Pathogenesis Of Graft-versus-host Disease Research Articles

Sigle Agent of Posttransplant Cyclophosphamide Without Calcineurin Inhibitor Controls Severity of Experimental Chronic GVHD.

Chronic graft-versus-host disease (GVHD) is a major cause of late death and morbidity following allogeneic hematopoietic cell transplantation (HCT), but its pathogenesis remains unclear. Recently, haplo-identical HCT with post-transplant cyclophosphamide (Haplo-HCT with PTCY) was found to achieve a low incidence rate of acute GVHD and chronic GVHD. However, while the pathogenesis of acute GVHD following Haplo-HCT with PTCY has been well investigated, that of chronic GVHD remains to be elucidated, especially in HLA-matched HCT with PTCY. Based on its safety profile, PTCY is currently applied for the human leucocyte antigen (HLA)-matched HCT setting. Here, we investigated the mechanisms of chronic GVHD following HLA-matched HCT with PTCY using a well-defined mouse chronic GVHD model. PTCY attenuated clinical and pathological chronic GVHD by suppressing effector T-cells and preserving regulatory T-cells compared with a control group. Additionally, we demonstrated that cyclosporine A (CsA) did not show any additional positive effects on attenuation of GVHD in PTCY-treated recipients. These results suggest that monotherapy with PTCY without CsA could be a promising strategy for the prevention of chronic GVHD following HLA-matched HCT.

Molecular Imaging of Acute Graft-Versus-Host Disease.

Noninvasive molecular imaging of acute graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation has great potential to detect GvHD at the early stages, aid in grading of the disease, monitor treatment response, and guide therapeutic decisions. Although the specificity of currently available tracers appears insufficient for clinical GvHD diagnosis, recently, several preclinical studies have identified promising new imaging agents targeting one or more biologic processes involved in GvHD pathogenesis, ranging from T-cell activation to tissue damage. In this review, we summarize the different approaches reported to date for noninvasive detection of GvHD using molecular imaging with a specific focus on the use of PET. We discuss possible applications of molecular imaging for the detection of GvHD in the clinical setting, as well as some of the predictable challenges that are faced during clinical translation of these approaches.

Severity and organ distribution of chronic graft-versus-host disease with posttransplant cyclophosphamide-based versus methotrexate/calcineurin inhibitor-based allogeneic hematopoietic cell transplantation.

The reduced risk of chronic graft-versus-host-disease (GVHD) with posttransplant cyclophosphamide (ptCy) in the setting of haploidentical related donor and more recently, with HLA-matched related and matched and mismatched unrelated donor allogeneic transplantation has been established. There is, however, paucity of data to show if ptCy impacts chronic GVHD pathogenesis, its phenotype and evolution after HCT regardless of the donor status. We examined the differences in chronic GVHD incidence and presentation in 314 consecutive patients after receiving their first allogeneic transplantation (HCT) using ptCy-based GVHD prophylaxis (ptCy-HCT; n = 120; including 95 with haploidentical related donor) versus conventional calcineurin inhibitor-based prophylaxis (CNI-MUD; n = 194) between 2012 and 2019. The 1-year cumulative incidence of all-grade chronic GVHD and moderate/severe chronic GVHD was 24% and 12%, respectively, after ptCy-HCT and 40% and 23% in the CNI-MUD recipients (p = 0.0003 and 0.007). Multivariable analysis confirmed that use of CNI-based GVHD prophylaxis and peripheral blood stem cell graft as the risk factors for chronic GVHD. The cumulative incidence of visceral (involving ≥1 of the following organs: liver, lungs, gastrointestinal tract, serous membranes) chronic GVHD was significantly higher with CNI-MUD vs. ptCy-HCT (27% vs. 15% at 1 year, p = 0.009). The incidence of moderate/severe visceral chronic GVHD was 20% in CNI-MUD group vs. 7.7% in the ptCy-HCT group at 1 year (p = 0.002). In addition, significantly fewer ptCy-HCT recipients developed severe chronic GVHD in ≥3 organs (0.8%) vs. 8.8% in the CNI-MUD group at 1-year posttransplant (p = 0.004). There was no significant different in relapse, non-relapse mortality, and relapse-free and overall survival between the two groups. Further investigation is needed to confirm that reduced risk and severity of chronic GVHD, less visceral organ distribution with ptCy-HCT leads to improved quality of life.

