Pathogenesis Of Graft-versus-host Disease Research Articles

Long-term treatment with the P2X7 receptor antagonist Brilliant Blue G reduces liver inflammation in a humanized mouse model of graft-versus-host disease

Allogeneic haematopoietic stem cell transplantation (HSCT) is a frequent curative therapy for numerous haematological malignancies. However, HSCT is limited by the occurrence of graft-versus-host disease (GVHD), with current therapies restricted to general immunosuppression. Activation of the P2X7 receptor by extracellular adenosine triphosphate (ATP) causes inflammation and tissue damage in GVHD. Short-term pharmacological blockade of P2X7 has been shown to reduce clinical disease and/or reduce inflammatory markers in allogeneic and humanized mouse models of GVHD. The current study demonstrates that long-term P2X7 blockade by intra-peritoneal injection of Brilliant Blue G (BBG) thrice weekly for up to 10 weeks did not impact human (h) peripheral blood mononuclear cell (PBMC) engraftment, predominantly T cells, in blood at 3 weeks post-hPBMC injection or in spleens at end-point in humanized mice. Histological analysis demonstrated long-term BBG treatment reduced leukocyte infiltration in the livers of humanized mice. Immunohistochemical analysis demonstrated that BBG treatment reduced liver apoptosis. Long-term BBG treatment did not alter clinical disease, mRNA expression of pro-inflammatory markers in tissues or serum human interferon (IFN)-γ concentrations. Therefore, this study demonstrates that P2X7 activation plays a role in GVHD pathogenesis in the livers of humanized mice, supporting a role for this receptor in GVHD development in HSCT recipients.

Research Progress on Separating Graft Versus Host Disease and Graft Versus Tumor Effect--Review

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been the only method for curing many hematopoietic benign or malignant diseases, from which a number of patients obtain long-term survival. However, graft versus host disease (GVHD) deeply hinders the development and clinical use of HSCT. Up to now, immune response triggered by donor's T cell has been considered to be the pathogenesis of GVHD. On the other side, donor's T cell has indispensable effect in competing with the tumor cells and reducing the disease recurrence rate. So, how to prevent GVHD, meanwhile, to retain graft versus tumor (GVT) effect, has become one of the challenging problems after HSCT. Recently, drugs such as trametinib and echinomycin, expanding regulatory T cells in vitro and advanced techniques such as conformal nanoencapsulation have shown optimistic results in experiments of separating GVHD and GVT effect, which are hopefully to be utilized in clinic in the future.

Interferon Regulatory Factor 4 Is a Key Factor in Regulation of Thelper Cell Polarization in Graft-Versus-Host Disease

Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for severe malignant diseases. Following allo-HSCT, donor T cells are the driving force for eradication of the remaining malignant cells known as graft-versus-tumor (GVT) effect. However, these alloreactive T cells are also responsible for induction of graft-versus-host disease (GVHD). To date, the role of the different Thelper (Th) subsets in the pathogenesis of GVHD is not completely understood. Interestingly, these subsets share expression of a transcription factor called Interferon Regulatory Factor 4 (IRF4), which is proposed as a master regulator of cell fate decision of T cells. This project aims to analyze the role of IRF4 in Th cell polarization during the development of GVHD.Methods: In mixed lymphocyte reaction (MLR), we analyzed the proliferation capacity of CFSE-labelled IRF4-deficient (IRF4-/-) T cells upon allogeneic stimulation by LPS-induced dendritic cells (DC). For analyzing the impact of IRF4 in vivo, we used previously published complete MHC-mismatched murine GVHD model (Ullrich et al., J Clin Invest, 2018). Herein, we investigated the alloreactivity of the transplanted donor T cells towards GVHD target organs with focus on colonic tissue. Additionally, RNA sequencing of re-isolated and high purity FACS-sorted donor Th cells were performed to get a deep insight into the IRF4-mediated regulation of Th cell polarization.Results: In the MLR setting, reduced CFSE dilution indicated a diminished proliferative capacity of both CD4+ and CD8+ IRF4-/- T cells compared to the corresponding WT (IRF4+/+) T cell subsets upon allogeneic stimulation (Figure 1A). Furthermore, while alloreactive WT CD4+ T cells induced severe forms of GVHD in vivo, clinical GVHD symptoms of recipients transplanted with IRF4-/- CD4+ T cells were significantly reduced and these mice showed prolonged overall survival (Figure 1B). Analyzing the mechanism, we found that the frequency of in vivo circulating donorCD4+ IRF4-/- T cells was reduced compared to transplanted WT Th cells, especially in the GVHD target organs such as the colon. However, IRF4-/- Th cells persisted in spleen, lung and colon even if they showed a reduced proliferative capacity. In line with that, colonoscopy of mice transplanted with IRF4-deficient Th cells revealed a significant reduction of GVHD associated colitis. Transcriptome analysis of re-isolated and high purity FACS-sorted donor Th cells depicted an altered gene expression profile in donor IRF4-/- Th cells compared to donor WT Th cells. Specifically, master regulators of Th cell subsets like T-bet (Th1), RORγt (Th17) and to some amount also GATA-3 (Th2) were downregulated in donor IRF4-/- Th cells whereas FoxP3, the master regulator of regulatory T cells (Treg cells), was significantly upregulated. Along the same line cytokines associated with Th1, Th2 and Th17 cell subsets such as IFN-γ, IL-21, IL-6 and IL-13 were also significantly downregulated. Besides genes that are associated with Treg cell function like Helios, FR4 (folate receptor 4) and Neuropilin 1, a transcriptional repressor, Bach2, which regulates the formation of Treg cells and suppresses Th1, Th2 and Th17 subset differentiation was highly upregulated (Tsukumo et al., Proc Natl Acad Sci U S A, 2013; Kim et al., J Immunol, 2014 ; Roychoudhuri et al., Nature, 2013 ; Vahedi et al., Nature, 2015). Along with the upregulation of Bach2 and the significant downregulation of Blimp1, another transcriptional repressor involved in T cell homeostasis and function as well as direct target of Bach2, we hypothesize that IRF4 might compete with BACH2 for the binding to BATF. These hypotheses also rely on our previous finding of BATF as critical mediator of GVHD colitis and are currently under further evaluation (Ullrich et al., J Clin Invest, 2018).Conclusion: In summary, our results indicate that IRF4 plays a key role in regulation of the Th cell polarization and therefore also in the development of GVHD. Thus, IRF4 in its interplay with BATF might be considered as a clinically relevant target for GVHD therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Signal Transducing Adaptor Protein (STAP) Family Accelerates Gut and Thymic Graft-Versus-Host-Disease in Murine Model

