Blockade Of Jak2 Protects Cardiac Allografts From Chronic Rejection By Attenuating Th1 Response Along With Increased Regulatory T Cells

1. Background and AimCardiac transplantation is a common clinical strategy for treatment of patients with end-stage heart failure. Although the survival rate for cardiac allografts has recently been greatly improved, long-term survival, however, remains disappointed due to the toxicity of anti-rejection drugs and shortage of effective therapeutic strategies to manage chronic rejection. More recently, regulatory T cell (Treg) based therapies appeared to be a promising therapeutic alternative to attenuate chronic rejection while free of side effect. However, problems with the expansion, instability and antigen specificity of Tregs hindered its clinical applications. Therefore, a better understanding of the underlying molecular mechanisms would be instrumental for the development of Treg based strategies to achieve clinical transplant tolerance.As non-receptor tyrosine kinases, the mammalian Janus kinase (Jak) family comprises four evolutionarily conserved members, Jakl, Jak2, Jak3 and tyrosine kinase 2 (Tyk2). Upon the engagement of cytokines with cell surface receptors, Jaks undergo autophosphorylation on tyrosine residues, which generates docking sites to phosphorylate signal transducers and activators of transcription (STAT). Jak phosphorylated STAT members next form homo-or heterodimers along with nuclear translocation to regulate the expression of immune responsive genes. It is noteworthy that selective Jak3 inhibitors have recently been shown to be noninferior to currently used immunosuppressants for the treatment of transplant rejection and autoimmune disorders. Nevertheless, graft protective Tregs were also significantly suppressed due to blockage of the IL-2-Jak3-STAT5 axis, which significantly limited their application in the setting of chronic rejection.As an important member in Jak family, Jak2 is involved in the regulation of various processes relevant to cell survival, proliferation, activation and differentiation. Unlike Jak3, the research interest for Jak2 was mainly focused on its role in hematologic malignancies relevant to the V617F gain-of-function mutation. Preceding studies on cell lines revealed that Jak2 plays an indispensable role in interleukin-3, interleukin-5, interleukin-12 (IL-12), interferon-y (IFN-y) and granulocyte-macrophage colony-stimulating factor signaling. Given that mice deficient in Jak2 are embryonic lethal, the above observations might not fully resemble the enzymatic coupling that happens in vivo. Recently, we demonstrated that loss of Jak2 in adult mice impairs dendritic cell (DC) development and maturation, while its role in adaptive immune response, particularly in T helper 1 (Th1) response, is yet to be fully addressed. We thus in the current report induced Jak2 deficiency in adult mice and then assessed its role in adaptive immune response in the setting of cardiac allograft rejection. Loss of Jak2 significantly suppressed Thl development, which led to a preferential increase of Tregs and, as a result, cardiac allografts were protected from chronic rejection.2. Methods and ResultsJak2 deficiency was induced in 8wk old Cre++ -Jak2fl fl mice by i.p. injection of tamoxifen for 5 consecutive days, and Jak2 depletion was firstly confirmed by genotyping of tail blood DNA for the presence of floxed null allele. Indeed, Jak2 was undetectable in the lysates of spenocytes by Western blot analysis, demonstrating that tamoxifen efficiently abrogated Jak2 expression. In consistent with our previous studies, mice deficient in Jak2 manifested a significant reduction for the total number of splenocytes. Interestingly, the myeloid lineage cells in Jak2-/- spleens were nearly disappeared on the forward and side scatter image of flow cytometry data along with a significant increase for the proportion of lymphoid lineage. In line with this observation, the proportion of CD4+T cells in pan splenic cells was elevated by 1-fold. The significant elevation for the proportion of lymphoid cells and CD4+T cells were also seen in peripheral blood.We next sought to address the impact of Jak2 deficiency on T cell development. Loss of Jak2 did not result in a perceptible change for the CD4+or CD8+T cell ratio in total CD3+splenic cells, and similar results were found in the lymph nodes and peripheral blood. However, Jak2 deficiency resulted in a 1-fold decrease for the CD4+CD44highCD62Llow effector/memory T cells (TEM cells) along with a 1.5-fold reduction for the number of CD4+IFN-y producing Th1 cells. On the contrary, the proportion of CD4+Foxp3+Tregs was noted to be slightly but statistically significant higher in Jak2-- mice as compared with