Functional, Antigen-Specific Stem Cell Memory (TSCM) CD4+ T Cells Are Induced by Human Mycobacterium tuberculosis Infection

Abstract

Background: Maintenance of long-lasting immunity is thought to depend on stem cell memory T cells (T_SCM), which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (T_CM) or effector (T_EFF) T cells. Our knowledge of T_SCM derives primarily from studies of virus-specific CD8⁺ T_SCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis, generates antigen-specific CD4⁺ T_SCM and to characterize their functional ontology.

Methods: We studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT) converters and three cross-sectional QFT⁺ adult cohorts; and to bacillus Calmette-Guerin (BCG) vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer⁺ CD4⁺ T_SCM (CD45RA⁺ CCR7⁺ CD27⁺) were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.

Results: M. tb-specific T_SCM were not detected in QFT-negative persons. After QFT conversion frequencies of T_SCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces T_SCM cells. Gene expression (GE) profiling of tetramer⁺ T_SCM showed that these cells were distinct from bulk CD4⁺ naïve T cells (T_N) and shared features of bulk T_SCM and M. tb-specific tetramer⁺ T_CM and T_EFF cells. These T_SCM were predominantly CD95⁺ and CXCR3⁺, markers typical of CD8⁺ T_SCM. Tetramer⁺ T_SCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk T_N and T_SCM cells. M. tb-specific T_SCM were also functional, producing IL-2, IFN-γ, and TNF-α upon antigen stimulation, and their frequencies correlated positively with long-term BCG-specific CD4⁺ T cell proliferative potential after infant vaccination.

Conclusion: Human infection with M. tb induced distinct, antigen-specific CD4⁺ T_SCM cells endowed with effector functions, including expression of cytotoxic molecules and Th1 cytokines, and displayed chemokine receptor profiles consistent with memory Th1/17 cells. Induction of CD4⁺ T_SCM should be considered for vaccination approaches that aim to generate long-lived memory T cells against M. tb.

Keywords: LTBI; Mycobacterium tuberculosis; QuantiFERON conversion; TSCM; memory T cells.

Figure 1

Transcriptional profile of Mycobacterium tuberculosis (M. tb)-specific T_SCM. Expression of 96 genes was measured by microfluidic qRT-PCR on bulk or M. tb-tetramer⁺ CD4⁺ T cells expressing a naïve (T_N, CD45RA⁺ CCR7⁺ CD27⁺ CD95⁻, labeled as naïve-like memory T_NLM for M. tb-tetramer⁺), T_SCM (CD45RA⁺ CCR7⁺ CD27⁺ CD95⁺, bulk CD4⁺ cells only), T_CM (CD45RA⁻ CCR7⁺ CD27⁺), or T_EFF (CD45RA⁻ CCR7⁻) phenotype, sorted from M. tb-infected adults (n = 7). Analyses shown in this figure were built on transcripts (22 mRNA) that were differentially expressed [Kruskal–Wallis H test p < 0.05 and false discovery rate (FDR) < 0.05] between bulk CD4⁺ T_N, T_SCM, T_CM, and T_EFF cells. (A) Principal component analysis (PCA) of sorted bulk T_N (green), T_SCM (indigo), T_CM (magenta), and T_EFF (purple). PCA loadings—PC1 and PC2—defined on bulk CD4⁺ subsets were applied to M. tb-tetramer⁺ T_NLM (light blue triangles) CD4⁺ T cells. (B) Unsupervised heat map of 22 transcripts differentially expressed between bulk T_N, T_SCM, T_CM, and T_EFF subsets. Bulk T_N, T_SCM, and M. tb-tetramer⁺ T_NLM represented as green, indigo, and light blue, respectively. Gene expression (GE) is reported as delta Et values [40 − threshold cycle (Ct) values]. (C) Supervised heat map of 20 transcripts differentially expressed (Kruskal–Wallis H test p < 0.05 and FDR < 0.2) between M. tb-tetramer⁺ T_NLM (light blue), T_CM (magenta), and T_EFF (purple) CD4 T cell subsets. GE is reported as delta Et values (40 − Ct values).

Figure 2

Mycobacterium tuberculosis (M. tb)-specific T_SCM are detected in recently M. tb-infected adolescents. (A) Median (error bars denote IQR) of Tuberculin Skin Test (TST) and QuantiFERON-TB Gold In-Tube (QFT) performed at the indicated time points. A 5 mm and 0.35 IU/ml cutoff were used to define positive TST and QFT, respectively (n = 19 participants). (B) Representative flow cytometry plots showing peripheral blood mononuclear cells staining with two identical major histocompatibility complex (MHC) class II tetramers conjugated to PE and APC to detect CD4⁺ T cells specific for CFP-10. Tetramers matched by MHC allele but loaded with a self-peptide were used as negative control. Number indicates percentage of tetramer⁺ CD4 T cells. (C) Representative flow cytometry plots for memory markers CD45RA, CCR7, CD27, and CD95 gated on bulk (pseudo-color and gray) and M. tb-specific CD4⁺ T cells (red dots). (D) Median (error bars denote IQR) frequencies of M. tb-specific T_SCM (CD45RA⁺ CCR7⁺ CD27⁺, light blue), T_CM (CD45RA⁻ CCR7⁺, magenta), and T_EFF (CD45RA⁻ CCR7⁻, purple) during primary M. tb infection (n = 12 participants).

