Targeting JAK2 By Gene Knockout or Pacritinib Treatment Reduces Gvhd and Xenograft Rejection By Promoting Induced Treg Differentiation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1874-1874 ◽  
Author(s):  
Brian C Betts ◽  
David Bastian ◽  
Hung Nguyen ◽  
Jessica Lauren Heinrichs ◽  
Yongxia Wu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Human Skin ◽  
Skin Graft ◽  
Graft Rejection ◽  
Cd4 T Cells ◽  
Vehicle Control ◽  
Jak2 Inhibitor ◽  
Th17 Differentiation ◽  
Jak2 Inhibition

Abstract Janus kinase 2 (JAK2) signal transduction is a critical mediator of immune response. JAK2 activation promotes T-cell allosensitization, as well as Th1 and Th17 differentiation. JAK2-mediated STAT3 phosphorylation limits the generation of induced regulatory T cells (iTregs) by disrupting interactions between STAT5 and Foxp3. JAK2 is implicated in the onset of graft-versus-host disease (GVHD), which is a significant cause of transplant-related mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Using murine models of allo-HCT we show here that transfer of donor JAK2-/- T cells is associated with significantly less GVHD, compared with wild-type or JAK2 replete donors (Figure 1, P =.003 and 0.01, respectively). Th1 differentiation among JAK2-/- T-cells is dramatically decreased, compared with controls. Conversely, iTreg polarization and stability are significantly increased among the JAK2 deficient T cells. To investigate the effect of pharmacologic JAK2 inhibition on T-cell alloresponses, pacritinib (supplied by CTI BioPharma), was chosen as it does not induce myelosuppression or increase risk for opportunistic infections in myelofibrosis patients - unlike JAK1/JAK2 inhibitors. Pacritinib potently inhibits JAK2, but also has suppressive activity toward JAK3, CSF1R, and IRAK1. Pacritinib was administered at 100mg/kg twice a day by oral gavage for 4 weeks beginning on the day of MHC-disparate allo-HCT, significantly reducing GVHD in recipient mice compared with methylcellulose vehicle control. In allogeneic mixed leukocyte reactions using human cells, pacritinib (2.5μM) significantly reduces T-cell proliferation after 5 days of culture (P <.0001). In vitro studies verified pacritinib eliminates STAT3 activity by IL-6 in human CD4+ T-cells, while permitting IL-2 induced STAT5 phosphorylation despite its effects on JAK3. This offers a platform to reduce Th17 development, while promoting iTreg populations. As such, pacritinib dramatically suppresses Th17 differentiation reflected by RORgammaT expression among naïve CD4+ T cells stimulated by allogeneic dendritic cells (DC), compared with DMSO (P <.0001). iTregs were generated with allogeneic DCs in the presence of pacritinib or DMSO for 5 days, and then cultured with self T-cell responders and fresh DCs without additional drug exposure. The suppressive potency of either pacritinib- or DMSO-treated iTregs was similar, suggesting JAK2 is not required for iTreg function. We then evaluated whether JAK2 inhibition could prevent human skin graft rejection in an NSG mouse xenograft model. A 1x1 cm split-thickness human skin graft was transplanted onto the animal dorsally. The mice rested for 30 days after surgery to permit engraftment, then received 5x106 allogeneic peripheral blood mononuclear cells (PBMC) by intraperitoneal injection. Pacritinib was administered at the same dose and schedule for 2 weeks beginning at time of PBMC injection as tolerated by the NSG mice. The treatment significantly delayed allograft rejection by the human donor PBMCs, compared with vehicle control (Figure 2: Median graft survival 32.5 v 51 days, Day +60 survival 0% v 33.3%, pooled data from 2 experiments, n=5-6 mice per arm, P =.0011). Collectively, these data clearly identify JAK2 as a therapeutic target to control donor alloreactivity and promote iTreg responses after HCT or solid organ transplantation. *BCB and DB contributed equally to this work. Figure 1. Decreased murine GVHD with JAK2 deficient T-cells Figure 1. Decreased murine GVHD with JAK2 deficient T-cells Figure 2. Decreased human skin graft rejection with JAK2 inhibitor, pacritinib Figure 2. Decreased human skin graft rejection with JAK2 inhibitor, pacritinib Disclosures Singer: CTI BioPharma, Corp: Employment, Equity Ownership.

