ALBERT

Description: Janus kinase 2 (JAK2) signal transduction is a critical mediator of the immune response. 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). Transfer of JAK2−/− donor T cells to allogeneic recipients leads to attenuated GVHD yet maintains graft-versus-leukemia. Th1 differentiation among JAK2−/− T cells is significantly decreased compared with wild-type controls. Conversely, iTreg and Th2 polarization is significantly increased among JAK2−/− T cells. Pacritinib is a multikinase inhibitor with potent activity against JAK2. Pacritinib significantly reduces GVHD and xenogeneic skin graft rejection in distinct rodent models and maintains donor antitumor immunity. Moreover, pacritinib spares iTregs and polarizes Th2 responses as observed among JAK2−/− T cells. Collectively, these data clearly identify JAK2 as a therapeutic target to control donor alloreactivity and promote iTreg responses after allo-HCT or solid organ transplantation. As such, a phase I/II acute GVHD prevention trial combining pacritinib with standard immune suppression after allo-HCT is actively being investigated (https://clinicaltrials.gov/ct2/show/NCT02891603).

Description: Graft-versus-host disease (GVHD) remains one of the major complications after allogeneic bone marrow transplantation (allo-BMT). Sirtuin-1 (Sirt-1) plays a crucial role in various biological processes including cellular senescence, metabolism, and inflammatory responses. Sirt-1 deacetylation regulates different transcription factors that are important for modulating immune responses. In the current study, we addressed the role of Sirt-1 in GVHD induction by employing Sirt-1 conditional knockout mice as well as a pharmacological Sirt-1 inhibitor. Using major histocompatibility complex (MHC)–mismatched and MHC-matched murine BMT models, we found that Sirt-1−/− T cells had a reduced ability to induce acute GVHD (aGVHD) via enhanced p53 acetylation. Sirt-1-deficient T cells also promoted induced regulatory T cell (iTreg) differentiation and inhibited interferon-γ production after allo-BMT. Sirt-1 deletion in iTregs increased Foxp3 stability and restrained iTreg conversion into pathogenic T cells. Furthermore, we found that administration with a Sirt-1 inhibitor, Ex-527, significantly improved recipient survival and clinical scores, with no signs of tumor relapse. These results indicate that Sirt-1 inhibition can attenuate GVHD while preserving the graft-versus-leukemia effect. Consistently, Sirt-1-deficient T cells also displayed a remarkably reduced ability to induce chronic GVHD (cGVHD). Mechanistic studies revealed that Sirt-1 deficiency in T cells enhanced splenic B-cell reconstitution and reduced follicular T helper cell development. Sirt-1 deficiency in T cells modulated donor B-cell responses reducing both B-cell activation and plasma cell differentiation. In addition, therapeutic Sirt-1 inhibition could both prevent cGVHD and reduce established cGVHD. In conclusion, Sirt-1 is a promising therapeutic target for the control of aGVHD and cGVHD pathogenesis and possesses high potential for clinical application.

