Dynamic MAIT cell response with progressively enhanced innateness during acute HIV-1 infection

Abstract

Mucosa-associated invariant T (MAIT) cell loss in chronic HIV-1 infection is a significant insult to antimicrobial immune defenses. Here we investigate the response of MAIT cells during acute HIV-1 infection utilizing the RV217 cohort with paired longitudinal pre- and post-infection samples. MAIT cells are activated and expand in blood and mucosa coincident with peak HIV-1 viremia, in a manner associated with emerging microbial translocation. This is followed by a phase with elevated function as viral replication is controlled to a set-point level, and later by their functional decline at the onset of chronic infection. Interestingly, enhanced innate-like pathways and characteristics develop progressively in MAIT cells during infection, in parallel with TCR repertoire alterations. These findings delineate the dynamic MAIT cell response to acute HIV-1 infection, and show how the MAIT compartment initially responds and expands with enhanced function, followed by progressive reprogramming away from TCR-dependent antibacterial responses towards innate-like functionality.

Fig. 1. MAIT cell activation and dynamics in the RV217 acute HIV-1 infection cohort.

a Representative flow cytometry plots of a pre-infection time point from one individual enrolled in the RV217 acute capture cohort showing identification of MAIT cells as CD161++Vα7.2+ of CD3+CD14-CD19- live lymphocytes in PBMC. Confirmation of MAIT cell identification using the 5-OP-RU loaded MR1 tetramer, and the relative distribution of CD8 and CD4 positivity within the MAIT cell gate is displayed. b MAIT cell frequency as a percentage of CD3+ T cells is shown longitudinally in individuals with acute HIV-1 infection (gray), with median frequency (blue) (n = 29). c Median absolute counts (cells/μl of blood) of MAIT cells relative to conventional CD4 and CD8 T cells and HIV-1 viral load displayed over time in acute HIV-1 infection (n = 29). d MAIT cell absolute counts in 9 donors where matching data points were available from days 1 and 43 after HIV-1 infection. e MAIT cell percentages out of total CD3+ cells isolated from rectal biopsies from individuals with acute HIV infection (n = 7), and matched uninfected controls (n = 17). f MAIT cell count per gram of rectal biopsy tissue from individuals with acute HIV infection (n = 7), and matched uninfected controls (n = 17). g Median MAIT cell subset distribution displayed over time in acute HIV-1 infection. CD161, CD8, CD4, and double negative expression in MR1 tetramer-defined MAIT cells displayed (n = 19). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. In d, statistical analysis was performed using Wilcoxon Signed Rank test; in e and f using the Mann-Whitney test; in g the nonparametric Friedman test with the Dunn’s multiple comparison test. PBMC, Peripheral blood mononuclear cells. MAIT cells are identified as CD161++Vα7.2+ within CD3+CD14-CD19- live lymphocytes, except in e, where MAIT cells are identified using the 5-OP-RU loaded MR1 tetramer of CD3+CD14-CD19- live lymphocytes. 5-OP-RU 5-(2-oxopropylideneamino)−6-d-ribitylaminouracil. VL viral load. The source data underlying b, c, and g are provided as a Source Data file.

Fig. 2. MAIT cell activation in acute HIV-1 infection.

