Compartmentalization of immune responses in human tuberculosis: few CD8+ effector T cells but elevated levels of FoxP3+ regulatory t cells in the granulomatous lesions

Abstract

Immune responses were assessed at the single-cell level in lymph nodes from children with tuberculous lymphadenitis. Tuberculosis infection was associated with tissue remodeling of lymph nodes as well as altered cellular composition. Granulomas were significantly enriched with CD68+ macrophages expressing the M. tuberculosis complex-specific protein antigen MPT64 and inducible nitric oxide synthase. There was a significant increase in CD8+ cytolytic T cells surrounding the granuloma; however, CD8+ T cells expressed low levels of the cytolytic and antimicrobial effector molecules perforin and granulysin in the granulomatous lesions. Quantitative real-time mRNA analysis revealed that interferon-gamma, tumor necrosis factor-alpha, and interleukin-17 were not up-regulated in infected lymph nodes, but there was a significant induction of both transforming growth factor-beta and interleukin-13. In addition, granulomas contained an increased number of CD4+FoxP3+ T cells co-expressing the immunoregulatory cytotoxic T-lymphocyte antigen-4 and glucocorticoid-induced tumor necrosis factor receptor molecules. Low numbers of CD8+ T cells in the lesions correlated with high levels of transforming growth factor-beta and FoxP3+ regulatory T cells, suggesting active immunosuppression at the local infection site. Compartmentalization and skewing of the immune response toward a regulatory phenotype may result in an uncoordinated effector T-cell response that reduces granule-mediated killing of M. tuberculosis-infected cells and subsequent disease control.

Figure 1

Induction of tissue remodeling and altered cellular composition in TB-infected lymph nodes. A: The images demonstrate tissue morphology and CD3 immunohistological staining in TB-pos(+) and TB-neg(−) lymph nodes as well as uninfected control tonsil. Magnification = original ×50. B: Expression and distribution of CD68, CD8 and collagen type I in TB-pos(+) lymph nodes compared with uninfected control tonsil. Granulomatous lesions in TB-pos(+) lymphadenitis are marked with a solid line. Magnification = original ×125. GC indicates germinal centers or follicles in lymphoid tissue. Staining was performed using immunohistochemistry and positive cells are shown in brown (diaminobenzidine) whereas negative cells are counterstained blue with hematoxylin. C: Mean cellularity (±SD) of TB-pos(+) and TB-neg(−) lymphadenitis compared with uninfected control, respectively, was estimated in the analysis. Data are presented as % cellularity of lymphoid tissue, in a box and whisker plot. D: In situ computerized image analysis was used to assess median expression (±IQR) of the indicated APC markers and Mtb-specific antigen MPT64 or (E) T cell markers in TB-pos(+) compared with TB-neg(−) lymphadenitis as well as uninfected control tonsils. Data are presented as % positive area of the total cell area. Statistical significance of differences in tissue cellularity and protein expression was determined by a non-parametric Kruskal-Wallis test, TB-pos(+) lymphadenitis versus TB-neg(−) lymphadenitis versus uninfected control. The statistical significance of the indicated P values was determined as: *P < 0.05, **P < 0.01, ***P < 0.001 and P > 0.05 ns (not significant).

Figure 2

Activated T cells produce suboptimal levels of inflammatory cytokines and cytolytic effector molecules in TB infected lymph nodes. A: Cytokine profile and expression of T cell-associated cytolytic effector molecules in lymphoid tissue was determined using quantitative real-time PCR. Fold change of CD4 mRNA and pro-inflammatory cytokines IFN-γ, TNF-α, and IL-17, as well as CD8 mRNA and cytolytic effector molecules granzyme A, perforin, granulysin to ubiquitin C mRNA in TB-pos(+) lymph nodes was compared with that of TB-neg(−) lymph nodes and uninfected control tissue. Data are presented as fold change of mRNA in a box and whisker plot and the dotted line represents a relative difference of 1. B: In situ computerized image analysis was used to assess median expression (±IQR) of cytolytic and antimicrobial effector molecules in TB-pos(+) compared with TB-neg(−) lymphadenitis as well as uninfected control tonsils. Data are presented as % positive area of the total cell area. C: The expression and distribution of granzyme A, perforin, granulysin and iNOS in TB-pos(+) lymph nodes was compared with uninfected control tonsil. Staining was performed using immunohistochemistry and arrows indicate positive (brown staining) cells whereas negatively stained cells are counterstained with hematoxylin. Granulomatous lesions in TB-pos(+) lymphadenitis are marked with a solid line. GC indicates germinal centers or follicles in the tonsil tissue. Magnification = original ×125. D: Immunofluorescent staining and confocal microscopy showed local distribution and co-expression of CD8+ T cells (red; Alexa-594), granzyme A, perforin and granulysin (green; Alexa-488) in the parafollicular area of a TB-pos(+) lymph node. Arrows indicate double-positive cells in yellow. Magnification = original ×300. Statistical significance of differences in mRNA and protein expression was determined by a non-parametric Kruskal-Wallis test (TB-pos(+) lymphadenitis versus TB-neg(−) lymphadenitis versus uninfected control). The statistical significance of the indicated P values was determined as: *P < 0.05, **P < 0.01, ***P < 0.001 and P > 0.05 ns (not significant).