Description: The diversity and composition of T cell receptor (TCR) repertoire, which is the result of V, D and J gene recombination in TCR gene locus, has been found to impact immune responses in autoimmune and infectious diseases. The correlation of T-cell repertoire with the pathogenesis and outcome of graft-versus-host disease (GVHD) remain undefined. Here, by utilizing high-throughput sequencing of the gene encoding the TCRβ-chain, we comprehensively analyzed the profile of T-cell repertoire in host lymphoid and GVHD target organs after bone marrow transplantation (BMT). To understand whether T-cell repertoire is affected by different strength of alloantigen stimulation, we transferred same donor T cells derived from C57BL/6 (B6) mice into irradiated BALB/c (MHC-fully mismatched), B6D2F1 (MHC-haploidentical), BALB.b (MHC-matched ) and B6 recipients (syngeneic). Fourteen days later, T cells were isolated from recipient peripheral blood, spleen, peripheral lymphoid nodes (pLN), mesenteric lymphoid nodes (mLN), liver, lung, gut and skin for TCR sequencing. Clonality of donor T cells, which is inversely associated with TCR diversity, was significantly increased in either syngeneic or allogeneic recipients when compared with naïve donor T-cells, consistent with the concept that TCR diversity is reduced after T-cell activation and expansion. Increased TCR clonality was observed in lymphoid organs of allogeneic compared with syngeneic recipients, confirming that donor T cells were further activated in allogeneic recipients. However, decreased TCR clonality was observed in GVHD target organs of allogeneic compared with syngeneic recipients, suggesting that only limited donor T-cell clones were able to migrate in target organs in syngeneic compared to allogeneic recipients. The frequency of top clones in total productive rearrangements was increased in GVHD target organs especially liver of allogenic than syngeneic receipts. Interestingly, the frequency of top clones was positively associated with MHC disparity between donor and host, ranging from low to high in syngeneic, MHC-matched, haploidentical, and fully-mismatched recipients, respectively. To understand the extent to which TCR rearrangement is shared among different organs after BMT, we analyzed the overlap of TCR clones across different organs in the same recipients. T-cell clones were highly overlapping across organs, especially among GVHD target organs, in the same recipients after allogeneic BMT, although much lower overlapping in recipients after syngeneic BMT. The results suggest that alloantigen stimulation selectively activate certain T-cell clones and enrich antigen specific clones. On the other hand, much fewer shared clones were found among different recipients within the same group, regardless of MHC-disparity between donor and host. These results suggest that specific T-cell clones activated and expanded by alloantigens stimulation were highly different in individual recipients even with the same MHC-disparity between donor and host. Interestingly, the levels of clone overlapping were different in distinct organs among individual recipients. The level of T-cell clone overlapping was found high in liver of individual recipients regardless of the strength of alloantigen stimulation. The level of T cell clone overlapping was relatively high in pLNs and skin of the recipients after haploidentical BMT; whereas the level of T cell clone overlapping was relatively high in mLNs and gut of the recipients after MHC-matched BMT. These results suggest that skin may be a dominant target in haploidentical BMT and gut as a dominant target in MHC-matched BMT; whereas liver is a common target organ regardless. In conclusion, the current study establishes the association between MHC disparity, T-cell activation, and GVHD development in the level of donor T-cell repertoire. While TCR repertoire of donor T cells in peripheral blood or lymph nodes likely is representative in any individual recipient/patient, it is nearly impossible to identify T-cell clones that are pathogenic and shared among groups of recipients/patients even with the same MHC-disparity between donor and host. Disclosures No relevant conflicts of interest to declare.

Description: The successful treatment of hematologic malignances with allogeneic hematopoietic cell transplantation (allo-HCT) is limited by acute graft-versus-host disease (GvHD). The complement system has been shown to modulate adaptive immunity via interaction between complement activation products and their receptors expressed on both innate and adaptive immune cells. Complement receptors play an important role in pathogen and danger sensing by translating information gathered by complement fluid phase sensors into cellular responses. The anaphylatoxins, C3a and C5a, are key effector molecules of the complement system. C3aR/C5aR signaling plays an important role in the survival, maturation and differentiation of antigen presenting cells (APCs), as well as for effective antigen presentation to T cells and the subsequent modulation of T-cell proliferation, differentiation and function. Given that host APCs play a crucial role in priming alloreactive donor T cells to induce and intensify aGvHD, we evaluated the role of C3aR/C5aR in the induction of aGvHD via the regulation of host APC function. Using the two distinct well-defined, clinically relevant