Graft-versus-host-disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the most frequent complication and one of the major causes of non-relapse mortality. However, its pathogenesis has not yet been fully understood. We cloned signal-transducing adaptor protein (STAP)-2 as a c-fms binding protein from a fetal liver library in 2003. The family that contains STAP-1 and STAP-2 has a pleckstrin homology (PH) and Src-homology 2 (SH2)-like domains, suggesting that this adapter protein functions as an immune and inflammatory regulator. Indeed, STAP-2 regulates adhesion and chemotaxis in T cells (Sekine et al., J Immunol. 2009). In this study, we aimed to elucidate the roles of STAP family in GVHD.First, we examined the expression of STAP-1 and STAP-2 mRNA in various human hematopoietic subsets, including CD34+ CD38- hematopoietic stem cells (HSCs), CD34+ CD38+ hematopoietic progenitor cells (HPCs), CD19+ CD27- naïve B cells, CD19+ CD27+ memory B cells, CD3+ CD4+ helper T-cells, and CD3+ CD8+ cytotoxic T lymphocytes, using real-time PCR. As a result, STAP-1 and STAP-2 were expressed in lymphoid cells, as well as HSCs and HPCs. STAP-2 mRNA was highly expressed in T cells. Next, to investigate the role of STAPs in GVHD, we made an experimental murine model. To study the pathogenesis of immune reconstitution and tolerance after allo-HSCT, lethally irradiated BALB/c mice were injected with T and B cell-depleted bone marrow cells (5×106 cells) derived from syngeneic BALB/c or allogeneic C57BL/6 mice on day 0. Co-transplantation of splenocytes was not adapted in this model. Survival and clinical degree of GVHD were assessed by a scoring system that sums changes in 5 clinical parameters: body weight (BW) loss, posture, activity, fur texture, and skin integrity. Recipients transplanted from allogenic wild type (WT) C57BL/6 donor survived without suffering from severe GVHD symptoms, owing to development of immune tolerance against allogeneic antigens. However, compared to syngeneic transplanted mice, these recipients started to show gradual BW loss and GVHD score was increased approximately 28 days after allo-HSCT, indicating the existence of an allogeneic immune reaction. To evaluate the role of STAPs in GVHD, we generated transgenic mice (Tg) that overexpress STAP under the control of Em enhancer and Lck proximal promoter. The promoter could drive expression of the inserted cDNA in lymphoid lineage cells from the common lymphoid progenitor (CLP) stage. When STAP-2 Tg marrow was used as a donor source, we found that the overall survival of STAP-2 Tg recipients was significantly lower than that of WT recipients (22.2% and 91.7%, respectively; p<0.001) on day 60. STAP-2 Tg recipients showed decreased BW and had a higher clinical GVHD score with statistical significance compared to control WT recipients. The overexpression of STAP-1 also exacerbated the severity of GVHD. At day 42, BW was decreased by 16.3% in WT recipients. In contrast, recipients of STAP-1 Tg and the STAP-2 Tg donor showed more severe BW loss along with diarrhea (23.3% and 29.8%, respectively). STAP-1 as well as STAP-2 Tg recipients showed significantly worsened GVHD scores, and this lasted until day 90 at the end of follow-up. In histologic examination of both STAP Tg recipients, inflammatory damages with lymphocyte infiltration were most notably observed in the colon. Interestingly, we found that thymus was atrophic or indistinguishable and the cortico-medullary junction disappeared. Moreover, compared to control WT recipients, the number of CD4+ CD25+ regulatory T (Treg) cells in the peripheral blood was significantly low in STAP-2 Tg recipients on day 60 (WT vs STAP-2 Tg; 44.0 /μL vs 18.2 /μL; p<0.05).In this study, we show that STAPs in reconstituted lymphocytes after allo-HSCT regulate the pathogenesis of GVHD. Our results suggest that STAP activation in lymphocytes during immune reconstitution accelerates gut and thymic GVHD. Severe thymic damage induced by STAP overexpression might contribute to impairment of immune tolerance such as a decreased number of Treg cells as well as dysfunction of thymic negative selection of host-reactive T cells after allo-HSCT, which is involved in persistence of GVHD. Future study should further elucidate the detailed molecular mechanisms involved. DisclosuresIchii:Celgene K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Novartis Pharma K.K.: Speakers Bureau. Shibayama:Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Jansen Pharmaceutical K.K: Honoraria; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:Novartis Pharma: Speakers Bureau. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

