Unconventional Human T Cells Accumulate at the Site of Infection in Response to Microbial Ligands and Induce Local Tissue Remodeling

Abstract

The antimicrobial responsiveness and function of unconventional human T cells are poorly understood, with only limited access to relevant specimens from sites of infection. Peritonitis is a common and serious complication in individuals with end-stage kidney disease receiving peritoneal dialysis. By analyzing local and systemic immune responses in peritoneal dialysis patients presenting with acute bacterial peritonitis and monitoring individuals before and during defined infectious episodes, our data show that Vγ9/Vδ2(+) γδ T cells and mucosal-associated invariant T cells accumulate at the site of infection with organisms producing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and vitamin B2, respectively. Such unconventional human T cells are major producers of IFN-γ and TNF-α in response to these ligands that are shared by many microbial pathogens and affect the cells lining the peritoneal cavity by triggering local inflammation and inducing tissue remodeling with consequences for peritoneal membrane integrity. Our data uncover a crucial role for Vγ9/Vδ2 T cells and mucosal-associated invariant T cells in bacterial infection and suggest that they represent a useful predictive marker for important clinical outcomes, which may inform future stratification and patient management. These findings are likely to be applicable to other acute infections where local activation of unconventional T cells contributes to the antimicrobial inflammatory response.

FIGURE 1.

Proinflammatory migratory profile of unconventional T cells. (A) Total cell counts and concentration of the neutrophil-attracting chemokine CXCL8 in the peritoneal effluent of stable PD patients and patients presenting with acute peritonitis. (B) Total numbers of Vγ9⁺CD3⁺ T cells and Vα7.2⁺CD3⁺ T cells within the peritoneal cell population in stable PD patients and during acute peritonitis. (C) Representative example for the coexpression of CCR2, CCR5, and CCR6 with CD161 on blood Vα7.2⁺CD3⁺ T cells in a stable PD patient. (D) Percentage of CCR2⁺, CCR5⁺, and CCR6⁺ cells among Vγ9⁻ and Vγ9⁺CD3⁺ T cells (upper panels) or among Vα7.2⁺CD161⁻ and Vα7.2⁺CD161⁺CD3⁺ T cells in the blood of stable PD patients (lower panels). (E) Concentration of the indicated chemokines in the effluent of patients presenting with acute peritonitis; upper limits of detection for CCL3 and CCL4 were 4.12 and for 4.32 ng/ml, respectively. Data were analyzed using Mann–Whitney tests (in the case of CCL2 after normalization). Each data point represents an individual patient, error bars depict the median ± interquartile range.

FIGURE 2.

Systemic and local levels of unconventional T cells in stable PD patients and during acute peritonitis. (A) Blood (PBMC) and peritoneal dialysis effluent (PDE) were analyzed by flow cytometry for the proportion of Vγ9/Vδ2 T cells (identified as Vγ9⁺; left) and MAIT cells (Vα7.2⁺CD161⁺; right), expressed as percentage of all CD3⁺ T cells. Samples were collected from stable PD patients and patients presenting with acute peritonitis (day 1), before commencing antibiotic treatment. Data were analyzed using Kruskal–Wallis tests combined with Dunn’s multiple comparisons tests. Each data point represents an individual patient; asterisks indicate significant differences between groups. (B) Local levels of unconventional T cells in the effluent of two PD patients while the individuals were stable and when they presented with distinct peritonitis episodes caused by bacteria capable or not of producing HMB-PP or vitamin B2 (day 1).

FIGURE 3.

Matched levels of unconventional T cells in blood and effluent of PD patients before and during acute peritonitis. Blood and peritoneal effluent samples from the same individuals were analyzed by flow cytometry for the proportion of Vγ9/Vδ2 T cells (identified as Vγ9⁺) (A–C) and MAIT cells (Vα7.2⁺ CD161⁺) (D–F), expressed as percentage of all CD3⁺ T cells. Samples were collected while patients were stable and when they presented with acute peritonitis (day 1), before commencing antibiotic treatment. (A and D) Unconventional T cell levels in blood and effluent of stable individuals. (B and E) Unconventional T cell levels in blood and effluent of all patients with acute peritonitis (left) and in subgroups of patients with confirmed infections by bacteria capable or not of producing HMB-PP or vitamin B₂ (middle, right). (C and F) Local unconventional T cell levels in the effluent of PD patients before and during acute peritonitis (left) and in subgroups of patients with infections by bacteria producing HMB-PP and/or vitamin B₂ (middle, right). Data were analyzed using Wilcoxon matched–pairs signed rank tests. Each data point represents an individual patient.

