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

Study approval

Recruitment of PD patients and healthy volunteers for this study was approved by the South East Wales Local Ethics Committee under reference numbers 04WSE04/27 and 08/WSE04/17, respectively, and conducted according to the principles expressed in the Declaration of Helsinki. All individuals provided written informed consent. The PD study was registered on the U.K. Clinical Research Network Study Portfolio under reference numbers 11838 “Patient immune responses to infection in Peritoneal Dialysis” (PERIT-PD) and 11839 “Leukocyte phenotype and function in Peritoneal Dialysis” (LEUK-PD). Fresh omentum samples from consented patients were obtained from the Wales Kidney Research Tissue Bank.

Patient samples

The local study cohort comprised 101 adults PD patients admitted to the University Hospital of Wales, Cardiff, on day 1 of acute peritonitis between September 2008 and April 2016. Forty-one stable individuals receiving PD for at least 3 mo and with no previous infection served as noninfected controls. Subjects known to be positive for HIV or hepatitis C virus were excluded. Clinical diagnosis of acute peritonitis was based on the presence of abdominal pain and cloudy peritoneal effluent with >100 WBCs/mm³. According to the microbiological analysis of the effluent by the routine Microbiology Laboratory, Public Health Wales, episodes of peritonitis were defined as culture negative (with unclear etiology) or as confirmed bacterial infections caused by specific subgroups of Gram-positive and Gram-negative organisms. The distribution of the nonmevalonate (HMB-PP) and vitamin B₂ pathways across microbial species was determined based on the absence or presence of the enzymes HMB-PP synthase (EC 1.17.7.1) and 6,7-dimethyl-8-d-ribityllumazine (DMRL) synthase (EC 2.5.1.78), respectively, in the corresponding genomes, according to the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg). Cases of fungal infection and mixed or unclear culture results were excluded from this analysis.

Outcome analysis

Microbiological and clinical outcome data were obtained from 5071 adult patients of the Australia and New Zealand Dialysis Transplant (ANZDATA) Registry who developed first-time peritonitis between April 2003 and December 2012, excluding cases of fungal infection, polymicrobial infection, or unrecorded culture results. Clinical outcomes examined were cessation of therapy because of catheter removal in 90 d, transfer to permanent hemodialysis (HD) in 90 d, transfer to interim HD for at least 30 d and death in 30 d, as well as the individuals outcomes combined to yield the overall technique failure. Outcome predictors were determined using binary logistic regression and forward elimination of data. Survival analyses were performed using the Kaplan–Meier approach, and differences between groups were assessed using log-rank tests.

Media, reagent, and Abs

Peritoneal leukocytes were cultured in RPMI 1640 medium supplemented with 2 mM l-glutamine, 1% sodium pyruvate, 50 μg/ml penicillin/streptomycin, and 10% FCS (Life Technologies). Mesothelial cells were cultured in Earle’s buffered Medium 199 (Life Technologies) containing 10% FCS, and peritoneal fibroblasts were cultured in a 1:1 (v/v) mixture of DMEM and Ham’s F-12 nutrients (Life Technologies) with 20% FCS; both media were supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine (Life Technologies) as well as 5 μg/ml transferrin, 5 μg/ml insulin, and 0.4 μg/ml hydrocortisone (all from Sigma-Aldrich). Synthetic (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) was provided by Dr. H. Jomaa (University of Giessen, Giessen, Germany) and synthetic DMRL by Dr. B. Illarionov (Hamburg School of Food Science, Hamburg, Germany). Biotinylated monomers of human MHC-related protein 1 (MR1) loaded with reduced 6-hydroxymethyl-8-(1-D-ribityl) lumazine (active ligand) or 6-formylpterin (negative control) were provided by Dr. L. Kjer-Nielsen (University of Melbourne, Melbourne, VIC, Australia) and reconstituted as described before (16). rIFN-γ, rTNF-α, and rIL-1β were purchased from Miltenyi Biotec. Human T-Activator CD3/CD28 Dynabeads were purchased from Life Technologies. Blocking reagents used included anti-BTN3 (103.2; Dr. D. Olive, Université de la Méditerranée, Marseille, France); anti-MR1 (26.5; Dr. T. Hansen, Washington University School of Medicine, St. Louis, MO); anti–IFN-γ (B27) and anti–IL-1β (H1b-27) (BioLegend); and sTNFR p75-IgG1 fusion protein (etanercept/Enbrel; Amgen).