Monocyte Subpopulations and Their Expression of HLA-DR and CD300e after Allogeneic Stem Cell Transplantation in Patients with or without GvHD

Introduction: Graft versus host disease (GvHD) is a complex immune process that can affect up to 50% of patients undergoing allogeneic stem cell transplantation (allo-SCT). Even though prophylactic immunosuppressive regimens have been established over the last decades, GvHD is still one of the main causes of morbidity and mortality among these patients. Monocytes are cells of the innate immune system, that represent, as professional antigen-presenting cells, a connection to the adaptive immune system, and play a role in several autoimmune disorders. They are classified according to the expression of CD14 and CD16 into classical (CD14++/CD16-), intermediate (CD14++/CD16+), and non-classical (CD14+/CD16++) monocytes. Previous research has shown higher levels of intermediate monocytes in pediatric patients with GvHD. Furthermore, non-classical monocytes in allogenic bone marrow grafts have also been correlated with the development of GvHD. The expression of other surface markers such as HLA-DR and CD300e (IREM-2) are related to monocyte function and engagement in inflammatory processes. Nevertheless, the role of CD300e in GvHD remains unclear. Here, we present a description of monocyte subgroups and their expression of HLA-DR and CD300e in allo-SCT patients with or without GvHD as well as in healthy subjects. Methods: Transversal observational study with patients undergoing allo-SCT. Peripheral blood of patients was collected once after engraftment. Both acute and chronic GvHD patients were enrolled. Five healthy volunteers served as controls. Flow cytometry was performed to determine the immunophenotype of monocytes according to their expression of CD14 and CD16 stratifying into classical, intermediate, and non-classical subgroups as described above. In addition, the expression of HLA-DR and CD300e was analyzed for all monocytes and individual subgroups. Clinical characteristics are described in the table below. Statistical analysis was performed using GraphPad Prism 9. Comparisons between groups were made using non-parametrical tests and tests for multiple comparisons (Mann-Whitney and Kruskal-Wallis respectively). Results: 29 patients were enrolled. Two patients with GvHD were excluded due to neutropenia. 19 patients presented GvHD grade II or higher. Of those, ten had acute GvHD, five had prolonged acute GvHD, two had late onset acute GvHD, and two had chronic GvHD. Eight patients did not show GvHD or had only mild skin disease (grade one). The mean age was 60 years for all patients, 62.5 and 58.5 years among GvHD and non-GvDH patients, respectively. The image summarizes patient characteristics and comparisons. We compared transplant recipients with and without GvHD which each other and with healthy controls. In single comparisons, patients with GvHD showed significantly lower percentages of total monocytes as well as CD14+/HLA-DR+, total HLA-DR+, and intermediate monocytes compared to patients without GvHD. In addition, we observed a trend toward lower levels of CD16+/HLA-DR+ and CD300e-expressing monocytes in GvHD patients. When compared to healthy controls, allogeneic recipients without GvHD showed significantly higher levels of total monocytes, intermediate monocytes, and CD16+/HLA-DR+ monocytes and lower levels of classical and non-classical monocytes. Compared to controls, patients with GvHD showed significantly lower levels of total monocytes expressing CD300e (also true in all subgroups) as well as lower levels of classical monocytes, but higher levels of intermediate and non-classical monocytes. P values are shown in Table 1. Means and ranges for all groups are represented in Image 1. Conclusions: In our cohort, patients with GvHD showed higher levels of intermediate and non-classical monocytes compared to controls, however, these populations were lower when compared to allogeneic recipients without GvHD. This could indicate a consumption process similar to that observed in COVID-19 patients and a direct involvement of these monocytes in the GvHD process. In addition, we report for the first time the involvement of CD300e in GvHD, which was significantly lower expressed in the GvHD patients, compared to controls. However, there was only a strong trend compared to transplanted non-GvHD patients. This indicates a possible contribution of CD300e in the pathogenesis of GvHD. Further studies are needed in order to confirm these findings.

Diverse macrophage populations contribute to distinct manifestations of human cutaneous graft-versus-host disease.

