Lethal Graft-versus-host Disease Research Articles

An Early Biomarker Algorithm Predicts Lethal Graft-Versus-Host Disease and Survival after Allogeneic Hematopoietic Cell Transplantation

No laboratory test can predict non-relapse mortality (NRM) after hematopoietic cellular transplantation (HCT) prior to the onset graft-versus-host disease (GVHD). Recently, we have shown that a signature of three GVHD plasma biomarkers (TNFR1, ST2, and REG3α) can predict response to GVHD therapy and NRM at the onset of clinical GVHD (Levine, Lancet Haem, 2015). Our goal in the current study was to identify a blood biomarker signature that could predict lethal GVHD and six-month NRM well in advance of the onset of GVHD symptoms. Patient samples on day +7 after HCT were obtained from 1,287 patients from 11 HCT centers in the Mount Sinai Acute GVHD International Consortium (MAGIC). Samples from two large centers (n = 929) were combined and randomly assigned to a training set (n = 620) and test set (n = 309). 358 patients from nine others centers constituted an independent validation set. The overall cumulative incidences of 6-month NRM were 11%, 12%, and 13% for the training, test, and validation sets respectively. The incidence of lethal GVHD, defined as death without preceding relapse while under steroid treatment for acute GVHD, were 18%, 24%, and 14% in the same groups, respectively. The median day of GVHD onset was 28 days in the training set and 29 days in the test and validation sets. We measured four GVHD related biomarkers [ST2, REG3α, TNFR1, and IL2Rα] in all samples and used the training set alone to develop competing risks regression models that used all 13 possible combinations of one to four biomarkers to predict 6-month NRM. The best algorithm, which we rigorously confirmed through Monte Carlo cross-validation of 75 different combinations of training sets, included ST2 and REG3α. No combination of one, three, or four biomarkers was superior to the combination of these two biomarkers. The day 7 algorithm identified high risk (HR) and low risk (LR) groups with 6-month NRMs of 28% and 7%, respectively (p<0.001) (Fig 1A). The relapse rates did not differ between risk groups so that overall survival (OS) was 60% for HR and 84% for LR (p<0.001) (Fig 1B). When applied to the test set (Fig 1C/D), the algorithm identified 54/309 (17%) of the patients as HR with an NRM of 33% vs 7% for LR patients (p<0.001) and 6-month OS of 57% and 81% for HR and LR patients, respectively (p<0.001). In the independent validation set (Fig 1 E/F), the algorithm identified 72/358 (20%) of the patients as HR with an NRM of 26% vs 10% for LR patients (p<0.001) and OS of 68% and 85% for HR and LR patients, respectively (p<0.001). High risk patients were three times more likely to die from GVHD than LR patients in each cohort (p<0.001) (Fig 2). The GI tract is the GVHD target organ that is most resistant to treatment and represents a major cause of NRM, and we observed twice as much severe GI GVHD (stage 3 or 4) in HR patients as in LR patients (p<0.001, data not shown). The algorithm successfully separated HR and LR strata for 6 month NRM in several groups with differing risks for GVHD and NRM, including donor type, degree. of HLA-match, age group, and conditioning regimen intensity (Fig 3).In conclusion, we have developed a blood biomarker algorithm that predicts the development of lethal GVHD seven days after HCT, which performed successfully in large multicenter validation sets. The GVH reaction is already in progress by day +7, even though clinical symptoms may not occur until days or weeks later. We speculate that the blood biomarker concentrations at this early time point reflect subclinical GI pathology, a notion that is reinforced by the fact that ST2 and REG3α, the two biomarkers in the algorithm, are closely associated with GI GVHD. The algorithm identified HR and LR strata in several patient groups with different overall risk for lethal GVHD (donor, HLA match, conditioning regimen intensity, age). This day +7 algorithm should prove useful in clinical BMT research by identifying patients at high risk for lethal GVHD who might benefit from aggressive preemptive treatment strategies. [Display omitted] [Display omitted] [Display omitted] DisclosuresChen:Novartis: Research Funding; Incyte Corporation: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Jagasia:Therakos: Consultancy. Kitko:Therakos: Honoraria, Speakers Bureau. Kroeger:Novartis: Honoraria, Research Funding. Levine:Viracor: Patents & Royalties: GVHD biomarkers patent. Ferrara:Viracor: Patents & Royalties: GVHD biomarkers patent.

Third Party Cytokine-Induced Killer Cells Eliminate Highly Activated T-Cells, Leading to Protect from Murine Lethal Graft-Versus-Host Disease

