key: cord-0776819-x6crg57j authors: Peter, Lena; Wendering, Désirée Jacqueline; Schlickeiser, Stephan; Hoffmann, Henrike; Noster, Rebecca; Wagner, Dimitrios Laurin; Zarrinrad, Ghazaleh; Münch, Sandra; Picht, Samira; Schulenberg, Sarah; Moradian, Hanieh; Mashreghi, Mir-Farzin; Klein, Oliver; Gossen, Manfred; Roch, Toralf; Babel, Nina; Reinke, Petra; Volk, Hans-Dieter; Amini, Leila; Schmueck-Henneresse, Michael title: Tacrolimus-resistant SARS-CoV-2-specific T-cell products to prevent and treat severe COVID-19 in immunosuppressed patients date: 2022-02-26 journal: Mol Ther Methods Clin Dev DOI: 10.1016/j.omtm.2022.02.012 sha: e6996ee8823004bcb2f31c6006f2e65c1d37129e doc_id: 776819 cord_uid: x6crg57j Solid organ transplant (SOT) recipients receive therapeutic immunosuppression that compromises their immune response to infections and vaccines. For this reason, SOT patients have a high risk of developing severe COVID-19 and an increased risk of death from SARS-CoV-2 infection. Moreover, the efficiency of immunotherapies and vaccines is reduced due to the constant immunosuppression in this patient group. Here, we propose adoptive transfer of SARS-CoV-2-specific T-cells made resistant to a common immunosuppressant, Tacrolimus, for optimized performance in the immunosuppressed patient. Using a ribonucleoprotein approach of CRISPR-Cas9 technology, we have generated Tacrolimus-resistant SARS-CoV-2-specific T-cell products from convalescent donors and demonstrate their specificity and function through characterizations at the single cell level, including flow cytometry, scRNA-CITE and TCR sequencing analyses. Based on the promising results, we aim for clinical validation of this approach in transplant recipients. Additionally, we propose a combinatory approach with Tacrolimus, to prevent an overshooting immune response manifested as bystander T-cell activation in the setting of severe COVID-19 immunopathology, and Tacrolimus-resistant SARS-CoV-2-specific T-cell products, allowing for efficient clearance of viral infection. Our strategy has the potential to prevent severe COVID-19 courses in SOT or autoimmunity settings and to prevent immunopathology whilst providing viral clearance in severe non-transplant COVID-19 cases. ). Naïve control donors were defined as being seronegative 152 for IgG and IgA targeting SARS-CoV-2 Spike S1 as detected by ELISA ( Supplementary Fig. 1A , 153 B). The SARS-CoV-2-specific T-cell responses were evaluated by stimulating PBMCs with 154 overlapping peptide pools (15-mers, 11aa overlap), encompassing the amino acid sequence of 155 structural proteins (NCAP, Spike S1 + S2, VEMP, VME1) and accessory proteins (AP3a, NS6, 156 NS7a, NS7b, NS8, ORF9b, ORF10, Y14) of SARS-CoV-2. Cells were stimulated for 16 h to 157 analyze the reactivity of T-cells by flow cytometry using a set of markers for T-cell activation 158 and effector cytokine production ( Fig. 1A-C) . In all SARS-CoV-2 convalescent donors we 159 observed upregulation of CD137 (4-1BB) and production of either IFN-, TNF- or both 160 cytokines ( Fig. 1D-G) , which is consistent with effector T-cell activation following SARS-CoV-161 2-specific stimulation. Furthermore, we found SARS-CoV-2-specific CD4 + and CD8 + T-cells 162 responded to different viral antigens. CD4 + T-cells predominantly reacted to NCAP, Spike S1 163 and S2 and to a lesser extent to VME1 (Fig. 1D, E) . In contrast, CD8 + We have previously described a vector-free protocol for electroporation and ribonucleoprotein 173 (RNP)-based KO of FKBP12 to generate Tac-resistant antiviral TCPs, which was now applied 174 to generate SARS-CoV-2-specific Tac-resistant TCPs from eight convalescent donors (CD 1-3, 175 15, 17-20 [Supplementary Table 1] ). 