTY720 treatment during VP2 121-503 130 injection reduced the overall CD4 T cell counts in the brain (VP2 vs. VP2+FTY720). G) 504 Frequencies (top) of antigen specific CD8 T cells, D b : VP2 121-130 tetramer + , slightly decreased in 505 VP2 121-130 injected group compared to E7 control, while it did not change between E7+FTY720 506 and VP2+FTY720 treated groups. Absolute numbers (bottom) of D b : VP2 121-130 specific CD8 T 507 cells were comparable amongst all previously infected groups. H left) absolute counts of iv-508 labeled CD4 T cells in the brain increased between E7 vs. VP2 groups but did not change 509 between E7+FTY720 and VP2+FTY720 groups. H right) Absolute counts of iv-labeled CD8 T 510 cells in the brain increased between E7 vs. VP2 groups. I left) In VP2 121-130 peptide injected 511 mice, frequencies (top) and numbers (bottom) of D b : VP2 121-130 tetramer + antigen specific CD8 T 512 cells increase when compared to E7 control. FTY720, however, prevented activation of antigen 513 specific CD8 T cells in the spleen. I right) In aCD8LD treated mice, spleens of VP2 121-130 514 injected mice do not support antigen specific expansion of memory T cells due to the extent of 515 depletion. 516 J-L) Reactivation of memory T cells following VP2 121-130 injection induces detectible blood brain 517 barrier permeability as measured by gadolinium leakage in MRI images despite aCD8LD and 518 FTY720 treatment. This indicates that reactivation of brain resident, and not peripheral, memory 519 T cells is responsible for the neuroinflammatory sequalae observed following VP2 121-130 injection 520 in TMEV infected mice. J shows 3D rendering of representative MRI images while K shows 2D 521 representatives. Quantification of MRI analysis is shown in L. In A-D, and F-I, data is pooled data from 2 independent experiments. Data points from male 523 mice are represented as grey while non-grey symbols represent data from female mice. 524 Experiments were performed once in males and once in females. Experiments in J-L were 525 performed once in female mice. Data are shown as individual mice with mean. Error bars 526 represent standard deviation. N=5-9 mice per group. Data were tested for normal distribution 527 using Shapiro-Wilk test first. If data was normally distributed, a one-way Anova test was used to 528 compare between groups. If data was not normally distributed, non-parametric test of Kruskal-529 Wallace test was performed comparing between groups. Either Sidak's test (post Anova for 530 selected pairs) or Dunn's test (post Kruskal-Wallace for selected pairs) or Tukey's test (when 531 comparing every group to every other group) was used to compared between groups. When 532 certain groups were selected for statistics analysis, all comparisons are shown on the graph. If 533 every group was compared to every other group, only statistically significant data are shown on 534 the graph. ns or nothing for P > 0.05, * for P ≤ 0.05, ** for P ≤ 0.01, *** for P ≤ 0.001, **** for P ≤ 535 0.0001. A) gating strategy for T cells is shown in the spleen. Only a minority of spleen T cells express 547 CD69 suggesting the majority is not tissue resident. This is as opposed to the brain where 548 majority of T cells express CD69. B) Gating strategy for CD45 is shown in the brain. CD45 hi 549 cells are infiltrating immune cells including T cells whereas CD45 mid cells are resident immune 550 cells including microglia. Over 90% of CD45 mid cells in the brain express CD11b consistent with 551 them being myeloid in origin . 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 Figure S9 : Both antiCD8 LD and FTY720 treatments deplete peripheral CD8 T cells in the blood during antigen specific T cell reactivation Figure S9 : Both antiCD8LD and FTY720 treatments deplete peripheral CD8 T cells in the Selective expression of the interleukin 7 receptor identifies effector CD8 T cells 1014 that give rise to long-lived memory cells Cutting edge: rapid in vivo killing by memory CD8 T 1016 cells Tissue-Resident Memory T Cells and Fixed Immune Surveillance in Nonlymphoid 1018 Organs Tissue patrol by resident memory CD8(+) T cells in human skin The emerging role of resident memory T cells in protective immunity 1022 and inflammatory disease Tissue-resident memory T cells Tissue-resident memory T cells: local specialists in immune 1026 defence Resident Memory and Recirculating Memory T Cells Cooperate to 1028 Maintain Disease in a Mouse Model of Vitiligo