Mtb-Specific HLA-E-Restricted T Cells Are Induced during Mtb Infection but Not after BCG Administration in Non-Human Primates and Humans

Abstract

Background: Novel vaccines targeting the world's deadliest pathogen Mycobacterium tuberculosis (Mtb) are urgently needed as the efficacy of the Bacillus Calmette-Guérin (BCG) vaccine in its current use is limited. HLA-E is a virtually monomorphic unconventional antigen presentation molecule, and HLA-E-restricted Mtb-specific CD8⁺ T cells can control intracellular Mtb growth, making HLA-E a promising vaccine target for Mtb. Methods: In this study, we evaluated the frequency and phenotype of HLA-E-restricted Mtb-specific CD4⁺/CD8⁺ T cells in the circulation and bronchoalveolar lavage fluid of two independent non-human primate (NHP) studies and from humans receiving BCG either intradermally or mucosally. Results: BCG vaccination followed by Mtb challenge in NHPs did not affect the frequency of circulating and local HLA-E-Mtb CD4⁺ and CD8⁺ T cells, and we saw the same in humans receiving BCG. HLA-E-Mtb T cell frequencies were significantly increased after Mtb challenge in unvaccinated NHPs, which was correlated with higher TB pathology. Conclusions: Together, HLA-E-Mtb-restricted T cells are minimally induced by BCG in humans and rhesus macaques (RMs) but can be elicited after Mtb infection in unvaccinated RMs. These results give new insights into targeting HLA-E as a potential immune mechanism against TB.

Keywords: BCG; HLA-E; T cells; tuberculosis; vaccine.

Figure 1

HLA-E–Mtb and CMV CD4⁺ and CD8⁺ T cell frequencies in the circulation remain stable after receiving BCG in humans and RMs. Peptide pools for HLA-E TM staining on human and RM samples are shown in Table 1. (A) HLA-E*01:03 and *01:01 CD4⁺ and CD8⁺ T cell frequencies for CMV (left) and the Mtb pool (right) at week 0 and 3, 5, and 52 weeks after intradermal BCG revaccination in healthy TST⁺ HIV⁻ volunteers (n = 20); (B) HLA-E*01:01 (left) and *01:03 (right) CD4⁺ and CD8⁺ T cell frequencies for the Mtb pool on day 0 (circles) and 7 (squares) and 14 (triangles) days after aerosol BCG inhalation (n = 12) or intradermal BCG administration (n = 12) in healthy volunteers; (C) HLA-E*01:03 CD4⁺ and CD8⁺ T cell frequencies for CMV (left), Mtb pool 1 (middle), and 2 (right) in RM study 1 at the pre-vaccination time point (white circles) and 10 (blue circles) and 16 weeks post-vaccination (blue triangles); (D) Same as C, but for RM study 2 at the pre-vaccination time point (white circles) and 9 weeks post-vaccination (blue circles); (E) Correlating HLA-E CD4⁺ T cell frequencies (X-axis) and HLA-E CD8⁺ T cell frequencies (Y-axis) at all time points in the human studies for the Mtb pool (left) and in RM study 1 and 2 combined for Mtb pool 2 (right). Shaded bars represent the median frequency, and the error bars represent the 95% confidence interval. Significance was tested using a repeated measures (RM) two-way ANOVA with multiple comparison correction (A–D) and a Spearman’s rank correlation (E). **** = p < 0.0001.

Figure 2

Mtb challenge only or following BCG vaccination in RMs results in stable HLA-E–Mtb and CMV CD4⁺ and CD8⁺ T cell frequencies in the circulation. Peptide pools for HLA-E TM staining on RM samples are shown in Table 1. (A) HLA-E*01:03 CD4⁺ and CD8⁺ T cell frequencies for CMV (upper panel) and Mtb pool 2 (lower panel) in RM study 1 at the pre-challenge time point (blue triangles) and 2 (orange circles), 6 (orange triangles), and 10 weeks (orange squares) post-Mtb challenge; (B) Same as A, but in RM study 2 at the pre-challenge time point (blue circles) and 3 (orange circles) and 7 weeks (orange triangles) post-Mtb challenge. Shaded bars represent the median frequency, and the error bars represent the 95% confidence interval. Significance was tested using a repeated measures (RM) two-way ANOVA with multiple comparison correction.

