Human thymic MR1-restricted MAIT cells are innate pathogen-reactive effectors that adapt following thymic egress

Abstract

Human mucosal-associated invariant T (MAIT) cells express the semi-invariant T-cell receptor (TCR) Vα7.2 and are restricted by the major histocompatibility complex-Ib molecule MR1. While MAIT cells share similarities with other innate T cells, the extent to which MAIT cells are innate and their capacity to adapt is unknown. We evaluated the function of Vα7.2(+) T cells from the thymus, cord blood, and peripheral blood. Although antigen-inexperienced MAIT cells displayed a naïve phenotype, these had intrinsic effector capacity in response to Mycobacterium tuberculosis (Mtb)-infected cells. Vα7.2(+) effector thymocytes contained signal joint TCR gene excision circles (sjTRECs) suggesting limited replication and thymic origin. In evaluating the capacity of Mtb-reactive MAIT cells to adapt, we found that those from the peripheral blood demonstrated a memory phenotype and had undergone substantial expansion, suggesting that they responded to antigenic stimulation. MAIT cells, an evolutionarily conserved T-cell subset that detects a variety of intracellular infections, share features of innate and adaptive immunity.

Figure 1. CD8 co-receptor blockade prevents the detection of Mtb-infected DC by human MR1-restricted Mtb-reactive CD8⁺ T cell clones

D466 DC were left uninfected (U) or infected with Mtb (MOI 30) overnight and incubated (10,000/well) with Mtb-reactive MR1-restricted CD8⁺ T cell clones (D426B1, D466A3, D466F5) (10,000/well) or the Mtb-specific CD4⁺ T cell clone (D466G8) (5000/well) in the presence of CD8 blocking antibody LT8 or control msIgG1 antibody (2.5ug/ml each). IFN-γ production was detected the following day by ELISPOT. Each bar and error bars represent the mean and SEM respectively of IFN-γ spot forming units (SFU) from duplicate wells. Similar results were obtained in 3 independent experiments.

Figure 2. Human MAIT cells from the thymus and cord blood have innate ex vivo effector capacity

CD4-depeletd T cells from the thymus, cord blood, and peripheral blood (500,000/well) were incubated with stimulator A549 cells left uninfected or infected with Mtb (1.5e6/2 cm² well) and tested for the intracellular production of TNF-α as described in material and methods. Briefly, after overnight incubation, a protein transport inhibitor was added for the final 6 hrs of the assay after which cells were surface stained for expression of the Vα7.2 TCR and CD8, then fixed and permeabilized before staining for intracellular TNF-α. As TNF-α+ Vα7.2⁺ is not present in the CD8-negative subset, all frequencies were determined from CD8 positive gated events only. A) Dot plot analyses from the intracellular cytokine staining assay demonstrating representative responses to uninfected A549 (left column) or Mtb-infected A549 (right column) from an individual thymus, cord blood and adult donor. Cells are gated on CD8⁺ events. The x-axis represents TNF-α; the y-axis represents Vα7.2. Numbers represent the frequency of CD8⁺ events within each gate. B) Frequencies of Vα7.2⁺ CD8⁺ cells from the thymus (n = 6; mean: 1.8; SEM: 0.6), cord blood (n = 4; mean 4.2; SEM: 1.0) and peripheral blood (n = 6; mean: 4.3; SEM: 1.0). Results for each donor were derived using the frequencies of cells derived from the red gate (right panel). Significant differences were detected between thymus and cord blood (P = 0.05) and thymus and peripheral blood (P = 0.04). C) Frequencies of Vα7.2⁺ CD8⁺ cells that produced TNF-α in response to Mtb-infected A549 cells. Thymus (n = 6; mean: 3.02; SEM: 0.58), cord blood (n = 4; mean 0.92; SEM: 0.52) and peripheral blood (n = 6; mean: 6.20; SEM: 2.80). Results for each donor were derived using the percentage of cells in the black gate as a proportion of those in the red gate (right panel). Significant differences were observed between thymus and cord blood (P =0.04). D) Frequencies of Vα7.2⁺ cells among Mtb-reactive TNFα⁺ CD8⁺ cells. Thymus (n = 6; mean: 6.07; SEM: 1.14), cord blood (n = 4; mean 15.68; SEM: 5.89) and peripheral blood (n = 6; mean: 26.60; SEM: 5.12). Results for each donor were derived using the percentage of cells in the black gate as a proportion of those in the red gate (right panel). Significant differences were observed between thymus and peripheral blood (P =0.002). The unpaired two-tailed, t-test was used to assess significant differences between groups. A single asterisk represents a P value < 0.05 and two asterisks < 0.01.

Figure 3. Human Vα7.2⁺ thymocytes and functional MAIT thymocytes retain sjTREC

A) sjTREC content per 10⁴ cells from the following FACS-sorted subsets; total CD8⁺ thymocytes (n = 2); paired CD8⁺Vα7.2⁺ and Vα7.2⁻ populations from thymus (n = 7), cord blood (n = 3) and PBMC (n = 3). B) Analysis of thymocyte data in A) demonstrating that CD8⁺ Vα7.2⁺ thymocytes have significantly less sjTREC than their paired CD8⁺ Vα7.2⁻ thymocytes (P= 0.02). C Representative dot plot of Vα7.2 and LAMP expression on CD8⁺ CD4⁻, PI⁻, γδTCR⁻ thymocytes stimulated without (left) or with (right) PMA/ionomycin for 2 hours. D) sjTREC analysis from FACS-sorted subsets. Significant differences (P = 0.02) were observed between the LAMP⁺ and LAMP⁻ CD8⁺ CD4⁻, PI⁻, γδTCR⁻ thymocytes that were Vα7.2⁺ but not Vα7.2⁻. The paired two-tailed, t-test was used to assess significant differences between groups. A single asterisk represents a P value < 0.05.

Figure 4. Antigen-inexperienced MAIT cells are inherently innate effectors with a naïve phenotype while peripheral blood MAIT cells display a phenotype associated with antigen exposure

The intracellular cytokine-staining assay was performed as described in figure 1. In addition, cells were surface stained with antibodies for CD45RA, CD62L, and CCR7. Frequencies and representative histograms represent expression of CD45RA (left column), CCR7 (middle column) or CD62L (right column) on total CD8⁺ cells (solid line), Vα7.2⁺ CD8⁺ cells (dashed line), and TNFα⁺ Vα7.2⁺ CD8⁺ cells (bold line) from thymus (n = 6), (top row), cord blood (n = 4), (middle row) and peripheral blood (n = 6) (bottom row). Within each graph frequencies from each donor are represented by the same symbol. The paired two-tailed, t-test was used to assess significant differences between groups. A single asterisk represents a P value < 0.05; two asterisks < than 0.01; and three asterisks < 0.001.

Figure 5. Endogenous MR1 is expressed at high levels on a subset of CD45⁺ CD3⁺ thymocytes

Representative analysis of single cell suspensions of thymocytes (n = 4) stained with antibodies for pan-CD45, CD3, CD4, CD8 and MR1 (clone 26.5) and analyzed by flow cytometry. A) Dot plots of CD45 expression (y-axis) and msIgG2a isotype control or MR1 (x-axis) from live cells. B) Histogram of CD3 expression on MR1⁺ cells. C) Dot plot of CD8 (x-axis) and CD4 expression (y-axis) from total live thymocytes (grey) or MR1-gated cells (red). The mean frequency of MR1⁺ cells from total live thymocytes (n =4) was 0.37% (SEM: 0.02). Similar results were seen from all 4 thymocyte donors.