Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, highly resistant to standard chemotherapy and immunotherapy. Regulatory T cells (Tregs) expressing tumor necrosis factor α receptor 2 (TNFR2) contribute to immunosuppression in PDAC. Treg infiltration correlates with poor survival and tumor progression in patients with PDAC. We hypothesized that TNFR2 inhibition using a blocking monoclonal antibody (mAb) could shift the Treg-effector T cell balance in PDAC, thus enhancing antitumoral responses. Method: To support this hypothesis, we first described TNFR2 expression in a cohort of 24 patients with PDAC from publicly available single-cell analysis data. In orthotopic and immunocompetent mouse models of PDAC, we also described the immune environment of PDAC after immune cell sorting and single-cell analysis. The modifications of the immune environment before and after anti-TNFR2 mAb treatment were evaluated as well as the effect on tumor progression. Results: Patients with PDAC exhibited elevated TNFR2 expression in Treg, myeloid cells and endothelial cells and lower level in tumor cells. By flow cytometry and single-cell RNA-seq analysis, we identified two Treg populations in orthotopic mouse models: Resting and activated Tregs. The anti-TNFR2 mAb selectively targeted activated tumor-infiltrating Tregs, reducing T cell exhaustion markers in CD8⁺ T cells. However, anti-TNFR2 treatment alone had limited efficacy in activating CD8⁺ T cells and only slightly reduced the tumor growth. The combination of the anti-TNFR2 mAb with agonistic anti-CD40 mAb promoted stronger T cell activation, tumor growth inhibition, and improved survival and immunological memory in PDAC-bearing mice. Conclusion: Our data suggest that combining a CD40 agonist with a TNFR2 antagonist represents a promising therapeutic strategy for patients with PDAC.

Treg cells hold enormous promise for therapeutic application in GVH disease, a lethal complication of allogeneic HSC transplantation. Mouse studies showed that donor‐derived recipient‐specific Treg (rsTreg) cells are far more efficient than polyclonal Treg cells in suppressing GVH disease. However, clinical grade preparations of rsTreg cells carries the risk of containing significant numbers of highly pathogenic recipient‐specific effector T cells. We hypothesized that an alternative approach using Treg cells specific for an exogenous (i.e. nondonor, nonrecipient) Ag (exoTreg cells) can overcome this risk by taking advantage of the bystander suppressive effect of Treg cells. For this, we used a murine model for aggressive GVH disease. We expanded ex vivo exoTreg cells that are primed against the HY Ag, which is only expressed in males. ExoTreg cells supressed GVH disease as efficiently as rsTreg cells in recipient male mice. We also applied this strategy in female mice that do not express this Ag. While exoTreg cells were not effective in female recipients when applied alone, providing the cognate HY Ag in vivo along side effectively activated exoTreg cells and completely abrogated GVH disease, establishing a targeted on/off system to provide a suppressive effect on alloreactive effector T cells.

Background: The pancreatic ductal adenocarcinoma (PDAC) microenvironment is highly fibrotic and hypoxic, with poor immune cell infiltration. Recently, we showed that nucleolin (NCL) inhibition normalizes tumour vessels and impairs PDAC growth. Methods: Immunocompetent mouse models of PDAC were treated by the pseudopeptide N6L, which selectively inhibits NCL. Tumour-infiltrating immune cells and changes in the tumour microenvironment were analysed. Results: N6L reduced the proportion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) and increased tumour-infiltrated T lymphocytes (TILs) with an activated phenotype. Low-dose anti-VEGFR2 treatment normalized PDAC vessels but did not modulate the immune suppressive microenvironment. RNAseq analysis of N6L-treated PDAC tumours revealed a reduction of cancer-associated fibroblast (CAF) expansion in vivo and in vitro. Notably, N6L treatment decreased IL-6 levels both in tumour tissues and in serum. Treating mPDAC by an antibody blocking IL-6 reduced the proportion of Tregs and MDSCs and increased the amount of TILs, thus mimicking the effects of N6L. Conclusions: These results demonstrate that NCL inhibition blocks the amplification of lymphoid and myeloid immunosuppressive cells and promotes T cell activation in PDAC through a new mechanism of action dependent on the direct inhibition of the tumoral stroma.