Bacteria

Clinical isolates of Escherichia coli (Gram⁻HMB-PP⁺vit.B2⁺), Klebsiella pneumoniae (Gram⁻HMB-PP⁺vit.B2⁺), Pseudomonas aeruginosa (Gram⁻HMB-PP⁺vit.B2⁺), Corynebacterium striatum (Gram⁺HMB-PP⁺vit.B2⁺), Listeria monocytogenes (Gram⁺HMB-PP⁺vit.B2⁻), Staphylococcus aureus (Gram⁺HMB-PP⁻vit.B2⁺), Streptococcus pneumoniae (Gram⁺HMB-PP⁻vit.B2⁻), and Enterococcus faecalis (Gram⁺HMB-PP⁻vit.B2⁻) were grown in Luria–Bertani broth, harvested at an OD₆₀₀ of 0.5–0.8, and sonicated in 1/10 (v/v) PBS (pH 8). Insoluble debris was removed by centrifugation, the supernatants were passed through 0.1-μm sterile filter units (Millipore), and the protein concentrations were determined using the BCA protein assay kit (Pierce). Low molecular mass fractions were obtained using cellulose filters with a molecular mass cutoff of 3 kDa (Millipore). Bacterial extracts were used in cell culture at dilutions corresponding to protein concentrations of the original samples (before 3-kDa filtration) of 60–100 μg/ml.

T cells

PBMC were isolated from peripheral blood of healthy volunteers using Lymphoprep (Axis-Shield). Vγ9⁺ T cells (>98%) were isolated from PBMC using mAbs against Vγ9-PECy5 (Beckman Coulter) and anti-PE magnetic microbeads (Miltenyi Biotec); Vα7.2⁺ T cells (>98%) were isolated using anti–Vα7.2-allophycocyanin (BioLegend) and anti-allophycocyanin microbeads (Miltenyi Biotec). To generate unconventional T cell–conditioned medium, purified blood Vγ9/Vδ2 T cells and MAIT cells were incubated for 24 h in the presence of 10 nM HMB-PP and anti-CD3/CD28 Dynabeads at 0.5 beads/cell, respectively. Human peritoneal leukocytes were harvested from overnight dwell effluents of stable PD patients (13) and cultured in the absence or presence of 1–100 nM HMB-PP, 100 μM DMRL, or bacterial extracts at dilutions corresponding to protein concentrations of 60–100 μg/ml. For blocking experiments, anti-BTN3 and anti-MR1 were used at 10 μg/ml and added 30 min before stimulating the cells.

Mesothelial cells and peritoneal fibroblasts

Human peritoneal mesothelial cells were obtained from fresh omental samples after two cycles of tissue digestion in the presence of trypsin (15 min each); peritoneal fibroblasts were obtained after a third digestion cycle lasting 1 h (17–19). All data presented are from experiments performed with confluent mesothelial cells and fibroblasts between the first and third passage. Mesothelial cells were growth arrested for 48 h in serum-free medium prior to treatment; fibroblasts were growth arrested in medium containing 0.2% FCS. After starvation, cells were exposed for 24 h to T cell–conditioned medium at the indicated dilutions; rTNF-α and rIFN-γ were used as controls. Cell-free peritoneal effluent from stable and infected patients (n = 3–4) was added to cell cultures at a dilution of 1:4. In blocking experiments, T cell–conditioned medium or peritoneal effluent were pretreated for 30 min with anti–IFN-γ, anti–IL-1β, and sTNFR, either alone or in combination at 10 μg/ml. Supernatants were harvested and assessed by ELISA; cells were analyzed by quantitative PCR.