Protecting from lethal graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects by adoptive immunotherapy would be an ideal goal in allogeneic hematopoietic stem cell transplantation (HSCT), especially using third party derived cells, because the use of third party cells would open a huge source of availability and feasibility across major histocompatibility barriers. We have demonstrated that third party cytokine-induced killer (CIK) cells protected from murine lethal GVHD with faster hematological recovery, which are ex vivo-expanded T lymphocytes expressing both natural killer and T-cell markers with strong cytotoxic activity both against autologous and allogeneic tumor cells. However the mechanism responsible for ameliorating acute GVHD by third party CIK cells are still unknown.First, lethally irradiated Balb/c (H-2d) mice were given C3H (H-2k) bone marrow cells with C3H whole splenocytes to induce lethal GVHD with/without third party (C57/BL6 (H-2b)) CIK cells or third party whole splenocytes on day 0 or day4. Mice receiving third party CIK cells showed much less GVHD and significant survival benefit compared those with third party splenocytes as shown below (p<0.05). On day7 after BMT, the percentage of CD69 on CD8 T-cells in peripheral blood (PB) in the mice receiving third party CIK cells was significantly lower than those in GVHD control mice as shown below (p<0.05), meanwhile donor derived not-activated T-cells were preserved in both CD4 and CD8 T-cells. From these results, we hypothesized that third party CIK cells prevent acute GVHD by selectively depleting highly proliferative alloreactive T-cells stimulated early after transplant, while sparing naïve T-cells. To test this, we performed mixed lymphocyte reaction (MLR) by adding third party CIK cells or third party splenocytes (Stimulator: Balb/c splenocytes, Responder: C3H splenocytes, third party cells: C57/BL6). The percentage of CD69 on responder derived CD8 T-cells in the culture mixture with third party CIK cells was much less compared to that with third party splenocytes as shown below (p<0.05). We have previously shown that allogeneic CIK cells had cytotoxicity against cultured dendritic cells (DCs) by 51Cr release assay. Therefore reduced alloreaction by third party CIK cells might be due in part to the elimination of antigen presenting cells such as DCs. Next, to demonstrate that third party CIK cells could eliminate responder cells directly and then reduce the alloreaction, we performed MLR like experiments using PHA to activate T-cells strongly instead of antigen presenting cells as a stimulator. As shown below, we have demonstrated that the percentage of highly activated T-cells (CD8+CD69+) in third party CIK added plates was significantly lower compared to that in third party splenocytes added plates, indicating that third party CIK cells could have direct effects against highly activated T-cells.To further clarify the mechanisms of how third party CIK cells reduce the highly activated T-cells, we performed MLR as mentioned above with/without the neutralizing antibody against NKG2D, which is an activator receptor expressed on NK cells. Although CIK cells have cytotoxicity against tumor cells via NKG2D signaling pathway in part, adding NKG2D antibody did not show a statistical significant difference in MLR assays. It suggested that third party CIK cells could suppress alloreactive T-cells by several pathways other than NKG2D signaling pathway.Finally, we identified the in vivo kinetics of third party CIK cells after BMT. Third party derived CIK cells could be identified as a rare subpopulation in recipient peripheral blood for some days after CIK infusion, but all of them could not be detected anymore by day 7 after infusion. Whereas third party whole splenocytes expanded in recipient peripheral blood and mice received third party splenocytes with donor BM and splenocytes died until day7 because of GVHD induced by third party splenocytes.In conclusion, infusion of third party CIK cells has strong potential to prevent murine lethal GVHD probably due to selectively depleting highly proliferative alloreactive T-cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reduced Non Relapse Mortality (NRM) and Survivable Acute Graft Versus Host Disease (GVHD) in the Current Era of Myeloablative Ex Vivo T-Cell Depleted (TCD) Transplantation

Introduction:Little is known about the lethality of acute GVHD (aGVHD) in T -cell depleted (TCD) allogeneic hematopoietic stem cell transplantation (HSCT). We examined the incidence of aGVHD and its relative contribution to non relapse mortality (NRM) in a cohort of consecutive TCD HSCT at a single institute.Methods:We report 132 consecutive subjects who had undergone TCD HLA-identical sibling HSCT between 2006 and 2016 for hematologic malignancies. All subjects received conditioning with Fludarabine 125mg/m2, Cytoxan 120 mg/kg and 1200 cGy total body irradiation (TBI) (<age 55 years) or 400-600 cGy (³age 55) followed by either a CD34+ selected (n=112) or CD3/19 depleted (n=20) peripheral blood stem cell graft with a T cell addback of 5-50x10e4CD3+/kg (Miltenyi CliniMACS, resulting in 3.5 to 4-log TCD). Low dose cyclosporine was used until day 21 for GVHD prophylaxis.Thirty-six subjects were over the age of 55 years, 66 were females and 66 were males. Transplant indications were acute leukemia (92), myelodysplastic / myeloproliferative syndromes (26), lymphoid malignancies (7) and chronic myelogenous or myelomonocytic leukemia (7). Fifty-three percent of subjects were at a high risk of relapse. The median follow-up post HSCT was 4.3 years.Results:The incidence of Grade 1-2 and 3-4 aGVHD were 51.6% and 19%, respectively. Of these, 15% of aGVHD was steroid refractory and was treated with infliximab/basiliximab or with mesenchymal stromal cells. Extensive or limited chronic GvHD was observed in 30% and 24% of subjects. 74% of those at risk developed cytomegalovirus (CMV) reactivation. The Kaplan-Meier (KM) estimate for overall survival (OS), NRM and cumulative incidence of relapse (CIR) for the entire cohort was 71%, 14.6% and 24% respectively at one year, and 52%, 32.5% and 39% at 5 years. We considered the impact of CMV reactivation, slow donor lymphoid chimerism and steroid refractory aGVHD on NRM. However there was no significant impact from CMV reactivation or slow (> 31 days) achievement of complete donor lymphoid chimerism. Significantly improved outcomes were noted for those transplanted beyond 2012: OS, NRM and CIR being 82%,6.2% and 20% at one year, and 68%, 6.2% and 41% at 3 years. Cox proportionate hazard modeling identified steroid refractory aGVHD (HR 4.0, P=0.007) and transplant prior to 2012 (HR 6.7, P=0.001) as significant factors impacting NRM.Conclusions:T cell addback after ex vivo TCD HSCT was associated with a significant burden of aGVHD. Steroid refractory aGVHD impacted NRM, but slow lymphoid engraftment, disease risk, CMV reactivation and age did not. Significant improvements in NRM in the current era suggest greatly improved salvage of steroid refractory aGVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Blockade of interleukin-27 signaling reduces GVHD in mice by augmenting Treg reconstitution and stabilizing Foxp3 expression