Graft-versus-host disease (GvHD) is a major life-threatening complication of allogeneic haematopoietic stem cell transplantation (HSCT), limiting the broad application of HSCT for haematological malignancies. Cutaneous GvHD is described as a post-transplant inflammatory reaction by skin-infiltrating donor T cells and remaining recipient tissue-resident memory T cells. Despite the major influence of lymphocytes on GvHD pathogenesis, the complex role of mononuclear phagocytes (MNPs) in tissues affected by GvHD is increasingly appreciated. To characterize the identity, origin and functions of MNPs in patients with acute cutaneous GvHD. Using single-cell RNA sequencing and multiplex tissue immunofluorescence, we identified an increased abundance of MNPs in skin and blood from 36 patients with acute cutaneous GvHD. In cases of sex-mismatched transplantation, we used expression of X-linked genes to detect rapid tissue adaptation of newly recruited donor MNPs resulting in similar transcriptional states of host- and donor-derived macrophages within GvHD skin lesions. We showed that cutaneous GvHD lesions harbour expanded CD163+ tissue-resident macrophage populations with anti-inflammatory and tissue-remodelling properties including interleukin-10 cytokine production. Cell-cell interaction analyses revealed putative signalling to strengthen regulatory T-cell responses. Notably, macrophage polarization in chronic cutaneous GvHD types was proinflammatory and drastically differed from acute GvHD, supporting the notion of distinct cellular players in different clinical GvHD subtypes. Overall, our data reveal a surprisingly dynamic role of MNPs after HSCT. Specific and time-resolved targeting to repolarize this cell subset may present a promising therapeutic strategy in combatting GvHD skin inflammation.

Intestinal Epithelial-Derived IL-34 Regulates Macrophage Polarization in an Apolipoprotein E Dependent Manner and Mitigates the Severity of Graft Versus Host Disease

Acute graft versus host disease (GVHD) of the gastrointestinal (GI) tract is a major complication of allogeneic bone marrow transplantation. While donor T cells are the proximate trigger of GVHD, innate cells also contribute to disease pathogenesis, although the role of these cell populations is not as well defined. Macrophages, for example, are known contributors in the pathophysiology of chronic GVHD, but their role in acute GVHD is not well delineated. Fundamentally, macrophages are dependent upon exposure to m-CSF and IL-34 which serve as critical trophic factors necessary for their development through binding to the CSF-1R. IL-34 is a more recently discovered cytokine produced by macrophages, but whose role in GVHD pathogenesis is unknown. To examine this question, we employed a murine bone marrow transplantation model (B10.BR [H-2k]®B6[H-2b]) and observed that that there was no difference in GVHD-induced lethality when recipients were reconstituted with marrow grafts from wild type (WT) or IL-34 -/- donors. In contrast, recipient IL-34 -/- animals had significantly worse survival compared to WT mice indicating that host IL-34 expression protected animals from lethal GVHD. We observed no difference in the absolute number of donor macrophages in the colons of WT versus IL-34 -/- mice; however, IL-34 -/- recipient mice had a significant increase in the number of inflammatory Ly6C + macrophages, and a corresponding decrease in Ly6C - cells. Single cell RNA sequence (scRNAseq) analysis of donor macrophages from the colons of WT and IL-34 -/- recipients revealed that macrophages from WT animals had increased expression of anti-inflammatory genes (i.e., CD83, ApoE, Selenop, Lyz1 and Mrc1), whereas macrophages from IL-34 -/- recipients had increased expression of pro-inflammatory genes (i.e., Ifitm1, S100A8, S100A9 and GOs2), indicating that absence of host IL-34 skewed donor macrophages towards an inflammatory phenotype. Since the CSF-1R is not expressed on T cells, we sought to determine the effect of IL-34/CSF-1R signaling on proinflammatory T cells in GVHD target organs. scRNAseq analysis of colonic CD4+ T cells revealed that Csf2 (GM-CSF) was the most differentially expressed gene in IL-34 -/- CD4+ T cells. Functionally, this was confirmed by the observation of a significantly increased number of CD4 + GM-CSF+ T cells in the colons of these mice. Notably, there was no difference in proinflammatory CD8 + T cells, nor any effect on T cell phenotype in the liver or lung, demonstrating that the mitigating effect of host IL-34 expression was confined to the GI tract. To determine the cellular source of IL-34 production, we employed IL-34 LacZ reporter mice and showed that β-Gal co-localized with the epithelial marker, E-cadherin, indicating IL-34 is produced primarily by intestinal epithelial cells. Additional studies using chimeric mice in which IL-34 was restricted to either the hematopoietic or non-hematopoietic compartmentrevealed that absence of IL-34 in the non-hematopoietic compartment phenocopied findings observed with global IL-34 -/- animals, confirming that intestinal epithelial cells were the primary source of this cytokine. The administration of exogenous IL-34 prolonged survival, abrogated pathological damage in the colon and resulted in a corresponding decrease in pro-inflammatory macrophages and T cells, demonstrating this pathway could be pharmacologically targeted. Re-analysis of the scRNAseq data on donor macrophages from the colons of WT and IL-34 -/- mice revealed that apolipoprotein E ( Apoe) was the most differentially expressed gene in macrophages from WT GVHD animals. To confirm a functional role for ApoE, mice were transplanted with T cell depleted (TCD) BM from either WT or ApoE -/- mice and treated with IL-34. These studies revealed that the protective effect conferred by IL-34 was abrogated in animals that received TCD ApoE -/- BM grafts, indicating that ApoE expression in donor macrophages was necessary for agonist driven IL-34-mediated regulation of GVHD. Finally, we confirmed these studies have translational relevance by demonstrating IL-34 and Apoe have increased expression in intestinal epithelial cells and macrophages, respectively, in the colons of humans with GVHD. Thus, these results identify IL-34 as a novel intestinal epithelial cell-derived cytokine that regulates macrophage polarization and mitigates GVHD in an ApoE-dependent manner.