Figure 3

HLA-E–Mtb and CMV CD4⁺ and CD8⁺ T cell frequencies are increased after Mtb challenge in BAL of BCG-unvaccinated RMs. Peptide pools for HLA-E TM staining on RM and human samples are shown in Table 1. (A) Density plots for HLA-E p44, CMV, and Mtb pool 1 and 2 CD4⁺ and CD8⁺ T cell frequencies in a BAL sample of one representative unvaccinated RM of study 2 post-Mtb challenge. HLA-E TMs in PE are shown on the X-axis and CD4 and CD8 are shown on the Y-axis; (B) HLA-E*01:03 CD4⁺ and CD8⁺ T cell frequencies for CMV and Mtb pool 1 and 2 in BAL (orange) and PBMCs (blue), 8 and 7 weeks, respectively, post-Mtb challenge in unvaccinated RMs of RM study 2 (n = 8); (C) HLA-E*01:01 (triangles) and *01:03 (circles) CD4⁺ and CD8⁺ T cell frequencies in BAL (orange) and in PBMCs (blue) for the Mtb pool, 14 days after aerosol BCG inhalation in healthy volunteers (n = 12); (D) HLA-E*01:03 CD4⁺ and CD8⁺ T cell frequencies in BAL for CMV (left) and Mtb pool 2 (right) in RM study 2, 12 weeks post-BCG vaccination (blue) and 8 weeks post-Mtb challenge (orange); (E) correlating the CFU counts (Y-axis) and the PA scores (X-axis) in the total lung (left), primary lobe (middle), and secondary lobe (right) for RM study 2. Grey dots represent the unvaccinated group, blue dots the intradermal BCG vaccinated group, and orange dots the mucosal BCG vaccinated group. Dotted lines represent the 95% confidence interval; (F) Same as E, but correlating HLA-E*01:03 Mtb CD3⁺ T cell frequencies in BAL (Y-axis) and the PA scores in the total lung (left) and primary lobe (right) (X-axis) 8 weeks post-Mtb challenge. Shaded bars represent the median frequency, and the error bars represent the 95% confidence interval. Significance was tested using a repeated measures (RM) two-way ANOVA with multiple comparison correction (A–D) and a Spearman’s rank correlation (E,F). * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Figure 4

Phenotypic analysis of HLA-E–Mtb CD4⁺ and CD8⁺ T cells after BCG vaccination and Mtb challenge in RMs. Peptide pools for HLA-E TM staining on RM samples are shown in Table 1. (A) Memory subset identification of HLA-E*01:03 Mtb and CMV CD3⁺ T cells relative to total CD3⁺ T cells in the circulation of RM study 1 (n = 19), 4 weeks pre-BCG vaccination (left), 16 weeks post-BCG vaccination (middle), and 10 weeks post-Mtb challenge (right); (B) Same as A, but in RM study 2 (n = 24) 2 weeks pre-BCG vaccination (left), 9 weeks post-BCG vaccination (middle), and 7 weeks post-Mtb challenge (right); (C) CCR4, CCR6, and CXCR3 expression in HLA-E*01:03 CMV (blue circles) and Mtb pool 1 (orange circles) and 2 (orange triangles) CD3⁺ T cells relative to total CD3⁺ T cells (white circles) in the circulation 16 weeks post-BCG vaccination in RM study 1 (n = 19); (D) Same as C, but for RM study 2 (n = 24) 9 weeks post-BCG vaccination; (E) Same as D (without CCR6), but in the BAL 12 weeks post-BCG vaccination; (F) CCR4, CCR6, and CXCR3 expression in HLA-E*01:03 CMV (upper panel) and Mtb pool 2 (lower panel) CD3⁺ T cells in the circulation 16 weeks post-BCG vaccination (blue) and 10 weeks post-Mtb challenge (orange) in RM study 1; (G) Same as F, but for RM study 2, 9 weeks post-BCG vaccination (blue) and 7 weeks post-Mtb challenge (orange); (H) Same as G, but in the BAL 12 weeks post-BCG vaccination (blue) and 8 weeks post-Mtb challenge (orange). Shaded bars and stacked bars represent the median and mean frequency, respectively, and the error bars represent the 95% confidence interval. Significance was tested using a repeated measures (RM) two-way ANOVA with multiple comparison correction (C–H). * = p < 0.05, ** = p < 0.01 *** = p < 0.001, **** = p < 0.0001.

Figure 5

Effect of Mtb–SIV co-infection on HLA-E–Mtb and CMV CD4⁺ and CD8⁺ T cell frequencies in cynomolgus macaques (CMs). Peptide pools for HLA-E TM staining on CM samples are shown in Table 1. Three CMs were vaccinated with BCG and were challenged with Mtb followed by SIV infection, three CMs were challenged with Mtb followed by SIV infection without BCG vaccination, and three CMs were challenge with Mtb only. (A) HLA-E*01:03 CMV (left), Mtb pool 1 (middle) and 2 (right) CD4⁺ and CD8⁺ T cell frequencies 6 weeks post-Mtb challenge and 4 weeks post-SIV challenge in unvaccinated (white) and BCG-vaccinated (blue) CMs; (B) Same as A, but 4 weeks post-SIV challenge in unvaccinated and Mtb-challenged CMs, either without (white) or with (purple) subsequent SIV infection; (C) Same as A, but pre-Mtb challenge (white circles) and 6 (orange circles) and 14 weeks (orange triangles) post-Mtb challenge (before SIV infection) in six unvaccinated CMs. Shaded bars represent the median frequency, and the error bars represent the 95% confidence interval. Significance was tested using a repeated measures (RM) two-way ANOVA with multiple comparison correction.