FIGURE 4.

Peritoneal unconventional T cell responses to microbial metabolites. (A) Activation of peritoneal Vγ9⁺ γδ T cells and Vα7.2⁺CD161⁺ MAIT cells from stable PD patients upon overnight stimulation with HMB-PP (n = 4 individual patients) or DMRL (n = 3), as analyzed by flow cytometry and expressed as proportion of γδ or MAIT cells coexpressing CD69 and TNF-α (means ± SEM). (B) Activation of peritoneal Vγ9⁺ γδ T cells and Vα7.2⁺CD161⁺ MAIT cells upon overnight stimulation in the presence of extracts from different clinical isolates that produce (filled symbols) or do not produce (empty symbols) the corresponding ligands (median ± interquartile range): E. coli (HMB-PP⁺vit.B2⁺), K. pneumoniae (HMB-PP⁺vit.B2⁺), P. aeruginosa (HMB-PP⁺vit.B2⁺), C. striatum (HMB-PP⁺vit.B2⁺), L. monocytogenes (HMB-PP⁺ vit.B2⁻), S. aureus (HMB-PP⁻vit.B2⁺), E. faecalis (HMB-PP⁻vit.B2⁻), and S. pneumoniae (HMB-PP⁻vit.B2⁻). Data were analyzed using Kruskal–Wallis tests combined with Dunn’s multiple comparisons tests. Each data point represents an individual patient. (C) Activation of total peritoneal leukocytes by extracts of the indicated bacteria, in the absence or presence of anti-BTN3 blocking Abs, shown as coexpression of CD69 and TNF-α (left) or IFN-γ (right) by Vγ9⁺ T cells after overnight stimulation. Data were analyzed using Wilcoxon matched–pairs signed rank tests. Each data point represents an individual patient; asterisks depict significant differences of anti-BTN3–treated samples compared with untreated controls.

FIGURE 5.

Ex vivo responsiveness of peritoneal leukocytes to pathogenic bacteria. Peritoneal cells were obtained from the effluent of stable patients and exposed overnight to extracts prepared from the indicated bacterial species. (A) Representative example of an intracellular staining of TNF-α in peritoneal leukocytes cultured in the absence (medium; top panel) or presence of E. coli extract (middle panel), as analyzed by flow cytometry within the CD3⁺ gate. Bottom panel, distribution of Vα7.2⁺ and Vγ9⁺ cells within all CD3⁺TNF-α⁺ peritoneal cells after stimulation with E. coli extract. (B) Proportion of Vα7.2⁺ (black) and Vγ9⁺ cells (shaded) T cells among peritoneal T cells producing or not TNF-α and IFN-γ in response to E. coli, as analyzed by flow cytometry in nine stable individuals. (C) Overnight secretion of IFN-γ, CXCL10, and CXCL8 by peritoneal cells in response to bacteria that produce (S. aureus, C. striatum; filled circles) or do not produce (E. faecalis; empty circles) ligands for Vγ9/Vδ2 T cells and/or MAIT cells, as analyzed by ELISA (median ± interquartile range). Data were analyzed using Kruskal–Wallis tests combined with Dunn’s multiple comparisons tests. Each data point represents an individual patient; asterisks indicate significant differences compared with medium controls (triangles). (D) Specific inhibition of IFN-γ secretion by peritoneal leukocytes in response to bacterial extracts, in the absence or presence of anti-BTN3 and anti-MR1 blocking Abs, alone or in combination. Data shown are means ± SEM from independent experiments with three omental donors. ND, not done.

FIGURE 6.