aGVHD models, B6 (H2b)-〉BALB/c (H2d) and FVB (H2q)-〉B6 (H2b), we observed that host C3aR/5aR deficiency led to a significantly reduced aGvHD as indicated by a higher survival rate and milder GvHD clinical scores as compared to WT recipients (Figure 1A). The ameliorated GVHD in C3aR/C5aR-/- recipients was associated with reduced donor T-cell activation, survival, bioenergetic capacity and Th1 differentiation, and with increased iTreg generation. Donor T cells in C3aR/C5aR-/- recipients express lower levels of chemokine receptors, CXCR3 and CCR6, which is likely responsible for the decreased migration of the effector T cells to GvHD target organs. Utilizing BM chimeras to distinguish the role of C3aR/C5aR on host hematopoietic cells vs. parenchymal tissues in the development of GVHD, we found that C3aR/C5aR expressed on recipient hematopoietic APCs was primarily responsible for donor T-cell response and pathogenicity in aGvHD. Among APCs, DCs are considered to be the most efficacious APCs due to their superior ability to take up antigen, express co-stimulatory molecules, and produce pro-inflammatory cytokines to polarize alloreactive T cells. C3aR/C5aR expression was drastically upregulated in DCs after total body irradiation. In comparison with WT DCs, irradiated C3aR/C5aR-/- DCs displayed higher apoptosis which is associated with DC upregulation of Fas expression, higher autophagy, and decreased activation and antigen presenting capacity reflected by lower IFN-γ positive DC cells and reduced MHCII expression. Notably, while C3aR/C5aR on hematopoietic cells was required for GVHD development, it was largely dispensable for the graft-versus-leukemia (GVL) effect. For translational application, we evaluated the effect of systemic administration of a combination of C3aR and C5aR antagonists (C3aRA/C5aRA) to block C3aR/C5aR signaling, on GVHD and GVL activity. We found that prophylactic treatment of recipients with C3aRA/C5aRA prior to transplant effectively prevented GvHD while preserving GVL effect (Figure 1B). Taken together, the current work provides a strong rationale and demonstrates the feasibility to target host C3aR/C5aR for the control of GVHD while preserving GVL activity after allo-HCT. Disclosures No relevant conflicts of interest to declare.

Description: Allogeneic hematopoietic cell transplantation (HCT) can cure a variety of benign and malignant hematopoietic disorders, but graft-versus-host disease (GVHD) remains a significant source of transplant-related mortality and morbidity. Mature T cells in donor stem cell graft are primarily responsible for the development of acute GVHD. PIM kinases are a family of serine/threonine kinases, including PIM1, PIM2 and PIM3, which are expressed following T-cell activation. The PIM kinases have been shown to inhibit apoptosis as well as to stimulate the cell metabolism and protein synthesis. In the current study, we investigated how PIM kinases regulate T-cell responses to alloantigens and GVHD development. Using genetically modified mice on an FVB background that are deficient for either single, double, or triple kinases of the PIM family, we evaluated the role of PIM kinases on T-cell alloresponses. T cells deficient for PIM1/PIM3 kinases had reduced survival and proliferation upon stimulation with alloantigens in vitro, whereas T cells without the PIM2 kinase displayed increased survival and IFNγ production as compared to WT T cells. After being transferred to lethally irradiated allogeneic mice in vivo, PIM1/PIM3 double knockout (KO) T cells had reduced expression of IFNγ in spleen, whereas PIM2 KO T cells showed decreased IL-4/5 expression, although similar IFNγ production when compared with WT T cells. These data suggest PIM2 kinase plays a distinct role by negatively regulating T-cell alloresponses. To test the role of PIM kinases on T-cell ability to induce GVHD, we transferred WT, PIM2 single KO, PIM1/PIM2 double KO, PIM1/PIM3 double KO, PIM2/PIM3 double KO, or PIM1/PIM2/PIM3 triple KO T cells together with WT bone marrow cells into lethally irradiated B6 mice. While PIM1/PIM3 double KO T cells induced milder GVHD, PIM2 KO T cells induced much more severe GVHD when compared to WT T cells. In addition, any types of T cells deficient for PIM2 caused significantly more severe GVHD than their PIM2-replete counterparts. Furthermore, restoration of PIM2 expression by gene transfection reduced the ability of PIM2-deficient T cells in the induction of GVHD. Enhanced pathogenicity of PIM2-deficient T cells was also confirmed in another murine model of allogeneic HCT. These results indicate that PIM2 plays a dominant role among PIM kinases in negatively regulating T-cell alloresponses and GVHD induction. To validate the data obtained from KO mice, we silenced the PIM2 kinase in PIM1/PIM3 KO T cells by administration of a PIM kinase pan-inhibitor (AZD, AstraZeneca R&D, Waltham, MA), and we observed exacerbated GVHD induced by PIM1/PIM3 KO T cells after PIM2 kinase silencing. Our study demonstrates the important result that PIM2 negatively regulates T-cell mediated alloresponse and GVHD development. The finding uncovers a novel biological function of PIM kinases as well as urges caution in inhibiting PIM kinases for the treatment of hematologic malignances in patients after allogeneic HCT. YW and AD contributed equally to this work. Disclosures No relevant conflicts of interest to declare.