AbstractReestablishment of competent regulatory pathways has emerged as a strategy to reduce the severity of graft-versus-host disease (GVHD), and recalibrate the effector and regulatory arms of the immune system. However, clinically feasible, cost-effective strategies that do not require extensive ex vivo cellular manipulation have remained elusive. In the current study, we demonstrate that inhibition of the interleukin-27p28 (IL-27p28) signaling pathway through antibody blockade or genetic ablation prevented lethal GVHD in multiple murine transplant models. Moreover, protection from GVHD was attributable to augmented global reconstitution of CD4+ natural regulatory T cells (nTregs), CD4+ induced Tregs (iTregs), and CD8+ iTregs, and was more potent than temporally concordant blockade of IL-6 signaling. Inhibition of IL-27p28 also enhanced the suppressive capacity of adoptively transferred CD4+ nTregs by increasing the stability of Foxp3 expression. Notably, blockade of IL-27p28 signaling reduced T-cell–derived-IL-10 production in conventional T cells; however, there was no corresponding effect in CD4+ or CD8+ Tregs, indicating that IL-27 inhibition had differential effects on IL-10 production and preserved a mechanistic pathway by which Tregs are known to suppress GVHD. Targeting of IL-27 therefore represents a novel strategy for the in vivo expansion of Tregs and subsequent prevention of GVHD without the requirement for ex vivo cellular manipulation, and provides additional support for the critical proinflammatory role that members of the IL-6 and IL-12 cytokine families play in GVHD biology.

Effects of Donor Vitamin A Deficiency and Pharmacologic Modulation of Donor T Cell Retinoic Acid Pathway on the Severity of Experimental Graft-versus-Host Disease

Graft-versus-host disease (GVHD) is the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). A combination of genetic and nongenetic factors dictates the incidence and severity of GVHD. Recent studies have identified the potential role of the retinoic acid (RA)/retinoic acid receptor (RAR) pathway in the pathogenesis of GVHD. RA is the active metabolite of vitamin A. Thus, a clinically relevant question is whether HSCT donor and/or recipient vitamin A status affects the development of GVHD. It has been previously reported that recipient vitamin A deficiency is associated with reduced intestinal GVHD and prolonged overall survival after experimental allogeneic HSCT. However, it is still unknown whether donor vitamin A status influences GVHD development. In the current study, we report that chronic vitamin A deficiency changes the composition of T cell compartment of donor mice with a reduction in the percentage of CD4+ T cells. We showed that although vitamin A deficiency does not affect donor T cell alloreactivity on a per cell basis, a decreased proportion of donor CD4+ T cells in marrow graft inoculums leads to reduced incidence and severity of GVHD. Furthermore, our proof of principle studies using a pan-RAR antagonist demonstrated that transient inhibition of donor T cell RAR signaling can reduce T cell alloreactivity and their ability to cause lethal GVHD. Our studies provide preclinical evidence that donor vitamin A deficiency may be a nongenetic factor that can modulate the severity of GVHD and pharmacologic interfering RA/RAR pathway in donor T cells might be a valuable approach for mitigating GVHD after allogeneic HSCT.

Targeting T Cell Bioenergetics by Modulating P-Glycoprotein Selectively Depletes Alloreactive T Cells To Prevent Graft-versus-Host Disease.

T lymphocytes play a central role in many human immunologic disorders, including autoimmune and alloimmune diseases. In hematopoietic stem cell transplantation, acute graft-versus-host-disease (GVHD) is caused by an attack on the recipient's tissues from donor allogeneic T cells. Selectively depleting GVHD-causing cells prior to transplant may prevent GVHD. In this study, we evaluated 24 chalcogenorhodamine photosensitizers for their ability to selectively deplete reactive T lymphocytes and identified the photosensitizer 2-Se-Cl, which accumulates in stimulated T cells in proportion to oxidative phosphorylation. The photosensitizer is also a potent stimulator of P-glycoprotein (P-gp). Enhanced P-gp activity promotes the efficient removal of photosensitizer not sequestered in mitochondria and protects resting lymphocytes that are essential for antipathogen and antitumor responses. To evaluate the selective depletion of alloimmune responses, donor C57BL/6 splenocytes were cocultured for 5 d with irradiated BALB/c splenocytes and then photodepleted (PD). PD-treated splenocytes were infused into lethally irradiated BALB/c (same-party) or C3H/HeJ (third-party) mice. Same-party mice that received PD-treated splenocytes at the time of transplant lived 100 d without evidence of GVHD. In contrast, all mice that received untreated primed splenocytes and third-party mice that received PD-treated splenocytes died of lethal GVHD. To evaluate the preservation of antiviral immune responses, acute lymphocytic choriomeningitis virus infection was used. After photodepletion, expansion of Ag-specific naive CD8(+) T cells and viral clearance remained fully intact. The high selectivity of this novel photosensitizer may have broad applications and provide alternative treatment options for patients with T lymphocyte-mediated diseases.

