BackgroundCross-presentation of exogenous antigens in HLA-class I molecules by professional antigen presenting cells (APCs) is crucial for CD8+ T cell function. Recent murine studies show that several non-professional APCs, including cancer-associated fibroblasts (CAFs) also possess this capacity. Whether human CAFs are able to cross-present exogenous antigen, which molecular pathways are involved in this process and how this ultimately affects tumor-specific CD8+ T cell function is unknown.MethodsIn this study, we investigated the ability of human colorectal cancer (CRC)-derived CAFs to cross-present neoantigen-derived synthetic long peptides (SLPs), corresponding to tumor-derived mutant peptides, and how this affects tumor-specific T-cell function. Processing of the SLP was studied by targeting components of the cross-presentation machinery through CRISPR/Cas9 and siRNA-mediated genetic ablation to identify the key molecules involved in fibroblast-mediated cross-presentation. Multispectral flow cytometry and killing assays were performed to study the effect of fibroblast cross-presentation on T cell function.ResultsHere, we show that human CRC-derived CAFs display an enhanced capacity to cross-present neoantigen-derived SLPs when compared with normal colonic fibroblasts. Cross-presentation of antigens by fibroblasts involved the lysosomal protease cathepsin S. Cathepsin S expression by CAFs was detected in situ in human CRC tissue, was upregulated in ex vivo cultured CRC-derived CAFs and showed increased expression in normal fibroblasts after exposure to CRC-conditioned medium. Cognate interaction between CD8+ T cells and cross-presenting CAFs suppressed T cell function, reflected by decreased cytotoxicity, reduced activation (CD137) and increased exhaustion (TIM3, LAG3 and CD39) marker expression.ConclusionThese data indicate that CAFs may directly suppress tumor-specific T cell function in an antigen-dependent fashion in human CRC.

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Locally applied mesenchymal stromal cells (MSCs) have the capacity to promote the healing of perianal fistulas in Crohn’s disease (CD) and are under clinical development for the treatment of proctitis in ulcerative colitis (UC). Despite these clinical advances, the mechanism of action of local MSC therapy in inflammatory bowel disease (IBD) is largely unknown. We hypothesized that the local cytokine environment in IBD patients affects the immunomodulatory properties of MSCs. To evaluate this, 11 cytokines were analyzed in inflamed tissues obtained from CD and UC patients. Based on the identified cytokine profiles 4 distinct cytokine mixtures that mimic various inflammatory IBD environments were established. Next, MSCs were cultured in the presence of either of these 4 cytokine mixtures after which the expression of immunomodulatory and tissue regenerative molecules and the capacity of MSCs to modulate T-cell proliferation and dendritic cell (DC) differentiation were assessed. Our data show that MSCs respond, in a cytokine-specific manner, by upregulation of immunomodulatory and tissue regenerative molecules, including cyclooxygenase-2, indoleamine 2,3-dioxygenase, and transforming growth factor-β1. Functional studies indicate that MSCs exposed to a cytokine profile mimicking one of the 2 UC cytokine milieus were less effective in inhibition of DC differentiation. In conclusion, our data indicate that cytokine mixes mimicking the local cytokine milieus of inflamed UC colonic or CD fistulas tissues can differentially affect the immunomodulatory and tissue regenerative characteristics of MSCs. These data support the hypothesis that the local intestinal cytokine milieu serves as a critical factor in the efficacy of local MSC treatment.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation of the intestinal tract with currently not well-understood pathogenesis. In addition to the involvement of immune cells, increasing studies show an important role for fibroblasts in the pathogenesis of IBD. Previous work showed that glycolysis is the preferred energy source for fibroblasts in fibrotic diseases. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) is a key kinase supporting glycolysis. Increased expression of PFKFB3 in several cancers and inflammatory diseases has been previously reported, but the metabolic status of fibroblasts and the role of PFKFB3 in patients with IBD are currently unknown. Therefore, in this study, we evaluated the role of glycolysis and PFKFB3 expression in IBD. Single-sample gene set enrichment analysis (ssGSEA) revealed that glycolysis was significantly higher in IBD intestinal samples, compared to healthy controls, which was confirmed in the validation cohorts of IBD patients. Single-cell sequencing data indicated that PFKFB3 expression was higher in IBD-derived stromal cells. In vitro, PFKFB3 expression in IBD-derived fibroblasts was increased after the stimulation with pro-inflammatory cytokines. Using seahorse real-time cell metabolic analysis, inflamed fibroblasts were shown to have a higher extracellular acidification rate and a lower oxygen consumption rate, which could be reversed by inhibition of JAK/STAT pathway. Furthermore, increased expression of pro-inflammatory cytokines and chemokines in fibroblasts could be reverted by PFK15, a specific inhibitor of PFKFB3. In vivo experiments showed that PFK15 reduced the severity of dextran sulfate sodium (DSS)- and Tcell transfer induced colitis, which was accompanied by a reduction in immune cell infiltration in the intestines. These findings suggest that increased stromal PFKFB3 expression contributes to inflammation and the pathological function of fibroblasts in IBD. Inhibition of PFKFB3 suppressed their inflammatory characteristics.

Background Local mesenchymal stromal cell (MSC)-therapy is approved for the treatment of Crohn’s disease-associated perianal fistulas. However, little is known about the working mechanism of local MSC-therapy. For the first time we evaluated engraftment and immunoregulatory effects of local MSC-therapy in patients with refractory proctitis. To do so, we analyzed biopsies and serum from patients with ulcerative proctitis before and after treatment with endoscopically injected MSCs in a phase IIa clinical trial (EudraCT number 2017-003524-75). Methods Thirteen therapy-refractory ulcerative proctitis patients were endoscopically injected bone marrow-derived allogeneic MSCs from healthy donors. Clinical efficacy was evaluated by the endoscopic and full Mayo score. Engraftment of the MSCs was investigated using fluorescence in-situ hybridization (FISH) of sex chromosomes on post-treatment biopsies. The presence of anti-HLA-antibodies against the MSC-donor was determined in the serum. Changes in immune cell subsets were evaluated using cytometry-by-time-of-flight (CyTOF) analysis. Results Thirteen patients with an endoscopic Mayo score of 2 (n=3) or 3 (n=10) of the rectum were treated with local MSC-therapy. Although complete remission was not achieved, full Mayo score was improved at week 6 (median 8 [IQR 6–10]) compared to baseline (median 11 [IQR 9.5–12]) (p=0.001). Preliminary data using FISH on the Y-chromosome, indicated the presence of MSCs in the rectum biopsies of female patients treated with male donor derived-MSCs at week 6. At baseline, HLA-antibodies were present in four patients. Six weeks after local injection of the MSCs, two out of thirteen patients developed new class I and II HLA-antibodies against the MSCs. Interestingly, in two patients pre-existing HLA-antibodies showed increased/boosted levels after local MSC-therapy, while one additionally developed new HLA-antibodies. CyTOF analysis of inflamed rectal biopsies 6 weeks after MSC treatment revealed significantly increased frequencies of several myeloid subsets (i.e. CD11b+CD14+CCR7+/-CD127+CD25+HLADR+ and CD14+HLA-DR-CD123-CCR7+) and a subset of CD4+ memory T cells with a more exhausted/regulated phenotype (PD-1+TIGIT+CD69+CD38+CD69). Conclusion Local MSC-therapy in patients with refractory proctitis changed the rectal immune profile characterised by a significant increase in a subset of effector memory CD4+ cells and several myeloid subsets, which might be associated with immune modulation. These results provide the basis for future studies on the mechanism of action of MSCs on rectal mucosa. New anti-HLA class antibodies developed in 2/13 patients after local administration. Whether these latter results have consequences for MSC-donor selection deserves further study.