Antigen-independent differentiation and maintenance of effector-like resident 1030 memory T cells in tissues CD4(+) resident memory T cells dominate immunosurveillance and orchestrate 1032 local recall responses Resident memory T cells in the skin mediate durable immunity to melanoma Influenza-specific lung-resident memory T cells are proliferative and 1036 polyfunctional and maintain diverse TCR profiles Antibody-targeted vaccination to lung dendritic cells generates tissue-1038 resident memory CD8 T cells that are highly protective against influenza virus infection. Mucosal 1039 Immunol Cutting Edge: Tissue-Resident Memory T Cells Generated by Multiple 1041 Immunizations or Localized Deposition Provide Enhanced Immunity A Novel Vaccination Strategy Mediating the Induction of Lung-Resident Memory 1044 CD8 T Cells Confers Heterosubtypic Immunity against Future Pandemic Influenza Virus Vaccine-generated lung tissue-resident memory T cells 1047 provide heterosubtypic protection to influenza infection Lung-resident memory CD8 T cells (TRM) are indispensable for optimal cross-1049 protection against pulmonary virus infection Human skin is protected by four functionally and phenotypically discrete 1052 populations of resident and recirculating memory T cells Brain-resident memory CD8(+) T cells induced by congenital CMV infection 1054 prevent brain pathology and virus reactivation Memory T cells in nonlymphoid tissue that provide enhanced local immunity 1056 during infection with herpes simplex virus Airway T cells protect against RSV infection in the absence of antibody CD4 T cells control development and maintenance of brain-resident CD8 T 1060 cells during polyomavirus infection Tissue-resident memory T cells populate the human brain Memory T cells persisting within the brain 1064 after local infection show functional adaptations to their tissue of residence The molecular signature of tissue resident memory CD8 T cells isolated from 1067 the brain Derivation and maintenance of virtual memory CD8 T 1069 cells Cutting edge: Central memory CD8 T cells in aged mice are virtual memory cells The antigen-specific CD8+ T cell repertoire in unimmunized mice includes 1073 memory phenotype cells bearing markers of homeostatic expansion Virtual memory cells make a major contribution to the response of aged 1076 influenza-naive mice to influenza virus infection Virtual memory CD8 T cells display unique functional properties Age-Related Decline in Primary CD8(+) T Cell Responses Is Associated with the 1080 Development of Senescence in Virtual Memory CD8(+) T Cells Virtual memory T cells develop and mediate bystander protective immunity in an 1083 IL-15-dependent manner The compartmentalized inflammatory response in the multiple 1085 sclerosis brain is composed of tissue-resident CD8+ T lymphocytes and B cells Resident memory T cells in human health and disease Non-equivalent antigen presenting capabilities of dendritic cells and 1089 macrophages in generating brain-infiltrating CD8 (+) T cell responses Immunomodulation Mediated by Anti-angiogenic Therapy Improves CD8 T Cell 1092 Immunity Against Experimental Glioma Prevalent class I-restricted T-cell response to the Theiler's virus epitope 1094 Db:VP2121-130 in the absence of endogenous CD4 help, tumor necrosis factor alpha, gamma 1095 interferon, perforin, or costimulation through CD28 Superior isolation of antigen-specific brain infiltrating T cells using 1097 manual homogenization technique Brain cancer induces systemic immunosuppression through release of non-1099 steroid soluble mediators Parabiosis in Mice: A Detailed Protocol Perforin Expression by CD8 T Cells Is Sufficient To Cause Fatal Brain Edema 1103 during Experimental Cerebral Malaria GM-CSF inhibition reduces cytokine release syndrome and 1107 neuroinflammation but enhances CAR-T cell function in xenografts Modulatory effects of perforin gene dosage on pathogen-associated 1110 blood-brain barrier (BBB) disruption Cutting edge: intravascular staining redefines lung CD8 T cell responses Virus-specific memory T cells populate tumors and can be repurposed for 1116 tumor immunotherapy Immune-cell crosstalk in multiple sclerosis Antigen-Specific CD8+ T Cells Mediate a Peptide-Induced Fatal Syndrome. 1119 The Journal of Immunology The immune modulator FTY720 targets sphingosine 1-phosphate receptors. 