Immunosuppressive therapy alleviates murine cytomegalovirus recurrence by reducing TNF-α post cell transplantation with lethal GVHD

Cytomegalovirus (CMV) reactivation leads to obvious morbidity and mortality in patients after allogeneic hematopoietic cell transplantation (allo-HCT). Current immunosuppressive therapy reduces the frequency of graft-versus-host disease (GVHD), and is generally accepted as a high risk factor for viral recurrence. In the present study, we investigated the influence of cyclosporin A (CsA) and rapamycin (RAPA), two commonly used immunosuppressive agents, on murine cytomegalovirus (MCMV) recurrence by two allo-HCT models: one with mild and one with severe GVHD. In models with mild GVHD and partial immune recovery, transplanted mice with CsA and RAPA showed a higher viral load and impaired CMV-specific immune recovery compared to those with placebo. In contrast, in HCT models with severe GVHD, groups treated with immunosuppressive therapy showed alleviation of viral recurrence as well as GVHD-related symptoms. In addition, no CMV-specific immune reconstitution was found in any group, implying immunosuppressive therapy was not relevant to antiviral response. Furthermore, a significant correlation between MCMV DNA copies and tumor necrosis factor alpha (TNF-α) was found, and recipients with immunosuppressive therapy showed a lower level of TNF-α. Finally, using lenalidomide (an inhibitor of TNF-α), a lower viral load was found in animals with lenalidomide. Having received lenalidomide, recipients showed no statistical difference in viral load between groups with and without immunosuppressive therapy. Taken together, we provide evidence of the dual effect of immunosuppressive therapy on viral reactivation. Importantly, we found that immunosuppressive therapy had the ability to alleviate viral load by reducing TNF-α in a mouse model with severe GVHD.

Peri-alloHCT IL-33 administration expands recipient T-regulatory cells that protect mice against acute GVHD

AbstractDuring allogeneic hematopoietic cell transplantation (alloHCT), nonhematopoietic cell interleukin-33 (IL-33) is augmented and released by recipient conditioning to promote type 1 alloimmunity and lethal acute graft-versus-host disease (GVHD). Yet, IL-33 is highly pleiotropic and exhibits potent immunoregulatory properties in the absence of coincident proinflammatory stimuli. We tested whether peri-alloHCT IL-33 delivery can protect against development of GVHD by augmenting IL-33–associated regulatory mechanisms. IL-33 administration augmented the frequency of regulatory T cells (Tregs) expressing the IL-33 receptor, suppression of tumorigenicity-2 (ST2), which persist following total body irradiation. ST2 expression is not exclusive to Tregs and IL-33 expands innate immune cells with regulatory or reparative properties. However, selective depletion of recipient Foxp3+ cells concurrent with peri-alloHCT IL-33 administration accelerated acute GVHD lethality. IL-33–expanded Tregs protected recipients from GVHD by controlling macrophage activation and preventing accumulation of effector T cells in GVHD-target tissue. IL-33 stimulation of ST2 on Tregs activates p38 MAPK, which drives expansion of the ST2+ Treg subset. Associated mechanistic studies revealed that proliferating Tregs exhibit IL-33–independent upregulation of ST2 and the adoptive transfer of st2+ but not st2− Tregs mediated GVHD protection. In total, these data demonstrate the protective capacity of peri-alloHCT administration of IL-33 and IL-33–responsive Tregs in mouse models of acute GVHD. These findings provide strong support that the immunoregulatory relationship between IL-33 and Tregs can be harnessed therapeutically to prevent GVHD after alloHCT for treatment of malignancy or as a means for tolerance induction in solid organ transplantation.

Genetically modified human CD4(+) T cells can be evaluated invivo without lethal graft-versus-host disease.

Adoptive cell immunotherapy for human diseases, including the use of T cells modified to express an anti-tumour T-cell receptor (TCR) or chimeric antigen receptor, is showing promise as an effective treatment modality. Further advances would be accelerated by the availability of a mouse model that would permit human T-cell engineering protocols and proposed genetic modifications to be evaluated invivo. NOD-scid IL2rγ(null) (NSG) mice accept the engraftment of mature human T cells; however, long-term evaluation of transferred cells has been hampered by the xenogeneic graft-versus-host disease (GVHD) that occurs soon after cell transfer. We modified human primary CD4(+) T cells by lentiviral transduction to express a human TCR that recognizes a pancreatic beta cell-derived peptide in the context of HLA-DR4. The TCR-transduced cells were transferred to NSG mice engineered to express HLA-DR4 and to be deficient for murine class II MHC molecules. CD4(+) T-cell-depleted peripheral blood mononuclear cells were also transferred to facilitate engraftment. The transduced cells exhibited long-term survival (up to 3months post-transfer) and lethal GVHD was not observed. This favourable outcome was dependent upon the pre-transfer T-cell transduction and culture conditions, which influenced both the kinetics of engraftment and the development of GVHD. This approach should now permit human T-cell transduction protocols and genetic modifications to be evaluated invivo, and it should also facilitate the development of human disease models that incorporate human T cells.