Regulatory T Cells Inhibit CD8+ T-Cell Tissue Invasion in Human Skin Graft-Versus-Host Reactions

2012 ◽  
Vol 94 (5) ◽  
pp. 456-464 ◽  
Author(s):  
Emily Mavin ◽  
Shaheda S. Ahmed ◽  
Graeme O’Boyle ◽  
Brie Turner ◽  
Stephen Douglass ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Human Skin ◽  
Skin Graft ◽  
Cd8 T Cell ◽  
Graft Versus Host ◽  
Tissue Invasion ◽  
Cell Tissue

The ICOS:ICOSL Costimulatory Pathway Plays an Important Role in GVHD and Bone Marrow (BM) Graft Rejection.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 591-591 ◽  
Author(s):  
Patricia Taylor ◽  
Angela Panoskaltsis-Mortari ◽  
Gordon Freeman ◽  
Arlene Sharpe ◽  
Randolph Noelle ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Family Member ◽  
Cd8 T Cells ◽  
Graft Rejection ◽  
Cd4 T Cells ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Activated T Cells ◽  
Effector Cells

Abstract ICOS, a CD28/CTLA-4 family member, is expressed on activated T cells. ICOS Ligand, a B7 family member, is constitutively expressed on B cells, monocytes and some T cells. Through the use of blocking anti-ICOS mAb and ICOS deficient (−/−) mice, we found that ICOS:ICOSL interactions play an important role in GVHD and BM graft rejection. Anti-ICOS mAb (given d-1 to d28 post BMT) significantly delayed or reduced mortality at 2 different T cell doses in a full MHC-disparate GVHD model. ICOS−/− T cells led to delayed or reduced mortality at 3 different cell doses compared to wild-type T cells. ICOS−/− CD4+ or CD8+ T cells infused into class II- or class I-disparate recipients, respectively, revealed that ICOS:ICOSL interactions regulate both CD4+ and CD8+ T cell alloresponses. Anti-ICOS inhibited GVHD in a CD28-independent fashion. Anti-ICOS inhibited GVHD mediated by either stat 4−/− or stat 6−/− T cells indicating that the ICOS pathway regulates both Th2 and Th1-mediated GVHD. In contrast to blockade of the B7:CD28/CTLA-4, CD40L:CD40 or the OX40:OX40L pathway, anti-ICOS mAb inhibited GVHD even when delayed until d5 post BMT, a time when substantial T cell expansion has occurred. A TCR transgenic model of GVHD was used to further study effects of ICOS:ICOSL blockade. All CB6 F1 recipients of anti-host alloreactive 2C CD8+ and TEa CD4+ T cells succumbed to GVHD mortality by d18 after transfer of cells. In contrast, 88% of anti-ICOS-treated mice survived long-term. Evaluation of spleens early after transplant revealed that anti-ICOS mAb reduced the number of TEa CD4+ cells by 44% and 2C CD8+ cells by 83%. Green fluorescent protein (GFP) 2C CD8+ and GFP TEa CD4+ T cells were infused into irradiated CB6 F1 mice and irrelevant or anti-ICOS mAb was administered. Mice were imaged on d4, 7 and 12 after T cell transfer. By d7, pronounced infiltration of GFP+ cells was noted in the peripheral and mesenteric LN, spleen, Peyer’s patches (PP), skin, gingiva, liver, kidney, lung, ileum, and colon of GVHD control mice. In contrast, there were fewer GFP+ cells in the spleen, ileum, colon, kidney, lung, skin and gingiva of anti-ICOS-treated mice, although there was no decrease in GFP+ cells in LNs or PP. To study the role of host ICOS expression in BM graft rejection, wild-type or ICOS−/− mice were sublethally irradiated and given allogeneic BM and evaluated for donor chimerism at 6 weeks post BMT. Five of 10 wild type mice engrafted (ave − 26% donor) in contrast to all 10 of ICOS−/− mice (ave − 71% donor). Collectively, these data indicate that ICOS:ICOSL interactions play an important role in GVHD, whether mediated by CD4+ Th1 or Th2 T cells or CD8+ T cells. Importantly, blockade of ICOS:ICOSL after initiation of alloresponses inhibited GVHD, in contrast to blockade of other costimulatory pathways, suggesting that the ICOS pathway may be a novel therapeutic target in primed transplantation situations. Anti-ICOS interfered with expansion of donor T cells in the spleen early after transplant and reduced the number of effector cells in several GVHD target tissues. These data suggest this pathway may be indicated for therapeutic targeting for the inhibition of GVHD and BM graft rejection.