63 We isolated SARS-CoV-2-specific T-cells with a high 176 purity from PBMC, based on their IFN- secretion after stimulation with SARS-CoV-2 peptide 177 pools (NCAP, Spike S1 + S2, VEMP, VME1, AP3a, NS6, NS7a, NS7b, NS8, ORF9b; ORF10, Y14) 178 ( Fig. 2A-C) . These SARS-CoV-2-specific T-cells were expanded and split on day 7. One half of 179 the culture was subsequently electroporated with RNP complexes of Cas9 and a single guide 180 (sg)RNA, whereas the other half served as the unmodified control ( Fig. 2A) . The unmodified 181 and FKBP12 KO SARS-CoV-2-specific T-cells were expanded for two more weeks ( Fig. 2A ). Expansion rates and cell yields were similar for both fractions at days 14 and 21, illustrated in 183 Figures 3A and 3D . Similarly, the expansion rates and total counts of CD4 + the TCPs at day 21 were comparable between unmodified and FKPB12 KO fractions (Fig. 3B, E) . Although on day 0 we found the CD4 + /CD8 + presented with an overall high frequency of TNAIVE, followed by TEMRA and TEM, whereas TCM as 227 well as TSCM were present at lower frequencies (Fig. 4E, G) . After SARS-CoV-2-specific 228 enrichment, the CD8 + T-cells contained a high proportion of TEMRA and TEM. On day 21 of 229 expansion, SARS-CoV-2-specific CD8 + T-cells expressed a similar phenotype, with the majority 230 being TEMRA and TEM in both bulk and CD137 + & IFN- + T-cells (Fig. 4F, H) . Overall, the FKBP12 KO did not have a major effect on T-cell differentiation and the subset composition of the TCPs 232 nor did it confer a discernible advantage to any particular subset. 233 234 Effector cytokine production in the presence of tacrolimus is rescued by FKBP12 KO in SARS- CoV-2-specific T-cell products 236 To demonstrate both efficacy against SARS-CoV-2 and to confirm Tac-resistance of our 237 FKBP12 KO TCPs, we re-stimulated the distinct TCPs with SARS-CoV-2 peptide pools and Since targeted elimination of virus-infected cells is an essential characteristic of antiviral T-274 cells, we tested the cytotoxic killing capacity of SARS-CoV-2-specific TCPs. Although short-275 term incubation with the IS Tac showed a strong effect on antiviral cytokine production in 276 TCPs (Fig. 5) , we found that the T-cell-mediated cytotoxic killing of target cells loaded with 277 SARS-CoV-2 peptides was not affected by short-term treatment with Tac, neither in 278 unmodified nor in FKBP12 KO TCPs ( Fig. 6A-C) . To identify the dominant antigens driving T-cell-mediated killing of SARS-CoV-2 peptide 280 loaded target cells, we analyzed the killing capacity of TCPs with regard to individual antigens 281 of SARS-CoV-2. Both unmodified control and FKBP12 KO TCPs showed efficient killing of NCAP as well as AP3a peptide loaded target cells, followed by target cells loaded with VME1 283 and Spike S1 and S2 peptides (Fig. 6D ). We also found T-cell-mediated killing toward target 284 cells loaded with peptides from the accessory proteins NS7a and ORF9b for some of the TCPs, 285 but not towards target cells loaded with peptides from the remaining accessory proteins NS6, 286 NS7b, NS8, ORF10 and Y14 or the structural protein VEMP (Fig. 6D) . This was in contrast to 287 antiviral cytokine production, which we did not observe in response to accessory protein- we detected the most efficient killing of NCAP, AP3a and SARS-CoV-2 peptide pool loaded 295 target cells, followed by target cells loaded with VME1, Spike S1 and Spike S2 peptide pools 296 TCPs can recognize and kill these cells, we co-transfected target cells with a plasmid encoding 299 the full sequence of the SARS-CoV-2 wild-type Spike protein (pSpike) ( Supplementary Fig. 4A ) 300 and a plasmid encoding GFP (pmaxGFP TM by Lonza). We sorted for GFP + target cells and co-301 cultured them with the distinct SARS-CoV-2-specific TCPs to determine the T-cell-mediated 302 cytotoxicity. Expression of Spike was confirmed by flow cytometry (Supplementary Fig. 4B ). 