a Median expression of markers of activation and exhaustion (HLA-DR, PD-1, CD38, TIGIT, and GrzB) in MAIT cells in PBMC as assessed by flow cytometry displayed over time in acute HIV-1 infection (n = 19). b The fold change of gene expression compared to pre-infection of individual genes (CD38, HLADRA, TIGIT, PDCD1, GZMB, MKI67, and IRF4) in three post-infection time points in acute HIV-1 infection from bulk sorted MAIT cells (n = 20). c Correlation of IRF4 and d, MKI67 gene expression in bulk sorted MAIT cells with the protein expression of markers activation (HLA-DR, PD-1, and CD38) at the post-infection time point corresponding with peak VL (median 16 days since first positive test for HIV-1 RNA) (n = 20). e Correlation of gene expression of IRF4 in sorted MAIT cells with MAIT cell absolute counts, or f, MAIT cell frequency at two post-infection time points corresponding with set point VL (median 43 days since first positive test for HIV) or early chronic infection (n = 20). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. In a, statistical analysis was performed using the nonparametric Friedman test with the Dunn’s multiple comparison test; in c, d, e, and f correlative analyses were performed using Spearman Rank correlation test. In a, significance indicated is valid for all markers displayed. PBMC, Peripheral blood mononuclear cells. MAIT cells are identified as CD161 + + Vα7.2 + within CD3 + CD14-CD19- live lymphocytes. VL viral load. The source data underlying a and b are provided as a Source Data file.

Fig. 3. The transcriptional signature of MAIT cells before and during acute HIV-1 infection.

RNA-Seq was performed on sorted MAIT cells from the PBMC of longitudinal samples corresponding to one pre-infection and three post-infection time points in the acute capture cohort (n = 9). a Volcano plots depict upregulated (red) or downregulated (blue) genes compared to pre-infection at three post-infection time points in acute HIV-1 infection. Individual genes listed in Supplementary Table 4. Highlighted genes have a –Log₁₀ p-value ≥ 3 and a Log₂ fold change of 0.5 or −0.5 (corresponding to p ≤ 0.001, and fold change of 1 or −1, in a generalized linear model). b The temporal dynamics of the upregulated and downregulated genes shown longitudinally in acute HIV-1 infection, together with plasma VL. c Shared and unshared differently expressed genes compared to pre-infection between all three post-infection time points in acute HIV infection are highlighted as a Venn diagram, and listed in Supplementary Table 4. d Gene expression patterns were subjected to Gene Set Enrichment Analysis (GSEA), and upregulated and downregulated pathways in post-infection time points compared to pre-infection are displayed as a Normalized Enrichment Score (NES) heat map. Enrichment plots from three selected post-infection upregulated pathways compared to pre-infection are shown; e, negative regulation of viral entry into host cell, f, regulation of IFNγ production, and g, natural killer cell mediated immunity. Genes contributing to enrichment plots are listed in Supplementary Table 5. PBMC, Peripheral blood mononuclear cells. VL, viral load. MAIT cells are identified as CD161 + + Vα7.2 + cells within CD3 + CD14-CD19- live lymphocytes.

Fig. 4. TCR repertoire diversity of MAIT cells before and after acute HIV-1 infection.

RNA-Seq was performed on sorted MAIT cells from PBMC samples at pre-infection and early chronic post-infection time points in the acute capture cohort (n = 6). a TCRα and TCRβ clonal sequence diversity of MAIT cells before and after HIV-1 infection in one representative donor. b Change in the number of MAIT cell TCRα and TCRβ clones detected before and after HIV-1 infection. *p < 0.05, Wilcoxon Signed Rank test. MAIT cells are identified as CD161++Vα7.2 + cells within CD3+CD14-CD19- live lymphocytes. Donors analyzed here are a subset of donors analyzed in Fig. 2. Post-infection time point corresponds with early chronic HIV infection (85 days since first positive test for HIV-1 RNA).

Fig. 5. MAIT cells display increased innate-like properties in acute HIV infection.