Programming of donor T cells using allogeneic δ-like ligand 4–positive dendritic cells to reduce GVHD in mice

AbstractAlloreactive T cells play a critical role in eliminating hematopoietic malignant cells but are also the mediators of graft-versus-host disease (GVHD), a major complication that subverts the success of allogeneic hematopoietic stem cell transplantation (HSCT). However, induction of alloreactive T cells does not necessarily lead to GVHD. Here we report the development of a cellular programming approach to render alloreactive T cells incapable of causing severe GVHD in both major histocompatibility complex (MHC)–mismatched and MHC-identical but minor histocompatibility antigen–mismatched mouse models. We established a novel platform that produced δ-like ligand 4–positive dendritic cells (Dll4hiDCs) from murine bone marrow using Flt3 ligand and Toll-like receptor agonists. Upon allogeneic Dll4hiDC stimulation, CD4+ naïve T cells underwent effector differentiation and produced high levels of interferon γ (IFN-γ) and interleukin-17 in vitro, depending on Dll4 activation of Notch signaling. Following transfer, allogeneic Dll4hiDC-induced T cells were unable to mediate severe GVHD but preserved antileukemic activity, significantly improving the survival of leukemic mice undergoing allogeneic HSCT. This effect of Dll4hiDC-induced T cells was associated with their impaired expansion in GVHD target tissues. IFN-γ was important for Dll4hiDC programming to reduce GVHD toxicities of alloreactive T cells. Absence of T-cell IFN-γ led to improved survival and expansion of Dll4hiDC-induced CD4+ T cells in transplant recipients and caused lethal GVHD. Our findings demonstrate that Dll4hiDC programming can overcome GVHD toxicity of donor T cells and produce leukemia-reactive T cells for effective immunotherapy.

Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality.

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1-/- donors. PD-L1-deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1-/- donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell-mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.

In vivo engineering of mobilized stem cell grafts with the immunomodulatory drug FTY720 for allogeneic transplantation.

The immunological attributes of stem cell grafts play an important role in the outcome of allogeneic stem cell transplants. Currently, ex vivo manipulation techniques such as bulk T-cell depletion or positive selection of CD34(+) cells are utilized to improve the immunological attributes of grafts and minimize the potential for graft-versus-host disease (GvHD). Here, we demonstrate a novel graft engineering technique, which utilizes the immunomodulatory drug FTY720 for in vivo depletion of naïve T (TN ) cells from donor G-CSF-mobilized grafts without ex vivo manipulation. We show that treatment of donor mice with FTY720 during mobilization depletes grafts of TN cells and prevents lethal GvHD following transplantation in a major mismatch setting. Importantly, both stem cells and NK cells are retained in the FTY720-treated grafts. FTY720 treatment does not negatively affect the engraftment potential of stem cells as demonstrated in our congenic transplants or the functionality of NK cells. In addition, potentially useful memory Tcells may be retained in the graft. These findings suggest that FTY720 may be used to optimize the immunological attributes of G-CSF-mobilized grafts by removing potentially deleterious TN cells which can contribute to GvHD, and by retaining useful cells which can promote immunity in the recipient.

LRBA is essential for allogeneic responses in bone marrow transplantation

Abstract Members of the PH-BEACH-WD40 repeat gene family (PBW) play a role in coordinating receptor signaling and intracellular vesicle trafficking. LPS-Responsive Beige-like Anchor (LRBA) is a PBW protein whose immune function has not been defined. Here we show that LRBA−/− mice are viable with normal and healthful life span, but exhibit compromised rejection of allogeneic, xenogeneic and missing self bone marrow grafts. Further we demonstrate that LRBA−/− Natural Killer (NK) cells exhibit impaired signaling by the key NK activating receptors, NKp46 and NKG2D. However, induction of γ-IFN by co-culture with IL12 and IL18 remains intact, indicating LRBA selectively facilitates signals by receptors for ligands expressed on the surface of NK targets. Surprisingly, LRBA limits immunoregulatory cell numbers in tissues where Graft versus Host Disease (GvHD) is primed or initiated, and consistent with this LRBA−/− mice demonstrate resistance to lethal GvHD. These findings demonstrate that LRBA is redundant for host longevity while being essential for both host and donor-mediated immune responses in allogeneic transplantation and thus represent a unique and novel molecular target in transplant immunology.

Murine allogeneic CD19 CAR T cells harbor potent antileukemic activity but have the potential to mediate lethal GVHD

AbstractAcute lymphoblastic leukemia (ALL) persisting or relapsing following bone marrow transplantation (BMT) has a dismal prognosis. Success with chimeric antigen receptor (CAR) T cells offers an opportunity to treat these patients with leukemia-redirected donor-derived T cells, which may be more functional than T cells derived from patients with leukemia but have the potential to mediate graft-versus-host disease (GVHD). We, together with others, have previously demonstrated tumor-specific T-cell dysfunction in the allogeneic environment. Here, we studied CAR T-cell function following BMT using an immunocompetent murine model of minor mismatched allogeneic transplantation followed by donor-derived CD19-CAR T cells. Allogeneic donor-derived CD19-CAR T cells eliminated residual ALL with equal potency to those administered after syngeneic BMT. Surprisingly, allogeneic CAR T cells mediated lethal acute GVHD with early mortality, which is atypical for this minor mismatch model. We demonstrated that both allogeneic and syngeneic CAR T cells show initial expansion as effector T cells, with a higher peak but rapid deletion of allogeneic CAR T cells. Interestingly, CAR-mediated acute GVHD was only seen in the presence of leukemia, suggesting CAR-target interactions induced GVHD. Indeed, serum interleukin (IL)-6 was elevated only in the presence of both leukemia and CAR T cells, and IL-6 neutralization ameliorated the severity of GVHD in a delayed donor lymphocyte infusion model. Finally, allogeneic CD4+ CAR T cells were responsible for GVHD, which correlated with their ability to produce IL-6 upon CAR stimulation. Altogether, we demonstrate that donor-derived allogeneic CAR T cells are active but have the capacity to drive GVHD.

