MR1-restricted mucosal-associated invariant T (MAIT) cells respond to mycobacterial vaccination and infection in nonhuman primates

Abstract

Studies on mucosal-associated invariant T cells (MAITs) in nonhuman primates (NHP), a physiologically relevant model of human immunity, are handicapped due to a lack of macaque MAIT-specific reagents. Here we show that while MR1 ligand-contact residues are conserved between human and multiple NHP species, three T-cell receptor contact-residue mutations in NHP MR1 diminish binding of human MR1 tetramers to macaque MAITs. Construction of naturally loaded macaque MR1 tetramers facilitated identification and characterization of macaque MR1-binding ligands and MAITs, both of which mirrored their human counterparts. Using the macaque MR1 tetramer we show that NHP MAITs activated in vivo in response to both Bacillus Calmette-Guerin vaccination and Mycobacterium tuberculosis infection. These results demonstrate that NHP and human MR1 and MAITs function analogously, and establish a preclinical animal model to test MAIT-targeted vaccines and therapeutics for human infectious and autoimmune disease.

Figure 1. MR1 sequence polymorphisms explain species-specific tetramer reactivity

a) The amino acid alignment of the MR1 α1 and α2 domains is displayed. Identity to the human reference sequence is indicated by a “-”. Differences are indicated by the specific amino acid. Lowercase indicates heterozygosity. b) A ribbon diagram of the α1 and α2 domains of the MR1 molecule (from above). TCR contact residues are highlighted in blue. c) Left panel - Negative ion MS1 extracted ion chromatogram of rRL-6-CH2OH ([M-H]^- = 329.1100) from macaque MR1 co-cultured with E. coli (black) or the macaque MR1 mock control (blue). Middle panel - Composite MS1 spectra from 11.25-12.00 minutes of ion 329.1100. The black trace is macaque MR1 co-cultured with E. coli and the macaque MR1 mock control is blue. Right panel - Fragment spectrum of ion 329.1105 at 11.51 minutes from the E. coli sample. The precursor and product ions are consistent with the published fragment spectra for synthetic rRL-6-CH2OH. Possible fragment ion structures are shown with their corresponding chemical formula, calculated mass, and observed ion. d) Left Panel - Negative ion MS1 extracted ion chromatogram of HMRL ([M-H]^- = 327.0946) from macaque MR1 co-cultured with E. coli (black) or the macaque MR1 mock control (blue). Second from left panel - Composite negative ion MS1 spectra from 10.00-10.80 minutes of ion 327.0948. The black trace is macaque MR1 co-cultured with E. coli and the macaque MR1 mock control is blue. Second from right panel - Fragment spectrum of ion 329.0948 at 10.38 minutes from the E. coli sample. The precursor and product ions are consistent with the fragment spectra of synthetic HMRL. Possible fragment ion structures are shown with their corresponding chemical formula, calculated mass, and observed ion. Right panel – Fragment spectrum of synthetic HMRL. e) Tetramer staining of PBMC from each of the indicated species is plotted against staining by the anti-Vα7.2 antibody. Plots progressively gated on lymphocytes, singlets, live, CD3+, CD8+ cells.

Figure 2. Macaque MR1 tetramer+ cells are phenotypically and functionally similar to human and mouse MAITs