1121 The sphingosine 1-phosphate receptor modulator 1123 fingolimod as a therapeutic agent: Recent findings and new perspectives Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor 1126 agonists Microglia and Perivascular Macrophages Act as Antigen Presenting Cells to 1128 Pro-inflammatory functions of astrocytes correlate 1130 with viral clearance and strain-dependent protection from TMEV-induced demyelinating disease. 1131 Virology Preservation of neurologic function during inflammatory 1133 demyelination correlates with axon sparing in a mouse model of multiple sclerosis. 1134 Neuroscience Modulatory effects of perforin gene dosage on pathogen-associated 1136 blood-brain barrier (BBB) disruption Age-Associated Resident Memory CD8 T Cells in the Central Nervous System 1138 Are Primed To Potentiate Inflammation after Ischemic Brain Injury Neuroinflammation in the normal aging hippocampus Functional virus-specific memory T cells survey glioblastoma Peripherally induced brain tissue-resident memory CD8(+) T cells mediate 1145 protection against CNS infection Modulators of IgG penetration through the blood-brain barrier: 1147 Implications for Alzheimer's disease immunotherapy Blood-brain barrier disruption during spontaneous canine visceral 1149 leishmaniasis Adsorptive-Mediated Endocytosis of Sulfo-Cy5-Labeled IgG 1151 Causes Aberrant IgG Processing by Brain Endothelial-Like Cells Delivery of chemotherapeutics across the blood-brain barrier: challenges 1154 and advances Meningeal lymphatic dysfunction exacerbates traumatic brain injury 1156 pathogenesis Functional aspects of meningeal lymphatics in ageing and Alzheimer's 1158 disease Neuroimmunology in 2017: The central nervous system: privileged by 1160 immune connections Understanding the functions and relationships of the glymphatic system and 1162 meningeal lymphatics VEGF-C-driven lymphatic drainage enables immunosurveillance of brain tumours. 1164 Nature The CNS Immune-Privilege Goes Down the Drain(age) Sequestration of T cells in bone marrow in the setting of glioblastoma 1168 and other intracranial tumors Systemic immune suppression in glioblastoma: the interplay between 1170 CD14+HLA-DRlo/neg monocytes, tumor factors, and dexamethasone Brain Ischemia Suppresses Immunity in the Periphery and Brain via Different 1173 Neurogenic Innervations Translocation and dissemination of commensal bacteria in post-stroke 1175 infection Functional Innervation of Hepatic iNKT Cells 1177 Is Immunosuppressive Following Stroke Post-injury immunosuppression and secondary infections are caused by an AIM2 1179 inflammasome-driven signaling cascade Brain MRI in SARS-CoV-2 pneumonia patients with newly developed 1181 neurological manifestations suggestive of brain involvement Neurological Manifestations in COVID-19 Infection: A Systematic Review 1184 and Meta-Analysis Longitudinal Analysis of T and B Cell Receptor Repertoire Transcripts Reveal 1186 Dynamic Immune Response in COVID-19 Patients. Front Immunol Psychiatric and neuropsychiatric presentations associated with severe 1188 coronavirus infections: a systematic review and meta-analysis with comparison to the COVID-19 1189 pandemic Persistent symptoms and lab abnormalities in patients who recovered from 1191 COVID-19. Sci Rep Neuroinvasion, neurotropic, and neuroinflammatory events of SARS-CoV-2: 1193 understanding the neurological manifestations in COVID-19 patients Neuroinvasion of SARS-CoV-2 in human and mouse brain SARS-CoV-2 is associated with changes in brain structure in UK Biobank. 1198 Nature Endothelial cell damage is the central part of COVID-19 and a mouse model 1200 induced by injection of the S1 subunit of the spike protein COVID-19-related anosmia is associated with viral persistence and 1205 inflammation in human olfactory epithelium and brain infection in hamsters Mild respiratory SARS-CoV-2 infection can cause multi-lineage 1208 cellular dysregulation and myelin loss in the brain Subjective neurological symptoms frequently occur in patients with SARS-CoV2 1210 infection Divergent and self-reactive immune responses in the CNS of COVID-19 patients 1212 with neurological symptoms Longitudinal characteristics of lymphocyte responses and cytokine profiles in the 1214 peripheral blood of SARS-CoV-2 infected patients. EBioMedicine within the brain. M) Absolute counts of total CD8 T cells in the brain are shown and found to 69 increase as a function of age. N) Data from a representative mouse demonstrate increased CD8 70 CD103 + T cells in 1 year old mice. O) CD8 CD69 + CD103 + T cells are quantified in the brain. 