Disease-specific T cell receptors maintain pathogenic T helper cell responses in postinfectious Lyme arthritis.

BACKGROUND: Antibiotic-Refractory Lyme Arthritis (ARLA) involves a complex interplay of T cell responses targeting Borrelia burgdorferi antigens succeeding towards autoantigens by epitope spreading. However, the precise molecular mechanisms driving the pathogenic T cell response in ARLA remain unclear. Our aim was to elucidate the molecular program of disease-specific Th cells. METHODS: Using flow cytometry, high-throughput T cell receptor (TCR) sequencing and scRNA-seq of CD4+ Th cells isolated from the joints of European ARLA patients, we aimed at inferring antigen specificity through unbiased analysis of TCR repertoire patterns, identifying surrogate markers for disease-specific TCRs and connecting TCR specificity to transcriptional patterns. RESULTS: PD-1hiHLA-DR+CD4+ effector T cells were clonally expanded within the inflamed joints and persisted throughout disease course. Among these cells, we identified a distinct TCRß motif restricted to HLA-DRB1*11 or *13 alleles. These alleles, being underrepresented in North American ARLA patients, were unexpectedly prevalent in our European cohort. The identified TCRß motif served as surrogate marker for a convergent TCR response specific to ARLA, distinguishing it from other rheumatic diseases. In the scRNA-seq dataset, the TCRß motif particularly mapped to peripheral T helper (TPH) cells displaying signs of sustained proliferation, continuous TCR signaling, and expressing CXCL13 and IFN-γ. CONCLUSION: By inferring disease-specific TCRs from synovial T cells we identified a convergent TCR response in the joints of ARLA patients that continuously fueled the expansion of TPH cells expressing a pathogenic cytokine effector program. The identified TCRs will aid in uncovering the major antigen targets of the maladaptive immune response. FUNDING: Supported by the German Research Foundation (DFG) MO 2160/4-1; the Federal Ministry of Education and Research (BMBF; Advanced Clinician Scientist-Program INTERACT; 01EO2108) embedded in the Interdisciplinary Center for Clinical Research (IZKF) of the University Hospital Würzburg; the German Center for Infection Research (DZIF; Clinical Leave Program; TI07.001_007) and the Interdisciplinary Center for Clinical Research (IZKF) Würzburg (Clinician Scientist Program, Z-2/CSP-30).

Positive resection margins in Crohn's disease are a relevant risk factor for postoperative disease recurrence.

Postoperative disease recurrence in Crohn's disease represents a relevant issue despite recent advancements in surgical and medical therapies. Additional criteria are necessary to improve the identification of patients at risk and to enable selective therapeutic approaches. The role of resection margins on disease recurrence remains unclear and general recommendations are lacking. A single-center retrospective analysis was performed including all patients who received ileocecal resection due to Crohn's disease. Resection margins were analyzed by two independent pathologists and defined by histopathological criteria based on previous consensus reports. 158 patients were included for analysis with a median follow up of 35 months. While postoperative morbidity was not affected, positive resection margins resulted in significantly increased rates of severe endoscopic recurrence at 6 months (2.0% versus 15.6%, p = 0.02) and overall (4.2% versus 19.6%, p = 0.001), which resulted in significantly increased numbers of surgical recurrence (0% versus 4.5%, p = 0.04). Additionally, positive margins were identified as independent risk factor for severe endoscopic disease recurrence in a multivariate analysis. Based on that, positive margins represent an independent risk factor for postoperative endoscopic and surgical disease recurrence. Prospective studies are required to determine whether extended resection or postoperative medical prophylaxis is beneficial for patients with positive resection margins.

DARPins detect the formation of hetero-tetramers of p63 and p73 in epithelial tissues and in squamous cell carcinoma.

The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p632/p732 hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p632/p732 hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma.

USP28 controls SREBP2 and the mevalonate pathway to drive tumour growth in squamous cancer.

SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.

RNF4~RGMb~BMP6 axis required for osteogenic differentiation and cancer cell survival.

Molecular understanding of osteogenic differentiation (OD) of human bone marrow-derived mesenchymal stem cells (hBMSCs) is important for regenerative medicine and has direct implications for cancer. We report that the RNF4 ubiquitin ligase is essential for OD of hBMSCs, and that RNF4-deficient hBMSCs remain as stalled progenitors. Remarkably, incubation of RNF4-deficient hBMSCs in conditioned media of differentiating hBMSCs restored OD. Transcriptional analysis of RNF4-dependent gene signatures identified two secreted factors that act downstream of RNF4 promoting OD: (1) BMP6 and (2) the BMP6 co-receptor, RGMb (Dragon). Indeed, knockdown of either RGMb or BMP6 in hBMSCs halted OD, while only the combined co-addition of purified RGMb and BMP6 proteins to RNF4-deficient hBMSCs fully restored OD. Moreover, we found that the RNF4-RGMb-BMP6 axis is essential for survival and tumorigenicity of osteosarcoma and therapy-resistant melanoma cells. Importantly, patient-derived sarcomas such as osteosarcoma, Ewing sarcoma, liposarcomas, and leiomyosarcomas exhibit high levels of RNF4 and BMP6, which are associated with reduced patient survival. Overall, we discovered that the RNF4~BMP6~RGMb axis is required for both OD and tumorigenesis.