a Flow cytometry was performed on PBMC from individuals in the acute capture cohort to investigate changes in CD56 expression within MAIT cells, and concatenation of all individuals examined are shown over time in acute HIV-1 infection (n = 19). b CD56 expression in MAIT cells is displayed longitudinally at the protein level measured by flow cytometry, and c, at the gene expression level as measured by targeted transcriptomics (Et = #of qPCR cycles–Ct) (n = 19). d The fold change compared to pre-infection of innate-like genes after targeted transcriptomics of bulk sorted MAIT cells at three post-infection time points in acute infection (n = 20). e Example flow cytometry staining of IFNγ production in CD56− or CD56+ MAIT cells from a pre-infection time point from one donor in the acute capture cohort after stimulation of PBMC with IL-12 and IL-18 (n = 10). f IFNγ production in CD56 + and CD56− MAIT cells after stimulation of PBMC with IL-12 and IL-18 at one pre-infection and one post-infection time point (n = 10). *p ≤ 0.05, **p ≤ 0.01. In b and c, statistical analysis was performed using the nonparametric Friedman test with the Dunn’s multiple comparison test; in f, using Wilcoxon Signed Rank test. PBMC, Peripheral blood mononuclear cells. MAIT cells are identified as CD161 + + Vα7.2 + cells within CD3 + CD14-CD19- live lymphocytes. Post-infection time point corresponds with early chronic HIV infection (85 days since first positive test for HIV-1 RNA). The source data underlying d are provided as a Source Data file.

Fig. 6. MAIT cells in acute HIV-1 infection become dysfunctional in early chronic infection.

PBMC from one pre-infection and three post-infection time points from the acute capture cohort study subjects (n = 20) were stimulated with E. coli, PMA/ionomycin, or without stimulation, to examine expression of markers of cytotoxicity (CD107a and GrzB) and cytokine production (IFNγ and TNF) in MAIT cells. a Example flow cytometry gating showing the functional read out within the MAIT cell gate in unstimulated (black) and stimulated (red) cells. b MAIT cell functionality after stimulation with mildly fixed E. coli, or c PMA/ionomycin is displayed longitudinally with data from one pre-infection and three post-infection time points in acute infection as the percentage of MAIT cells positive for functional markers, and the median of these markers at pre-infection is shown dashed black line. Longitudinal median values shown as a solid red or turquoise line, respectively. d The percentage of MAIT cells from b and c expressing at least one function is shown longitudinally. *p ≤ 0.05. In b, c, and d, statistical analysis was performed using the nonparametric Friedman test with the Dunn’s multiple comparison test. PBMC, Peripheral blood mononuclear cells. MAIT cells are identified as CD161++Vα7.2+ cells within CD3+CD14-CD19- live lymphocytes. VL viral load. The source data underlying d are provided as a Source Data file.

Fig. 7. Plasma soluble factor associations with MAIT cell phenotype and function in acute HIV-1 infection.

a The fold change compared to pre-infection of plasma soluble factors (IFABP, sCD14, IL-6, and CRP) and total MAIT cell function after E. coli or PMA/ionomycin stimulation from individuals enrolled in the acute capture cohort, displayed longitudinally (n = 20). b Correlative analysis of plasma sCD14 fold over baseline at peak VL (median 16 days since first positive test for HIV-1 RNA) or set point VL (median 43 days since first positive test for HIV-1 RNA) with either the expression of activation markers (HLA-DR and PD-1) on MAIT cells, or the production of TNF and IFNγ in MAIT cells after PMA/ionomycin stimulation, respectively (n = 20). c Predictive correlation of plasma CRP at the peak VL time point with MAIT cell functionality after PMA/ionomycin stimulation at either the set point VL time point (median 43 days since first positive test for HIV-1 RNA), or the early chronic infection time point (median 85 days since first positive test for HIV-1 RNA) (n = 10). **p ≤ 0.01, ***p ≤ 0.001. In b and c, correlative analyses were performed using Spearman Rank correlation test. PBMC, Peripheral blood mononuclear cells. MAIT cells are identified as CD161++Vα7.2+ cells within CD3+CD14-CD19- live lymphocytes. VL viral load. IFABP intestinal fatty acid binding protein. sCD14 soluble CD14. CRP C-reactive protein. The source data underlying a are provided as a Source Data file.

Fig. 8. Dynamic response of the MAIT cells compartment during acute HIV-1 infection.

Schematic view of the complex response pattern of MAIT cells from pre-infection, to peak viremia, over the viral load set-point, into early chronic infection.