Bendamustine Conditioning Alters Host Dendritic Cell Composition and Function

BackgroundA largely understudied strategy for reducing graft-versus-host disease (GvHD) is the modification of conditioning regimens. We have demonstrated that pre-transplant bendamustine (BEN) used in combination with total body irradiation (TBI) significantly reduces GvHD compared to the more commonly used cyclophosphamide (CY) + TBI. The short half-lives of BEN/CY (<4h), which are given 48h prior to transplant, indicate that these drugs do not directly affect the donor cells. Therefore, the striking improvement in GvHD by substituting CY with BEN must be due to differential effects on the host cells. Conditioned host immune cells diminish over time, but are the majority early after transplant and are able to critically interact with the infused donor cells at the earliest stages of GvHD pathogenesis.MethodsUsing an MHC-mismatched murine model of bone marrow transplantation (BMT) with C57BL/6 donors into BALB/c recipients (H2Kb into H2Kd), we evaluated the differential effects of BEN conditioning on host dendritic cells (DCs) early after transplant, as compared to CY conditioning. Transplant recipient mice were conditioned with BEN or CY on day -2 pre-BMT, and supplemented with 400cGy of total body irradiation on day -1. On days 1, 3, and 5 post-BMT, splenic pan-DCs were isolated and analyzed by flow cytometry to evaluate host (H2Kb-) DC phenotype and activation state. Further, on day 0 (without BMT), circulating leukocytes and splenic pan-DCs were isolated from conditioned mice and analyzed by flow cytometry, qPCR and allogeneic mixed leukocyte reactions.ResultsWe found that BEN-conditioned pan DCs are less stimulatory of allogeneic T-cells than CY-treated DCs. BEN conditioning significantly alters host DC composition compared to CY conditioning, resulting in a significantly higher frequency and number of host CD8α+ DCs on days 1 and 3 post-BMT. BEN-conditioned CD8α+ DCs also display a highly regulatory phenotype (CD80highCD86low) and have greater suppressive ability compared to CY-conditioned CD8α+ DCs. Analysis of host DCs on day 0 (without BMT) revealed a significantly greater percentage of BEN-conditioned DCs positively expressing the Flt3 receptor compared to CY.ConclusionsThese findings advocate a potential role of Flt3L-dependent DCs, particularly CD8α+ DCs, in the mechanism by which BEN alters GvHD pathogenesis to limit morbidity and mortality. Thus, BEN may have advantages as an alternative agent to CY for pre-transplant conditioning in allogeneic BMT. DisclosuresNo relevant conflicts of interest to declare.

Host MyD88 signaling protects against acute graft-versus-host disease after allogeneic bone marrow transplantation.

Recent experimental strategies to reduce graft-versus-host disease (GVHD) have focused largely on modifying innate immunity. Toll-like receptor (TLR)-driven myeloid differentiation primary response 88 (MyD88)-dependent signalling pathways that initiate adaptive immune function are also critical for the pathogenesis of GVHD. This study aimed to delineate the role of host MyD88 in the development of acute GVHD following fully major histocompatibility complex-mismatched allogeneic bone marrow transplantation (BMT). When myeloablated BALB/c MyD88 knock-out recipients were transplanted with C57BL/6 (B6) donor cells, they developed significantly more severe GVHD than wild-type (WT) BALB/c hosts. The increased morbidity and mortality in MyD88-/- mice correlated with increased serum levels of lipopolysaccharide and elevated inflammatory cytokines in GVHD target organs. Additionally, MyD88 deficiency in BMT recipients led to increased donor T cell expansion and more donor CD11c+ cell intestinal infiltration with apoptotic cells but reduced proliferation of intestinal epithelial cells compared with that in WT BMT recipients. Decreased expression of tight junction mRNA in epithelial cells of MyD88-/- mice suggested that MyD88 contributes to intestinal integrity. Cox-2 expression in the GVHD-targeted organs of WT mice is increased upon GVHD induction, but this enhanced expression was obviously inhibited by MyD88 deficiency. The present findings demonstrate an unexpected role for host MyD88 in preventing GVHD after allogeneic BMT.

