key: cord-0312620-om65waen
authors: Sakai, Shunsuke; Lora, Nickiana E.; Kauffman, Keith D.; Dorosky, Danielle E.; Oh, Sangmi; Namasivayam, Sivaranjani; Gomez, Felipe; Fleegle, Joel D.; Lindestam Arlehamn, Cecilia S.; Sette, Alessandro; Sher, Alan; Freeman, Gordon J.; Via, Laura E.; Barry, Clifton E.; Barber, Daniel L.
title: Functional inactivation of pulmonary MAIT cells following 5-OP-RU treatment of non-human primates
date: 2021-01-31
journal: bioRxiv
DOI: 10.1101/2021.01.29.428844
sha: 441a2b6490dd702ff5168160e50fd7a203202b10
doc_id: 312620
cord_uid: om65waen

Targeting MAIT cells holds promise for the treatment of different diseases and infections. We previously showed that treatment of Mycobacterium tuberculosis infected mice with 5-OP-RU, a major antigen for MAIT cells, expands MAIT cells and enhances bacterial control. Here we treated M. tuberculosis infected rhesus macaques with 5-OP-RU intratracheally but found no clinical or microbiological benefit in M. tuberculosis infected macaques. In fact, after 5-OP-RU treatment MAIT cells did not expand, but rather upregulated PD-1 and lost the ability to produce multiple cytokines, a phenotype resembling T cell exhaustion. Furthermore, we show that vaccination of uninfected macaques with 5-OP-RU+CpG instillation into the lungs also drives MAIT cell dysfunction, and PD-1 blockade during vaccination partly prevents the loss of MAIT cell function without facilitating their expansion. Thus, in rhesus macaques MAIT cells are prone to the loss of effector functions rather than expansion after TCR stimulation in vivo, representing a significant barrier to therapeutically targeting these cells.

I-like molecule MR1 and express TCRs specific for small molecule metabolites produced by microbes (1, 2) . 5-OP-RU, a derivative of intermediates produced during bacterial riboflavin biosynthesis, is major MR1 ligand and stimulatory MAIT cell antigen that is recognized by a majority of MR1-restricted T cells (3) (4) (5) . MAIT cells display pro-inflammatory, cytotoxic, as well as tissue-repair properties (6) (7) (8) . Given the non-polymorphic nature of MR1 there is interest in targeting MAIT cells through vaccination or as therapies for the treatment of various conditions including cancer and infections. Indeed, using mouse models, MAIT cells have been shown to be important for the control of certain bacterial infections (9) (10) (11) , and they can easily be driven to expand to large numbers in vivo via vaccination with antigen and adjuvant (7, 12) .

Targeting MAIT cells may be useful during Mycobacterium tuberculosis (Mtb) infection (13) . Recently, it was shown that MR1 deficient mice have no defect in bacterial control or survival after Mtb infection (14, 15) , and the presence of large populations of MAIT cells at the time of Mtb exposure has no impact on host resistance (14) (15) (16) . Therefore, the murine model data indicate that pre-infection vaccination of MAIT cells may not be beneficial for Mtb infection.

However, we showed that treatment of mice harboring a chronic Mtb infection with 5-OP-RU was able to reduce bacterial loads ~10 fold in 3 weeks in a manner dependent on IL-17A (14) , indicating that post-exposure stimulation of MAIT cells may be a promising host directed therapy for tuberculosis. While there is significant overlap in the gene expression patterns of human and mouse MAIT cells (7) , there are several key differences between murine and nonhuman primate (NHP)/human MAIT cells. For example, the majority of mouse MAIT cells are CD4 -CD8 -IL-17A-producing MAIT17 cells, while most human and macaque MAIT cells are CD8 + IFN-γ-producing MAIT1 cells (8, (17) (18) (19) . Moreover, mice have several limitations in their ability to model human tuberculosis (TB). In contrast, Mtb infection of macaques recapitulates most features of human TB, and macaques are considered the gold standard pre-clinical model of TB (20) . Therefore, we tested the potential therapeutic efficacy of 5-OP-RU instillation into the lungs of Mtb infected rhesus macaques.