71Data are shown as individual mice with mean. Error bars represent standard deviation. Data 72were tested for normal distribution using Shapiro-Wilk test first. If data was normally distributed, 73 a one-way anova test was used to compare between groups. If data was not normally 74 distributed, non-parametric test of Kruskal-Wallace was performed. For aging studies in H- O, 75 only results that were significant are shown on the graph. Designation of symbols is as follows: 76 ns or not shown for P > 0.05, * for P ≤ 0.05, ** for P ≤ 0.01, *** for P ≤ 0.001, **** for P ≤ 0.0001. Aging experiments were repeated twice, and similar results were obtained. N=3-5 per group . 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 growing glioma through expansion. 104 Experiments with intracranial PBS injection: Mice were intracranially injected with 2 µl of sterile 105 PBS and euthanized and perfused at various time points post injections. Numbers of CD69 + 106 CD103 -CD4 and CD8 T cells were quantified using a LSRII flow cytometer. A) Expansion of 107CD4 + CD69 + CD103 -cells on day 1 post injection is shown. B) CD4 + CD69 + CD103 -cells are 108 quantified at various time points post PBS injection in the brain. C) Expansion of CD8 + CD69 + 109 CD103 -cells on day 1 post injection is shown. D) CD8 + CD69 + CD103 -cells are quantified at 110 various time points post PBS injection in the brain. N=10-33 to compare naïve to day 1 post 111 PBS injection. N=5-10 for days 3-28 post PBS injection time points. Data are shown as 112individual mice with mean. Error bars represent standard deviation. Data were tested for normal 113 distribution using Shapiro-Wilk test first. If data was normally distributed, a one-way anova test 114 was used to compare three or more groups and an Unpaired t test was used to compare 115 between two groups. If data was not normally distributed, non-parametric test of Wallace test was performed if comparing 3 or more groups and a Mann-Whitney test was used 117when comparing between two groups. Only results that were significant are shown on the 118 graph. ns or not shown for P > 0.05, * for P ≤ 0.05, ** for P ≤ 0.01, *** for P ≤ 0.001, **** for P ≤ 119 0.0001. These experiments were repeated 2-3 times and similar results were obtained. Data are 120 pooled from all experiments. Experiments with intracranial gliomas: Numbers of CD4 + and CD8 + CD69 + CD103 -and CD103 + 122 resident memory T cells were quantified at various times points post implantation of a luciferase 123 expressing GL261 glioma cell line in the brain. Bioluminescence imaging was performed and 124 mice without a detectable tumor were excluded from analysis. E) Absolute numbers of CD4 + 125 CD69 + CD103 -T cells in the brain expand beginning on day 7 and remain detectable at various 126 time point post GL261 implantation. F) Indicates that absolute numbers of CD4 + CD69+ CD103 + 127T cells in the brain begin to increase beginning at day 14 post tumor implant. These TRM cells 128 remain detectable at various time point post GL261 implantation. G) Shows absolute numbers 129 of CD8 + CD69 + CD103 -T cells in the brain accumulate at various time point post GL261 130 implantation similar to CD4 TRM cells. H) Absolute numbers of CD8 + CD69 + CD103 + T cells in 131 the brain becomes detectable and expands between days 14-28 post GL261 implantation. Data 132for E-H was generated using a LSRII flow cytometer. N=15-25. Data are shown as individual 133 mice with mean. Error bars represent standard deviation. Data were tested for normal 134 distribution using Shapiro-Wilk test first. If data was normally distributed, a one-way anova test 135 was used to compare between groups. If data was not normally distributed, non-parametric test 136of Kruskal-Wallace test was performed comparing between groups. Only results that were 137 significant are shown on the graph. ns or not shown for P > 0.05, * for P ≤ 0.05, ** for P ≤ 0.01, 138 *** for P ≤ 0.001, **** for P ≤ 0.0001. I) Mice with intracranial gliomas were euthanized and 139 perfused at moribund. Brain infiltrating cells were isolated and analyzed using high parameter 140 flow cytometry using a Cytek Aurora spectral cytometer. As control, naïve mice were used. I) 141CD45 + populations from glioma-bearing mice were concatenated and a UMAP was built. UMAP 142analysis ( Brains of mice described in Figure 6 in the cohort with aCD8LD treatment were analyzed 383 further. A) Brains of mice from the cohort that included aCD8LD treatment was