Description: Graft-versus-host disease (GvHD) remains a leading cause of non-relapse mortality after allogeneic hematopoietic cell transplantation (allo-HCT). The imbalance between T effector (Teffs) and regulatory cells (Tregs) is a defined characteristic of GvHD. Therefore, promoting Tregs for controlling GvHD has drawn an intense interest in preclinical and clinical studies of allo-HCT. Although CD4 Tregs (CD4+Foxp3+) have been shown to effectively prevent GvHD, they impaired graft-versus-leukemia (GVL) effect. Our lab has previously demonstrated that CD8 iTregs (CD8+Foxp3+) not only suppressed allogeneic T-cell responses, but also possessed the GVL activity themselves. However, instability of Foxp3 in CD8 iTregs is a major obstacle to translate this Treg population into clinic application. Vitamin C has been reported to promote Foxp3 stability by accelerating its demethylation. In the current study, we asked whether vitamin C could stabilize CD8 iTregs and enhance their therapeutic potential in controlling GvHD. Addition of vitamin C in the culture significantly increased the generation of allo-reactive CD8 iTregs in vitro (Fig. 1A). These CD8 iTregs generated with vitamin C had strikingly increased demethylation on Foxp3 CpGs (Fig. 1B) and superior suppressive activity (data not shown) compared to control-treated CD8 iTregs. We further evaluated the capability of vitamin C-treated CD8 iTregs in preventing GvHD in allo-HCT. Using a MHC-mismatched murine BMT model, vitamin C-treated CD8 iTregs could significantly alleviate GvHD reflected by a marked reduction of recipient mortality (Fig. 1C) and clinical scores (data not shown). To determine the Foxp3 stability in vivo, we analyzed the transferred CD8 iTregs and allogeneic Teffs in recipient spleens 7 days after allo-HCT. Vitamin C-treated CD8 iTregs (Ly5.2+) remarkably suppressed Teff (Ly5.1+) expansion and maintained higher Foxp3 expression compared to the control iTregs (Fig. 1D). To address a critical question whether vitamin C-treated CD8 iTregs can preserve the GVL activity, we performed a haploidentical transplant with a leukemia relapse model, and found that vitamin C-treated CD8 iTregs not only attenuated GvHD severity, but also prevented recipients from tumor relapse (Fig. 1E). In conclusion, our studies provide a rationale and mean to use stabilized CD8 iTregs with vitamin C for controlling GvHD and leukemia relapse in the clinic. Disclosures No relevant conflicts of interest to declare.

Description: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for many hematological malignancies due to its potent graft-versus-leukemia (GVL) effect; yet its clinical application is limited by the development of graft-versus-host-disease (GVHD). Currently, the separation of GVHD and GVL is a great challenge in the field. Complement can be activated via three different pathways: the classical (CP), lectin (LP) and alternative pathway (AP). The AP can spontaneously activate, but it also serves as an amplification loop for the other pathways, and hence plays a central role in the development of autoimmune diseases such inflammatory bowel disease, which shares certain pathological similarities with intestinal GVHD. Complement activation has been implicated in GVHD development, yet how complement regulates GVHD is not known. In current study, deposition of complement activation product C3d occurs within GVHD target organs (Fig. 1A), suggesting that local complement activation contributes to GVHD pathogenesis. To functionally dissect the roles of each complement pathway, we used mice deficient in fB (no AP) or C1q/MBL (no CP or LP) as recipients in MHC-mismatched murine models of GVHD. GVHD severity and mortality was significantly reduced in fB, but not C1q/MLB, deficient recipients compared to wild type (WT) counterparts, suggesting a crucial role for the AP in GVHD (Fig. 1B). Furthermore, using chimeric mice with a fB-deficiency specifically in the hematopoietic compartment, we demonstrated that the AP in hematopoietic cells is required for GVHD development, but not for the GVL effect (Fig. 1D). For translational purposes, we evaluated GVHD and GVL responses using CR2-fH, an AP-specific protein inhibitor (Fig. 1C). CR2-fH consists of the N-terminus of mouse complement factor H, which contains the AP-inhibitory domain, linked to complement receptor 2 (CR2) targeting fragment that binds the complement activation product C3d. CR2-fH has been shown to specifically co-localize to sites of injury and C3d deposition. In recipients harboring C1498 acute myeloid leukemia, systemic treatment of recipients with CR2-fH significantly ameliorated GVHD with no associated tumor relapse (Fig. 1D), suggesting CR2-fH does not