In contrast to mice, there was no therapeutic benefit observed with 5-OP-RU treatment of macaques with TB. MAIT cells in these animals failed to expand, upregulated high levels of PD-1 and became functionally impaired. To ask if the loss of function was due to Mtb infection or

To examine the therapeutic efficacy of MAIT cell stimulation during Mtb infection, macaques were treated intratracheally with PBS or 5-OP-RU from week 6 to week 14 post-infection ( Figure 1A ). Necropsies were planned for week 15-16. All five PBS treated control animals survived until the pre-determined endpoint, however, three of the five 5-OP-RU treated animals developed acute signs of disease (cough and labored breathing) and were humanly euthanized early ( Figure 1B ). All animals in both groups had relatively stable lung disease as measured by Figure 1E ). However, when control animals were compared to just the two 5-OP-RU treated macaques which continued until the week15/16 pre-determined endpoint, there was a slight decrease in the numbers of bacteria in treated animals ( Figure 1E ). Two of the 5-OP-RU treated macaques that were euthanized early (DHLR at week 7 and DHAJ at week 10) displayed slightly increased bacterial loads in the granulomas, but we could not distinguish whether the increase in the number of bacteria was due to the earlier necropsy or 5-OP-RU treatment ( Figure 1E ). Moreover, there was no significant difference in the number of bacteria in the spleens of both groups ( Figure 1F ). Since 3 of the 5-OP-RU treated macaques (DHLR, DHAJ, DHMK) had acute clinical signs after treatment, we assessed the levels of airway constriction in the animals by measuring the maximum diameter of bronchi on CT images collected at baseline and during Mtb infection.

The three 5-OP-RU treated animals that were euthanized early showed rapid reductions in airway diameter after 5-OP-RU treatment relative to most of the PBS control treated animals and other 5-OP-RU treated 2 animals that did not develop severe signs of distress (Figure 2A and B, S2). The bronchoconstriction was not associated with either higher bacterial loads in the pulmonary lymph nodes (LNs) ( Figure 2C ) or overall LN [ 18 F]-FDG uptake on PET/CT scans ( Figure 1C and 2D ). However, a trend was observed in which animals with bronchoconstriction had at least one very cellular pulmonary LN at necropsy relative to the 5-OP-RU treated animals that did not develop respiratory distress or the PBS treated animals ( Figure 2E ). On visual inspection at necropsy, it was apparent that the enlarged LNs were impinging on the bronchus. Therefore, it seems likely that lymphadenopathy-associated airway constriction and not expansive tubercular lung lesions was responsible for the acute clinical signs that led to the early euthanasia of these three macaques treated with 5-OP-RU. Rhesus macaques have been shown previously to be particularly susceptible to lethal LN pathology (21, 22) , so it is not clear if the 5-OP-RU treatment was the direct cause of the bronchoconstriction. Regardless, 5-OP-RU treatment during Mtb infection did not have a clinical or microbiological benefit in rhesus macaques.

We next measured the frequency of MAIT cells in the bronchoalveolar lavage (BAL) and peripheral blood using rhesus macaque MR1/5-OP-RU tetramers. Consistent with the previous findings (23), the frequency of MAIT cells in the airway and blood did not change in the PBS treated group after Mtb infection ( Figure 3A Figure   3E ). PD-1 is induced by TCR signaling, so we next examined its expression to ask if MAIT cells were stimulated through their TCR after infection and 5-OP-RU treatment. While PD-1 expression by MAIT cells was not changed in the BAL or blood of control animals, 5-OP-RU treatment led to a striking upregulation of PD-1 by MAIT cells in both compartments ( Figure 3H to J), indicating that MAIT cells clearly received antigen stimulation through their TCRs after 5-OP-RU treatment. At necropsy, we examined multiple tissue sites to ask if MAIT cells expanded in any tissue. There was also no impact of 5-OP-RU administration on the frequency of MAIT cells in the thymus, spleen, pulmonary LNs, granulomas or instillation site lesions ( Figure 3K ).

Overall, these data demonstrate that 5-OP-RU treatment in Mtb infected rhesus macaques indeed activates MAIT cells via TCR stimulation but fails to induce expansion of MAIT cells in the tissues.

We next evaluated the impact of 5-OP-RU treatment on the function of MAIT cells. Cells from the BAL or blood were stimulated with 5-OP-RU or PMA/ionomycin, and production of IFN-γ, TNF-, IL-17A and GM-CSF was measured by intracellular cytokine staining ( Figure 4A ). The vast majority of MAIT cells in both blood and BAL produced cytokines after stimulation with PMA/ionomycin ( Figure 4B to D). However, prior to instillation of 5-OP-RU, there were major differences in the responses of BAL vs blood MAIT cells after in vitro 5-OP-RU stimulation.

Approximately 75% of MAIT cells in the BAL produced cytokines upon 5-OP-RU stimulation while only ~10% of circulating MAIT cells were able to respond ( Figure 4B to D). After the macaques were treated, BAL MAIT cells in animals receiving 5-OP-RU rapidly lost the ability to respond to in vitro restimulation with 5-OP-RU, while MAIT cells in PBS treated macaques maintained their ability to produce cytokines ( Figure 4B to D). In contrast, PMA/ionomycin induced similar levels of cytokine production by MAIT cells in both PBS and 5-OP-RU treated macaques, although there was a trend for a reduction at very late time points ( Figure 4B to D).