further analyzed 384 using unbiased UMAP analysis (using Euclidean methods with default 15 nearest neighbors, 385 minimum distance of 0.5 and 2 set as the number of components). UMAPs were built based on 386 SSC-A and the following compensated parameters: CD103, MHCII, CD8, CD44, Ly6G, CD4, 387Thy1.2 iv, Ly6C, CD69, CD45, CD11b, TCRβ, Db: VP2 tetramer, B220, and CD62L. Naïve mice 388were excluded from this analysis. Following downsampling of data, equal numbers of CD45 + 389 cells from all previously infected groups of mice were concatenated into one file and UMAP 390 analysis was performed. E7 and E7+aCD8LD groups cluster together while VP2 and 391VP2+aCD8LD groups appear similar. This further indicates that majority of changes within the 392 brain during antigen specific reactivation are independent of peripheral CD8 T cells. B) Total 393 CD45 + brain infiltrate is further divided into resident (CD45 mid ) and infiltrating (CD45 hi ) immune MHCII upregulation on a sub-population. The MHCII hi population of brain resident immune cells 397is being contributed only from VP2 groups and is missing from control E7 groups indicating this 398 change is specific to T cell reactivation and independent of aCD8LD treatment. D) The CD45 hi 399 population is further analyzed and shown to be comprised of B cells, T cells, CD11b + , and 400CD11b -major populations of infiltrating immune cells. E) The TCRβ + population is comprised of 401 CD4 T cells, D b : VP2 121-130 + and D b : VP2 121-130 -populations of CD8 T cells in the brain. F) The 402CD11b + population is mostly comprised of MHCII hi and MHCII low , and Ly6G + MHCIIpopulations. 403 G) Manual gating on the TCRβ -B220 -population confirms that the major infiltrating cell following 404 VP2 121-130 peptide injection, regardless of aCD8LD treatment, is inflammatory monocytes 405 identified by CD11b + and MHCII hi expression. H) Expression levels of activation, residency, and 406 memory markers on the brain resident CD8 T cell population is evaluated. I) Reactivation of 407 memory T cells in the brain leads to increased infiltration of CD45 hi immune cells into the brain 408 despite aCD8LD treatment as both VP2 and VP2+aCD8LD groups had comparable increases in 409 overall immune infiltrate. J) CD11b + MHCII hi (B220 -TCRβ -) myeloid cells infiltrate the brain upon 410 memory T cell reactivation and aCD8LD does not impact their infiltration as VP2 and 411VP2+aCD8LD groups had comparable increases in this population. K) Representative 412 histograms indicate high expression of MHCII and Ly6C on myeloid cells and increased side 413 scatter in CD8 T cells in both VP2 and VP2+aCD8LD treated groups compared to E7 controls. 414 L) Memory T cell reactivation through VP2 121-130 peptide injection induced CD8 T cell activation 415and blasting in the brain as measured by increased side scatter in the VP2 groups despite 416 aCD8LD treatment. M) Frequencies of CD4 T cells (left) in previously infected groups are 417 comparable but are higher than naïve unmanipulated controls. Absolute counts of CD4 T cells 418(right) in the brains of mice with T cell reactivation is higher than naïve controls, regardless of 419 treatment. N) Frequencies of CD8 T cells (left) in the brain are decreased in VP2+aCD8LD 420 compared to E7+aCD8LD indicating a portion of infiltrate was contributed from the circulation 421 and was effectively depleted by this treatment. Absolute numbers of CD8 T cells (right) 422 between E7 and E7+aCD8LD groups are comparable indicating that CD8 T cell depletion did 423 not significantly impact the brain resident memory T cell pool in the absence of antigen specific 424 reactivation. However, when comparing E7+aCD8LD to VP2+aCD8LD groups, we detected a 425 significant decrease in total CD8 T cell counts indicating this difference is due to the absence of 426 circulating T cells that infiltrate upon reactivation. E7, VP2, and E7+aCD8LD groups all had 427higher numbers of CD8 T cells in their brains when compared to naïve mice. O) Frequencies 428(left) of antigen specific CD8 T cells slightly decreased in VP2 121-130 injected mice compared to 429 E7 control, while it did not change between E7+aCD8LD and VP2+aCD8LD groups. Absolute 430 numbers of D b : VP2 121-130 specific CD8 T cells were comparable amongst E7, VP2, and 431E7+aCD8LD groups. However, total numbers of antigen specific CD8 T cells decreased in the 432VP2+aCD8LD group indicating a portion of the infiltrate during antigen