Detailed characterization of human Mycobacterium tuberculosis specific HLA-E restricted CD8+ T cells

Abstract

HLA-E presented antigens are interesting targets for vaccination given HLA-Es' essentially monomorphic nature. We have shown previously that Mycobacterium tuberculosis (Mtb) peptides are presented by HLA-E to CD8⁺ effector T cells, but the precise phenotype and functional capacity of these cells remains poorly characterized. We have developed and utilized in this study a new protocol combining HLA-E tetramer with intracellular staining for cytokines, transcription factors and cytotoxic molecules to characterize these cells in depth. We confirm in this study the significantly increased ex vivo frequency of Mtb-peptide/HLA-E-TM⁺ CD8⁺ T cells in the circulation of patients with active tuberculosis (TB). HLA-E restricted CD8⁺ T cells from TB patients produced more IL-13 than cells from controls or subjects with latent tuberculosis infection (LTBI). Compared to total CD8⁺ T cells, HLA-E restricted cells produced more IFNγ, IL-4, IL-10, and granulysin but less granzyme-A. Moreover, compared to "classical" Mtb specific HLA-A2 restricted CD8⁺ T cells, HLA-E restricted CD8⁺ T cells produced less TNFα and perforin, but more IL-4. In conclusion, HLA-E restricted- Mtb specific cells can produce Th2 cytokines directly.

Keywords: Active TB; CD8+ T cells; Cytokines; Tetramers; Th2.

Figure 1.

Ex vivo frequency of HLA-E restricted CD8⁺ T cells in HDs, LTBI, TB, and TB-HIV subjects. PBMCs were stained using TMs followed by viability staining and cell surface marker staining. Each experiment contained samples from different clinical groups, a total of 21 experiments were performed to analyze all samples. (A) Gating strategy: Initial gate was on single cells, followed by selection of live cells using VIVID cell viability dye and gating on lymphocytes on the basis of forward-side scatters. CD3⁺ cells were gated as T cells and further selected for CD8, but not CD4 expression. (B) Representative dot plot of ex vivo TM staining, with a control (CMV) TM on the y-axis and the combined Mtb TMs on the x-axis. Dot plots show results of one HD, one LTBI, one TB-HIV coinfected patient and 3 TB patients. (C) Frequency and (D) memory profile analysis of ex vivo HLA-E restricted CD8⁺ T cells in HDs, LTBI, TB, and TB-HIV patients. Memory populations were defined based on the expression of CCR7 and CD45RA. N = naïve T cells; EM = effector memory T cells; CM = central memory T cells; TEMRA = T effector memory recently activated cells. For ex vivo analysis, event counts below <100 were not considered a TM⁺ population and therefore not included in the downstream analysis of the memory compartment. 16 out 26 HDs (11 Dutch and 5 Italian HDs), 11 out 14 LTBI, 20 out 24 TB and 5 out 5 HIV-TB subjects were included in the analysis of memory subsets. Horizontal lines (C) represent median values. Black circles: Dutch HDs, open circles: Italian HDs, black triangles: LTBI; black squares: TB patients, black stars: HIV-TB. Bars represent mean values. Data shown in A-B is representative of 21 independent experiments with in total 71 samples and data shown in C and D are pooled from 21 independent experiments with —three to four donors per experiment. P-values were calculated using the Kruskal-Wallis test, including multiple test correction, ***p<0.001, ****p<0.0001. TB, LTBI and TB-HIV groups were each compared to the HD control group.

Figure 2.

HLA-E/Mtb TM⁺ CD8⁺ T-cell frequency after expansion. PBMCs were expanded using mitogenic stimulation, followed by magnetic bead separation of CD8⁺ T cells and specific peptide stimulation. Cells were stained with TMs first before staining with cell surface markers and intracellular cytokine staining. (A) Representative plots of TM staining of CD8⁺ T cells after control or combined p62 and p68 stimulation. Dot plots are representative of one HD and one TB patient that were analyzed in the same experiment. (B) Frequency of HLA-E/Mtb TM⁺ CD8⁺ T cells after PHA expansion and p62 and p68 peptide re-stimulation. 26 out 26 HDs (13 Dutch and 13 Italian HDs), 12 out 14 LTBI, 17 out 24 active TB and 4 out 5 HIV-TB had sufficient cells for in vitro expansion. Black circles: Dutch HDs, open circles: Italian HDs, black triangles: LTBI; black squares: TB patients, black stars: HIV-TB. Each symbol represents one patient. P-values were calculated using Kruskal–Wallis test including multiple-test correction. *** p<0.001. TB, LTBI and TB-HIV groups were compared to HD control group. —Two to three donors from different clinical groups were included per experiment. (C) Absolute count of TM⁺ events from TB patients before (ex vivo) and after PHA expansion and p62 and p68 re-stimulation. An average fold increase of 4.89 was achieved after expansion. Data were pooled from 13 culture experiments. The dashed horizontal line represents the cut off of 1000 cells required for in-depth analysis of the TM+ population, the p-value was calculated using a Wilcoxon signed rank test.****p<0.0001. (D) HLA-E restricted CD8⁺ T cells expanded with PHA and with p62 and p68 Mtb- specific peptides were characterized for expression of TCR-Vβ16. These results were obtained from six independent experiments in which 6 HDs (all Dutch), 7 LTBI and 8 active TB patients were included. Each symbol represents one sample and the p-value was calculated using a Wilcoxon-signed-rank-test. ****p<0.0001. (E) Memory profile analysis of HLA-E/Mtb TM⁺ CD8⁺ T cells upon PHA expansion and p62 and p68 re-stimulation. Samples with TM⁺ counts <1000 were not considered for analysis of the memory compartment: 14 out 26 HDs (10 Dutch and 4 Italian), 6 out 13 LTBI, 15 out 17 TB subjects were considered in the memory analysis. Bars represent mean values.