Figure 3

Mycobacterium tuberculosis (M. tb)-specific T_SCM are not T_N and express distinct homing and cytotoxic profiles from bulk T_SCM. Expression of chemokine receptors and cytotoxic molecules was measured in remotely M. tb-infected (QFT⁺) adults (n = 28) when at least 20 events were detected in each tetramer⁺ memory subset. (A) Representative flow cytometry plots of chemokine receptor expression on bulk (pseudo-color and gray) and M. tb-specific (red dots) CD4⁺ T cells. Box and whisker plots represent the median proportion, IQR, and range of expression of chemokine receptor (n = 28). (B) Box and whisker plot depicting the proportion of chemokine receptor-expressing bulk T_N (CD45RA⁺ CCR7⁺ CD27⁺ CD95⁻, n = 28, green), T_SCM (CD45RA⁺ CCR7⁺ CD27⁺ CD95⁺, n = 28, blue), and M. tb-specific T_SCM (CD45RA⁺ CCR7⁺ CD27⁺, n = 15, light blue) CD4⁺ T cells. p-Values were calculated using Wilcoxon signed-rank test and corrected for multiple comparison using the Benjamini–Hochberg method with an false discovery rate (FDR) of 0.05. Adjusted p-values <0.05 were considered significant. (C) Pie chart showing the median proportions (slices) of bulk T_SCM (n = 28) and M. tb-specific T_SCM (n = 15) CD4⁺ T cells co-expression of CD95, CCR4, CCR5, CCR6, and/or CXCR3, denoted by arcs. p-Value was calculated using non-parametric permutation test comparing the overall distribution between pies. (D) Representative flow cytometry plots of cytotoxic molecule expression on total (pseudo-color and gray) and M. tb-specific (red dots) CD4⁺ T cells. Box and whisker plots represent the median proportion, IQR, and range of expression of cytotoxic molecules (n = 20). (E) Proportion of granzyme (grn) A, grnB, grnK, granulysin, and perforin expression in bulk T_N (n = 20, green), T_SCM (n = 20, blue), and M. tb-specific T_SCM (n = 5, light blue) CD4⁺ T cells. p-Values were calculated with the Mann–Whitney test, corrected for multiple comparisons with the Benjamini–Hochberg method with an FDR of 0.05. Adjusted p-values <0.05 were considered significant. (F) Pie chart showing the median proportions (slices) of bulk T_SCM (n = 20) and M. tb-specific T_SCM (n = 5) CD4⁺ T cells co-expression grnA, grnB, grnK, granulysin, and/or perforin, denoted by arcs. p-Value was calculated using non-parametric permutation test comparing the overall distribution between pies.

Figure 4

Mycobacterium tuberculosis (M. tb)-specific T_SCM display early memory tissue homing and cytotoxic profiles. Expression of chemokine receptors and cytotoxic molecules was measured in remotely M. tb-infected (QFT⁺) adults (n = 28) when at least 20 events were detected in each tetramer⁺ memory subset. (A) Box and whiskers plots depicting the proportion of chemokine receptors expression of M. tb-specific T_SCM (n = 15, light blue), T_CM (n = 27, magenta), and T_EFF (n = 23, purple) CD4⁺ T cells. p-Values were calculated using Wilcoxon signed-rank test and corrected for multiple comparison using the Benjamini–Hochberg method with a false discovery rate (FDR) of 0.05. Adjusted p-values <0.05 were considered significant. (B) Pie chart showing the median proportions (slices) of M. tb-specific T_SCM (n = 15), T_CM (n = 27), and T_EFF (n = 23) CD4⁺ T cells co-expression of CD95, CCR4, CCR5, CCR6, and/or CXCR3, denoted by arcs. p-Values were calculated using non-parametric permutation test comparing the overall distribution between pies. (C) Box and whiskers plots depicting the proportion of grnA, grnB, grnK, granulysin, and perforin expression in M. tb-specific T_SCM (n = 5, light blue), T_CM (n = 19, magenta), and T_EFF (n = 17, purple) CD4⁺ T cells. p-Values were calculated using Mann–Whitney test and corrected for multiple comparison using the Benjamini–Hochberg method with an FDR of 0.05. Adjusted p-values <0.05 were considered significant. (D) Pie chart showing the median proportions (slices) of M. tb-specific T_SCM (n = 5), T_CM (n = 19), and T_EFF (n = 17) CD4⁺ T cells co-expression of grnA, grnB, grnK, granulysin, and/or perforin, denoted by arcs. p-Values were calculated using non-parametric permutation test comparing the overall distribution between pies.

Figure 5

Mycobacterium tuberculosis (M. tb)-specific T_SCM express Th1 cytokines. Fresh whole blood from remotely M. tb-infected (QFT⁺) adults (n = 13) was left unstimulated (gray) or stimulated with peptide pools spanning M. tb antigens, Ag85B (green), ESAT-6 (orange), or CFP-10 (purple), or whole bacillus Calmette–Guerin (red) for 12 h. Box and whisker plots depict frequencies of CD4⁺ T_SCM (RA⁺ R7⁺) T cells expressing IFN-γ (A), TNF-α (B), or IL-2⁺ (C). p-Values were calculated using the Wilcoxon matched pairs test, and p-values <0.0125 were considered significant (corrected for multiple testing using the Bonferroni method).

Figure 6

Bacillus Calmette–Guerin (BCG)-specific T_SCM are associated with long-term CD4⁺ T cell proliferation after vaccination. Whole blood from 1-year-old infants (n = 23) was stimulated with BCG for 12 h to measure the frequencies of cytokine-producing T_SCM (CD45RA⁺, CCR7⁺), T_CM (CD45RA⁻, CCR7⁺), and T_EFF (CD45RA⁻, CCR7⁻). In parallel, whole blood was stimulated with BCG for 7 days, and the frequency of proliferating CD4⁺ cells was assessed by upregulation of Ki-67. Correlations between the frequencies of BCG-specific CD4⁺ memory T cell subsets and those of proliferating CD4⁺ T cells were calculated by Spearman test 10 months postvaccination.