their counterparts.To confirm the above data, CD4+CD44lowCD62Lhigh naive T cells isolated from Jak2-/- and control mice were cultured under Thl and Treg condition as described, followed by flow cytometry analysis of IFN-y+Th1 cells and Foxp3+Tregs. Indeed, IFN-y secreting Th1 cells were successfully induced in control cells, while loss of Jak2 almost completely abolished the production of IFN-y producing Thl cells. More importantly, addition of IFN-y (50ng/ml) markedly augmented Thl differentiation in control cells, but the repressed Thl differentiation was not restored by IFN-y in Jak2-/- cells. Interestingly, Jak2 deficiency did not affect the production of Foxp3+Tregs, rather the percentage of Foxp3+Tregs was even higher than that of control cells. Particularly, anti-CD28 stimulation substantially increased Foxp3 expression in both Jak2-/-and control cells, while the percentage of Foxp3+cells was still higher in Jak2-/-cells than that of control cells. Collectively, these results indicate that loss of Jak2 only selectively impairs Thl development.The above findings prompted us to examine the impact of Jak2 deficiency on allograft chronic rejection, a process predominantly mediated by CD4+T cells. For this purpose, BABL/c (H-2d)-derived cardiac grafts were heterotopically transplanted into the abdomen of Jak2 deficient mice or their control littermates (H-2b). Remarkably, loss of Jak2 significantly prolonged allograft survival [median survival time (MST) 58±30.6 days vs.7±0.3 days, P< 0.001]. Particularly,4 out of 13 Jak2 deficient recipients (30%) showed long-term acceptance of allografts as manifested by the graft survival time>100 days. To exclude the potential effect of tamoxifen induction on allograft survival, tamoxifen induced Cre-ERT2 transgenic mice (H-2h) were transplanted with BABL/c (H-2d)-derived cardiac grafts as well. No perceptible difference in terms of allograft survival time was noted between vehicle treated control mice and tamoxifen-induced Cre-ERT2 mice (MST 7±0.3 days vs.7.5±0.4 days).Histological analysis was next conducted to further confirm the above data. Indeed, H&E staining of cardiac allograft sections originated control recipients after day 6 of transplantation revealed rigorous inflammatory infiltration along with cardiomyocyte destruction. In sharp contrast, no significant inflammatory infiltration was noted in the graft sections derived from Jak2-/-recipients along with well reserved myocardium. The attenuated allograft rejection was also confirmed by the differences of mRNA levels for inflammatory cytokines and chemokines. Specifically, allografts originated from control recipients manifested significantly higher levels of IFN-y, Tumor Necrosis Factor-a (TNF-a), IL-2, IL-6, IL-12p40 and CC chemokine ligand 2 (CCL-2) expression as compared with grafts from Jak2-/- recipients. Particularly, IFN-? and IL-12p40 were almost undetectable in the grafts derived from Jak2-- recipients.Since loss of Jak2 altered Th program, we next compared the difference between Jak2-- and control recipients for IFN-? producing Thl cells after day 6 of transplantation. Flow cytometry analysis of splenic CD4+T cells revealed that control recipients manifested a 1.9-fold higher IFN-?+ Th1 cells than that of Jak2-- recipients. However, higher proportion of splenic Tregs was noted in Jak2-- recipients as compared with that of control recipients. Especially, Jak2-- recipients manifested a 5% higher proportion of Tregs than that of control recipients in the draining lymph nodes (DLN).To demonstrate whether Tregs deficient in Jak2 were still functionally competent, Treg suppressive assays were next carried out based on CFSE labeling. CD4+ responders (Tresp) and non-T accessory cells were obtained from WT mice, while Jak2-- and WT Tregs were added in different proportions into the cultures to repress Tresp proliferation following anti-CD3 stimulation. Interestingly, Jak2-/- Tregs exhibited a comparable suppressive kinetics as that of WT Tregs, suggesting that loss of Jak2 does not affect the functionality of Tregs.The next important question is whether Jak2 deficiency impacts the intrinsic capability of CD4+ T cells for proliferation. To address this issue, splenic CD4+ T cells originated from Jak2-/- and control mice were labeled with CFSE and then stimulated with anti-CD3/anti-CD28 antibodies or low dose phorbol-12-myristate-13-acetate (PMA)/Ionomycin as described, respectively. In line with our expectation, Jak2-- CD4+T cells manifested similar proliferation potency as that of control CD4+T cells in response to anti-CD3/anti-CD28 or PMA/Ionomycin stimulation, indicating that Jak2-- deficiency does not affect their intrinsic proliferative capability. To further demonstrate this question, we