Figure 3.

HLA-E/Mtb TM⁺ CD8⁺ T cells cytokine production, transcription factors, and cytolytic molecules. PBMCs were expanded using mitogenic stimulation, followed by magnetic bead separation of CD8⁺ T cells and specific peptide stimulation. Cells were stained with TMs first before staining with cell surface markers and intracellular cytokine staining. Events count below <1000 were not included for analysis of HLA-E restricted CD8⁺ T cells’ properties; 14 out 26 HDs (10 Dutch, 4 Italian), 6 out 13 LTBI, 15 out 17 active TB subjects were analyzed. P-values were calculated using a two-way ANOVA non-parametric Dunnet’s test multiple comparison of LTBI and TB with HD control group. Black circles: Dutch HD; Open circles: Italian HD; black triangles: LTBI; black squares: TB patients. (A) A representative plot from 21 independent experiments with —two to four samples per experiment show HLA-E restricted CD8⁺ T cells upon p62 and p68 peptide stimulation in a TB patient, dot plots show TM⁺ cells producing cytokines; transcription factors and cytotoxic molecules. Blue indicates expanded cells not re-stimulated with specific peptides (unstimulated control), red is the sample following overnight peptide stimulation. Data are shown for total CD8⁺ T cells, with the tetramer positive population on the x-axis. Donor TB1 is shown for IFN-γ, TNF-α, IL-4, IL-13 and the transcription factors, whereas donor TB12 is shown for IL-10 and the cytolytic molecules. (B) Frequency of TM⁺ CD8⁺ T cells producing IFNγ, TNFα, IL-4, IL-13, and IL-10 upon p62 and p68 peptide stimulation in HDs, LTBIs and TB samples. 15 out 17 TB patients were analyzed, only for IL-10 (included in Panel B) 14 out 17 TB patients were evaluated. (C) Frequency of TM⁺ CD8⁺ T cells expressing T-bet, gata3 and FoxP3 transcription factors upon expansion in HDs, LTBI and TB subjects. (D) Frequency of TM⁺ CD8⁺ T cells producing Granulysin (Gnly), Granzyme A (GzmA), Granzyme B (GzmB) and Perforin (Prf). upon expansion in HDs, LTBIs and TB samples. In Panel B (including cytotoxic molecules) 14 out 17 active TB patients were analyzed. Each symbol represents one sample. Data shown in B and D are pooled from 21 independent experiments with —two to four patients per experiment. the p-value was calculated using a two-way ANOVA with Dunnets’multiple comparison correction *p<0.05, **p<0.01, ***p<0.001.

Figure 4.

Comparison of HLA-E TM⁺ with total CD8⁺ T cells. PBMCs were expanded usingmitogenic stimulation, followed by magnetic bead separation of CD8⁺ T cells and peptide stimulation. Cells were stained with TMs before staining cell surface markers and intracellular cytokine staining. Total CD8⁺ T-cell production of IFN-γ, TNF-α, IL-4, IL-13, and IL-10 (A), expression of T-bet, Gata3 and FoxP3 transcription factors (B), and Granulysin, Granzyme A, Granzyme B and Perforin (C) was compared with the proportion of cells within the HLA-E/Mtb TM⁺ CD8⁺ T cells upon p62 and p68 stimulation. Lines connect cytokine/transcription factor expression in the same patient sample. Data are pooled from 21 independent experiments with —two to four patients per experiment, p-values were calculated using the Wilcoxon-signed-rank-test. Black stars indicate p values calculated for all samples (n = 37) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; stars in square brackets are p-values calculated for TB subjects only (n = 15). Circles: HD; squares: TB patients; triangles: LTBI.

Figure 5.

Comparison of HLA-A*0201 and HLA-E TM⁺ CD8⁺ T cells. PBMCs were expanded using mitogenic stimulation, followed by magnetic bead separation of CD8⁺ T cells and specific peptide stimulation. Cells were stained with TMs first before staining with cell surface markers and intracellular cytokine staining. Frequency of HLA-A*0201/Mtb and HLA-E/Mtb TM⁺ CD8⁺ T cells producing IFNγ, TNFα, IL-4, IL-13, and IL-10 (A), expressing T-bet, Gata3 and FoxP3 transcription factors (B) and producing Granulysin, Granzyme A, Granzyme B and Perforin (C). 3 out 26 HDs (all Dutch), 3 out 13 LTBI, 7 out 17 active TB patients for panel A and 6 out 17 active TB patients for panel B were HLA-A*0201⁺. Data are pooled from 10 independent experiments with 1–3 HLA-A2⁺ samples per experiment. P-values were calculated using a Wilcoxon-signed-rank-test. p-values calculated for all samples (n = 14; except for cytotoxic molecules & IL-10 n = 13) *p<0.05, **p<0.01, ***p<0.001; stars in square brackets: p-values for TB subjects (n = 7). Circles: HD; squares: TB patients; triangles: LTBI.