The in Vitro and In Vivo Effects of DPP-4 Inhibition with Sitagliptin, Alone and in Combination with Bortezomib, on T Cell Activation: Rationale for GvHD Prevention

Introduction: Dipeptidyl peptidase-4 (DPP-4; also known as CD26) is a homodimeric type II transmembrane receptor expressed on a variety of cell types and is present in an enzymatically active form in plasma. DPP-4/CD26 is involved in a number of biological processes, including T cell activation. The interaction of DPP-4/CD26 on T cells with its ligand caveolin-1 on antigen-presenting cells (APC) enhances T cell activation, proliferation, and cytokine production and also upregulates CD86 on APC resulting in co-stimulation. We have recently shown that DPP-4 inhibition using the specific inhibitor sitagliptin is associated with remarkably low rates of acute graft-versus-host disease (Farag et al. NEJM 2021, 384:11-19). Herein we describe the in vitro and in vivo effects of DPP-4 inhibition alone and in combination with bortezomib, as a rationale for further studies in GvHD prevention.

Methods: One-way mixed lymphocyte reactions (MLR) using PBMC obtained from random donor buffy coats were used to evaluate the effect of the specific DPP-4 inhibitor sitagliptin alone and in combination with bortezomib, on T cell activation and proliferation in vitro. Cytokines in MLR supernatant were assayed using Luminex multiplex assay. MHC-mismatched mouse transplant (BL6 → BALB/c) model using CD26 gene knockout or wild type mice donors were used to investigate the effect of DPP-4 on GvHD in vivo. RNA sequencing (RNA-seq) was used to profile gene expression in CD3 positive cells from 72 hours of MLR groups.

Results: Sitagliptin induced a dose-dependent inhibition on T-cell activation and proliferation in 96h MLR using concentrations readily obtainable clinically (400-800 µg/ml) as shown in Fig.1, as well as reduction in the production of Th1 cytokines, IFN-γ, TNF-α and IL-1β, Th2 cytokine, IL-10, and Th17 cytokines, IL-17A and IL-17F in MLR supernatants.

Compared with wild-type (CD26^+/+) counterparts, mice receiving CD26^-/- BM and splenic T cells developed significantly less acute GvHD as measured by mortality and clinical score. In our transplant model, recipients of WT BL6 BM + CD3⁺ cells developed focal and mild liver and skin pathogenic but moderate gastrointestinal toxicity, which included crypt loss in the colon and villous blunting, crypt loss in the small intestine. CD26^-/- BL6 BM + CD3⁺ cells reduced the intestinal injury, which showed normal colon and small intestine structure. The Histopathological scores of GvHD between recipients of WT BL6 BM + CD3⁺ cells and that of CD26^-/- BL6 BM + CD3⁺ cells were 2.67±0.67 vs 0.58±0.12 (p<0.05) in colon, 2.67±0.76 vs 0.88±0.18 (p<0.05) in small intestine, and 0.73 ±0.06 vs 0.65±0.64 (p>0.05) in liver. Also, recipient mice transplanted with BL6 CD26^-/- BM and spleen cells showed significantly improved survival compared to those receiving wild type (CD26^+/+) cells.

The combination of sitagliptin (400 µg/ml or 500 µg/ml) and bortezomib (4.5 nM) showed synergistic inhibition on T-cell proliferation (Fig. 2) and reduction on IFN-γ, IL-10, IL-17A and IL-17F production in MLR. Bulk sequencing of RNA from CD3+ cells obtained after 72-hour MLR showed a significant change in the JAK-STAT signaling pathway, including downregulation of STAT1, IL7R,PIK3R1, IL6ST, IL6R, SOS1 and PIAS1 genes with sitagliptin alone (400 µg/ml). The combination of sitagliptin (400 µg/ml) and bortezomib (4.5 nM) reduced expression in genes associated with DNA replication (including MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, DNA2, PCNA, LIG1, and other genes), cell cycle (including CDC6, CDC7, CDC20, CDC25A, CDC45, CDK1, CDK2 and other genes), and Th17 cell differentiation (including IL17F, IL21, IL21R, and IL23R genes).

Conclusions: Our studies show that the specific DPP-4 inhibitor sitagliptin results in a dose dependent reduction in T cell activation and proliferation in MLR, and that DPP-4 elimination using CD26 gene knockout donor cells is associated with protection from GvHD in mouse transplant. The findings are consistent with our recently published clinical trial demonstrating low incidence of acute GVHD with high dose sitagliptin in combination with standard prophylaxis of tacrolimus and sirolimus. Furthermore, we demonstrate in vitro synergy of sitagliptin with bortezomib in inhibiting T cell activation and proliferation. Our data provides rationale for further clinical testing of sitagliptin for acute GvHD prevention, including in combination with bortezomib.

No relevant conflicts of interest to declare.