conducted mixed lymphocyte reaction (MLR) assays to assess the differences for allo-antigen stimulated proliferation. BALB/c-derived bone marrow dendritic cells (BMDCs) were treated with Mitomycin C and then employed for stimulation of CFSE-labeled Jak2'and control CD4+T cells, respectively. The purity of CDllc positive dendritic cell (DC) was about 95% as manifested by flow cytometric analysis. Similar as above, Jak2-- CD4+T cells displayed comparative proliferative capability as that of their control counterparts in response to allo-antigen stimulation.To dissect the mechanisms by which Jak2 deficiency impairs the balance between Th1 and Treg program, we first examined cytokine-stimulated Jak2 activity in CD4+T cells. WT CD4+T cells were stimulated with IFN-y, IL-12 and IL-2, followed by analysis of the levels for phosphorylated Jak2 (p-Jak2). High levels of p-Jak2 were detected in both IFN-y and IL-12 stimulated cells, and in sharp contrast, p-Jak2 was almost undetectable in IL-2 stimulated CD4+ cells.The above results prompted us to check the activities of Jak2 downstream signaling molecules following cytokine stimulation. Under steady condition, phosphorylated STAT4 (p-STAT4) was undetectable both in WT and Jak2-- CD4+T cells, while high levels of p-STAT4 were noted in WT CD4+T cells following IL-12 stimulation. However, IL-12 failed to induce the expression of p-STAT4 in Jak2-- CD4+ T cells. Similarly, IFN-y induced high levels of phosphorylated STAT1 (p-STAT1) in WT CD4+T cells, but p-STAT1 remained undetectable in Jak2-- CD4+ T cells following IFN-y stimulation. Interestingly, unlike STAT4 and STAT1, both WT and Jak2-- CD4+T cells manifested low levels of expression for the phosphorylated STAT5 (p-STAT5) under steady condition, and more importantly, IL-2 stimulation induced high levels of p-STAT5 both in WT and Jak2-- CD4+T cells. Taken together, these data suggest that loss of Jak2 repressed IL-12/STAT4 and IFN-y/STAT1 signaling, which then attenuated naive CD4+T cells toward to IFN-y producing Th1 cell polarization. On the contrary, Jak2 is dispensable for the IL-2/STAT5 signaling, and as a result, Jak2 deficiency did not affect Treg development. Since Jak2 deficiency did not impact IL-2/STAT5 signaling, higher percentage of Tregs noted in Jak2-- mice is likely caused by the impaired IL-12 and IFN-y signaling, in which Jak2-/- naive CD4+T cells preferentially differentiate into Foxp3+Tregs in the absence of IFN-y.To dissect the mechanisms by which loss of Jak2 impairs Thl development, we first checked transcription factors T box 21 (T-bet) and H 2.0-like homeobox (Hlx), in which T-bet serves as a Th1 "master regulator", while Hlx drives optimal IFN-y expression to stabilize Th1 phenotype. To this end, naive T cells isolated from Jak2-/- and control mice were cultured under Thl condition and then subjected to Western blot analysis of T-bet and Hlx expression. Unlike WT T cells which manifested high levels of T-bet expression, T-bet was hardly detectable in Thl polarized Jak2-- T cells, and remarkably, Hlx was completely absent in Thl polarized Jak2-- T cells. To further confirm this observation, we examined Runt-Related Transcription Factor 3 (Runx3), another transcription factor that acts in cooperation with T-bet to ensure Thl polarization, and similar results were obtained. During Thl development the IL-12R?2 subunit for IL-12 receptor is rapidly induced following the expression of T-bet. Unexpectedly, the abrogated T-bet expression did not couple with diminished induction of IL-12R?2 as manifested by the detection of relatively lower levels of IL-12R?2 in JakT2-/- CD4 T cells. Collectively, these data suggest that loss of Jak2 attenuates the induction of T-bet/Hlx and the related transcription factor Runx3, which then impairs Th1 polarization.3. ConclusionIn summary, we demonstrated that loss of Jak2 led to diminished Thl cell development along with a preferential increase of functionally competent Tregs. As a result, cardiac allografts were significantly protected from chronic rejection in recipients deficient in Jak2. Cellular studies revealed that Jak2 deficiency did not impact the intrinsic proliferative capability for CD4+T cells in response to nonspecific polyclonal and allogenic stimulation. Mechanistic studies documented that the impaired Thl development was caused by the attenuated IFN-y/STAT1 and IL-12/STAT4 signaling along with repressed expression of Th1 transcription factors T-bet, Hlx and Runx3. However, the IL-2/STAT5 signaling remained intact, which ensured normal Treg development in Jak2-/- naive CD4 T cells. Together, our data support that that suppression of Jak2 may have therapeutic potential for prevention and treatment of allograft rejection in clinical settings.