Flow cytometry

Cells were acquired on an eight-color FACSCanto II (BD Biosciences) and analyzed with FlowJo 10.1 (Tree Star), using mAbs against CD3 (SK7), CD69 (FN50), CCR4 (1G1), CCR5 (2D7), and CCR6 (11A9) from BD Biosciences; anti–TCR-Vγ9 (Immu360) from Beckman Coulter; and anti-CD161 (HP-3G10), CCR2 (K036C2), anti–TCR-Vα7.2 (3C10) (BioLegend), together with appropriate isotype controls. Anti-mouse beads were used to set compensation (Life Technologies). Intracellular cytokines were detected using anti–IFN-γ (B27; BioLegend) and anti–TNF-α (188; Beckman Coulter). For detection of intracellular cytokines, 10 μg/ml brefeldin A (Sigma-Aldrich) was added to cultures 5 h prior to harvesting. Leukocyte populations were gated based on their appearance in side scatter and forward scatter area/height and exclusion of live/dead staining (fixable Aqua; Invitrogen). Unless stated otherwise, peritoneal γδ T cells were defined as Vγ9⁺CD3⁺ lymphocytes. Peritoneal MAIT cells were defined as Vα7.2⁺CD161⁺CD3⁺ lymphocytes; control stainings using MR1 tetramers as reference confirmed the validity of this approach (data not shown).

ELISA

Cell-free peritoneal effluents were analyzed on a SECTOR Imager 6000 (Meso Scale Discovery) for IFN-γ, TNF-α, IL-1β, CCL3, CCL4, and CXCL8. Conventional ELISA kits and a Dynex MRX II reader were used for CCL2 (eBioscience) and CCL20 (R&D Systems). Cell culture supernatants were analyzed using conventional ELISA kits for IFN-γ (BioLegend), TNF-α and CCL2 (eBioscience) as well as for CXCL8, CXCL10, and IL-6 (R&D Systems).

Real-time PCR

Total RNA was isolated from mesothelial cells cultured under the indicated conditions using TRIzol (Invitrogen). cDNA was generated from 0.5 μg of RNA using the high-capacity cDNA reverse transcription kit (Thermo Fisher), 100 mM 2′-deoxynucleoside 5′-triphosphates, 40 U/μl RNase inhibitor (New England Biolabs), 50 U/μl MultiScribe reverse transcriptase, and 1× random primers, according to the manufacturer’s recommendations. Quantitative PCRs were run on a ViiA7 real-time PCR system (Thermo Fisher), using the power SYBR green PCR master mix (Thermo Fisher) and 300 nM forward and reverse primers: 5′-TCCCAATACATCTCCCTTCACA-3′ and 5′-ACCCACCTCTAAGGCCATCTTT-3′ for E-cadherin; 5′-TAAATCCACGCCGGTTCCTGAAGT-3′ and 5′-AGGTGTCTCAAAGTTACCACCGCT-3′ for occludin; 5′-CCGAGGTTTTAACTGCGAGA-3′ and 5′-TCACCCACTCGGTAAGTGTTC-3′ for fibronectin; 5′-CGAGCCCACCGGGAACGAAA-3′ and 5′-GGACCGAAGGCGCTTGTGGAG-3′ for IL-6; 5′-CCTCTGACTTCAACAGCGACAC-3′ and 5′-TGTCATACCAGGAAATGAGCTTGA-3′ for GAPDH; 5′-TTTACCTTCCAGCAGCCCTA-3′ and 5′-GGACAGAGTCCCAGATGAGC-3′ for Snail; 5′-AACTGGGACGACATGGAAA-3′ and 5′-AGGGTGGGATGCTCTTCAG-3′ for α-smooth muscle actin; 5′-GGAGAGGTGTTCCGTGTTGT-3′ and 5′-GGCTAGCTGCTCAGCTCTGT-3′ for zona occludens-1; and 5′-CGGGTTGCTTGCAATGTGC-3′ and 5′-CCGGCGACAACATCGTGAC-3′ for claudin-1. Ten nanograms of total RNA were used for cellular microRNAs (miRs) using the TaqMan Universal Master Mix II and specific primers for miR-21 and miR-191 (Applied Biosystems). mRNA and miR expression levels were normalized to the endogenous controls GAPDH and miR-191, respectively.

Statistics

Statistical analyses were performed using GraphPad Prism 6.0 software. Data distributions were analyzed using D’Agostino–Pearson omnibus normality tests. Data were analyzed using two-tailed Student t tests for normally distributed data and two-tailed Mann–Whitney tests for non-parametric data. Differences between groups were analyzed using one-way ANOVA with Holm–Sidak’s post tests for multiple comparisons of parametric data, or Kruskal–Wallis tests combined with Dunn’s post tests for nonparametric data. Matched data were analyzed using paired t tests or Wilcoxon matched-pairs tests for two groups, or Friedman tests combined with Dunn’s multiple comparisons tests for more than two groups. Differences were considered statistically significant as indicated in the figures and tables: *p < 0.05, **p < 0.01, and ***p < 0.001.