Extended CCR5 Blockade for Graft-versus-Host Disease Prophylaxis Improves Outcomes of Reduced-Intensity Unrelated Donor Hematopoietic Cell Transplantation: A Phase II Clinical Trial

Graft-versus-host disease (GVHD) remains the most common treatment-related complication after allogeneic hematopoietic cell transplantation (allo-HCT). Lymphocyte migration plays a critical role in the pathogenesis of GVHD. A previous phase I/II trial demonstrated that CCR5 blockade with maraviroc in the first 30days after allo-HCT resulted in a low incidence of early acute GVHD, primarily in visceral organs, but with no impact on late acute or chronic GVHD. We conducted a phase II trial to examine the efficacy of an extended course of maraviroc, administered through post-transplantation day +90 in addition to standard prophylaxis in 37 recipients of reduced-intensity-conditioned unrelated donor allo-HCT performed to treat hematologic malignancies. Extended maraviroc treatment was safe and feasible. The primary study endpoint, day +180 rate of grade II-IV acute GVHD, was 22 ± 7%, liver GVHD was not observed, and gut GVHD was uncommon. The day +180 rate of grade III-IV acute GVHD was 5 ± 4%. The 1-year rate of moderate to severe chronic GVHD was 8 ± 5% and that of disease relapse was 30 ± 8%. Overall survival at 1 year was 70 ± 8%. Compared with the previously studied short course of maraviroc, the extended course resulted in a significantly higher GVHD-free, relapse-free survival (adjusted hazard ratio [HR], .45; 95% confidence interval [CI], .25 to .82; P = .009) and overall survival (adjusted HR, .48; 95% CI, .24 to .96; P = .037). A combined analysis of both trials showed that high maraviroc trough concentrations on the day of hematopoietic cell infusion were associated with lower rates of acute GVHD. An extended course of maraviroc after reduced-intensity-conditioned unrelated donor allo-HCT is safe and effective in preventing acute and chronic GVHD and is associated with favorable survival.

Xenogeneic Graft-Versus-Host Disease in Humanized NSG and NSG-HLA-A2/HHD Mice.

Despite the increasing use of humanized mouse models to study new approaches of graft-versus-host disease (GVHD) prevention, the pathogenesis of xenogeneic GVHD (xGVHD) in these models remains misunderstood. The aim of this study is to describe this pathogenesis in NOD/LtSz-PrkdcscidIL2rγtm1Wjl (NSG) mice infused with human PBMCs and to assess the impact of the expression of HLA-A0201 by NSG mice cells (NSG-HLA-A2/HHD mice) on xGVHD and graft-versus-leukemia (GvL) effects, by taking advantage of next-generation technologies. We found that T cells recovered from NSG mice after transplantation had upregulated expression of genes involved in cell proliferation, as well as in TCR, co-stimulatory, IL-2/STAT5, mTOR and Aurora kinase A pathways. T cells had mainly an effector memory or an effector phenotype and exhibited a Th1/Tc1-skewed differentiation. TCRβ repertoire diversity was markedly lower both in the spleen and lungs (a xGVHD target organ) than at infusion. There was no correlation between the frequencies of specific clonotypes at baseline and in transplanted mice. Finally, expression of HLA-A0201 by NSG mice led to more severe xGVHD and enhanced GvL effects toward HLA-A2+ leukemic cells. Altogether our data demonstrate that the pathogenesis of xGVHD shares important features with human GVHD and that NSG-HLA-A2/HHD mice could serve as better model to study GVHD and GvL effects.

Host-Derived Serine Protease Inhibitor 6 Provides Granzyme B–Independent Protection of Intestinal Epithelial Cells in Murine Graft-versus-Host Disease

Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic cell transplantation (allo-HCT) that limits the therapeutic potential of this treatment. Host antigen-presenting cells (APCs) play a vital role in activating donor T cells that subsequently use granzyme B (GzmB) and other cytotoxic molecules to damage host normal tissues. Serine protease inhibitor 6 (Spi6), known as the sole endogenous inhibitor of GzmB, has been implicated in protecting T cells and APCs against GzmB-inflicted damage. In this study we used murine models to examine the previously unknown role of host-derived Spi6 in GVHD pathogenesis. Our results indicated that host Spi6 deficiency exacerbated GVHD as evidenced by significantly increased lethality and clinical and histopathologic scores. Using bone marrow chimera system, we found that Spi6 in nonhematopoietic tissue played a dominant role in protecting against GVHD and was significantly upregulated in intestinal epithelial cells after allo-HCT, whereas Spi6 in hematopoietic APCs surprisingly suppressed alloreactive T cell response. Interestingly, the protective effect of Spi6 and its expression in intestinal epithelial cells appeared to be independent of donor-derived GzmB. We used in silico modeling to explore potential targets of Spi6. Interaction tested in silico demonstrated that Spi6 could inhibit caspase-3 and caspase-8 with the same functional loop that inhibits GzmB but was not capable of forming stable interaction with caspase-1 or granzyme A. Using an in vitro co-culture system, we further identified that donor T cell–derived IFN-γ was important for inducing Spi6 expression in an intestinal epithelial cell line. Altogether, our data indicate that host Spi6 plays a novel, GzmB-independent role in regulating alloreactive T cell response and protecting intestinal epithelial cells. Therefore, enhancing host-derived Spi6 function has the potential to reduce GVHD.

Unraveling the Mechanisms of Cutaneous Graft-Versus-Host Disease.

The skin is the most common target organ affected by graft-versus-host disease (GVHD), with severity and response to therapy representing important predictors of patient survival. Although many of the initiating events in GVHD pathogenesis have been defined, less is known about why treatment resistance occurs or why there is often a permanent failure to restore tissue homeostasis. Emerging data suggest that the unique immune microenvironment in the skin is responsible for defining location- and context-specific mechanisms of injury that are distinct from those involved in other target organs. In this review, we address recent advances in our understanding of GVHD biology in the skin and outline the new research themes that will ultimately enable design of precision therapies.