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease.

The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.

Third Party Cytokine-Induced Killer Cells Protect from Murine Lethal Graft-Versus-Host Disease

Cell adoptive immunotherapy protect from lethal graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects is ideal in allogeneic hematopoietic stem cell transplantation (HSCT) in patients with hematologic malignancies. Moreover, the use of third party cells would open a huge source of availability and feasibility across major histocompatibility barriers. In fact, some studies demonstrated that infusion of third party derived cells such as whole bone marrow cells or iNKT cells could ameliorate lethal GVHD in a murine model.Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer and T-cell markers. We have previously reported that donor-type CIK cells have potential to separate graft-versus-tumor effects from GVHD by eliminating host dendritic cells due to the enhanced killing activity by interferon-gamma. Actually, donor-typed CIKs infusion for refractory hematological malignancies after HSCT has been started in some clinical trails. In the present study, we examine the effect of third party cells against GVHD protection, in particular third party CIK cells for prevention from lethal GVHD in a murine model of allogeneic HSCT.To test this, lethally irradiated host Balb/c (H-2d) mice were given C3H (H-2k) or B6 (H-2b) bone marrow cells and splenocytes to induce lethal GVHD with/without third-party cells (C57/BL6 or C3H) such as CIK cells, whole splenocytes, or bone marrow derived dendritic cells (BMDCs) on day 0, which were cultured from bone marrow cells with GM-CSF. Mice receiving third party CIK cells showed much less GVHD and significant survival benefit compared those with third party splenocytes, or BMDCs as shown in the figure below. Interestingly, when infusion of third party splenocytes was delayed until day 4 after bone marrow transplantation, host mice receiving splenocytes survived much better compared with mice receiving splenocytes on day 0, even though reduced number of third party splenocytes was used. This result indicated that timing of third party cell infusion was quite important, especially when third party splenocytes were used. As expected, the mice given reduced number of third CIK cells also showed excellent survival with much less GVHD. Taken together, our results clearly demonstrated that third party CIK cells have strong potential to prevent lethal GVHD without attention to timing of cell infusion.Next, to further investigate the advantage of third party CIK cells rather than whole splenocytes, we compared the hematological reconstitution between the mice given third party CIK cells on day 4 after BMT and whole splenocytes. Both mice showed much less GVHD with the same level, however the recovery of white blood cell count on day 21 after BMT was significantly better in the mice receiving third party CIK cells.In conclusion, infusion of third party CIK cells has strong potential to prevent lethal GVHD with faster hematological recovery after HSCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Programming of Donor T Cells Using Allogeneic Delta-like Ligand 4-Positive Dendritic Cells to Reduce Gvhd but Retain GVL Activity

Host antigen-presenting cells (APCs) are critical for inducing a potent graft-versus-leukemia (GVL) response after allogeneic hematopoietic stem-cell transplantation (allo-HSCT). In this setting, host APCs activate donor T cells to become effector T cells that recognize and react to antigens in malignant cells. However, alloreactive T cells also mediate graft-versus-host disease (GVHD), which causes significant morbidity and mortality after allo-HSCT. Many studies suggest that if alloreactive T cells have reduced capacity to expand in local tissues, they will be unable to trigger severe GVHD. Thus, it is possible that host APC induction of qualitative changes in donor T cells can potentially modify their anti-host toxicities while retaining the GVL effect.Here we report the establishment of a cellular programming approach that reduces the GVHD toxicity of donor T cells using host dendritic cells (DCs) that express high levels of Dll4 (named Dll4hi DCs). We have previously identified inflammatory Dll4hi DCs. They occurred in HSCT mice early during GVHD induction and had a greater ability than Dll4-negative DCs to induce IFN-γ and IL-17 in alloantigen-activated T cells. However, only approximately 0.03 X 105 Dll4hi DCs were recovered from one HSCT mouse. To provide adequate numbers of Dll4hi DCs for therapeutic translation, we developed a novel culture system capable of producing large number of Dll4hi DCs (about 100.0 X 105) from the bone marrow (BM) of one mouse using Flt3L and the TLR agonists lipopolysaccharide (LPS) and R848, which activate TLR4 and TLR7/8, respectively. Dll4hi DCs showed significantly different phenotype as compared to conventional DCs derived from GM-CSF-stimulated BM cells (named GM-DCs), as evidenced by expressing higher levels of Dll4, Ifnb, Il4, Il6 and Ido, and producing lower levels of iNOS and arginase I. When cultured with C57BL/6 (B6) mouse CD4+ T cells (H2b) at a T cell: DC ratio of 4:1 for 5 days, BALB/c mouse Dll4hi DCs (H2d) induced 3- to 5-fold more in frequency of alloreactive effector T cells producing high levels of IFN-γ and IL-17 compared to GM-DCs. Following transfer, allogeneic Dll4hi DC-induced CD4+ T cells were unable to mediate severe GVHD in BALB/c recipients, with all of them surviving 60 days after allo-HSCT. In contrast, both unstimulated B6 CD4+ T cells and allogeneic GM-DC-induced B6 CD4+ T cells caused lethal GVHD in all BALB/c recipients, indicating that GM-DCs could not be used for reducing the GVHD toxicity of donor CD4+ T cells. Mechanistic analysis showed that Dll4hi DC-induced CD4+ T cell recipients showed 2- to 6-fold less donor CD4+ T cells in the spleen, liver, and intestine 12 days after transplantation compared to unstimulated CD4+ T cell recipients. This reduction of Dll4hi DC-induced CD4+ T cells was associated with markedly increased apoptosis in recipient mice. IFN-γ production by Dll4hi DC-induced CD4+ T cells was essential for their anti-GVHD effects. Absence of T cell IFN-γ led to improved survival and expansion of Dll4hi DC-induced CD4+ T cells in transplant recipients and caused lethal GVHD. Finally, we demonstrated that Dll4hi DC-induced alloreactive T cells had acquired the ability to kill A20 leukemic cells in BALB/c recipients and control growth of P815 mastocytoma cells in the second model of BDF1 recipients, leading to significantly improved survival of mice receiving allo-HSCT. Furthermore, in the third mouse model of GVHD directed against minor histocompatibility antigens, B6 Dll4hi DC-induced C3H.SW CD8+ T cells produced high levels of IFN-γ, had reduced capacity to mediate GVHD in B6 recipients, but preserved GVL activity against C1498 myeloid leukemic cells.In summary, our findings demonstrate that in vitro Dll4hi DC programming represents a novel and effective platform to reduce toxicities of donor T cells. This strategy has several potential advantages compared to current and developing methods for the modification of donor T cells to reduce GVHD, including a relatively short period of culture, no requirement for T cell subset selection and no need of viral transduction. Importantly, this method may lead to new strategies that can produce large amount of leukemic cell-reactive donor T cells with decrease capability of causing severe GVHD. DisclosuresNo relevant conflicts of interest to declare.