303 We observed T-cell-mediated killing of pSpike transfected target cells by unmodified and 304 FKBP12 KO TCPs. Although the cytotoxic killing of SARS-CoV-2 peptide loaded target cells by 305 our TCPs was not affected by short-term incubation with the IS Tac (Fig. 6C ), we found that 306 cytotoxic elimination of target cells transfected with pSpike was reduced in presence of Tac for 307 unmodified but not FKBP12 KO TCPs (Fig. 6G ). SARS-CoV-2-specific unmodified and FKBP12 KO TCPs recognize Spike S1 and S2 of SARS-CoV-310 2 variants, but show little cross-reactivity to Spike S1 and S2 proteins of common endemic human 311 coronaviruses 312 Considering the ongoing occurrence of SARS-CoV-2 variants, it becomes increasingly 313 important that TCPs also recognize antigens of the mutant SARS-CoV-2 strains without the 314 need to determine the exact variant. Therefore, we re-stimulated the TCPs with peptide pools 315 of the distinct Spike proteins S1 and S2 of SARS-CoV-2 variants Alpha (B.1.1.7), Beta (B.1.351), 316 Delta (B.1.617.2) and Omicron (B.1.1.529) and analyzed production of antiviral cytokines (IFN-317  and TNF-). Upon re-stimulation with peptide pools of the Spike protein S1 and S2 of SARS-318 CoV-2 variants, frequencies of activated (CD137 + ) cytokine producers among CD4 + and CD8 + 319 T-cells of unmodified control and FKBP12 KO TCPs were comparable to those elicited by the 320 wild-type (WT) Spike S1 and S2 ( Supplementary Fig. 5A-H) . 321 Numerous studies have suggested SARS-CoV-2 cross-reactive T-cells in non-exposed 322 individuals directed against the S2 subunit of the spike protein occur due to its partial sequence 323 homology with common endemic human coronaviruses (HCoV) 24,25,65,66 . We investigated this 324 notion by re-stimulating unmodified control and FKBP12 KO TCPs with a peptide pool derived 325 from Spike S1 and S2 of HCoV-229E, HCoV-NL63, HCoV-OC43 and HKU1. We found that 326 unmodified control and FKBP12 KO TCPs showed little cross-reactivity towards Spike S1 and S2 both Spike S1 and S2 peptide pools of SARS-CoV-2 induced significantly higher frequencies of 329 activated IFN- as well as TNF- producers in unmodified control and FKBP12 KO TCPs 330 compared with Spike S1 and S2 peptide pools of common endemic HCoV ( Supplementary Fig. 331 6A, B). Among the CD8 + T-cells, the Spike S2 peptide pool of SARS-CoV-2 induced 332 significantly higher frequencies of activated IFN- as well as TNF- producers in unmodified 333 control and FKBP12 KO TCPs compared with Spike S1 and S2 peptide pools of common endemic To further characterize the proteomes of SARS-CoV-2-specific TCPs, we performed proteome 388 analysis based on mass spectrometry. We confirmed the gene knockout of FKBP12 on protein 389 level (Fig. 7D) . Furthermore, we detected 11 of the differentially expressed mRNA transcripts 390 (Supplementary Fig. 7A and B) in the proteome (Fig. 7D ). Among those, we observed increased 391 expression of DDX21, NAMPT, NCL, PGAM1 and PPA1 in SARS-CoV-2-activated unmodified but not FKBP12 KO TCPs (Fig. 7D) . In SARS-CoV-2-activated FKBP12 KO TCPs we found upregulated protein expression of RAB27A under Tac treatment (Fig. 7D ). High levels of GZMB 394 were detected in FKBP12 KO TCPs in the presence and absence of Tac, which was to lower 395 extend also observed in SARS-CoV-2-activated unmodified TCPs in the absence of IS (Fig. 7D) . 