A critical role of NLRP3 inflammasome in boosting antigen-specific immunity

Abstract While NLPR3 inflammasome is activated by pathogen signals and danger signals like ATP and MSU, the physiological triggers of NLRP3 inflammasome activation still remain to be explored. We identified perforin as a new physiological NLRP3 inflammasome activator. We found that NLRP3 inflammasome was activated during co-cultures of antigen-specific CD8+T cells (also called cytotoxic T lymphocytes, CTL) and antigen presenting cells (APCs) in both OT-1 CTL as well as allo-antigen CTL systems. We further found that antigen presentation was required for the NLRP3 inflammasome activation in the co-culture system in vitro and in mice in vivo. The NLRP3 inflammasome activation required the direct contact of CTLs and APCs. We demonstrated that perforin in CTLs activated NLRP3 inflammasome in APCs. Through utilizing Nlrp3, Caspase-1, IL1r or perforin-deficient mice, we demonstrated that perforin-activated NLRP3 inflammasome was critical for antigen-specific CTL-mediated anti-tumor immunity and GVHD (graft-versus-host disease) pathogenesis. However, the antigen-specific CTL-mediated NLRP3 inflammasome activation was not important for host defense against intracellular bacterial pathogen and virus infections. Thus, our study reveals a positive feedback loop of adaptive immunity to promote innate immunity to amplify antigen-specific adaptive immune responses in situations of antitumor immunity and GVHD pathogenesis where innate immunity is not strongly activated.

Neutrophils provide cellular communication between ileum and mesenteric lymph nodes at graft-versus-host disease onset

AbstractConditioning-induced damage of the intestinal tract plays a critical role during the onset of acute graft-versus-host disease (GVHD). Therapeutic interference with these early events of GVHD is difficult, and currently used immunosuppressive drugs mainly target donor T cells. However, not donor T cells but neutrophils reach the sites of tissue injury first, and therefore could be a potential target for GVHD prevention. A detailed analysis of neutrophil fate during acute GVHD and the effect on T cells is difficult because of the short lifespan of this cell type. By using a novel photoconverter reporter system, we show that neutrophils that had been photoconverted in the ileum postconditioning later migrated to mesenteric lymph nodes (mLN). This neutrophil migration was dependent on the intestinal microflora. In the mLN, neutrophils colocalized with T cells and presented antigen on major histocompatibility complex (MHC)-II, thereby affecting T cell expansion. Pharmacological JAK1/JAK2 inhibition reduced neutrophil influx into the mLN and MHC-II expression, thereby interfering with an early event in acute GVHD pathogenesis. In agreement with this finding, neutrophil depletion reduced acute GVHD. We conclude that neutrophils are attracted to the ileum, where the intestinal barrier is disrupted, and then migrate to the mLN, where they participate in alloantigen presentation. JAK1/JAK2-inhibition can interfere with this process, which provides a potential therapeutic strategy to prevent early events of tissue damage-related innate immune cell activation and, ultimately, GVHD.

Graft versus Host Disease: From Basic Pathogenic Principles to DNA Damage Response and Cellular Senescence.

Graft versus host disease (GVHD), a severe immunogenic complication of allogeneic hematopoietic stem cell transplantation (HSCT), represents the most frequent cause of transplant-related mortality (TRM). Despite a huge progress in HSCT techniques and posttransplant care, GVHD remains a significant obstacle in successful HSCT outcome. This review presents a complex summary of GVHD pathogenesis with focus on references considering basic biological processes such as DNA damage response and cellular senescence.

Host-Derived CD70 Suppresses Murine Graft-versus-Host Disease by Limiting Donor T Cell Expansion and Effector Function.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for hematologic and immunologic diseases. However, graft-versus-host disease (GVHD) may develop when donor-derived T cells recognize and damage genetically distinct normal host tissues. In addition to TCR signaling, costimulatory pathways are involved in T cell activation. CD27 is a TNFR family member expressed on T cells, and its ligand, CD70, is expressed on APCs. The CD27/CD70 costimulatory pathway was shown to be critical for T cell function and survival in viral infection models. However, the role of this pathway in allo-HCT is previously unknown. In this study, we have examined its contribution in GVHD pathogenesis. Surprisingly, Ab blockade of CD70 after allo-HCT significantly increases GVHD. Interestingly, whereas donor T cell- or bone marrow-derived CD70 plays no role in GVHD, host-derived CD70 inhibits GVHD as CD70-/- hosts show significantly increased GVHD. This is evidenced by reduced survival, more severe weight loss, and increased histopathologic damage compared with wild-type hosts. In addition, CD70-/- hosts have higher levels of proinflammatory cytokines TNF-α, IFN-γ, IL-2, and IL-17. Moreover, accumulation of donor CD4+ and CD8+ effector T cells is increased in CD70-/- versus wild-type hosts. Mechanistic analyses suggest that CD70 expressed by host hematopoietic cells is involved in the control of alloreactive T cell apoptosis and expansion. Together, our findings demonstrate that host CD70 serves as a unique negative regulator of allogeneic T cell response by contributing to donor T cell apoptosis and inhibiting expansion of donor effector T cells.