MAIT cells from pulmonary LNs, instillation site lesions in the lungs, the BAL and individually resected granulomas isolated at necropsy also showed a reduction in cytokine producing function after in vitro restimulation with 5-OP-RU and to a lesser extent after restimulation with PMA/ionomycin ( Figure 4E and F). Lastly, the functional defect can be overcome to a certain extent if the TCR is bypassed via stimulation with second messengers like PMA/ionomycin. Therefore, rather than the expected increase in MAIT cell responses after 5-OP-RU treatment, MAIT cells entered an exhaustionlike state.

We next examined the impact of 5-OP-RU treatment on the conventional adaptive immune response. We found that there was no difference in the kinetics of Mtb-specific CD4 T cell response in the airways as measured by intracellular cytokine staining for IFN-γ and TNF-after restimulation with Mtb peptide megapools ( Figure 5A and B). Likewise, there was no apparent impact of 5-OP-RU treatment on the kinetics of the Mtb-specific CD8 T cells in the BAL ( Figure   5C and D). There was also no impact of 5-OP-RU administration on the magnitude of Mtbspecific CD4 and CD8 T cells in the blood, spleen, LNs, granulomas or instillation site lesions at necropsy ( Figure 5E and F). A previous study found that MAIT cells may directly provide help to B cells in rhesus macaques (24), so we also measured Mtb-specific IgG responses in serum.

However, there was no clear difference in antibody responses between treated and untreated animals ( Figure 5G ). Collectively, these data show that stimulation of MAIT cells during Mtb infection did not boost Mtb-specific conventional adaptive immune responses.

Due to the role of MAIT cells at mucosal sites and the ability of MAIT cells to recognize microbe-derived molecules, a close interplay between MAIT cells and the microbiota has been hypothesized (25). Indeed, recent work has demonstrated that the microbiota plays a critical role in the development of MAIT cells in mouse models (6, 26) . Therefore, we sought to investigate if treatment with 5-OP-RU affects the microbiome. Fecal samples were collected from all 10 macaques prior to infection and following infection and treatment. The composition of the microbiota was characterized via 16S rRNA sequencing. We found that alpha-diversity (with-in sample diversity) of the microbiota varied over the course of the experiment in both PBS and 5-OP-RU groups (Supplemental Figure 1A , left panel). However, we did not find the alphadiversities of the microbiome from the 5-OP-RU or PBS treated timepoints to be significantly different from each other or from their respective pre-infection and infected microbiomes (Supplemental Figure 1A , right panel). Similarly, the community structure of the microbiota following 5-OP-RU treatment was not significantly different from that of PBS treated animals (Supplemental Figure 1B) . However, visualization of the relative abundance of microbial taxa over the experimental time course indicated alterations in the composition of the gut flora, albeit not consistent between animals within a treatment group (Supplemental Figure 1C ). Several taxa were identified to be differently abundant with comparing the PBS or 5-OP-RU treated animals to their respective pre-infection microbiomes (Supplemental Figure 1D ). Importantly, we found only three bacterial families, Gastranaerophilales, Family XIII and Paludibacteraceae to be enriched and one family, vadin BE97, to be depleted following 5-OP-RU treatment in comparison to PBS administration (Supplemental Figure 1D ). Overall, these analyses reveal that the 5-OP-RU treatment results in only minimal alterations in the intestinal microbiota of rhesus macaques.

In a separate experiment, we next vaccinated uninfected macaques with the same regimen of 5-OP-RU+CpG as we previously used in our murine model experiments ( Figure 6A ). This allowed us to test three possible explanations for the poor MAIT responses after 5-OP-RU treatment of Mtb infected macaques. First, treating uninfected macaques allowed us to ask if the Mtb infection itself inhibited the ability of MAIT cells to respond. Second, we lowered the dose tenfold to test the possibility that our previous results were due to a high zone tolerance-like effect. (10, 32, 35-38, 45, 46) . MAIT cells may accumulate to slightly higher frequencies in tissues during some infections as has been shown in the BAL during tuberculosis or peritoneal cavity during spontaneous bacterial peritonitis (47, 48) , but given the magnitude of changes reported this could be explained by recruitment from circulation rather than TCR-driven expansion. Based on the results presented here, it seems likely that MAIT cells do not mount large proliferative responses in humans and NHPs as part of their typical response to antigenic stimulation. In other words, our data raise the hypothesis that the lack of proliferation and downregulation of cytokine producing ability after strong antigenic challenge may not represent a defect but normal MAIT cell biology in vivo in humans and NHPs.

That is not to diminish the importance of MAIT cells in host defense, as human MAIT cell deficiency has been shown to lead to increase susceptibility to viral and bacterial infections (49) .

We suggest the lack of MAIT cell responses reported here and in the above-mentioned literature Each symbol represents an individual tissue sample from indicated animal. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. This is the volume of the bronchus directly constricted by the enlargement of the peri-carinal lymph nodes.