Hematopoietic Stem Cells Transplant (HSCT)-Related Chronic Pulmonary Diseases: An Overview.

Recipients of HSCT have a high risk of infective and non-infective pulmonary diseases. Most patients with pulmonary involvement present multiple pathogenetic mechanisms simultaneously with complex interactions. Therefore, it can be difficult to distinguish the contributions of each one and to perform studies on this subject. In this opinion article, we discuss only chronic pulmonary manifestations, focusing on LONIPCs (late-onset non-infectious pulmonary complications). This term embraces drug-related toxicity, allergies, and chronic pulmonary graft versus host disease (GvHD) in all its recently identified clinical variants. Among LONIPCs, GvHD represents the most critical in terms of morbidity and mortality, despite the rapid development of new treatment options. A recently emerging perspective suggests that pulmonary lung rejection in transplant patients shares striking similarities with the pathogenesis of GvHD. In a pulmonary transplant, the donor organ is damaged by the host immune system, whereas in GvHD, the donor immune system damages the host organs. It constitutes the most significant breakthrough in recent years and is highly promising for both hematologists and thoracic transplant surgeons. The number of patients with LONIPCs is scarce, with heterogenous clinical characteristics often involving several pathogenetic mechanisms, making it challenging to conduct randomized controlled trials. Therefore, the body of evidence in this field is scarce and generally of low quality, leading to jeopardized choices in terms of immunosuppressive treatment. Moreover, it risks being outdated by common practice due to the quick evolution of knowledge about the diagnosis and treatment of LONIPCs. The literature is even more pitiful for children with pulmonary involvement related to HSCT.

Abatacept and T-cell costimulation blockade—shifting the paradigm in the prevention of graft-versus-host disease

Despite advances in transplantation techniques and immunosuppressive therapies, graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality, necessitating the use of innovative strategies for its prevention. T-cell activation plays a crucial role in the pathogenesis of GVHD, and T-cell costimulation blockade (COSBL) has emerged as a promising approach to prevent this devastating condition. This review aims to explore the concept of COSBL and its potential as a paradigm-shifting strategy in the prevention of GVHD, in the context of the existing modalities for the prevention of GVHD and the preclinical and clinical studies on COSBL. The unique property of abatacept (CTLA4Ig) is not just limited to dampening T-cell activation. The salutary effect of abatacept on natural killer (NK) cells and Tregs alike provides a unique opportunity to dissociate T-cell-mediated GVHD from NK cell-mediated graft-versus-leukemia. Further research is warranted to explore other modalities of COSBL, determine the optimal dosing and combinations for COSBL, and identify predictive biomarkers for patient stratification, ultimately paving the way for improved outcomes in hematopoietic cell transplantation recipients.