PD-L1/CD80 and PD-L1/PD-1 Signaling Reciprocally Regulate Alloreactive CD8+ T Cell Glycolysis, Proliferation, Apoptosis and Gvhd-Inducing Capacity

Graft versus host disease (GVHD) remains the major obstacle for widespread application of allogeneic hematopoietic cell transplantation (HCT), a curative therapy for hematological malignancies. We and others have reported that while sorted CD4+ T cells induce severe acute GVHD, the same numbers of CD8+ T cells induce no acute GVHD. CD8+ T cells can facilitate donor cell engraftment and mediate graft versus leukemia (GVL) effect in the absence of GVHD, but the mechanisms remain largely unknown. Programmed death-ligand 1 (PD-L1) interacts with both CD80 and PD-1. We have recently reported that simultaneous PD-L1/CD80 and PD-L1/PD-1 signaling augments activated alloreactive CD4+ T cell proliferation and apoptosis and ameliorates acute GVHD. However, the impact of this PD-L1-mediated signaling on alloreactive CD8+ T cells has not been investigated. In addition, T cell glycolytic metabolism has been recently reported to be able to regulate acute GVHD, and PD-L1/PD-1 signaling inhibits CD4+ T cell glycolytic flux, but how PD-L1/CD80 modulates T cell metabolism remains unstudied. In the current studies, we evaluated the role of PD-L1/CD80 and PD-L1/PD-1 signaling on alloreactive CD8+ T cell glycolysis, proliferation, apoptosis and GVHD-inducing capacity, using a MHC-mismatched murine HCT model of C57BL/6 (H-2b) donor to BABL/c recipient (H-2d). We have observed the following early (3-5 days) after HCT: 1) In wild-type (WT) recipients, PD-1-/- CD8+ T cells significantly upregulate expression of Glucose Transporter I (Glut1) and exhibit much higher rates of glycolytic flux; PD-1-/- CD8+ T cells also show markedly enhanced proliferation and reduced apoptosis as well as up-regulation of gut tissue homing and chemokine receptors (i.e. α4β7 and CCR9) and induce lethal GVHD, as compared to WT CD8+ T cells that induce little signs of GVHD. 2) In PD-L1-/- recipients, PD-1-/- CD8+ T cells reduce expression of Glut1 and no longer exhibit enhanced glycolytic flux or reduced apoptosis; PD-1-/- CD8+ T cells transplanted into PD-L1-/- recipients also induce little GVHD, similar to WT CD8+ T cells. 3) After injection of anti-PD-L1 mAb (clone 43H12) that specifically blocks PD-L1/CD80 interaction and preserves PD-L1/PD-1 interaction, WT CD8+ T cells in WT recipients show drastically reduced expansion, as compared to control recipients treated with rat-IgG. Blockade of PD-L1/CD80 interaction reduces phosphorylation of key elements in the TCR signaling cascade and CD28 co-stimulatory pathway including ZAP-70, AKT, mTOR and rpS6. Taken together, these studies indicate that 1) PD-L1/CD80 signaling alone augments alloreactive CD8+ T cell expansion and GVHD-inducing capacity; 2) PD-L1/CD80 and PD-L1/PD-1 signaling reciprocally regulate CD8+ T cell glycolysis, proliferation, and apoptosis as well as their GVHD-inducing capacity, and a balance of the two signaling pathways would be required to allow donor CD8+ T cells to facilitate engraftment and mediate GVL effect without causing GVHD. This work was supported by Institutional Funds of The Beckman Research Institute of City of Hope. DisclosuresNo relevant conflicts of interest to declare.