Sixteen healthy rhesus macaques originally from the NIAID breeding colony on Morgan Island were selected for this study and were tuberculin skin test negative. Animals were housed in nonhuman primate biocontainment racks and maintained in accordance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals and all applicable regulations, standards, and policies in a fully AAALAC International accredited Animal Biosafety Level 3 vivarium. All procedures were performed utilizing appropriate anesthetics as listed in the NIAID DIR Animal Care and Use Committee (ACUC) approved animal study proposal LPD-25E.

Euthanasia methods were consistent with the AVMA Guidelines on Euthanasia and endpoint criteria listed in the NIAID DIR ACUC approved animal study proposal LPD-25E. 

Rhesus were imaged prior to infection and every two weeks beginning at 5 weeks post-infection for a maximum of 7 PET/CT scans ( Figure 1A) . The imaging studies were conducted with an optimized [ 18 F]-FDG dose (0.5 mCi/kg) administered intravenously as previously described (52) .

A 360-projection CT scan of the lungs was acquired during a ~50 second breath hold on a LFER 150 PT/CT scanner (Mediso Inc, Budapest, Hungary). A 20-minute PET dataset/per field of view was acquired during mechanical ventilation and the raw CT and PET data were reconstructed using the Nucline software (Mediso, Inc, Budapest, Hungary) to create individual DICOM files that were co-registered using MIM Maestro (v. 6.2, MIM Software Inc, Cleveland, Ohio). A lung volume of interest (VOI) was defined on the CT image and the VOI was transferred to the PET image as previously described to determine the total [ 18 F]-FDG uptake referred to as the lung total lesion glycolysis (TLG) (52) . 

Tetramer stains were performed by incubating 1x10 6 cells at 37°C for 30 minutes with rhesus macaque MR1/5-OP-RU tetramer in X-vivo 15 media containing 10% FCS and monensin.

Tetramers were produced by the NIAID tetramer core facility (Emory University, GA).

Fluorochrome-labeled antibodies used for flow cytometric analysis are listed in Supplemental Table 1 . Surface antigens and dead cells were stained in PBS + 1% FCS + 0.1% sodium azide for 20 minutes at 4 °C. For intracellular cytokine and transcription factor staining, cells were fixed and permeabilized with the Foxp3 Transcription Factor Staining Buffer Kit (eBioscience) and stained for 1 hour at 4 °C. Samples were acquired on a FACSymphony (BD Biosciences), and data were analyzed using FlowJo 10 (Treestar).

Ninety-six-well ELISA plates were coated with Mtb whole cell lysate (strain H37Rv, BEI Resources) at 10 μ g/ml diluted in PBS for 1 hour at 37°C. The plates were washed and blocked overnight at 4°C with block buffer (5% milk powder + 4% whey buffer in PBS Tween-20).

Plates were then washed, and plasma samples were added at a serial 1:3 dilutions starting at a 1:10 dilution with 4% whey buffer and incubated for 1 hour at 37°C. After washing, plates were incubated with goat anti-monkey IgG (H+L)-HRP (Novus Bio) was added at 1:1000 dilution in 4% whey buffer for 1 hour at 37°C. Plates were washed and 1-Step Ultra-TMB ELISA Substrate Solution (Thermo Scientific) was added to develop the plates. The reaction was stopped by adding 0.5 M sulfuric acid, and the OD measured at 450 nm. The OD value of each pre-infection baseline was subtracted from each post-infection timepoint sample in order to calculate Mtbspecific IgG levels.

One pre-infection fecal sample was collected 2 to 4 weeks prior to infection and another on the day of infection. Additional samples were collected at weeks 4, 8, 12 post-infection and at necropsy. All samples were stored at -80 °C until completion of experiment. DNA was extracted from ~0.05g of fecal material using QIAamp Fast DNA stool Mini kit (Qiagen, Hilden, Germany) and the V4 region of the 16s rRNA gene was amplified with primers 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA-3'and 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3' and sequenced as previously described (55) . The raw reads were demultiplexed, denoised and filtered for chimeras using the DADA2/QIIME2 pipeline (version 2-2020.2) (56). The processed data resulted in an average of ~55,000 reads/sample. Alpha and beta-diversity analyses were performed using Shannon and Bray-Curtis dissimilarity indices respectively on read data rarefied to a depth of 40,000 reads/sample. Taxonomic classification was performed utilizing QIIME2 and the Silva database release 132 (57) . Differentially abundant taxa were identified using Linear discriminant analysis (LefSe) and filtered for linear discriminant score (LDA) > 2 and p-value < 0.05 (58) .

All analyses were conducted using Prism 8 (GraphPad Software). Two-sample t test was used for two group comparisons and ANOVA was used for comparing multiple groups. Unless 

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