396 We additionally performed TCR repertoire analysis on the single cell level to determine the 397 effect of FKBP12 editing on TCR diversity. Comparing the TCR clonality and total number of T-cells before and after SARS-CoV-2-specific T-cell expansion was largely comparable for 433 CD4 + and CD8 + T-cells, except for AP3a, which was found to be a relevant driver of CD8 + T-434 cell expansion and was not affected by knockout of FKBP12. Supporting previous reports, AP3a. 72 The differences in antigen-specificity between SARS-CoV-2-specific CD4 + and CD8 + 438 However, it is also reported that apoptosis and autophagy are upregulated in PBMCs of SARS-440 CoV-2 infected individuals 73 , which may promote presentation of phagocytosed antigens via Cross-reactive SARS-CoV-2 T-cell epitopes with predominant spike-specificity have been 462 described in unexposed individuals, which might be due to previous infections with common healthy individuals from our study. There is evidence that pre-formed SARS-CoV-2-directed 466 immunity to structural proteins is not driven by cross-reactivity to common endemic HCoV 467 but rather by other frequently encountered pathogens. 83 Hence, the exact source of pre-formed 468 T-cell immunity in SARS-CoV-2 naïve individuals remains to be elucidated. To date, it remains 469 uncertain whether cross-reactive memory T-cells possess protective features to fight SARS-470 CoV-2 infection. 84 Our data from convalescent donors indicates little cross-reactivity of SARS-471 CoV-2-specific TCPs towards Spike S1 and S2 of common endemic HCoV after expansion. In 472 line with this, when stimulating PBMCs of convalescent SARS-CoV-2 infected donors with 473 spike peptide pools from either SARS-CoV-2, common endemic HCoV-229E or HCoV-OC43, 474 it was reported that frequencies of SARS-CoV-2 spike-specific CD4 + T-cells were significantly 475 higher than spike-299E-or spike-OC43-specific CD4 + T-cells. 74 This indicates that the T-cell 476 response towards spike protein is predominantly directed against SARS-CoV-2 in these donors. 477 for as long as four years 85 , the frequency of cross-reactive T-cells in the blood might be limited 479 since memory T-cells are known to reside in the bone marrow and without substantial viral 480 re-stimulation it is unlikely that they egress into the blood stream. 86 481 Patients who experience mild symptoms following SARS-CoV-2 infection show higher 482 proportions of CD8 + T-cell responses compared to those suffering from severe infection 42, 76 , 483 suggesting a potential protective role of CD8 + T-cell immunity against SARS-CoV-2. CD8 + T-484 cells are known to contribute to effective viral clearance to terminate acute viral infections, 485 whereas cytotoxic CD4 + T-cells are required to control chronic infections by e.g. human 486 immunodeficiency virus or herpes viruses. 87 We found that CD8 + T-cells were the main drivers 487 machinery. Interestingly, IL-2 was higher in SARS-CoV-2-stimulated FKBP12 KO CD8 + T-cells exposed to Tac compared to non-exposed FKBP12 KO CD8 + T-cells and unmodified controls. IL-2-producing CD8 + antiviral T-cells are associated with high proliferative potential and are 577 promising candidates to induce sustained immunity after adoptive transfer. 105 T-cell exhaustion 113 . The unmodified TCPs show higher TOX2 and EOMES expression 590 compared to FKBP12 KO TCPs indicating that the FKBP12 KO TCPs tend to be less exhausted, presumably due to reduced Ca 2+ influx into the cytoplasm and thus reduced Ca 2+ -dependent 592 activation. 