Key Points TNF-α produced during aGVHD is a strong and selective activator of CD4+CD25+FoxP3+ Tregs. In vitro TNF-α priming enhances CD4+CD25+FoxP3+ Treg proliferation and their ability to protect from GVHD.

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.

and 42 (20-98) days post UCBT, respectively. Percent of donor chimerism at day 180 was 97 AE5.4%. Probability of Grade II-IV AGVHD was 19.2% (CI 95 0.6-58.9) ( Figure 2). Probability of 6 month overall survival was 73.3% (CI 95 43.6-89.0). Twelve of sixteen patients are alive at 46-871 days post UCBT. Conclusions: These preliminary results suggest that single or double UCBT combined with unrelated HPDSC is safe, well tolerated, and have a lower than expected incidence of Grade II-IV AGVHD. A larger cohort and longer follow-up is required to determine the safety and clinical significance of these early findings.

Background Menopause is associated with an increase in the prevalence and severity of hypertension in women. Although premenopausal females are protected against T cell‐dependent immune activation and development of angiotensin II (Ang II) hypertension, this protection is lost in postmenopausal females. Therefore, the current study hypothesized that specific CD4 + T cell pathways are regulated by sex hormones and Ang II to mediate progression from premenopausal protection to postmenopausal hypertension. Methods and Results Menopause was induced in C57BL/6 mice via repeated 4‐vinylcyclohexene diepoxide injections, while premenopausal females received sesame oil vehicle. A subset of premenopausal mice and all menopausal mice were infused with Ang II for 14 days (Control, Ang II, Meno/Ang II). Proteomic and phosphoproteomic profiles of CD4 + T cells isolated from spleens were examined. Ang II markedly increased CD4 + T cell protein abundance and phosphorylation associated with DNA and histone methylation in both premenopausal and postmenopausal females. Compared with premenopausal T cells, Ang II infusion in menopausal mice increased T cell phosphorylation of MP2K2, an upstream regulator of ERK, and was associated with upregulated phosphorylation at ERK targeted sites. Additionally, Ang II infusion in menopausal mice decreased T cell phosphorylation of TLN1, a key regulator of IL‐2Rα and FOXP3 expression. Conclusions These findings identify novel, distinct T cell pathways that influence T cell‐mediated inflammation during postmenopausal hypertension.