Objective Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells.MethodsIsogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S. enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane.Results S. enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism.ConclusionsHLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention.

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We have assessed the utility of 3-dimensional (3D) in vitro human cell models to understand barriers to chimeric antigen receptor (CAR)-T cell activity in high-grade serous ovarian cancer, (HGSOC) a disease with a poor response to immunotherapy. We defined mucin-1 (MUC1) as a potential target in HGSOC biopsies and the HGSOC cell lines, OvCAR3 and G164. We then generated CAR-T cells against MUC1 and tested them in spheroid and collagen gel cultures. In spheroids, although CAR-T cells killed OvCAR3 cells, G164 cells failed to induce CAR-T cell activation or cytotoxicity. However, when we added primary omental fibroblasts from ovarian cancer patients to G164 spheroids, CAR-T cells were activated and cytotoxic. Fibroblast conditioned medium also activated CAR-T cells to kill G164 cells in spheroids and this was due to their production of C-C motif chemokine ligand 2 (CCL2). Further experiments revealed that CCL2 produced by fibroblast stimulated CCR2/4 positive CAR-T cells to a higher state of activation, which enhanced the cytotoxicity of CAR-T cells against G164 cells. We then investigated CAR-T cell activity in co-cultures of OvCAR3 or G164 cells and primary fibroblasts embedded in collagen. CAR-T cells migrated into OvCAR3 gels and killed the malignant cells during a three-day period. However, CAR-T cells failed to migrate into gels with G164 cells and there was no malignant cell killing. Gels containing G164 cells had denser extracellular matrix (ECM) than OvCAR3 gels, as measured by staining for collagens and fibronectin. Previously, we showed that transforming growth factor-beta (TGFβ) secreted by HGSOC cells acted on fibroblasts to induce the production of ECM in collagen gels.1 Treating G164 gels with the TGFβ receptor inhibitor SB431542 reduced ECM density, stimulated CAR-T cell migration and restored CAR-T cell cytotoxicity against G164 cells. Using these different human 3D models we have demonstrated that malignant cell intrinsic factors can cause resistance to CAR-T cells. Sensitivity to CAR-T cell killing can be modulated both positively and negatively by fibroblasts. Targeting ECM along with CAR-T cell therapy might improve the efficiency of CAR-T cells in solid tumors. 1Delaine-Smith et al, iScience, 2021 Citation Format: Joash Dominic Joy, Beatrice Malacrida, Florian Laforêts, Panoraia Kotantaki, Eleni Maniati, Sarah Hopkins, Ianire Calleja, Sara Brett, Takis Athanasopoulos, Sadfer Ali, Peter Emery-Billcliff, Ida Ricciardelli, Charlotte Kay, Jayne Colebrook, Magda Ali, Katherine Strong, Frances Balkwill. TGFβ-mediated targeting of the extracellular matrix enhances the migration and cytotoxicity of CAR-T cells in 3D models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 693.