Interleukin-6 in Allogeneic Stem Cell Transplantation: Its Possible Importance for Immunoregulation and As a Therapeutic Target.

Allogeneic stem cell transplantation is associated with a high risk of treatment-related mortality mainly caused by infections and graft-versus-host disease (GVHD). GVHD is characterized by severe immune dysregulation and impaired regeneration of different tissues, i.e., epithelial barriers and the liver. The balance between pro- and anti-inflammatory cytokine influences the risk of GVHD. Interleukin-6 (IL-6) is a cytokine that previously has been associated with pro-inflammatory effects. However, more recent evidence from various autoimmune diseases (e.g., inflammatory bowel disease, rheumatoid arthritis) has shown that the IL-6 activity is more complex with important effects also on tissue homeostasis, regeneration, and metabolism. This review summarizes the current understanding of how pro-inflammatory IL-6 effects exerted during the peritransplant period shapes T-cell polarization with enhancement of Th17 differentiation and suppression of regulatory T cells, and in addition we also review and discuss the results from trials exploring non-selective IL-6 inhibition in prophylaxis and treatment of GVHD. Emerging evidence suggests that the molecular strategy for targeting of IL-6-initiated intracellular signaling is important for the effect on GVHD. It will therefore be important to further characterize the role of IL-6 in the pathogenesis of GVHD to clarify whether combined IL-6 inhibition of both trans- (i.e., binding of the soluble IL-6/IL-6 receptor complex to cell surface gp130) and cis-signaling (i.e., IL-6 ligation of the IL-6 receptor/gp130 complex) or selective inhibition of trans-signaling should be tried in the prophylaxis and/or treatment of GVHD in allotransplant patients.

Altered homeostatic regulation of innate and adaptive immunity in lower gastrointestinal tract GVHD pathogenesis.

Lower gastrointestinal (GI) tract graft-versus-host disease (GVHD) is the predominant cause of morbidity and mortality from GVHD after allogeneic stem cell transplantation. Recent data indicate that lower GI tract GVHD is a complicated process mediated by donor/host antigenic disparities. This process is exacerbated by significant changes to the microbiome, and innate and adaptive immune responses that are critical to the induction of disease, persistence of inflammation, and a lack of response to therapy. Here, we discuss new insights into the biology of lower GI tract GVHD and focus on intrinsic pathways and regulatory mechanisms crucial to normal intestinal function. We then describe multiple instances in which these homeostatic mechanisms are altered by donor T cells or conditioning therapy, resulting in exacerbation of GVHD. We also discuss data suggesting that some of these mechanisms produce biomarkers that could be informative as to the severity of GVHD and its response to therapy. Finally, novel therapies that might restore homeostasis in the GI tract during GVHD are highlighted.

Fibroblastic niches prime T cell alloimmunity through Delta-like Notch ligands.

Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a major regulator of GVHD pathogenesis, little is known about the timing of essential Notch signals and the cellular source of Notch ligands after allo-BMT. Here, we have shown that critical DLL1/4-mediated Notch signals are delivered to donor T cells during a short 48-hour window after transplantation in a mouse allo-BMT model. Stromal, but not hematopoietic, cells were the essential source of Notch ligands during in vivo priming of alloreactive T cells. GVHD could be prevented by selective inactivation of Dll1 and Dll4 in subsets of fibroblastic stromal cells that were derived from chemokine Ccl19-expressing host cells, including fibroblastic reticular cells and follicular dendritic cells. However, neither T cell recruitment into secondary lymphoid organs nor initial T cell activation was affected by Dll1/4 loss. Thus, we have uncovered a pathogenic function for fibroblastic stromal cells in alloimmune reactivity that can be dissociated from their homeostatic functions. Our results reveal what we believe to be a previously unrecognized Notch-mediated immunopathogenic role for stromal cell niches in secondary lymphoid organs after allo-BMT and define a framework of early cellular and molecular interactions that regulate T cell alloimmunity.

Host Interleukin 6 and Indoleamine 2,3 Dioxygenase Regulate Inflammation in the Brain during Graft Versus Host Disease