a) TCRαV (top) and J (bottom) region usage from MR1 tetramer+ cells from both the blood (n=61 cells) and liver (n=44 cells) of a healthy macaque. b) Select CDR3α sequences of blood MAITs. The canonical ligand binding tyrosine is noted in bold/underlined. c) TCRβ V (top) and J (bottom) region usage from MR1 tetramer+ cells from blood (n=73 cells) and liver (n=64 cells). TRBV4-1, 4-2 and 4-3 were combined together since they are indistinguishable based on the sequence length of the single cell product. d) Representative nuclear staining for PLZF, RORγ, and T-bet. Plots were progressively gated on lymphocytes, singlets, live, CD3+, and CD8+ cells. e) PBMC from eight animals were stained for the nuclear proteins PLZF, RORγ, and T-bet. Mean, s.e.m. and p values (Wilcoxon Rank Sum test) are shown. f) Representative staining of MAITs (top) and non-MAIT CD8+ T cells (bottom) in a functional assay. g) Functional assay results examining activation of MAITs from spleen of eight animals to PFA fixed E. coli. Cells were stained with IFNγ and TNFα (left) and CD69 (right). Results from MR1 tetramer positive cells are shown in blue and MR1 tetramer negative cells in red. All blocking antibodies were added at a concentration of 100μg/ml. Conditions were plated in duplicate and averaged. Mean, s.e.m. and p values (Kruskal Wallis and Dunn's Multiple comparison post-test) are shown.

Figure 3. MAIT frequency and phenotype in macaque tissues

a) Representative tetramer staining of multiple macaque tissues. Plots gated on lymphocytes by FSC and SSC, singlets, live, CD3+, CD8+ cells. b) MAIT frequencies among extralymphoid tissues (top) and primary/secondary lymphoid tissues (bottom). 11 animals were stained except for colon which utilized 19 animals and samples were only included if there were at least 300 CD8+ T cells from which to gate tetramer+ cells. Mean, s.e.m. and p values (Kruskal Wallis test followed by Dunn's Multiple Comparison post test) are shown. c) Frequency of Ki-67+ MAITs (blue) and non-MAIT CD8+ T cells (red) in the extralymphoid (top) and primary/secondary lymphoid tissues (bottom). d) Frequency of CD69+ MAITs (blue) and non-MAIT CD8+ T cells (red) in the extralymphoid (top) and primary / secondary lymphoid tissues (bottom). Samples were included in the Ki-67 or CD69 analysis if there were at least 300 CD8+ T cells and at least 10 MR1 tetramer+ cells. Mean, s.e.m. and p values (Wilcoxon Rank Sum test) are shown.

Figure 4. MAITs respond to bacterial pathogens in vivo

Four macaques were challenged with BCG intradermally and monitored for 6 weeks. a) Frequency of MAITs in the blood of BCG vaccinated macaques. Mean and s.e.m. are shown. Kruskal Wallis test revealed no significant differences in MAIT frequencies. b) Frequency of Ki-67+ MAITs (blue) and non-MAIT CD8+ T cells (red) in the blood. Mean, s.e.m and p values (Friedman test with Dunn's Multiple comparison post test) are shown (*, p≤0.05, **, p≤0.01). Animals were revaccinated with BCG 9-weeks post-initial vaccination. These animals were brought to necropsy at days 13 and 14 for analysis. c) Representative MR1-tetramer, Ki-67, and GzmB staining from the pectoral (top, vaccine site) and inguinal (bottom, non-vaccine site) skin sections. d) Frequency of MAITs of CD8+ T cells in the chest and thigh skin sections (left) and in the axillary and inguinal lymph nodes (right). e) Percentage of Ki-67+ MAITs in the chest and thigh sections (left) and axillary and inguinal lymph nodes (right). f) Frequency of GzmB+ MAITs in the skin (left) and lymph nodes (right). g) Comparison of Ki-67+ frequencies among MAITs and non-MAIT CD8+ T cells in the chest (left), thigh (middle), and axillary lymph node (right). Mean, s.e.m. and p values (Wilcoxon Rank Sum test) are shown for parts d-g. h) Ki-67+ frequency of MAITs (blue) and not MAITs (red) after re-vaccination. i) MAIT frequencies of CD8+ T cells in blood after TB infection. j) MAIT and non-MAIT CD8+ T cell Ki-67 frequencies after Mtb infection. Mean, s.e.m and p values (Friedman test with Dunn's Multiple comparison post test) are shown (*, p≤0.05; **, p≤0.01; ***, p≤0.001).