scholarly journals Cells mediating graft rejection in the mouse. I. Lyt-1 cells mediate skin graft rejection.

1981 ◽  
Vol 153 (5) ◽  
pp. 1044-1057 ◽  
Author(s):  
B E Loveland ◽  
P M Hogarth ◽  
R Ceredig ◽  
I F McKenzie
Keyword(s):  
T Cells ◽  
T Cell ◽  
Skin Graft ◽  
Graft Rejection ◽  
Cytotoxic T Cells ◽  
Lymphoid Cells ◽  
Delayed Type Hypersensitivity ◽  
Cytotoxic T Cell ◽  
T Helper ◽  
Cytotoxic Lymphocyte

The Ly phenotype of cells mediating skin graft rejection was determined using monoclonal anti-Lyt-1.1 and Lyt-2.1 antibodies in CBA mice that received CBA lymphoid cells from mice sensitized to C57BL/6; i.e., alloantigenic differences arising from the H-2 and non-H-2 loci. It was clear that graft rejection was due wholly to the presence of Lyt-1 cells in the inoculum and that Lyt-123 or Lyt-23 cells had no effect. Furthermore, no synergism was noted between Lyt-1 and Lyt-2 cells. In this model, both the cytotoxic T cell and cytotoxic lymphocyte precursors were shown to be Lyt-123 and these could be depleted from sensitized Lyt-1 populations that mediated graft rejection. Thus cytotoxic T cells are not responsible for skin graft rejection, but rather, this is mediated by an Lyt-1 cell. Whether this T cell is distinct from other Lyt-1 cells (T helper, T cells mediating delayed hypersensitivity) is not clear at present, but other evidence, and traditional concepts, link graft rejection and delayed type hypersensitivity as being different manifestations of the same mechanism.

FTY720 Inhibits Graft-Versus-Host Disease (GVHD) and Allogeneic Bone Marrow (BM) Graft Rejection Independent of Effects on T Cell Egress from Lymphoid Tissue.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 450-450
Author(s):  
Patricia A. Taylor ◽  
Michael J. Ehrhardt ◽  
Angela Panoskaltsis-Mortari ◽  
Jonathan S. Serody ◽  
Volker Brinkmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Rejection ◽  
Cd4 T Cells ◽  
Single Agent ◽  
Lymphoid Tissues ◽  
Imaging Studies ◽  
Donor Chimerism ◽  
Cell Dose ◽  
Lymphocyte Egress