114 We also observed upregulation of some co-inhibitory receptors among CD4 + and 593 combined with high co-expression of inhibitory receptors. 115 Although we observed 598 upregulation of some exhaustion markers in both of our SARS-CoV-2-specific TCPs we also 599 found that they retain their antiviral effector function, furthermore, many of the "exhaustion The SARS-CoV-2-specific killing capacity was calculated according to the following formulas: 734 Ratio T-cell-free LCL mixtures: Spike S1 Spike S1 NCAP VME1 NCAP AP3a Spike S1 NCAP Spike S1 Spike S2 Spike S2 Spike S2 NCAM1 CD27 CD8A CD8B TIGIT CST7 IL2RB GNLY EOMES NKG7 GZMH PRF1 FCGR3A LGALS9 CD3D CCR1 CXCR6 STAT1 CD3E CD3G CXCR3 GZMK ID3 STAT4 RUNX3 HAVCR2 ID2 NR4A1 STAT3 XCL1 IRF8 XCL2 STAT5A IL2 IL23A BATF GZMB IFNG NR4A3 CD226 IL2RA IRF4 NFAT5 NR4A2 IL4 PDCD1 TNF CD58 CD69 KLRB1 IRF1 CD247 GZMA CD4 IL7R CD28 IL2RG MKI67 CX3CR1 SELL LTB S1PR4 CXCR4 COVID-19 in Solid Organ Transplantation: 911 Results of the National COVID Cohort Collaborative Targets of T Cell 950 Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Functional exhaustion of antiviral lymphocytes in COVID-19 patients Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study Hospitalized Patients with Covid-19 -Preliminary Report Dexamethasone in Hospitalized Patients with Covid-19 Risk Factors Associated With Acute Respiratory Distress Syndrome 1016 and Death in Patients With Coronavirus Disease The use of corticosteroid as treatment in 1020 SARS was associated with adverse outcomes: A retrospective cohort study Corticosteroid therapy 1024 for critically ill patients with middle east respiratory syndrome Choose Cyclosporin A as First-line Therapy in COVID-19 Pneumonia Viral shedding prolongation 1062 in a kidney transplant patient with COVID-19 pneumonia Long-term shedding of viable SARS-CoV-2 in kidney transplant recipients with 1066 COVID-19 Prolonged SARS-CoV-2 shedding and 1069 mild course of COVID-19 in a patient after recent heart transplantation Prolonged viral shedding of SARS-CoV-1073 2 in an immunocompromised patient CRISPR-Cas9 Tacrolimus-Resistant Antiviral T Cells for Advanced Adoptive Immunotherapy in 1077 Transplant Recipients Highly Efficient Genome 1080 Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9 Phenotype and kinetics of SARS-CoV-2-specific T cells in COVID-19 patients 1085 with acute respiratory distress syndrome SARS-CoV-2 Epitopes Are Recognized by a Public and Diverse Repertoire of CD226: a potent driver of antitumor immunity that needs to be 1091 maintained Structural and agonist properties of XCL2, the other member of the C-1094 chemokine subfamily P182 Role of IRF8 as a molecular integrator that orchestrates CD8 T 1096 effector differentiation Treatment of solid organ 1099 transplant recipients with autologous Epstein Barr virus-specific cytotoxic T 1100 lymphocytes (CTLs) SARS-CoV-2-specific T cell 1103 immunity in cases of COVID-19 and SARS, and uninfected controls The ORF3a protein of SARS-CoV-2 induces apoptosis in cells Transcriptomic Characteristics of Bronchoalveolar Lavage 1110 Fluid and Peripheral Blood Mononuclear Cells in COVID-19 Patients The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-1118 regulating MHC-Ι Broad and strong memory CD4+ and CD8+ T cells 1121 induced by SARS-CoV-2 in UK convalescent individuals following COVID-19 Ancestral SARS-CoV-2-specific T cells cross-recognize the Omicron variant T cell immune responses to SARS-CoV-2 1128 and variants of concern (Alpha and Delta) in infected and vaccinated individuals CoV-2 variants of concern partially escape humoral but not T-cell responses in COVID-19 convalescent