Graft versus host disease (GVHD) commonly induces pathological damage in peripheral target organs such as the skin, liver and gastrointestinal tract leading to well characterized organ-specific clinical manifestations. A number of studies, however, have shown that patients with GVHD can also have behavioral and mood alterations that can affect overall cognitive function and lead to significant impairments in quality of life. The extent to which GVHD contributes to cognitive dysfunction and induces inflammation within the central nervous system (CNS), however, has not been critically examined. To address this question, we conducted studies using two well-defined murine GVHD models [C57BL/6(H-2b)→Balb/c (H-2d) and B10.BR(H-2k)→B6 (H-2b)]. We observed that there was a significant increase in the number of donor-derived CD4+ and CD8+ T cells in the brains of GVHD recipients early (days 7 -14) and late (day 42) post transplantation compared to BM controls. Histological studies revealed activated microglial cells and CD3+ T cell infiltration in the periventricular regions of brains in GVHD recipients that were not present in BM animals. Real time q-PCR analysis also demonstrated significant increases in IFN-γ, TNF-α, and IL-6 mRNA expression indicative of a proinflammatory state. Notably, GVHD animals exhibited behavioral changes in the forced swim test and elevated plus maze which are validated assays of stress coping and anxiety, respectively. Since IL-6, in particular, plays a pivotal role in GVHD pathogenesis in murine models and humans, we examined whether blockade of IL-6 signaling altered neuroinflammation. Animals treated with an anti-IL-6R antibody had a significant reduction in the number of donor-derived CD4+ and CD8+T cells in the brain compared to isotype control-treated mice. Anti-IL-6R treatment of GVHD mice also resulted in significant reductions in IFN-γ, TNF-α, and IL-6 mRNA and normalized behavior in the forced swim test, indicative of a decreased inflammatory response. Since IL-6 is produced by a wide variety of cells, including microglial and T cells, both donor and recipient cells have the potential to modulate GVHD severity within the CNS. To define whether donor or host IL-6 production was most critical for inducing neuroinflammation, experiments were conducted employing IL-6-/- mice as either donors or recipients. Whereas the absence of IL-6 in donor-derived cells had no impact on the degree of inflammation within the CNS, recipient animals that lacked IL-6 had a significant decrease in the number of donor-derived T cells which accumulated in the brain as well as a marked reduction in inflammatory cytokines, indicating that host IL-6 production was critical. To define the downstream pathways of IL-6-mediated CNS inflammation, we examined the role of indoleamine 2,3-dioxygenase (IDO) since IL-6 has been shown to upregulate IDO-1 expression under inflammatory conditions. We observed that IDO-1 mRNA levels were significantly increased in the brains of GVHD animals, and that blockade of IL-6 signaling resulted in a marked decrease in IDO mRNA levels. Additionally, transplantation studies using IDO-/- mice revealed that host, but not donor, IDO production was required for maximal inflammatory effects. Serotoninergic projections to the prefrontal cortex (PFC), in particular, are sensitive to inflammation and contribute to stress coping behavior. Therefore, to further interrogate this pathway, we performed quantitative mass spectrometry of brain extracts from the PFC. We found that tryptophan and 5HT concentrations were not different between BM and GVHD groups. However, there was an increase in the IDO product, kynurenic acid, in GVHD recipients consistent with an increase in brain IDO expression. To provide additional support for the premise that IL-6 effects were mediated through the IDO pathway, recipient mice were treated with either 1-methyltryptophan (1-MT), a completive inhibitor of IDO, or a vehicle control. GVHD mice treated with 1-MT had decreased accumulation of T cells in the brain and normal behavior in the forced swim test, demonstrating that inhibition of IDO abrogated CNS inflammation and behavioral changes in the presence of intact IL-6 signaling. In summary, these studies demonstrate that host IL-6 and IDO regulate inflammation and adversely impact behavioral function within the brain during GVHD through the tryptophan metabolic pathway. DisclosuresNo relevant conflicts of interest to declare.

Interferon Gamma (IFNγ)/STAT1 Signaling in Host Antigen Present Cells Suppresses MHC Class II-Dependent Presentation of Self-Antigens and Development of Graft Versus Host Disease (GVHD)