Abstract FTY720 (FTY), a sphingosine-1-phosphate receptor agonist, inhibits lymphocyte egress from lymphoid tissues although the complete mechanism of its immunomodulatory effects is not well understood. We studied FTY in murine GVHD and engraftment models as a single agent and in combination with known beneficial strategies. FTY (3 mg/kg orally d0-28) inhibited GVHD, increasing the median survival time (MST) of lethally irradiated C57BL/6 (B6) mice given BALB/c BM and a moderate T cell dose from 30d to 71d (p&lt;.001) or a higher T cell dose from 7d to 32d (p&lt;.006). FTY was additive with ex vivo activated and expanded CD4+CD25+ regulatory T cells (Tregs) for GVHD inhibition. In contrast to control mice which all died of GVHD by d70 (MST = 37d), mice receiving FTY or Tregs or both had 100 day survival rates of 50%, 71% and 100%, respectively. The additive effects of FTY and Tregs were reproduced in a second strain combination. Although we hypothesized that FTY trapped Tregs in the lymph nodes allowing for increased contact time for Tregs to inhibit GVHD effector T cells, imaging studies of green fluorescent protein (GFP)+ Tregs did not support this hypothesis. Moreover, imaging studies of GFP+ effectors did not indicate that FTY inhibited donor T cell egress from lymphoid tissues. To study alloantigen-specific responses, B6 TCR Tg CD8+ and CD4+ T cells that are reactive against BALB/c alloantigen were adoptively transferred into B6 rag mice prior to sublethal irradiation and infusion with BALB/c BM. FTY reduced the expansion of Tg CD8+ and CD4+ T cells in the spleen 10d after BMT by 80% and 78%, respectively. In addition to GVHD inhibition, FTY also promoted donor BALB/c BM engraftment in sublethally (5.0 Gy) irradiated B6 mice although donor chimerism was not stable in all mice. In contrast to 4 of 26 water-treated controls, all 29 FTY-treated mice (3 mg/kg d0-13) were engrafted at 1 month as assessed by PBL phenotyping (ave of 9% vs 68% donor. p&lt;.001). However, by 3 months, the engraftment rate in FTY-treated mice had decreased to 12 of 28 mice. All 9 mice receiving FTY for an additional 2 weeks (d0-28) had high donor chimerism levels (&gt;96%) at 1 month but chimerism was lost in 3 mice by 6 months after BMT. In a different strain combination, even a 2 wk FTY course increased donor chimerism that remained stable for 6 months. Imaging studies revealed that FTY did not prevent the egress of adoptively transferred host-type GFP T cells in allogeneic BM recipients. FTY was also tested in combination with anti-CD40L mAb, a potent engraftment-promoting agent in mice. In contrast to anti-CD40L mAb, FTY did not promote engraftment in mice conditioned with very low levels of irradiation (0.5–2.0 Gy) although the combination of anti-CD40L mAb and FTY resulted in superior stable engraftment rates and levels compared to anti-CD40L mAb as a single agent. Interestingly, FTY increased donor chimerism in syngeneic, as well as, allogeneic recipients. These data indicate that FTY is efficacious as a single or adjunct agent for the prevention of GVHD and graft rejection and further indicate that inhibition of lymphocyte egress was not essential for the beneficial effects of FTY in BMT recipients.

Allospecific Effector Memory T Cells Are Able to Mediate Second-Set Skin Graft Rejection but Unable to Induce Graft-Versus-Host Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 232-232
Author(s):  
Ping Zhang ◽  
Jieying Wu ◽  
Divino Deoliveria ◽  
Nelson J. Chao ◽  
Benny J. Chen
Keyword(s):  
T Cells ◽  
T Cell ◽  
Skin Graft ◽  
Graft Versus Host Disease ◽  
Graft Rejection ◽  
Memory T Cells ◽  
Effector Memory ◽  
Memory Phenotype ◽  
Graft Versus Host ◽  
Effector Memory T Cells

