Notch Inhibition in Alloreactive CD4+ and CD8+ T Cells Blocks Graft-Versus-Host Disease by Inducing a Hyporesponsive Program with Features of T Cell Anergy
Authors: Sandy Ashley R , Chung Jooho , Tran Ivy T , Shan Gloria T , Friedman Ann , Blackwell Timothy S , Reddy Pavan , King Philip D , Maillard Ivan 
Published: October 2019

Abstract
Abstract 340
Graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality following allogeneic bone marrow transplantation (allo-BMT). We previously identified Notch signaling as an essential regulator of allogeneic CD4+ T cell responses mediating GVHD after allo-BMT. Alloreactive CD4+ T cells expressing the pan-Notch inhibitor DNMAML induced markedly less severe GVHD as compared to wild-type T cells, leading to improved survival of the recipients. Notch-deprived T cells had preserved in vivo expansion and cytotoxicity. However, alloreactive DNMAML CD4+ T cells produced markedly decreased amounts of multiple proinflammatory cytokines, including TNF-alpha, IFN-gamma, and IL-2. This was associated with increased expansion of Foxp3+ CD4+ T regulatory cells. Thus, Notch signaling is an attractive new therapeutic target to control GVHD without eliminating the anti-cancer activity of allo-BMT.
To elucidate the mechanisms of Notch action in GVHD, we studied the effects of Notch inhibition in alloreactive CD4+ and CD8+ T cells using minor and major histocompatibility antigen-mismatched models of allo-BMT. In the B6 anti-BALB/b minor antigen-mismatched model, recipients of B6 T cells were protected from lethal acute GVHD upon DNMAML expression in the CD4+, CD8+ or both T cell compartments. In the B6 anti-BALB/c MHC-mismatched model, DNMAML CD4+ or CD8+ T cells transplanted alone or in combination induced significantly less GVHD and resulted in improved survival compared to wild-type T cells. Upon ex vivo restimulation with anti-CD3/CD28 antibodies, both CD4+ and CD8+ DNMAML alloreactive T cells had markedly decreased production of IFN-gamma. These findings suggest that Notch signaling has parallel functions in CD4+ and CD8+ T cells. We then studied expression of Tbx21 (encoding T-bet) and Eomes, the key transcription factors regulating Ifng transcription in CD4+ Th1 and CD8+ T cells, respectively. DNMAML alloreactive T cells had preserved amounts of Tbx21 mRNA and T-bet protein, and increased levels of Eomes transcripts and protein. These data differ from past reports indicating that Notch signaling controls T cell differentiation through direct regulation of Tbx21 and Eomes expression. Ex vivo restimulation of DNMAML CD4+ and CD8+ T cells with PMA (diacylglycerol analog) and ionomycin (calcium ionophore) rescued IFN-gamma production by both T cell compartments and partially restored IL-2 production by CD4+ T cells, suggesting abnormal signaling downstream of the T cell receptor. After anti-CD3/CD28 restimulation, DNMAML alloreactive T cells showed markedly decreased phosphorylation of Mek1 and Erk1/2, indicating defective Ras/MAPK activation. PMA was sufficient to rescue Erk1/2 activation. NFkB activity was also significantly impaired in alloreactive DNMAML T cells as assessed with a NFkB-luciferase reporter transgene. Abnormal responsiveness was acquired in vivo during alloreactive T cell priming, since naïve DNMAML T cells had preserved Ras/MAPK activation. Moreover, alloreactive Notch-deprived T cells had elevated levels of intracellular cAMP and increased expression of the anergy-associated genes, Dgka and Egr3. Thus, alloreactive DNMAML T cells had features reminiscent of T cell anergy. Given that in vivo proliferation in irradiated recipients and cytotoxicity of DNMAML alloreactive T cells were largely preserved, our data suggest a “split anergy” phenotype with differential effects on distinct T cell effector functions.
Altogether, our results reveal a parallel role for Notch signaling in both the CD4+ and CD8+ T cell compartments that differ from all previous reports of Notch action in mature T cells. Understanding the role of Notch signaling in alloreactive T cells is essential for harnessing the therapeutic potential of Notch inhibition in GVHD.

Disclosures:
No relevant conflicts of interest to declare.