Blood unconventional T cells migrate early into the inflamed peritoneal cavity, at the time of peak neutrophil influx

Acute disease is characterized by a considerable influx of leukocytes to the site of infection, together with locally elevated levels of inflammatory mediators. In this study, the effluent of PD patients presenting with acute peritonitis, before commencing antibiotic treatment, contained increased levels of total cells and of chemokines like the neutrophil-attracting molecule CXCL8 (IL-8), compared with effluent from stable noninfected PD patients (Fig. 1A). Absolute numbers of both Vγ9/Vδ2 T cells and MAIT cells were increased during acute peritonitis compared with stable patients (Fig. 1B), indicating rapid corecruitment of unconventional T cells from blood to the inflamed peritoneal cavity, alongside neutrophils. These observations were in agreement with the migratory profiles of these cells. Circulating Vγ9/Vδ2 T cells and MAIT cells in stable PD patients preferentially expressed the chemokine receptors CCR2, CCR5, and CCR6, compared with their non-Vγ9 and non-MAIT counterparts (Fig. 1C, 1D). We detected markedly increased levels of the corresponding chemokines CCL2 (CCR2 ligand), CCL3 and CCL4 (CCR5 ligands), and CCL20 (CCR6 ligand) in early peritonitis (Fig. 1E), demonstrating a substantial production of inflammatory chemokines with the potential to attract unconventional T cells to the site of infection, thereby complementing the local pool of tissue-resident Vγ9/Vδ2 T cells and MAIT cells. In accordance, local frequencies of Vγ9/Vδ2 T cells and MAIT cells during peritonitis were generally higher than in blood (Fig. 2A). No systemic increase in blood unconventional T cell levels was seen in patients with peritonitis, in support of the locally confined nature of the infection.

Unconventional T cells are locally enriched during acute infections caused by bacterial pathogens producing the corresponding ligands

To avoid confounding resulting from the considerable biological variations between people and the underlying pathologies, we collected matched samples from the same individuals to examine systemic responses in blood and local responses in the peritoneal cavity, before and during episodes of peritonitis. Such investigations in acutely infected people have not been attempted before and are a unique advantage of studying individuals receiving PD. These matched analyses in fact identified further ligand-specific effects on local frequencies because Vγ9/Vδ2 T cells and MAIT cells were particularly elevated in peritonitis caused by bacteria producing HMB-PP and vitamin B₂, respectively, even in individuals with multiple infection episodes (Fig. 2B).

Across the cohort, proportions of Vγ9/Vδ2 T cells among all CD3⁺ T cells in blood and peritoneal cavity were comparable in the absence of infection, indicating that under homeostatic conditions Vγ9/Vδ2 T cells are not enriched locally (Fig. 3A). In contrast, on the day of presentation with acute peritonitis, local levels of Vγ9/Vδ2 T cells were elevated compared with blood, suggesting preferential recruitment and/or accumulation at the site of infection (Fig. 3B). This preferential increase in local Vγ9/Vδ2 T cell levels was apparent in patients infected with HMB-PP⁺ bacteria but not in patients with HMB-PP⁻ infections (Fig. 3B), despite highly elevated peritoneal chemokine levels in both HMB-PP⁺ and HMB-PP⁻ infections (data not shown). Finally, we performed a longitudinal study and observed a significant increase in local Vγ9/Vδ2 T cell levels in patients who developed acute peritonitis caused by HMB-PP⁺ organisms over preinfection baseline levels when the same individuals were stable (Fig. 3C). Because there was no such difference between preinfection and postinfection levels in individuals presenting with HMB-PP⁻ infections, these findings show that Vγ9/Vδ2 T cells accumulate locally at the site of infection in response to HMB-PP⁺ but not HMB-PP⁻ organisms.

