Janus Kinase-2 Inhibition Induces Durable Tolerance to Alloantigen by Dendritic Cell-Stimulated Human T Cells, Yet Preserves Immunity to Recall Antigen

Abstract 1903

Janus kinase 2 (JAK2) conveys signals from receptor binding by several inflammatory cytokines, including IL-6, IL-12, and IL-23, via phosphorylation of signal transducer and activator of transcription 3 (STAT3). These cytokines promote the development and expansion of T helper 1 (Th1) cells, which use IL-12, and Th17 cells, which use IL-6 and IL-23. Th1 and Th17 cells can in turn induce alloreactive end organ damage in GvHD. JAK2 is therefore a principle gatekeeper of alloreactivity and inflammation, and it represents an attractive target to control GvHD. We demonstrate that TG101348, a synthetic small molecule, highly selective JAK2 inhibitor, when present during initial and secondary encounters between human T cells and allogeneic monocyte-derived dendritic cells (moDCs), induces durable, profound, and specific T cell tolerance on reexposure to the same alloantigens. TG101348 ablates IL-6/JAK2-mediated phosphorylation of STAT3 in T cells (24% v 0%, P<0.05, n=5) but has no off-target effects on IL-2 or IL-15/JAK3/pSTAT5-dependent signaling, which would interfere with Tregs and other effector T cell responses. JAK2 inhibition preserves Treg numbers and thereby enhances the ratio of CD4⁺ Tregs to CD8⁺CD25⁺ effector T cells in favor of Tregs (Treg:Effector 1:2 vs 1:1, P<0.05, n=5). JAK2 inhibition also reduces the production of IL-6 (255 vs 157 pg/ml, P<0.05, n=5) and TNF-alpha (50 vs 15 pg/ml, P<0.05, n=4) in allogeneic MLRs, impairing the activation of central and effector memory T cells as well as the expansion of responder Th1 (24% vs 14%, P<0.05, n=4) and Th17 (2% vs 1%, P<0.05, n=4). T cells, stimulated by moDCs in the presence of TG101348, demonstrate a marked reduction in proliferative capacity upon reexposure to fresh allogeneic moDCs from the original stimulating party, even in the absence of the JAK2 inhibitor during the secondary MLR (P<0.05, n=4). Responses to stimulation de novo by influenza matrix peptide (fluMP), a pathogenic nominal antigen, remain intact (P=NS, n=4). Conversely, TG101348 also inhibits secondary MLR responses by T cells, which have already been sensitized to alloantigens in a primary MLR without the JAK2 inhibitor (P<0.05, n=4). The durable tolerance against full HLA disparities in each of these settings provides preclinical data in vitro that JAK2 represents a relevant biologic target for preventing or treating graft-versus-host disease (GvHD), or even solid organ allograft rejection, without broader immune impairment.