Inhibition of Jak1/Jak2 Is More Effective Than Inhibition of Jak3 in Protecting Mice From Acute Graft-Versus-Host Disease (aGvHD) by Significantly Decreasing Alloreactive CD4+ T-Cells.
Authors: Carniti Cristiana , Gimondi Silvia , Vaccaroli Raffaella , Vendramin Antonio , Bermema Anisa , Radaelli Enrico , Mariotti Jacopo , Corradini Paolo 
Published: October 2019

Abstract
Abstract 2997

Background:
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment for patients with hematological malignancies. However, its success is limited by a life-threatening complication: the graft-versus-host disease (GvHD). Although numerous studies have described immunosuppression protocols to mitigate acute GVHD (aGvHD), novel approaches are needed. Chemokines are well known inducers of leukocyte trafficking and activation. Stimulation of the chemokine receptor signaling pathway leads to initiation of the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway that contributes to the pathogenesis of GvHD. The key role of JAK signaling in normal and abnormal lymphocyte development and function, along with the cytotoxic effects of its inhibitor INCB18424 (Ruxolitinib) on leukemia cells, prompted us to hypothesize that this selective JAK1 and 2 inhibitor could be useful as anti-GvHD agent while maintaining antitumor activity. Since CP-690550, a more selective JAK3 inhibitor, was recently shown to protect against GvHD in mouse models, we also tested whether blocking the JAK1/JAK2 pathway could be more effective in preventing GvHD.

Methods:
To assess the therapeutic effect of pharmacologic modulation of JAK1 and 2 on GvHD, a major histocompatibility complex (MHC) mismatched HSCT mouse model was used. Recipient BALB/c mice were lethally irradiated and treated either with spleen and bone marrow (BM) cells from C57BL/6 (B6) donors (GvHD cohort, n=8), or with spleen and BM cells from B6 donors along with INCB18424 90mg/kg/day at days -1 to 13 (INCB18424 cohort, n=10) or with CP-690550 15mg/kg/day (CP-690550 cohort, n=8) at days -1 to 13. Syngeneic transplants (B6-B6, n=6) and BALB/c recipients treated with B6 BM cells only (control cohort, n=8) were also included as controls. Mice were characterized for GvHD by monitoring overall survival and weight loss. Recipient mice were sacrificed and tissues harvested on day 14 and 30 post transplant and GvHD confirmed by histology.

Results:
All mice in the GvHD cohort had clinical evidence of GvHD (weight loss, generalized erythema of the skin and poor fur quality) by day 14. The INCB18424 treated mice showed markedly reduced weight loss along the time of observation when compared to the GvHD cohort. Animals in the CP-690550 cohort tended to gain weight during the time of treatment (day-1 to 13), but thereafter they exhibited reduced body weight similar to that observed in the GvHD cohort. The histological examination of the stomach, liver, skin and intestine obtained at day 14 revealed no sign of GvHD in the control group as well as in the INCB18424 group. On the other hand, mild to moderate signs of GvHD were present in the tissues of CP-690550 treated mice and extensive inflammation and disruption of the normal architecture of the tissues was observed in the GvHD group. To determine whether INCB18424 treatment affected alloreactive CD4+ T cells, total spleen T cells were harvested at day 14 from the GvHD cohort and from recipients either of INCB18424 or CP-690550. Total spleen T cells were co-cultured with BM derived BALB/c (recipient-derived) or C57BL/6 (donor-derived) dendritic cells (DCs). After 24h, T cells alloreactivity was determined by IFN-γ production assessed by intracellular staining. As expected, T cells from GvHD mice showed significantly higher alloreactivity against BALB/c DCs compared to the reactivity observed against syngeneic B6 DCs (5.24% and 0.84% respectively, p<0.05). The alloreactivity observed when T cells from INCB18424 treated mice were stimulated with allogeneic BALB/c DCs was significantly lower than that in the GvHD group (0.64% and 5.24% respectively, p<0.05) and eas also lower than that of the CP-690550 group (1.43%). STAT phosphorylation analysis demonstrated that INCB18424 treatment was effective in vitro.

Conclusions:
The inhibition of Jak/STAT signaling using the sensitive and specific inhibitor of Jak1/Jak2, INCB18424, conferred effective protection from aGvHD in a HSCT mouse model. INCB18424 treatment was more effective than the targeting of JAK3 with CP-690550. In fact, CP-690550 administered during GvHD induction was not completely sufficient to restore the normal weight and to prevent the histological appearance of GvHD whereas INCB18424 was. INCB18424 protected mice against acute GvHD by significantly decreasing alloreactive CD4 T cells.

Disclosures:
No relevant conflicts of interest to declare.