Parallel studies on the distribution of MAIT cells revealed analogous patterns. Although in stable PD patients local MAIT cell frequencies in the peritoneal cavity were slightly higher than those in blood (Fig. 3D), such differences between anatomical sites were much more pronounced in acutely infected individuals (average 4.65% of all T cells in the peritoneal cavity versus 0.91% in blood), particularly during infections caused by vitamin B₂–producing (vit.B2⁺) bacteria (Fig. 3E). As observed for peritoneal Vγ9/Vδ2 T cell responses to HMB-PP⁺ bacteria, a comparison with matched preinfection values revealed a significant local increase in MAIT cell levels in individuals infected with vit.B2⁺ organisms (Fig. 3F), confirming the responsiveness of MAIT cells to vitamin B₂ derivatives in vivo. These findings on Vγ9/Vδ2 T-cells and MAIT cells support a ligand-induced local expansion of unconventional T-cells at the site of infection, in addition to the ligand-independent recruitment from the blood (Fig. 1B).

Peritoneal unconventional T cells respond to bacterial pathogens producing the corresponding ligands

To address the pathogen specificity of local unconventional T cell responses, we cultured peritoneal leukocytes from stable PD patients in the presence of different microbial stimuli. In agreement with our earlier data on blood cells (10), peritoneal Vγ9/Vδ2 T cells were highly specific for HMB-PP, whereas MAIT cells recognized the vitamin B₂ precursor, DMRL, as judged by upregulation of CD69 and production of TNF-α by responding cells (Fig. 4A). When testing extracts from defined clinical isolates covering the majority of pathogens associated with PD-related peritonitis, peritoneal Vγ9/Vδ2 T cells responded readily to HMB-PP–producing Gram-negative (E. coli, K. pneumoniae, and P. aeruginosa) and Gram-positive bacteria (C. striatum and L. monocytogenes) but not to HMB-PP–deficient E. faecalis and S. pneumoniae (Fig. 4B). Blocking experiments using neutralizing Abs confirmed that these HMB-PP–dependent responses were mediated via BTN3 (Fig. 4C). Strikingly, peritoneal Vγ9/Vδ2 T cells also responded to S. aureus despite this organism’s lack of HMB-PP, possibly via superantigens (20). Peritoneal MAIT cells were activated by the vit.B2⁺ bacteria E. coli, K. pneumoniae, C. striatum, and S. aureus but not by the vit.B2⁻ species L. monocytogenes, E. faecalis, and S. pneumoniae; responses to the vit.B2⁺ organism P. aeruginosa did not reach statistical significance (Fig. 4B).

Although Vγ9/Vδ2 T cells and MAIT cells constitute only minor proportions of the peritoneal T cell pool, these cells represented major producers of proinflammatory cytokines in response to bacterial extracts (Fig. 5A). Using E. coli as example of an organism producing both HMB-PP and vitamin B₂, responding peritoneal Vγ9/Vδ2 T cells and MAIT cells together made up a large fraction of TNF-α⁺ T cells (median 31.7%) and IFN-γ⁺ T cells (median 39.2%) despite considerable variability across PD patients (Fig. 5B). In contrast, both cell types constituted far lower proportions among TNF-α⁻ and IFN-γ⁻ T cells in E. coli–stimulated peritoneal leukocytes. Similar results were obtained using HMB-PP⁺vit.B2⁺ C. striatum extracts (data not shown). Analyses of supernatants from peritoneal leukocytes exposed to different bacteria demonstrated that only organisms producing HMB-PP and/or vitamin B₂ (S. aureus and C. striatum) but not HMB-PP⁻vit.B2⁻ E. faecalis induced secretion of IFN-γ and the IFN-γ–inducible chemokine CXCL10 (Fig. 5C). As control, levels of CXCL8 were comparable in response to all three bacterial species, demonstrating an equal potential to stimulate peritoneal leukocytes other than Vγ9/Vδ2 T cells and MAIT cells (data not shown). Secretion of TNF-α was not assessed in these experiments as any unconventional T cell–derived TNF-α would have been masked by peritoneal macrophages and neutrophils sensing diverse pathogen-associated molecular patterns. The contribution of Vγ9/Vδ2 T cells and MAIT cells to the overall secretion of IFN-γ by peritoneal leukocytes in response to HMB-PP⁺vit.B2⁺ organisms was confirmed by anti-BTN3 and anti-MR1 blocking Abs (Fig. 5D). Taken together, these findings show that peritoneal unconventional T cells are major producers of IFN-γ and TNF-α in response to a wide range of microbial pathogens and that inhibition of ligand recognition through BTN3 and MR1 abrogates this cytokine production.