Abstract Abstract 232 Several different groups have independently demonstrated that non-allospecific effector memory T cells do not induce graft-versus-host disease (GVHD). Limited data are available regarding the ability of allospecific effector memory T cells to induce GVHD. We first studied this question in the C57BL/6 into BALB/c model. Similar to the data previously published by other groups, purified CD62L- effector memory T cells isolated from donors, who were primed with the host antigens 8 weeks earlier, had decreased ability to induce acute GVHD compared with unseparated and CD62L+ T cells. Similar results were observed when the parous female mice, who were sensitized to the host antigens during pregnancy, were used as memory T cell donors. In order to study this question more definitely and to understand the mechanisms underlying these surprising observations, we further studied the ability of allospecific effector memory T cells to induce GVHD using a novel GVHD model mediated by transgenic TEa T cells. All TEa T cells are CD4+ and recognize the same peptide in the context of I-Ab. This peptide corresponds to positions 52-68 from the alpha-chain of I-E class II molecules and is expressed in all antigen presenting cells from H-2b/I-E+ strains such as CB6F1 mice. To generate memory T cells, naïve TEa cells were first parked in Rag1−/− mice and then immunized with irradiated CB6F1 spleen cells. More than eight weeks later, ∼98% of TEa cells obtained a memory phenotype (CD44high, CD45RB-.CD127+,CD11a bright, FasL bright, Ki67-, CD28-, KLRG-). Of them, about 93% were CD62L-CD44high effector memory T cells and 7% were CD62L+CD44high central memory T cells. These Rag1−/− mice that contained memory phenotype TEa cells rejected CB6F1 skin grafts much faster than naïve TEa mice did (median survival time: 6.5 vs. 13 days, P=0.01), suggesting that the memory phenotype T cells contained in these mice are functional. Moreover, CD62L-CD44high TEa cells purified from these mice mediated faster and stronger in vitro proliferative responses against alloantigens than naïve TEa cells did, further demonstrating that they are true functional effector memory T cells. We next tested the ability of these effector memory TEa cells to induce GVHD. Effector memory TEa cells were obtained after depletion of CD62L+ cells using magnetic beads and the purity was more than 99%. Transplantation of 1′105 TEa naïve T cells together with 1′107 T cell depleted bone marrow cells into lethally irradiated CB6F1 recipients induced lethal GVHD in all recipients and all animals in this group died within 35 days after transplantation. In contrast, none of the effector memory TEa cell recipients developed GVHD and all of them survived more than 100 days post transplantation (P<0.01, compared with naïve T cell control). To understand the mechanisms underlying these observations, we studied the kinetics of TEa proliferative responses upon challenge with alloantigens. The data indicated that effector memory TEa cells reached the peak responses faster than naïve TEa cells did. CFSE tracking experiment further confirmed this observation. Simultaneous staining with anti-Anexin V antibody and 7-AAD demonstrated that effector memory TEa cells undergone apoptosis and died faster than naïve T cells did. In conclusion, these data underscore the fundamental difference of alloresponses mediated by antigen-specific effector memory T cells in graft rejection and GVHD settings. The TEa transgenic T cell skin graft and GVHD models would allow further understanding of the unique alloresponses mediated by allospecific memory T lymphocytes in GVHD. Disclosures: No relevant conflicts of interest to declare.

CD4+ T Cell Functions Are Potently Suppressed By The Janus Kinase 1/2 (JAK1/JAK2) Inhibitor Ruxolitinib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2281-2281 ◽  
Author(s):  
Sowmya Parampalli Yajnanarayana ◽  
Isabelle Cornez ◽  
Annkristin Heine ◽  
Peter Brossart ◽  
Dominik Wolf
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Inflammatory Cytokines ◽  
Cd4 T Cells ◽  
Cell Function ◽  
Cd4 T Cell ◽  
Jak2 Inhibitor ◽  
Dose Dependent ◽  
Pro Inflammatory Cytokines