Pharmacological Inhibition of Hsp90 Destabilizes the Histone Methyltransferase Ezh2 in Alloreactive T Cells and Reduces Graft-Versus-Host Disease While Retaining Anti-Leukemic Effects in Mice

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). GVHD involves complex interactions of immune cells, induction of host-reactive donor effector T cells, and donor T cell-mediated injury to normal tissues. Epigenetic changes have been implicated in T cell-mediated GVHD. We previously described that genetic deletion of Ezh2, which catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3), reduced GVHD in mice but preserved graft-versus-leukemia (GVL) responses. Several selective inhibitors of Ezh2 have been recently discovered (e.g. GSK126, UNC1999 and EPZ6438), which specifically reduce the levels of H3K27me3 but not EZH2 protein. Unexpectedly, our preliminary studies showed that administration of GSK126 failed to prevent GVHD in mice. This stands in contrast to our findings that genetic deletion of T cell Ezh2 leads to GVHD inhibition, and suggest that Ezh2 may regulate GVHD through a mechanism independent of H3K27me3. Identifying an optimal method to target T cell Ezh2 for controlling GVHD remains an unmet need.Using experimental mouse models, we demonstrate that functional heat shock protein (Hsp)90 is critical for maintaining Ezh2 protein stability and function in activated T cells. Pharmacological inhibition of Hsp90 destablizes Ezh2 protein in alloreactive T cells, reduces GVHD but preserves GVL effects in mice. To determinethe molecule(s) that is critical for maintaining Ezh2 protein stablility in T cells, we performed mass spectrum (MS) analysis and identified 25 Ezh2-interacting proteins that showed higher intensities than others in T cell receptor (TCR)-activated CD8+ T cells. Among them, we found a group of proteins associated with protein folding and degradation, including Hsp90. Hsp90 is a molecular chaperone required for the stability and function of several key signaling intermediates (e.g., AKT, Raf1 and ERK1/2). Using reciprocal co-immunoprecipitation assay, we confirmed that Ezh2 and Hsp90 directly interacted with each other in TCR-activated CD8+ T cells. Pharmacological inhibition of Hsp90 using its specific inhibitor AUY922, which is currently in phase II clinical trials for cancer therapy, effectively reduced Ezh2 protein without decreasing H3K27me3 24 hours after treatment. This effect was accompanied by decreased proliferation and survival of TCR-activated T cells in vitro. Retroviral overexpression of Ezh2 in T cells markedly improved their proliferation in the presence of AUY922, suggesting that reducing Ezh2 by Hsp90 inhibition is an important mechanism that reduces proliferation and survival of activated CD8+ T cells. Building on these observations, we examined the impact of inhibiting Hsp90 on GVHD by administering AUY922 to B6 mice receiving MHC-identical minor histocompatibility antigen-mismatched C3H.SW mouse CD8+ T cells and T cell-depleted bone marrow (BM). While about 80% of control B6 recipients died from severe GVHD, 80% of AUY922-treated B6 recipients survived without clinical signs of severe GVHD by 84 days after transplantation. In vivo AUY922 administration reduced the survival and expansion of alloreactive T cells, and decreased the fequency of alloreactive T effector cells producing IFN-g and TNF-a. To rule out the model-specific effect of AUY922, we used a haplo-identical B6 into BDF1 mouse model of GVHD. Using CFSE-labeled donor T cells, we first validated that in vivo administration of AUY922 to unirradiated BDF1 mice receiving parent B6 T cells selectively reduced the expansion of alloantigen-reactive donor T cells, but did not impair the expansion and survival of donor T cells that did not respond to alloantigens. In lethally irradiated BDF1 mice receiving B6 T cells and BM, AUY922 administration reduces lethal GVHD, with approximately 50% of them surviving long-time. Importantly, AUY922 treatment preserved GVL activity of donor T cells, leading to significantly improved survival of BDF1 recipients challenged with A20 leukemic cells (Fig.1).Taken together, our findings identified a previously unrecognized molecular mechanism by which Ezh2 and Hsp90 are integrated to regulate alloreactive T cell responses and GVHD. Targeting the Ezh2-Hsp90 complex using AUY922 represents a novel and clinically relevant approach to reduce GVHD while preserving GVL effects, thereby improving the efficacy of allo-HSCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Granzyme B-Mediated Activation-Induced Death of CD4+ T Cells Inhibits Murine Acute Graft-versus-Host Disease.

Granzyme B (GzmB) has previously been shown to be critical for CD8(+) T cell-mediated graft-versus-host disease (GVHD) but dispensable for GVHD mediated by CD4(+) T cells. However, previous studies used high doses of CD4(+) T cells in MHC-mismatched models that caused rapid and lethal GVHD. Because of the hyperacute lethality, it is possible that the role of GzmB was concealed by the system. Therefore, in this study, we have titrated down the T cell dose to precisely determine the contribution of GzmB in GVHD mediated by CD4(+)CD25(-) T cells. Surprisingly, we have found that GzmB(-/-)CD4(+)CD25(-) T cells cause more severe GVHD compared with wild-type CD4(+)CD25(-) T cells in both MHC-matched and mismatched models. Mechanistic analyses reveal that although GzmB does not affect donor T cell engraftment, proliferation or tissue-specific migration, GzmB(-/-) CD4(+)CD25(-) T cells exhibit significantly enhanced expansion because of GzmB-mediated activation-induced cell death of wild-type CD4(+)CD25(-) T cells. As a result of enhanced expansion, GzmB(-/-) T cells produced higher amounts of proinflammatory cytokines (e.g., TNF-α and IFN-γ) that may contribute to the exacerbated GVHD. These results reveal that GzmB diminishes the ability of CD4(+) T cells to cause acute GVHD, which contradicts its established role in CD8(+) T cells. The differential roles suggest that targeting GzmB in selected T cell subsets may provide a strategy to control GVHD.