donors and vaccinees Selective and cross-reactive SARS-CoV-1136 2 T cell epitopes in unexposed humans T Cell Responses Induced by Attenuated 1139 Flavivirus Vaccination Are Specific and Show Limited Cross-Reactivity with Other 1140 Human CD8+ CD57-TEMRA 1176 cells: Too young to be called "old Ex vivo characterization of human CD8+ T subsets with distinct replicative history and partial effector functions Effector CD8 T cells dedifferentiate into 1183 long-lived memory cells Preferential Expansion of 1186 Human Virus-Specific Multifunctional Central Memory T Cells by Partial Targeting of 1187 the IL-2 Receptor Signaling Pathway: The Key Role of CD4 + T Cells Induction of apoptosis and modulation 1190 of activation and effector function in T cells by immunosuppressive drugs Inhibition of endogenous MHC class II-restricted antigen presentation 1194 by tacrolimus (FK506) via FKBP51 Cyclosporin A and tacrolimus, but not rapamycin, inhibit MHC-1197 restricted antigen presentation pathways in dendritic cells Cytokine-mediated regulation of plasma cell 1203 generation: IL-21 takes center stage Polyfunctional T cell 1206 responses are a hallmark of HIV-2 infection Heterogeneity of Memory CD4 T Cell Responses in Different Conditions of Antigen 1209 Exposure and Persistence IL-4: An important 1211 cytokine in determining the fate of T cells HIV-1-specific IFN-γ/IL-2-secreting CD8 T cells support CD4-independent 1214 proliferation of HIV-1-specific CD8 T cells T Cell-Intrinsic CX3CR1 Marks the Most Differentiated Effector CD4 + T 1218 Cells, but Is Largely Dispensable for CD4 + T Cell Responses during Chronic Viral 1219 Infection . ImmunoHorizons A role for cytomegalovirus-specific CD4 +CX3CR1 + T cells and 1222 cytomegalovirus-induced T-cell immunopathology in HIV-associated atherosclerosis IL-7 signaling imparts polyfunctionality and stemness 1226 potential to CD4+ T cells Transcriptional regulator Id2 1229 mediates CD8+ T cell immunity Phosphotyrosine-Dependent Coupling of Tim-3 Impaired expression of the CD3-zeta chain in peripheral blood T cells of 1235 patients with chronic myeloid leukaemia results in an increased susceptibility to 1236 apoptosis TOX transcriptionally and 1239 epigenetically programs CD8+ T cell exhaustion High Levels of Eomes Promote 1241 Exhaustion of Anti-tumor CD8+ T Immunophilin FK506 binding protein associated with inositol 1,4,5-1244 trisphosphate receptor modulates calcium flux Molecular and cellular insights into T cell 1246 exhaustion Elevated 1248 DDX21 regulates c-Jun activity and rRNA processing in human breast cancers NAMPT and NAPRT T cell-specific deletion of 1258 Pgam1 reveals a critical role for glycolysis in T cell responses Clinical significance and functional validation of PPA1 in various 1262 tumors Rab27a Is Required for Generation of HCMV-specific T-1268 cell lines from seropositive solid-organ-transplant recipients for adoptive T-cell 1269 therapy Low immunization rates among kidney transplant recipients 1271 who received 2 doses of the mRNA-1273 SARS-CoV-2 vaccine A high-fidelity Cas9 1275 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in 1276 human hematopoietic stem and progenitor cells B cells 1279 immortalized by a mini-Epstein-Barr virus encoding a foreign antigen efficiently 1280 reactivate specific cytotoxic T cells The VITAL assay: A versatile fluorometric 1286 technique for assessing CTL-and NKT-mediated cytotoxicity against multiple targets 1287 in vitro and in vivo The human liver matrisome -1290 Proteomic analysis of native and fibrotic human liver extracellular matrices for organ 1291 engineering approaches SARS-CoV-2 in severe COVID-19 induces