Abstract Introduction Recent discoveries of activating JAK mutations in patients with myeloproliferative diseases (MPNs) coupled with the so far known biology of JAKs in cytokine signaling provided the rationale for targeting these kinases in MPNs. Ruxolitinib (INCB018424) is the first JAK1/JAK2 inhibitor approved for treatment of patients with myelofibrosis (MF). Although ruxolitinib shows limited anti-clonal activity, a profound improvement of quality of life and splenomegaly in MF patients is observed and linked to a substantial reduction of MF-associated circulating pro-inflammatory cytokines and pro-angiogenic factors. JAK/STAT-signalling is known to be involved in the regulation of various immune cells including CD4+ T cells, which critically orchestrate inflammatory responses. To better understand how ruxolitinib is modulating CD4+ T cell response, we here provide an in depth analysis of CD4+ T cell function upon ruxolitinib exposure. Methods Highly purified CD4+ T cells isolated from healthy human PBMC from buffy coats were stimulated for 4 days with i) plate bound anti-CD3, ii) plate bound anti-CD3 and soluble anti-CD28 antibodies, iii) IL-2 in the presence of increasing concentrations of ruxolitinib (0.1µM – 10µM) or the respective vehicle control (DMSO). Phenotype and function were analyzed by flow cytometry. Cytokine production was quantified either by intracellular staining and subsequent flow cytometry or by flow-based bead assays (Human Th1/Th2 11plex FlowCytomix Multiplex). Proliferation was detected by CFSE dilution analysis using FACS. CD4+CD62L+ T cells obtained from C57BL/6 mice were isolated by using the CD4+CD62L+ T Cell Isolation Kit (Miltenyi Biotec) and subsequently differentiated into TH1, TH2, TH9, TH17 and iTreg. Polarization into the different CD4+ T cell subsets was induced by cytokine/antibody cocktails (TH1: IL-12 and anti-IL4; TH2: IL-4 and anti-IL12; TH9: IL-4, TGF-β and anti-IFNγ; iTreg: IL-2 and TGFβ; TH17: IL-6, TGFβ, IL-1b, anti-IFNγ and anti-IL4) together with anti-CD3 and anti-CD28. For analysis of apoptosis/necrosis induction, annexin/propidium iodide staining was applied. Signalling events were analyzed by phospho-flow technology to evaluate ruxolitinib-mediated changes of TCR- and/or cytokine-induced signalling cascades (using pS6, pSTAT1, pSTAT3, pSTAT5, pERK, pAKT, pP38, pFos, pJun and pZAP70 antibodies). Results CD4+ T cell proliferation is significantly and dose-dependently suppressed by ruxolitinib when T cells were activated by each of the three conditions tested. Of note, we could not detect any changes in the viability of ruxolitinib-exposed CD4+ T cells. In line with previous studies, production of pro-inflammatory cytokines such as IL-1β, IL-5, IL-6 and TNF-α were dose-dependently inhibited in ruxolitinib-exposed CD4+ T cells, although expression of the pro-inflammatory IL-8 was increased in a dose-dependent manner. Interestingly, despite the complete proliferation block, we also observed an increase in IL-2 and IFNγ particularly at the lower ruxolitinib concentrations (0.1μM) followed by a dose dependent reduction at higher dose-levels (10µM). After short-term activation of ruxolitinib-exposed CD4+ T cells by anti-CD3 and anti-CD28, proximal TCR signaling events (phosphorylation of SLP76 and ZAP70) were not affected, whereas a clear down-regulation of IL-2 induced STAT5 phosphorylation could be detected. After wash-out the ruxolitinib-induced inhibitory effects on CD4+ T cell function were fully reversible, as shown by induction of the T cell activation markers CD25 and CD69. Finally, we differentiated murine CD4+ naïve T cells into the various T Helper cell subsets and could provide clear evidence that the differentiation capacity of naïve CD4+ T cells into TH1, TH9, TH17 and iTreg was markedly reduced, whereas inhibition of Th2 differentiation was only marginally affected. The anti-inflammatory effects of ruxolitinib are currently tested in a TH9-dependent lung inflammation model in mice. Conclusion We could show that ruxolitinib potently affects CD4+ T cell biology. These data provide a rationale for testing JAK inhibitors in diseases triggered by hyperactive CD4+ T cells, such as autoimmune diseases. However, they also provide an explanation for the increased infection rates (i.e. viral reactivation and urinary tract infection) seen in ruxolitinib-treated patients. Disclosures: Wolf: Novartis: Honoraria, Research Funding.

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