specific reactivation 433 originates from circulation. P) Frequencies and absolute counts (Q) of iv-labeled CD4 T cells 434increased between E7 and VP2 and between E7+aCD8LD and VP2+aCD8LD groups. R) 435Frequencies and absolute counts (S) of iv-labeled CD8 T cells increased between E7 and VP2 436and between E7+aCD8LD and VP2+aCD8LD groups. However, the level of increase in 437VP2+aCD8LD treated group was lower than VP2 alone group due to depletion of peripheral 438 CD8 T cells. In I-J and L-O, data is pooled data from 2 independent experiments. Experiments 439were performed once in males and once in females. Data points from male mice are 440represented as grey while non-grey symbols represent data from female mice. Data are shown 441 as individual mice with mean. Error bars represent standard deviation. N=5-9 mice per group. were homogenized and subjected to Percoll centrifugation before flow cytometry (using LSRII 599 cytometer). A) Gating strategy for brain resident memory T cells is shown for naïve mice. Brain 600 resident memory T cells are CD69 + CD103 -CD44 hi CD62L low and can express CD49a. B) 601Frequency and C) numbers of brain resident CD69 + CD103 -CD4 and CD8 T cells are 602 quantified. Mice varying in age and sex all harbor TRM cell populations in their brain. D) 603Frequency of CD44 numbers of CD45 hi immune cells in the brain is shown to increase in mice beginning at 6 651 months of age. D) Frequencies and E) numbers of TCRβ + T cells is shown to increase in the 652 brain of mice with age. F-G) Frequencies of CD69 + CD103 -amongst CD8 and CD4 T cells is 653shown to be constant. H) Frequencies of CD69 + CD103 + of total CD8 T cells is shown in aging 654 mice. I) absolute numbers of CD4 + CD69 + CD103 + T cells do not change with age. Data are 655shown as individual mice with mean. Error bars represent standard deviation. Data were tested 656for normal distribution using Shapiro-Wilk test first. If data was normally distributed, a one-way 657anova test was used to compare between groups. If data was not normally distributed, non-658 parametric test of Kruskal-Wallace test was performed. Only results that were significant are 659shown on the graph. Designation of symbols is as follows: ns or not shown for P > 0.05, * for P 660 ≤ 0.05, ** for P ≤ 0.01, *** for P ≤ 0.001, **** for P ≤ 0. Figure S4 : Upregulation of CD103 on brain resident memory T cells may be delayed following a physical injury to the brain Figure S4 : Upregulation of CD103 on brain resident memory T cells may be delayed 699 following a physical injury to the brain 700As shown in A, absolute numbers of CD4 + CD69 + CD103 + (purple line) and CD8 + CD69 + 701 CD103 + (blue line) TRM populations in the brain appear to increase one month post PBS 702 injection. One-way anova with Dunn's multiple comparisons test was used to compare control 703 mice to PBS injected mice at various time points. N=5-27. Data is shown as average and 704standard deviation (only above). ns p ≥ 0.05, * p= 0.01 to 0.05, ** p=0.001 to 0.01, ***p=0.0001 705 to 0.001, **** p< 0.0001. 706 B) Mice with intracranial gliomas were euthanized and perfused at moribund. Brain infiltrating 707 cells were isolated and analyzed using high parameter flow cytometry using a Cytek Aurora 708 spectral cytometer. As control, naïve mice were used. Representative histograms demonstrate 709brain TRMs in glioma-bearing mice express higher levels of CD69 and CD103 compared to 710naïve TRMs while their expression of CD44 and CD62L remain comparable. Blood was analyzed from mice treated and described in Figure 5 . These mice were treated with 802 FTY720, or water as described in Figure 5 legend. These are the exact same mice as presented 803in Figure 5 . Blood analysis is shown here. A) Total numbers of cells in 100 µl of blood did not 804 change between groups. B) Frequencies and numbers of TCRβ + cells decreased in blood of 805 FTY720 treated mice when compared to controls indicating this treatment significantly reduces 806T cells in circulation. C) Frequencies and numbers of CD4 T cells was significantly reduced as 807 a result of FTY720 treatment. D) Frequencies and numbers of CD8 T cells was significantly 808reduced as a result of FTY720 treatment. E) Frequencies and numbers of Thy1.2 + iv-labeled T 809 cells in blood also reduced in both H 2 O and FTY720 treated TMEV infected groups when 810 compared to naïve controls indicating brain injury alone reduces circulating T cells. F) 