TNF-Alpha Priming Enhances CD4+ FoxP3+ Regulatory T Cell Suppressive Function in GvHD Prevention and Treatment

Abstract

CD4+ CD25+ FoxP3+ regulatory T cells (Treg) have been shown to effectively prevent graft versus host disease (GvHD) when adoptively transferred in murine models of hematopoietic cell transplantation (HCT) and phase I/II clinical trials. Critical limitations to the clinical application of Treg are the paucity of cells and limited knowledge of the mechanism(s) of in vivo function. In physiologic conditions Treg regulate immune responses during inflammation. We hypothesized that inflammatory conditions in GvHD modify Treg characteristics and function. To test this hypothesis, we primed Treg with irradiated (3000 cGy) peripheral blood from acute GvHD (aGvHD) affected mice for 20-24 hours and then transferred these cells in a mouse model of GvHD where allogeneic T cell depleted bone marrow (TCD BM) from C57BL/6 mice was transplanted into lethally irradiated (8 Gy) BALB/c recipients together with 7.5x105 to 1x106 /animal donor derived conventional CD4+ and CD8+ T cells (Tcon). C57BL/6 Treg primed with irradiated aGvHD peripheral blood were injected at day 0 after HCT for preventing GvHD or at day +7 or +8 as GvHD treatment. Their adoptive transfer resulted in improved survival in comparison to unprimed natural occurring Treg when used for both GvHD prevention (p=0.01) and treatment (p=0.04). Moreover treatment with irradiated aGvHD peripheral blood-primed Treg did not impact graft versus tumor effects in a mouse model of T cell mediated tumor killing. BLI demonstrated that injected allogeneic Tcon completely cleared previously infused luc+ A20 tumor cells even in the presence of primed Treg (primed Treg + Tcon + A20 vs A20 alone p<0.001). Irradiated aGvHD peripheral blood-primed Treg express increased levels of activation markers with suppressive function such as CTLA4 (p<0.001) and LAG3 (p<0.05) in comparison to unprimed Treg in vitro. We also found that Treg primed with irradiated cells of aGvHD affected animals after removing the serum did not enhance the expression of the same markers (p>0.05) demonstrating that serum from aGvHD animals is required for Treg priming and function. We further tested the ability of several inflammatory cytokines that are normally secreted during GvHD such as IFN-γ, IL-6, IL-12 and TNFα to induce similar in vitro Treg activation. We found that TNFɑ selectively activated Treg without impacting CD4+ FoxP3- T cells. TNFɑ-primed Treg have increased expression of activation markers such as CD69 (p<0.0001), CD25 (p<0.0001), and LAG3 (p=0.0002), produce a greater amount of suppressive cytokines such as IL-10 (p=0.03) and TGF-β (p=0.02), and enhance the expression of homing markers such as CD62L (p=0.005) that are required for in vivo function. TNFɑ-primed Treg had increased ability to proliferate (p=0.02) and, at the same time, to suppress Tcon proliferation (p=0.04) in a mixed lymphocyte reaction against irradiated allogeneic splenocytes, while, on the contrary, TNFɑ-primed Tcon had reduced ability to proliferate in similar conditions in comparison to unprimed Tcon (p=0.0004). To test the effect of TNFɑ priming on in vivo Tcon proliferation we injected TNFɑ-primed and unprimed luc+ Tcon in allogeneic BALB/c Rag2-/- γ-chain-/- immune deficient animals that were sublethally irradiated (400 cGy). BLI at day +7 after Tcon injection revealed reduced TNFɑ-primed Tcon in vivo proliferation (p=0.01) that resulted in milder GvHD symptoms (p=0.02). Finally, in a GvHD prevention mouse model TNFɑ-primed Treg infused at 1:10 Treg/Tcon ratio resulted in improved animal survival as compared to unprimed Treg (p=0.02), demonstrating enhanced efficacy of TNFɑ priming in the in vivo function of Treg. In summary, our study demonstrates that Treg respond to TNFɑ acquiring an activated status resulting in increased function. As TNFɑ is produced by several immune cells during inflammation, our work elucidates aspects of the physiologic mechanisms of Treg function. Furthermore TNFɑ priming of Treg in vitro provides a new tool to optimize Treg cellular therapies also allowing for the use of a reduced cell number for GvHD prevention and treatment. Disclosures No relevant conflicts of interest to declare.

Epidermal growth factor stimulates exosomal microRNA‐21 derived from mesenchymal stem cells to ameliorate aGVHD by modulating regulatory T cells

“…Interestingly, the inhibition of c‐Jun activity by siRNA was found to block the increase in TGF‐β1 expression 39 . Similar mechanism was reported in other studies, in which the function of Tregs was promoted, consistently suggesting Treg‐based cellular therapies to be new options in aGVHD 40 . More importantly, BMSCs‐derived exosomal miR‐21 protected C‐kit + cardiac stem cells through the PTEN/PI3K/AKT axis 41 …”

“…39 Similar mechanism was reported in other studies, in which the function of Tregs was promoted, consistently suggesting Treg-based cellular therapies to be new options in aGVHD. 40 More importantly, BMSCs-derived exosomal miR-21 protected C-kit + cardiac stem cells through the PTEN/PI3K/AKT axis. 41 The mRNA expression of Foxp3 was reduced in PB lymphocytes of grade I-II aGVHD patients relative to those without aGVHD after allogeneic bone marrow transplant.…”

TNFa and IL-6 promote ex-vivo proliferation of lineage-committed human regulatory T cells

“…In addition, TNFR2 expression defines a maximally suppressive subset of Tregs that accumulates intratumorally in a Lewis lung carcinoma model (34). Finally, TNFa-primed mouse Tregs have enhanced efficacy against graft-versus-host disease (GVHD) when compared with unprimed Tregs (35,36). Overall, these results suggest that TNFa negatively affects TGFbinduced Tregs, but exerts a positive effect on thymic derived Treg activation and function in mice.…”

Therapeutic use of regulatory T cells for graft‐versus‐host disease

“…A different strategy to overcome the low numbers of Treg cells is to enhance their suppressive activity. Priming of donor Treg cells in the presence of tumour necrosis factor‐α (TNF‐α) has been shown to increase FOXP3 expression, and to enhance Treg cell suppressive function in a GvHD model (Pierini et al , ). Alternatively, recipient Treg cells could be expanded in vitro before HSCT using a tumour necrosis factor receptor 2 (TNFR2) agonist (Chopra et al , ).…”