IFNγ signaling plays a critical role in the pathogenesis of GVHD. In this study, we observed that LPS-maturated bone marrow-derived dendritic cells (BMDCs) lacking IFNγ receptor (IFNγR, GRKO) or signal transducer and activator of transcription 1 (STAT1KO) had increased expression of major histocompatibility complex class II (MHC II), CD86, CD80, and enhanced allo-stimulatory capacity. This was further confirmed using fully MHC-mismatched bone marrow transplantation (BMT) studies. APC of GRKO or STAT1KO recipients had increased MHC II expression, which was associated with enhanced activation and expansion of donor CD4 and CD8 T cells and subsequently accelerated GVHD mortality compared to wild type (WT) controls. This increased GVHD mortality and increased MHC II expression on host APCs was further observed in the absence of recipient conditioning in the B6→CB6F1 mouse model. This was associated with increased presentation of host derived endogenous Eα52-68 peptide via I-Ab on recipient CD11c+ cells as detected by staining with the YA-e antibody. Furthermore, we could demonstrate that absence of IFNγR in BMDC promotes presentation Eα52-68 peptide and subsequently elicits pronounced activation, expansion and Th1 differentiation of TEa-TCR-tg CD4 T cells which recognize the Eα52-68 peptide presented by I-Ab. Next, we assessed the impact of this pathway on presentation of exogenous antigens. Interestingly, when lysate prepared from BALB/c splenocytes was incubated with BMDCs from B6 mice, Y-Ae expression on STAT1-/- BMDCs was significantly reduced compared to wild type BMDCs allowing us to hypothesize that IFNγ/STAT1 signaling may play an important role in promoting presentation of exogenous antigen while suppressing presentation of endogenous antigen. To further confirm this hypothesis, we used ovalbumin (OVA) as a second model antigen. To assess the impact of IFNγ/STAT1 signaling on presentation of exogenous antigen, WT, GRKO or STAT1KO BMDC were directly pulsed with OVA. To address the role in endogenous antigen presentation we studied act-mOVA-transgenic wildtype, act-mOVA.GRKO or act-mOVA.STAT1KO BMDCs. Transgenic OT-II CD4 T cells express a TCR specific for the OVA peptide 323-33 presented by I-Ab. The proliferation/activation of OT II T cells was monitored by flow cytometer as readout for effective Ag presentation. Our data demonstrated that IFNγR- or STAT1-deficient BMDCs loaded with exogenous intact OVA protein were compromised in promoting OT II proliferation. In contrast, responder OT-II CD4 T cells proliferated much more vigorously when stimulated with IFNγR/STAT1-deficient m-Act-OVA BMDCs compared to controls. We further observed significantly impaired OT-II cell proliferation in IFNγR or STAT1-deficient mice immunized with OVA indicating impaired presentation of exogenous antigens. However, OT-II CD4 T cells injected into lethally irradiated act-mOVA.STAT1KO transgenic mice proliferated more robustly and displayed increased Th1 differentiation compared to control mice when tested 3 days after OT II administration. We next started to assess several key factors (Ii [invariant chain, CD74], Cathepsin S [CTSS], H2-M, CIITA and MARCH1), known to be involved in the process of MHC class II antigen presentation and MHC II expression. We found retention of Invariant chain (CD74) expression as well as reduced CTSS and H2M expression in GRKO or STAT1KO BMDC following LPS-maturation. Furthermore, we observed significantly reduced lysosome formation/function in STAT1KO BMDCs compared to wild type BMDCs after LPS maturation. These data suggest that exogenous protein-derived peptide exchange in the MHCII compartment (MIIC) is impaired in STAT1KO BMDCs. Immature and LPS-maturated STAT1-/-BMDCs had significantly increased autophagy, which could explain enhanced endogenous Ag presentation since autophagy has been demonstrated to be critical in MHC II Ag presentation of cytoplasmic constituents. Finally, we found evidence of enhanced MHC II synthesis as supported by increased CIITA mRNA expression and conversely reduced MHC II degradation as indicated by reduced MARCH1 expression. In summary our data suggest that absence of IFNγR/STAT1 signaling in DC leads to abnormal surface MHC II turnover, promotes presentation of endogenous peptides and concomitantly impairs processing and presentation of exogenous antigens. DisclosuresLentzsch:BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria.

Mechanism of Suppression Effect of Myeloid-Derived Suppressor Cells on Hyperacute Graft-Versus-Host Disease

Allogeneic HSCT (allo-HSCT) is associated with serious side effects and its most common complication is graft-versus-host disease (GVHD). Hyperacute GVHD is a clinical syndrome that occurs within the first 14 days after allo-HSCT associated with significant morbidity and mortality. The large sample size of clinical study indicated that the incidence of hyperacute GVHD in patient who underwent an allo-HSCT was about 9%, but the pathological process and crucial factor of this complication have incompletely defined.Myeloid-derived suppressor cells (MDSCs) have been found that had a beneficial role in treatment of GVHD, on account of suppressing ability on alloreactive T-cell-response in vitro and in vivo. It was reported that reactive oxygen species (ROS) have been implicated in MDSCs-mediated T cell suppression and MDSCs from NOX2-deficient mice, chronic granulomatous disease (CGD) mice, failed to suppress T cell function. However, the investigation of whether and how MDSCs and ROS play in CGD mice receiving allo-HSCT is lacking.In our research, WT mice receiving allo-HSCT began to appear typical acute GVHD clinical manifestations in about 20 days and died within 30 days after transplantation, while CGD mice receiving allo-HSCT suddenly suffered from hyperacute GVHD at day 3 after allo-HSCT: performed continuous weight loss, demonstrated poor grooming and impairs movement with or without hunching or skin integrated and animals died within 2 days after onset of symptoms. Further study shown that the donor spleen derived T cells was indispensable for hyperacute GVHD of CGD mice after receiving allo-HSCT. T lymphocyte subsets and proportional change in bone marrow and spleen of each group were detected by flow cytommeter after transplantation. The percentage and absolute number of donor derived CD3+CD8+T cell from both BM and spleen of CGD were significant higher than that of WT mice received allo-HSCT. Moreover, cell size and expression of activation marker CD25, CD44, and CD69 of CD3+CD8+T cell from both BM and spleen of CGD mice were significant higher than that of WT mice. The killing ability of donor derived CD3+T cells was observed by the living cells workstation and it was obviously to see that allo-reactive T cells from CGD mice had stronger killing ability. The levels of different cytokines in serum of recipient mice were detected by protein chip at day 3 after allo-HSCT. Comparing to C57BL/6 mice, more than ten kinds of inflammatory factors, including IL-6, were increased in the serum of CGD mice, which indicated that the cytokine storm related to T cells might be occur during hyperacute GVHD. In addition, using this hyperacute mouse model, we revealed that application of ROS agonist, L-buthionine-S, R-sulfoximine (BSO), rescued the CGD mice receiving allo-HSCT from hyperacute GVHD.In General, this study pioneering established a stable murine model of hyperacute GVHD and proved that allo-reactive T cells massively activated and proliferated since ROS production defective MDSCs lose the ability of inhibiting T cell immune reaction and caused hyperacute GVHD. These data provided new insights into the pathogenesis of GVHD and may improve the clinical management of this common complication. DisclosuresNo relevant conflicts of interest to declare.