impact GVL activity. Mechanistic studies revealed that host AP deficiency significantly impacted the phenotype of immune cells in GVHD target organs rather than in lymphoid organs. Complement deposition was decreased in GVHD target organs of AP-deficient recipients. Gut integrity was significantly preserved in fB-/- compared to WT recipients (Fig. 1E). Activation and maturation of host DCs in the gut, lung, and thymus were also significantly decreased in fB-/- recipients, while GVHD-protective CD8+DCs were increased. In contrast, AP deficiency did not affect the phenotype of recipient DCs in the lymphoid organs. The numbers of donor T cells and particularly CD103+CD8+ T cells, a cell subset that critically mediates intestinal GVHD, were decreased in the gut of fB-/- recipients, which was associated with reduced expression of the gut homing chemokine CCR9 on donor T cells. Donor iTregs were increased in the liver of fB-/- recipients (Fig. 1F), while Th1 and Tc1 cells were significantly decreased; lower cytolytic activity was also seen in these Tc1 cells. Donor Th2 and Tc1 were diminished in the lung while thymic integrity was significantly improved in fB-/- recipients. We observed no differences in differentiation or function of donor T cells in lymphoid organs (Fig. 1G). These studies further indicate AP activation within target organs is important for GVHD pathogenesis. Taken together, the current data identifies a central role for the AP in GVHD and validates the AP as a therapeutic target for the control of GVHD. We provide evidence that local complement generation in GVHD target organs is important for GVHD development. Since site-specific AP inhibition is effective at controlling GVHD while preserving GVL activity, this study identifies a novel strategy to reduce GVHD and spare GVL effects. Because site-specific complement inhibition has been shown to reduce potentially dangerous side effects and since a humanized version of CR2-fH (TT30) has been shown to be safe and nonimmunogenic in humans, the current findings have a high potential for clinical translation. Disclosures No relevant conflicts of interest to declare.

Description: Adoptive regulatory T-cell (Treg) therapy has enhanced the outcome of patients suffering from graft-versus-host (GVH) disease following allogeneic hematopoietic stem cell transplantation (allo-HCT); however, fear of broad immune suppression and subsequent dampening of the beneficial graft-versus-leukemic (GVL) responses remains a challenge. In order to subvert broad immune suppression, we generated alloantigen-specific induced Tregs (iTregs) from resting CD4 or CD8 T cells and tested the ability of iTregs to suppress GVH and maintain GVL responses. We utilized a clinically relevant murine model of haploidentical-HCT with the addition of host-original leukemia cell line to evaluate the effects of CD4 and CD8 iTregs in GVH and GVL responses. While alloantigen-specific CD4 iTregs were effective in preventing GVHD (Fig. 1 A and C), they completely abrogated the GVL effect against aggressive leukemia resulting in 100% tumor mortality (Fig. 1 B and D). Mechanistically, these CD4 iTregs were found to potently suppress the expansion of effector T cells (Teffs) and their ability to secrete IFNγ and granzyme B in the recipient spleen and liver, which may contribute to the impaired GVL activity. Using similar approach, we generated alloantigen-specific CD8 iTregs and found they express higher levels of granzyme B and CTLA-4 compared to nTreg and CD4 iTregs. In vivo studies showed these CD8 iTregs moderately attenuated GVHD (Fig. 1 A and C)while completely sparing the GVL effect (Fig. 1 B and D). We thus further reasoned that the combination of CD4 and CD8 iTregs could achieve the optimal goal of allo-HCT: GVHD suppression with GVL preservation. Indeed, the combination therapy potently suppressed GVHD resulting in increased survival and decreased pathological injury to target organs than either CD4 or CD8 iTreg singular therapy (Fig. 1 A and C). More importantly, the combination therapy maintained potent GVL responses reflected by significantly decreased tumor mortality and load (Fig. 1 B and D).Mechanistically, we observed addition of CD8 iTregs maintained the suppression of Teff expansion but restored the ability of Teffs in producing inflammatory cytokines (e.g. IFNγ and TFNα) and cytolytic effector molecules (e.g. granzyme B and TRAIL). To our knowledge the current findings are the first to support the use of combinational iTreg therapy to achieve optimal suppression of GVHD while maintaining GVL responses. This work was supported by NIH grants: R01 CA118116 and R01 CA169116 Disclosures No relevant conflicts of interest to declare.