811Frequencies and numbers of B cells in the blood was reduced in both H 2 O and FTY720 treated 812TMEV infected groups when compared to naïve controls indicating brain injury reduces 813 circulating B cells. G) Frequencies and numbers of γδT cells were significantly reduced as a 814result of FTY720 treatment. Data are shown as individual mice with mean. Error bars represent 815standard deviation. Data were tested for normal distribution using Shapiro-Wilk test first. If data 816was normally distributed, a one-way anova test was used to compare between groups. If data 817was not normally distributed, non-parametric test of Kruskal-Wallace test was performed 818comparing between groups. Comparisons were made between each group and every other 819 group. ns or not shown for P > 0.05, * for P ≤ 0.05, ** for P ≤ 0.01, *** for P ≤ 0.001, **** for P ≤ 820 0.0001. N=9-10 per group. Data presented is pooled from two independent experiments. This 821 experiment was repeated at least 4 times and similar results were obtained . 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 These mice were treated with FTY720 or water and intracranially infected with TMEV as 853 described in Figure 5 legend. Analysis of brain is shown. These are the exact same mice as 854 presented in Figure 5 . Extra analysis of brain is shown here. presented out of CD45 hi infiltrating immune cells is slightly reduced in both TMEV+H 2 O and 856TMEV+FTY720 groups compared to naïve controls. This is because monocytes represent the 857 majority of the infiltrating immune cells at this time. B) When frequencies of T cells are 858 calculated out of total live cells, however, both TMEV infected groups, regardless of FTY720 859 treatment, have higher T cell frequencies when compared to naïve controls. C) Total numbers of 860CD4 Thy1.2 iv + T cells decrease in TMEV infected groups compared to naïve controls. Numbers of 873 iv-labeled T cells accumulated in the brain within 24 hours does not change between naïve and 874 TMEV infected groups. This further demonstrates that the expansion of resident memory T cells 875following TMEV infection is dependent on in situ proliferation within the brain and is not due to 876infiltration of circulating T cells. Each symbol represents one mouse. Open symbols represent 877 data from male mice. Data were tested for normal distribution using Shapiro-Wilk test first. If 878 data was normally distributed, a one-way anova test was used to compare between groups. If 879 data was not normally distributed, non-parametric test of Kruskal-Wallace test was performed 880 comparing between groups. Comparisons were made between each group and the naïve group. 881 ns for P > 0.05, * for P ≤ 0.05, ** for P ≤ 0.01, *** for P ≤ 0.001, **** for P ≤ 0.0001. N=9-10 per 882group. Data presented is pooled from two independent experiments. This experiment was 883 repeated at least 4 times and similar results were obtained. Figure S8 : Brain CD4 T cells ae comparable in previously infected brains that are currently quiescent (E7 groups) despite FTY720 treatment A B C Spleen Blood Brain Figure S8 : Brain CD4 T cells are comparable in previously infected brain that are 903 currently quiescent (E7 groups) despite FTY720 treatment. Experimental design is the same as described in Figure 6 . A) CD4 T cells are depleted from 905 blood of FTY720 treated mice, while they are comparable in aCD8LD treated mice when naïve, 906 E7, E7+aCD8LD, and E7+FTY720 groups are compared. B) absolute counts of splenic CD4 T 907 cells are comparable between naïve, E7, E7+aCD8LD, and E7+FTY720 treated groups. C) 908absolute counts of CD4 T cells in the brain are comparable between E7, E7+aCD8LD, and 909 E7+FTY720 treated groups, while all previously infected E7 treated groups have higher CD4 t 910cell count compared to naïve controls. Data is pooled from 2-4 experiments (naïve and E7are 911 pooled from 4 independent experiments while aCD8LD and FTY720 groups are pooled from 2 912 independent experiments each). This data is also presented in Figure S9 -S11 individually. Each 913 symbol represents one mouse. Data were tested for normal distribution using Shapiro-Wilk test 914first. If data was normally distributed, a one-way anova test was used to compare between 915groups. If data was not normally distributed, non-parametric test of Kruskal-Wallace test was 916performed comparing between groups. Comparisons were made between each group and the 917 naïve group. ns for P > 0.05, * for