Experiment models of xenogeneic graft-versus-host disease (xeno-GVHD), in which human immune cells are injected into immunodeficient mice, are increasingly used to study human immune cell behavior in vivo and to test therapeutic approaches. Today, the main, and more commonly accepted clinical parameters used to characterize xeno-GVHD are weight loss and mortality. These criteria do not provide an accurate and subtle assessment of the disease intensity, nor do they reflect the great variability of xeno-GVHD, which depends on the donor. Relying on previous work in which we described an original clinical grading system for assessing GVHD in mice, we propose an adaptation of this system for xeno-GVHD models. This simple, solid, and reproducible scoring system of xeno-GVHD is constituted of the binary (yes or no) evaluation of four easy-to-evaluate parameters that reflect the complexity of the disease without the need to sacrifice the mice. This scoring system is consistent with the gold standard histological grading of human GVHD and with numerous biomarkers characteristic of the disease. We propose this new clinical grading system to evaluate and compare the results obtained with a common tool, regardless of the experimenters and laboratories where the experiments would have been carried out and whatever the therapeutic strategy evaluated. Experiment models of xenogeneic graft-versus-host disease (xeno-GVHD), in which human immune cells are injected into immunodeficient mice, are increasingly used to study human immune cell behavior in vivo and to test therapeutic approaches. Today, the main, and more commonly accepted clinical parameters used to characterize xeno-GVHD are weight loss and mortality. These criteria do not provide an accurate and subtle assessment of the disease intensity, nor do they reflect the great variability of xeno-GVHD, which depends on the donor. Relying on previous work in which we described an original clinical grading system for assessing GVHD in mice, we propose an adaptation of this system for xeno-GVHD models. This simple, solid, and reproducible scoring system of xeno-GVHD is constituted of the binary (yes or no) evaluation of four easy-to-evaluate parameters that reflect the complexity of the disease without the need to sacrifice the mice. This scoring system is consistent with the gold standard histological grading of human GVHD and with numerous biomarkers characteristic of the disease. We propose this new clinical grading system to evaluate and compare the results obtained with a common tool, regardless of the experimenters and laboratories where the experiments would have been carried out and whatever the therapeutic strategy evaluated.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, highly resistant to standard chemotherapy and immunotherapy. Regulatory T cells (Tregs) expressing TNFα receptor 2 (TNFR2) contribute to immunosuppression in PDAC. Treg infiltration correlates with poor survival and tumor progression in PDAC patients. We hypothesized that TNFR2 inhibition using a blocking monoclonal antibody (mAb) could shift the Treg-effector T cell balance in PDAC, thus enhancing anti-tumoral responses. Method: To support this hypothesis, we first described TNFR2 expression in a cohort of 24 PDAC patients from publicly available single-cell analysis data. In orthotopic and immunocompetent mouse models of PDAC, we also described the immune environment of PDAC after immune cell sorting and single-cell analysis. The modifications of the immune environment before and after anti-TNFR2 mAb treatment were evaluated as well as effect on tumor progression. Results: PDAC patients exhibited elevated TNFR2 expression in Treg, myeloid cells and endothelial cells but low level in ductal cells. By flow cytometry and single cell RNAseq analysis, we identified two Treg populations in orthotopic mouse models: resting and activated Tregs. The anti-TNFR2 mAb selectively targeted activated tumor-infiltrating Tregs, reducing T cell exhaustion markers in CD8 ⁺ T cells. However, anti-TNFR2 treatment alone had limited efficacy in activating CD8 ⁺ T cells and only slightly reduced the tumor growth. The combination of the anti-TNFR2 mAb with agonistic anti-CD40 mAbs promoted stronger T cell activation, tumor growth inhibition, and improved survival and immunological memory in PDAC-bearing mice. Conclusion: Our data suggest that combining a CD40 agonist with a TNFR2 antagonist represents a promising therapeutic strategy for PDAC patients.