Cross-talk with local tissue amplifies the proinflammatory response to bacterial pathogens

Local activation of unconventional T cells in acute infection is likely to occur in close proximity to the peritoneal membrane, thus exposing the mesothelial cell layer that lines the peritoneal cavity to T cell–derived mediators. Using supernatants from activated unconventional T cells, our experiments demonstrate that Vγ9/Vδ2 T cells and MAIT cells induced secretion of CCL2, CXCL8, CXCL10, and IL-6 by omentum-derived primary mesothelial cells (Fig. 6A) and peritoneal fibroblasts (Fig. 6B). This activation of peritoneal tissue cells was dose dependent (data not shown). Neutralization of TNF-α and/or IFN-γ in the conditioned media prior to addition to peritoneal tissue cells attenuated these responses considerably, with the CXCL8 and IL-6 secretion being particularly sensitive to inhibition of TNF-α, whereas the CXCL10 secretion was mainly driven by IFN-γ (Fig. 6). These findings were in accordance with control experiments showing that rTNF-α was a potent inducer of CCL2, CXCL8, and IL-6 expression by mesothelial cells and fibroblasts (data not shown). rIFN-γ on its own was mainly effective at inducing CCL2 and, to a lesser extent, IL-6 expression by mesothelial cells and fibroblasts while having no effect on CXCL8. These findings identify unconventional T cell–derived TNF-α and IFN-γ as major stimulators of peritoneal tissue cells, which is likely to enhance local inflammation and contribute to further recruitment of monocytes, neutrophils and lymphocytes to the site of infection.

Activation of peritoneal tissue cells by effluent from PD patients with acute peritonitis

We next sought to address the physiological relevance of the findings above in more detail. Our previous work already demonstrated that peritoneal effluent of patients presenting with acute peritonitis can contain considerable levels of TNF-α and IFN-γ (13–15). We therefore tested whether cytokines released into the dialysis fluid during acute infection had similar bioactivity to unconventional T cell–conditioned media and the recombinant proteins themselves. In this study, the effluent from three patients with peritonitis induced CCL2 and CXCL8 secretion by mesothelial cells while effluent from stable patients showed only background activity (Fig. 7A). This chemokine production was in part blocked by combined pretreatment of the infected effluent with sTNFR and anti–IFN-γ (Fig. 7B). These experiments demonstrate that TNF-α and IFN-γ are produced locally in response to bacterial pathogens at concentrations that are sufficiently high to affect the cells lining the peritoneal cavity, and that targeting cytokine production by unconventional T cells may diminish local inflammation.

Clinical outcome from peritonitis depends on the capacity of the causative pathogen to produce unconventional T cell ligands

Acute inflammatory events make a major contribution to tissue fibrosis. In particular, peritonitis has a direct effect on peritoneal membrane morphology and function (21, 22) and is hence a major reason for technique failure in PD patients. Yet, little is known about the role of unconventional T cells in these processes. Given the contribution of Vγ9/Vδ2 T cells and MAIT cells to the local immune response to HMB-PP⁺ and vit.B2⁺ bacteria, respectively, we investigated the short- and midterm impact of infections by such organisms on the clinical outcome in 5071 PD patients presenting with first-time peritonitis recorded in the Australia and New Zealand Dialysis and Transplant (ANZDATA) Registry (Table I). In accordance with our earlier findings in a much smaller subgroup of the same cohort (14), infections by HMB-PP⁺ bacteria were associated with poorer outcomes as determined by higher rates of catheter removal, permanent transfer to HD, and overall technique failure, compared with HMB-PP⁻ infections (Fig. 8, Table II). This was true for episodes of peritonitis caused by both Gram⁺ and Gram⁻ species, thereby identifying the utilization of the nonmevalonate pathway of isoprenoid biosynthesis by the causative organism as useful predictive marker and implying that Vγ9/Vδ2 T cell–driven responses may contribute to overall clinical outcome. Within the HMB-PP⁺ group, Gram⁻ organisms caused even more severe complications than Gram⁺ species, including significant mortality within the first mo after the onset of acute peritonitis.