a TGF-β-dominated chronic immune response that does not target 1295 itself A step-by-step workflow for 1297 low-level analysis of single-cell RNA-seq data with Bioconductor scRepertoire: An R-based toolkit for 1300 single-cell immune receptor analysis SARS-CoV-2-specific stimulation of PBMC of convalescent SARS-CoV-2 + donors with individual structural and 1310 accessory proteins of SARS-CoV-2. n=20; *p< 0.05, **p < 0.01 -statistics refer to data of seronegative healthy donors 1311 in Supplementary Fig. 1C-F TCM = central memory T-cells (CCR7 + /CD45RA -), TEM = effector memory T-1374 cells (CCR7 -/CD45RA -), TEMRA = terminally differentiated effector memory T-cells (CCR7 -/CD45RA + ) H) Proportional distribution of T-cell memory subsets of either bulk or CD137 + & IFN- + CD4 + and CD8 + T-1376 cells of unmodified control and FKBP12 KO SARS-CoV-2-specific TCPs after 21 days of expansion Functional analysis of SARS-CoV-2-specific unmodified control and FKBP12 KO TCPs 1381 SARS-CoV-2-specific stimulation of unmodified control and FKBP12 KO TCPs on day 21 of culture Immunosuppressants were added where indicated: CsA Cyclosporine A A) Representative flow cytometry plots of antigen-reactive (CD137 + ) IFN- producers among CD4 + and CD8 + 1385 CoV-2-specific TCPs. IFN- production upon SARS-CoV-2-1386 specific re-stimulation of TCPs is shown in the presence or absence of the indicated immunosuppressants B) Quantified data for the IFN- production of SARS-CoV-2-activated (CD137 + ) CD4 + T-cells in unmodified 1388 and FKBP12 KO SARS-CoV-2-specific TCPs after 16 h of stimulation with SARS-CoV-2 peptide pool in the 1389 presence or absence of respective immunosuppressants C) Quantified data for the TNF- production of SARS-CoV-2-activated (CD137 + ) CD4 + T-cells in unmodified 1391 and FKBP12 KO SARS-CoV-2-specific TCPs after 16 h of stimulation with SARS-CoV-2 peptide pool in the 1392 presence or absence of respective immunosuppressants D) Quantified data for the IFN- & TNF- production of SARS-CoV-2-activated (CD137 + ) CD4 + T-cells in 1394 unmodified and FKBP12 KO SARS-CoV-2-specific TCPs after 16 h of stimulation with SARS-CoV-2 peptide 1395 pool in the presence or absence of respective immunosuppressants E) Quantified data for the IFN- production of SARS-CoV-2-activated (CD137 + ) CD8 + T-cells in unmodified 1397 and FKBP12 KO SARS-CoV-2-specific TCPs after 16 h of stimulation with SARS-CoV-2 peptide pool in the 1398 presence or absence of respective immunosuppressants F) Quantified data for the TNF- production of SARS-CoV-2-activated (CD137 + ) CD8 + T-cells in unmodified 1400 and FKBP12 KO SARS-CoV-2-specific TCPs after 16 h of stimulation with SARS-CoV-2 peptide pool in the 1401 presence or absence of respective immunosuppressants CoV-2-pool) and accessory proteins (AP3a) of SARS-CoV-2 by purified CD4 + T-cells of 1422 unmodified control and FKBP12 KO SARS-CoV-2-specific F) Percentage killing of autologous target cells loaded with indicated structural (NCAP, Spike S1 CoV-2-pool) and accessory proteins (AP3a) of SARS-CoV-2 by purified CD8 + T-cells of 1425 unmodified control and FKBP12 KO SARS-CoV-2-specific G) Percentage killing of autologous target cells transfected with a plasmid encoding the full Spike protein of 1427 SARS-CoV-2 by unmodified control and FKBP12 KO SARS-CoV-2-specific TCPs at 10:1 and 1:1 ratio (T-1428 cells:LCLs) in the presence or absence of Tac CoV-2-specific TCPs CITE-seq, proteome and TCR analysis of SARS-CoV-2-specific TCPs after SARS-CoV-2-specific re-stimulation at 1433 day 21 of culture. Immunosuppressant Tacrolimus (Tac) or Cyclosporin A (CsA) were added where indicated