p16Ink4a, a marker of cellular senescence, is associated with renal disease in the B6.NZMSle1/Sle2/Sle3 mouse model of lupus.

OBJECTIVES: Despite treatment, one-third of patients with lupus nephritis (LN) show a decline in renal function. Prognostic markers of poor outcome as well as novel therapeutic targets are therefore highly sought. We showed that p16INK4a, a marker of cellular senescence, is observed in baseline kidney biopsies from patients with LN, and is associated with renal disease. Here, we set out to assess for whether these findings are recapitulated in the B6.NZMSle1/Sle2/Sle3 (B6.Sle1.2.3) mouse model of spontaneous lupus. METHODS: We evaluated the occurrence and time of onset of p16Ink4a staining by immunohistochemistry on kidney sections, and tested for its association with multiple renal and systemic disease parameters, fibrosis and CD8+ T cell infiltration, in two cohorts of B6.Sle1.2.3 mice. RESULTS: The presence of p16Ink4a-positive cells in kidney was significantly associated with increased urine albumin/creatinine ratio, histopathological scores, CD8+ T cell infiltration and fibrosis, in both B6.Sle1.2.3 cohorts. In contrast, p16Ink4a staining was not associated with systemic disease parameters. A time course showed that systemic disease parameters as well as glomerular IgG deposits appeared in B6.Sle1.2.3 mice by 4 months of age; the appearance of p16Ink4a-positive cells occurred later, by 8 months of age, overlapping with renal disease. CONCLUSION: We report, for the first time, the presence of p16Ink4a-positive cells, a marker of cellular senescence, in the B6.Sle1.2.3 kidney, and their association with renal disease severity. This provides a preclinical model in which to test for the role of cellular senescence in the pathogenesis of LN, as a potential kidney-intrinsic disease mechanism.

Combined Blockade of GARP:TGF-ß1 and PD-1 Increases Infiltration of T Cells and Density of Pericyte-Covered GARP+ Blood Vessels in Mouse MC38 Tumors.

When combined with anti-PD-1, monoclonal antibodies (mAbs) against GARP:TGF-ß1 complexes induced more frequent immune-mediated rejections of CT26 and MC38 murine tumors than anti-PD-1 alone. In both types of tumors, the activity of anti-GARP:TGF-ß1 mAbs resulted from blocking active TGF-ß1 production and immunosuppression by GARP-expressing regulatory T cells. In CT26 tumors, combined GARP:TGF-ß1/PD-1 blockade did not augment the infiltration of T cells, but did increase the effector functions of already present anti-tumor T cells. Here we show that, in contrast, in MC38, combined GARP:TGF-ß1/PD-1 blockade increased infiltration of T cells, as a result of increased extravasation of T cells from blood vessels. Unexpectedly, combined GARP:TGF-ß1/PD-1 blockade also increased the density of GARP+ blood vessels covered by pericytes in MC38, but not in CT26 tumors. This appears to occur because anti-GARP:TGF-ß1, by blocking TGF-ß1 signals, favors the proliferation of and expression of adhesion molecules such as E-selectin by blood endothelial cells. The resulting densification of intratumoral blood vasculature probably contributes to increased T cell infiltration and to the therapeutic efficacy of GARP:TGF-ß1/PD-1 blockade in MC38. We conclude from these distinct observations in MC38 and CT26, that the combined blockades of GARP:TGF-ß1 and PD-1 can exert anti-tumor activity via multiple mechanisms, including the densification and normalization of intratumoral blood vasculature, the increase of T cell infiltration into the tumor and the increase of the effector functions of intratumoral tumor-specific T cells. This may prove important for the selection of cancer patients who could benefit from combined GARP:TGF-ß1/PD-1 blockade in the clinics.