P ≤ 0.05, ** for P ≤ 0.01, *** for P ≤ 0.001, **** for P ≤ 0. Experimental design is as described in Figure 6 . These data are blood results from same mice 956 whose brains were analyzed for Figures 6-7 . Blood of mice were analyzed using high parameter 957 flow cytometry using a Cytek Aurora cytometer. A) FTY720 treatment reduces T cell circulation 958 in blood. A representative flow plot is shown. B) FTY720 treatment reduced overall numbers of 959T cells in the blood. Of note, total T cell counts were also reduced in VP2 121-130 injected mice 960indicating lymphopenia. C) Total numbers of CD8 T cells are reduced in the blood of FTY720 961 treated mice. D) Total CD4 T cell counts are reduced in the blood of FTY720 treated mice. E) 962Frequencies of CD8 T cells are reduced in the blood of FTY720 treated mice compared to 963 controls. Data from naïve, E7, and VP2 groups shown in C-F, and I-L are combined and re-graphed to 973show the extent of lymphopenia in blood in Figure 6K -N. 974In B-F, and H-L, data is pooled from 2 independent experiments. Experiments were performed Experimental design is as described in Figure 6 . These data are results from spleens of the 1007 same mice whose brains were analyzed for Figures 6-7 groups. Tukey's test was then used to compare each group to every other group. Only 1030 statistically significant results are shown on the graph. nothing for P > 0.05, * for P ≤ 0.05, ** for 1031 P ≤ 0.01, *** for P ≤ 0.001, **** for P ≤ 0.0001 . 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 antigen specific T cell activation 1057Experimental design is as described in Figure 6 . These data are results from spleens of the 1058 same mice whose brains were analyzed for Figures 6-7 . Spleens of mice were analyzed using 1059 high parameter flow cytometry. A) Absolute counts of splenocytes are mostly comparable 1060 between groups. B) Frequencies (left) and absolute numbers (right) of T cells in the spleen are 1061 comparable between groups indicating FTY720 treatment does not reduce T cell counts in the 1062 spleen. C) Frequencies (left) and absolute numbers (right) of CD4 T cells in the spleen are 1063 comparable between groups indicating FTY720 treatment does not reduce CD4 T cell counts in 1064 the spleen. D) Frequencies (left) and absolute numbers (right) of CD8 T cells in the spleen are 1065 comparable between groups indicating FTY720 treatment does not reduce CD8 T cell counts in 1066 the spleen. E) Frequencies (top) and numbers (bottom) of iv-labeled CD4 1067(accumulated/recirculating from circulation in 24 hours) are reduced in VP2 and VP2+FTY720 1068groups mice compared to E7 control. F) Frequencies (top) and numbers (bottom) of iv-labeled 1069 CD8 (accumulated/recirculating from circulation in 24 hours) T cells in the spleen are largely 1070 unchanged amongst groups. G) Frequencies (left) and absolute counts (right) of CD11b + 1071 MHCII hi myeloid cells are largely unchanged in the spleen between all groups. H) In VP2 121-130 1072 peptide injected mice, frequencies of D b : VP2 121-130 tetramer + antigen specific CD8 T cells 1073increase when compared to E7 control. FTY720, however, prevents activation of antigen 1074 specific CD8 T cells in the spleen. I left) Frequencies of iv-labeled CD4 T cells in the brain did 1075 not change between E7 and VP2 and between E7+FTY720 and VP2+FTY720 groups. I right) 1076Frequencies of iv-labeled CD8 T cells in the brain increased between E7 vs. VP2 and between 1077 E7+FTY720 vs. VP2+FTY720 groups. Shaded grey area represents data presented from brain 1078tissue. Data is pooled from 2 independent experiments. Experiments were performed in male 1079 mice once and then repeated in females. Data points from male mice are represented as grey 1080while non-grey symbols represent data from female mice. Data are shown as individual mice 1081 with mean. Error bars represent standard deviation. N=3-9 mice per group. Data were tested for 1082 normal distribution using Shapiro-Wilk test first. If data was normally distributed, a one-way 1083Anova test was used to compare between groups. If data was not normally distributed, non-1084 parametric test of Kruskal-Wallace was performed comparing between groups. Tukey's test was 1085 then used to compare each group to every other group. Only statistically significant results are 1086shown on the graph. Ns or nothing for P > 0.05, * for P ≤ 0.05, ** for P ≤ 0.01, *** for P ≤ 0.001, 1087 **** for P ≤ 0.0001. 1088 1089