Activation of peritoneal tissue cells by γδ T cell– and MAIT cell–derived cytokines. Growth-arrested peritoneal mesothelial cells (A) or peritoneal fibroblasts (B) from human omentum were exposed to supernatants derived from activated Vγ9/Vδ2 T cells and MAIT cells at a dilution of 1:4, in the absence or presence of 10 μg/ml sTNFR and 10 μg/ml anti–IFN-γ, alone or together. Data shown are levels of CCL2, CXCL8, CXCL10, and IL-6 secreted into the culture medium over 24 h by ELISA (means ± SEM from independent experiments with four to seven omental donors). Data were analyzed using Friedman tests combined with Dunn’s multiple comparisons tests. Asterisks indicate significant differences compared with medium controls.

FIGURE 7.

Activation of peritoneal tissue cells by effluent from PD patients with acute peritonitis. (A) Growth-arrested peritoneal mesothelial cells from human omentum (n = 2–4) were exposed to peritoneal effluent obtained from three stable PD patients in the absence of any inflammation (#1–3) and from three patients presenting with acute peritonitis (#4: Enterobacter sp., #5: E. coli, and #6: Acinetobacter sp.). Data shown are levels of CCL2 and CXCL8 secreted into the culture medium over 24 h by ELISA (median ± interquartile range). Data were analyzed using Kruskal–Wallis tests combined with Dunn’s multiple comparisons tests. Asterisks indicate significant differences compared with medium controls (Ctrl). (B) Mesothelial cells were exposed to peritoneal effluent from patients presenting with peritonitis, in the absence or presence of 10 μg/ml sTNFR and 10 μg/ml anti–IFN-γ. Data shown are expressed as percent inhibition of CCL2 and CXCL8 secretion over 24 h, compared with untreated controls. Data were analyzed using Wilcoxon matched–pairs signed rank tests. Each data point represents an independent experiment.

FIGURE 8.

Association of first-time peritonitis caused by HMB-PP⁺ and vit.B2⁺ bacteria with poor clinical outcome. Cumulative rates of technique failure (top left), mortality (top right), catheter removal (middle), and transfer to permanent HD (bottom) of patients from the ANZDATA registry with first-time peritonitis, grouped into infections with Gram⁺HMB-PP⁻vit.B2⁻ (green), Gram⁺HMB-PP⁻vit.B2⁺ (gray), Gram⁺HMB-PP⁺vit.B2⁺ (blue), or Gram⁻HMB-PP⁺ vit.B2⁺ bacteria (red); episodes caused by Gram⁻HMB-PP⁻ (e.g., Legionella spp.) or Gram⁺HMB-PP⁺vit.B2⁻ species (e.g., L. monocytogenes) were not recorded and/or were too rare for this comparison. Numbers indicate the number of cases of acute peritonitis caused by the listed organisms. Comparisons were made using log-rank tests.

FIGURE 9.

Unconventional T cell–induced reprogramming of peritoneal mesothelial cells. Growth-arrested peritoneal mesothelial cells from human omentum were cultured in medium alone or exposed to supernatants derived from activated MAIT cells, in the absence or presence of 10 μg/ml sTNFR and 10 μg/ml anti–IFN-γ (A), or stimulated with 5 ng/ml TNF-α and IFN-γ, alone or in combination (B). Images were captured after 24 h in culture with a light microscope at original magnification ×20 and are representative of three to four individual donors. (C) Expression of epithelial (E-cadherin, occludin, zona occludens-1 [ZO-1], and claudin-1) and mesenchymal markers (fibronectin and Snail) by mesothelial cells after 24 h exposure to MAIT cell supernatants, as determined by quantitative PCR as relative expression compared with 1000 copies of GAPDH as housekeeping gene. (D) Expression of miR-21 by mesothelial cells after 24 h exposure to MAIT cell supernatants in the absence or presence of 10 μg/ml sTNFR and 10 μg/ml anti–IFN-γ, as determined by quantitative PCR as relative expression compared with miR-191 as reference miR. Data were analyzed using Wilcoxon matched–pairs signed rank tests or paired t tests. Each data point represents an individual patient.