Because all HMB-PP⁺ bacteria in the ANZDATA cohort were also positive for vitamin B₂ (and did not include L. monocytogenes as the only relevant HMB-PP⁺vit.B2⁻ pathogen in PD patients), outcome predictions based on the presence of the vitamin B₂ pathway followed closely those seen for HMB-PP⁺ organisms (Table II). However, differential analysis of HMB-PP⁻ infections allowed us to determine the clinical impact of vit.B2⁺ species in that patient subgroup. In this study, vit.B2⁺ infections showed a trend toward higher rates of catheter removal, compared with vit.B2⁻ infections, which was also reflected in total technique failure rates (Fig. 8). However, no such differences between vit.B2⁺ and vit.B2⁻ infections were seen with regard to transfer to HD or mortality. This relatively benign course of HMB-PP⁻vit.B2⁺ peritonitis may have been due to the high prevalence of infections caused by the comparatively avirulent skin commensal Staphylococcus epidermidis and related coagulase-negative species (67.6% of all infections in this group). As expected (23, 24), S. aureus infections were associated with a considerably greater risk of technique failure and catheter removal than coagulase-negative staphylococcal infections (data not shown). These findings indicate that although the presence of the vitamin B₂ pathway alone may not be sufficiently predictive of clinical outcome in that patient group, MAIT cells are nevertheless likely to make a contribution to the overall inflammatory response during acute peritonitis caused by vit.B2⁺ organisms.

Unconventional T cell–driven epithelial–mesenchymal transition of peritoneal mesothelial cells

Finally, we investigated the functional impact of activated unconventional T cells on the surrounding tissue. Inflammatory mediators including TNF-α have previously been associated with the induction of an epithelial–mesenchymal transition (EMT)–like process in mesothelial cells (25), and IFN-γ has been identified as major driver of tissue fibrosis in the peritoneal cavity (22). In this study, primary omentum-derived mesothelial cells exposed to supernatants from activated Vγ9/Vδ2 T cells (data not shown) and MAIT cells (Fig. 9A) underwent striking changes from an epithelial-like appearance to a spindled fibroblastic shape within 24 h. Such pronounced morphological changes were greatly diminished when neutralizing TNF-α and/or IFN-γ in the unconventional T cell–conditioned media prior to addition to mesothelial cells (Fig. 9A). As controls, similar effects were observed when mesothelial cells were cultured in the presence of TNF-α or IFN-γ, particularly when both cytokines were combined (Fig. 9B). In agreement with these morphological changes, the expression of epithelial cell–associated markers such as E-cadherin and occludin by mesothelial cells was downregulated upon exposure to MAIT cell–conditioned media, whereas the mesenchymal marker fibronectin was upregulated, as determined by RT-quantitative PCR (Fig. 9C). A similar upregulation of fibronectin expression was seen with Vγ9/Vδ2 T cell–conditioned medium, whereas effects on E-cadherin and occludin were less pronounced, most likely because of the generally lower levels of TNF-α and IFN-γ in the preparations used for those experiments, compared with MAIT cell–conditioned medium (data not shown). As viability control, expression of IL-6 was greatly enhanced by unconventional T cells at the mRNA level (data not shown), in agreement with the protein data. In addition to fibronectin, MAIT cell–stimulated mesothelial cells also expressed elevated levels of the miR miR-21, which has been linked to TGF-β induced EMT in mesothelial cells and to membrane fibrosis in patients receiving PD (M. Lopez-Anton and D.J. Fraser, unpublished observations). This miR-21 induction by MAIT cells was decreased upon neutralization of TNF-α and IFN-γ (Fig. 9D). Further epithelial (zona occludens-1; claudin) and mesenchymal markers (Snail, collagen-1, and α-smooth muscle actin) were not significantly affected by unconventional T cell–secreted factors (Fig. 9C, data not shown), indicating incomplete initiation of the EMT process under these conditions. Notably, although mesothelial cells treated with a combination of TNF-α and IFN-γ downregulated the expression of all epithelial markers tested (data not shown), only Vγ9/Vδ2 T cells and MAIT cells triggered a parallel upregulation of the mesenchymal marker fibronectin. These data imply that unconventional T cells may produce as yet unidentified mediators, in addition to TNF-α and IFN-γ, that may affect mesothelial cells and reprogram their morphology and phenotype. Taken together, these findings indicate that microbe-responsive unconventional T cells have the capacity to trigger local tissue remodeling with potential consequences for peritoneal membrane integrity and long-term clinical outcome.