Figure 3

Expression and distribution of APCs, mycobacterial antigens, T cells and cytolytic effector molecules in total TB-pos(+) lymph node tissue compared with granulomatous lesions. A: The Mtb-specific protein antigen, MPT64, was predominantly expressed inside tuberculous granulomas. An excluder function of the image analysis software program was used to determine protein expression in total TB-pos(+) lymph node tissue compared with the granulomatous lesions (solid line). B: In situ computerized image analysis was used to assess median expression (±IQR) of CD68+ macrophages, iNOS, and n-tyr, and mycobacterial antigens, BCG and MPT64, in total lymph node tissue as compared with protein expression in the granulomatous lesions. C: Confocal images reveal double-staining of CD68 or CD8 (red; Alexa 594) with Mtb-antigen MPT64 (green; Alexa-488). Arrows indicate double-positive cells in yellow and single-positive cells in red and green. Magnification = original ×200. D: In situ imaging was used to determine median expression (± IQR) of CD8+ and CD56+ T and NK cells, granzyme A, perforin and granulysin in total lymph node tissue compared with protein expression in the granulomatous lesions. The data are presented as % positive area of total cell area. E: Computerized image analysis was used to determine the relative expression of effectors and total CD3+ T cells in lymphoid tissue. The ratios of granzyme A (grzA), perforin (pfn) and granulysin (grs) expression to total CD3+ T cell expression in total TB-pos(+) lymph node tissue compared with granulomatous lesions and uninfected control are presented. The median values of paired expression of effectors and CD3+ T cells from all individual patients are shown. F: Confocal microscopy illustrate distribution and co-expression of perforin (green; Alexa-488) and granulysin (red; Alexa-594) in the granulomatous lesions and T cell rich areas of a TB-pos(+) lymph node. Arrows indicate single-positive cells inside the granuloma but also double-positive cells in yellow outside the granuloma. Note the granular and polarized co-expression of the cytolytic effector molecules in cells located outside the lesion. Magnification = original ×200 and ×600. Statistical significance of differences in protein expression was determined by a non-parametric Wilcoxon signed rank test (total TB-pos(+) lymph node tissue versus granulomatous lesions). The statistical significance of the indicated P values was determined as: **P < 0.01, ***P < 0.001 and P > 0.05 ns (not significant).

Figure 4

Expression and distribution of FoxP3 and TGF-β in total TB-pos(+) lymph node tissue compared with granulomatous lesions. A: Expression of immunoregulatory molecules and anti-inflammatory as well as Th2 cytokines in lymphoid tissue was determined using quantitative real-time PCR. Fold change of FoxP3 mRNA and cytokines TGF-β, IL-10, IL-13 to ubiquitin C mRNA in TB-pos(+) lymph nodes was compared with that of TB-neg(−) lymph nodes and uninfected control tissue. Data are presented as fold change of mRNA in a box and whisker plot and the dotted line represents a relative difference of 1. B: Immunohistochemical analysis of MTP64, CD8, FoxP3, and TGF-β expression in a TB-pos(+) lymph node; both granulomatous lesion (solid line) and non-granulomatous areas outside the granuloma are shown. Arrows indicate positive (brown staining) cells whereas negatively stained cells are counterstained with hematoxylin. Magnification = original ×125. C: Confocal analysis of Treg cell expression in a granuloma. Upper panel shows co-expression of CD4 (red; Alexa-594) and FoxP3 (green; Alexa-488) at a high (×125) and low (×600) magnification. Note the nuclear staining pattern of FoxP3, whereas CD4 is located at the surface of positive cells. Lower panel shows co-expression of CTLA-4 (red; Alexa-594) and GITR (green; Alexa-488) as well as CD4 (red; Alexa-594) and CTLA-4 (green; Alexa-488) inside a granuloma. Arrows indicate double-positive cells. Magnification = original ×200 and ×600. (D) Computerized image analysis was used to determine the median expression (±IQR) of CD4⁺ T cells, FoxP3 Treg cells, CTLA-4, GITR, and TGF-β in total lymph node tissue compared with protein expression in the granulomatous lesions. The data are presented as % positive area of total cell area. E: Computerized image analysis was used to determine the relative expression of CD8+ T cells and FoxP3+ Treg cells in lymphoid tissue. The ratios of total CD8+ T cells to total FoxP3+ Treg cells expressed in total TB-pos(+) lymph node tissue compared with granulomatous lesions as well as TB-neg(−) lymphadenitis and uninfected control are presented. The median values of paired expression of CD8+ T cells and FoxP3+ Treg cells from all individual patients are shown. *P < 0.05, **P < 0.01 and ***P < 0.001.