Description: PIM kinases, a family of serine/threonine kinases, play an important role in regulating cell survival, cell proliferation, transcription activation and protein translation. Of the three isoforms, PIM-1, PIM-2 and PIM-3, some of them are overly expressed in several hematological malignancies as well as solid tumors. Thus, these kinases have been targeted clinically in several cancer studies. However, the roles of PIM kinases in T cells from previous studies are inconclusive and the functions of each isoform in these cells are yet to be investigated. In our study, we focused on the role of PIM-2 kinase in T cell responses to allo-antigen and tumor using pre-clinical models. Using allogeneic bone marrow transplantation (allo-BMT) models, we transferred allogeneic T cells isolated from mice deficient for Pim-2 (Pim-2-/-, FVB background) in comparison to WT and PIM1/3double deficient mice (PIM1/3-/-) into lethally irradiated Balb/C recipients. Our data revealed that upon allogeneic stimulation, PIM-2-/- T cells induced accelerated graft-versus-host-disease (GVHD) compared to WT or PIM1/3-/- T cells (Fig.1A). In addition, PIM-2-/- donor T cells were highly activated with significantly increased proliferation and IFN-γ production in recipient spleen and GVHD target organs. Donor PIM-2-/- T cells also expressed high levels of chemokine receptor, CXCR3 and a4b7 integrin (gut homing receptor) that correlated with increased capacity of donor T-cell migration to GVHD target organs such as liver and gut. In graft-versus-leukemia (GVL) study, PIM-2-/- donor T cells had roughly 4-fold higher ability to mediate GVL effect to WT and PIM1/3-/- T cells (Fig.1B). Negative regulation of T-cell responses to allo-antigens by PIM-2 was independently verified by restoring PIM-2 expression on PIM-2-/- T cells or inhibiting PIM kinases in PIM1/3-/- T cells. Altogether, the data from allo-BMT studies suggested that PIM-2 isoform functions as a negative regulator in T cell allo-response in contrast to PIM-1 and PIM-3 kinases. We then focused on evaluating T-cell immunity against syngeneic tumor. By administering tumor (TS-1 breast tumor in FVB background) into WT and PIM-2-/- mice either subcutaneously or intravenously, we observed that PIM-2-deficient mice efficiently control tumor growth in sharp contrast to WT mice (Fig. 1C). Mechanistic study revealed that tumor rejection in PIM-2-/- recipient relies on CD8+ tumor infiltrating lymphocytes (TILs) with a significant up-regulation of IFN-g, TNF-a and Fas-L expression. Lower infiltration of regulatory T cells and down regulation of PD-1 expression on T cells recovered from tumor site were also observed in PIM-2 deficient mice. Moreover, the tumor growth rate in PIM-2-/- mice after T-cell depletion with antibody was similar to that of WT mice. To further exclude the effect of other immune cells involved in tumor regression, we adoptively transferred WT and PIM-2-/- CD8+ T cells into WT mice with pre-established TS-1 tumor. CD8+ T cells deficient for PIM-2 was more efficient in controlling of tumor growth than WT or PIM1/3-/- CD8+ T cells. These data indicate that inhibition of PIM-2 in T cells display superior anti-tumor activity. In conclusion, PIM-2 kinase possesses a distinct role in anti-tumor immunity compared to the other two isoforms and it represents a potential target to enhance anti-tumor activity of adoptive T-cell immunotherapy. Disclosures No relevant conflicts of interest to declare.