Role of Defibrotide in the Prevention of Murine Model Graft-versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Vascular endothelial cells are entirely exposed and damaged during the pathogenesis of acute GVHD. Defibrotide is a mixture of single-stranded oligonucleotides that has several pharmacological effects that contribute to its endothelial protective properties. B10.BR mice were conditioned followed by the infusion of donor C57BL/6J T-cell depleted (TCD) bone marrow cells with or without splenocytes. Animals were either treated with defibrotide or appropriate controls daily for the first week and then three times per week thereafter. Allogeneic defibrotide-treated recipients demonstrated significantly better survival with reduced clinical GVHD. Significantly reduced organ pathology in the gut was associated with significantly decreased T-cell infiltration in the ileum and colon on day +28. Serum cytokine analysis revealed significantly reduced level of TNF and IL-6 at day +7 and TNF at day +28 in allogeneic defibrotide treated recipients. Significantly reduced levels of ICAM-1, Angiopoietin-2 in serum and reduced VCAM-1 and HCAM in the ileum and colon of allogeneic defibrotide-treated recipients were observed. Improved survival was seen in the GVL model (C3H.SW into C57BL/6J mice with C1498-luc). Defibrotide treatment through its anti-inflammatory and endothelial-protective effects reduces the severity of acute GVHD, while not impairing GVL activity.

From support to therapy: rethinking the role of nutrition in acute graft-versus-host disease.

Allogeneic Hematopoietic stem cell transplantation (HSCT) offers a potential cure for patients with hematologic malignancies. Unfortunately, graft-versus-host disease (GVHD) remains a major obstacle to the greater success of this treatment. Despite intensive research efforts over the past several decades, GVHD is still a major cause of morbidity and mortality in patients receiving allogeneic HSCT. The genetic disparity between donor and recipient is the primary factor that dictates the extent of alloimmune response and the severity of acute GVHD (aGVHD). However, some nongenetic factors are also actively involved in GVHD pathogenesis. Thus, identifying host factors that can be readily modified to reduce GVHD risk is of important clinical significance. We are particularly interested in the potential role of nutrition, as a nongenetic factor, in the etiology and management of aGVHD. In this article, we summarize recent findings regarding how different routes of nutritional support and various dietary factors affect aGVHD. Since diet is one of the most important factors that shape gut microbiota, we also provide evidence for a potential link between certain nutrients and gut microbiota in recipients of allogeneic HSCT. We propose a shifting role of nutrition from support to therapy in GVHD by targeting gut microbiota.

After Bone Marrow Transplantation, the Cell-Intrinsic Th2 Pathway Promotes Recipient T Lymphocyte Survival and Regulates Graft-versus-Host Disease.

Recipient T cells can aggravate or regulate lethal and devastating graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). In this context, we have shown before that intestinal immune conditioning with helminths is associated with survival of recipient T cells and Th2 pathway-dependent regulation of GVHD. We investigated the mechanism of survival of recipient T cells and their contribution to GVHD pathogenesis in this helminth infection and BMT model after myeloablative preparation with total body irradiation in mice. Our results indicate that the helminth-induced Th2 pathway directly promotes the survival of recipient T cells after total body irradiation. Th2 cells also directly stimulate recipient T cells to produce TGF-β, which is required to regulate donor T cell-mediated immune attack of GVHD and can thereby contribute to recipient T cell survival after BMT. Moreover, we show that recipient T cells, conditioned to produce Th2 cytokines and TGF-β after helminth infection, are fundamentally necessary for GVHD regulation. Taken together, reprogrammed or immune-conditioned recipient T cells after helminth infection are crucial elements of Th2- and TGF-β-dependent regulation of GVHD after BMT, and their survival is dependent on cell-intrinsic Th2 signaling.

A prospective phase 2 clinical trial of a C5a complement inhibitor for acute GVHD with lower GI tract involvement.

Involvement of lower gastrointestinal tract (LGI) occurs in 60% of patients with graft-versus-host-disease (GVHD). Complement components C3 and C5 are involved in GVHD pathogenesis. In this phase 2a study, we evaluated the safety and efficacy of ALXN1007, a monoclonal antibody against C5a, in patients with newly diagnosed LGI acute GVHD receiving concomitant corticosteroid. Twenty-five patients were enrolled; one was excluded from the efficacy analysis based upon negative biopsy. Most patients (16/25, 64%) had acute leukemia; 52% (13/25) had an HLA-matched unrelated donor; and 68% (17/25) received myeloablative conditioning. Half the patients (12/24) had a high biomarker profile,Ann Arbor score 3; 42% (10/24) had high-risk GVHD per Minnesota classification. Day-28 overall response was 58% (13/24 complete response, 1/24 partial response), and 63% by Day-56 (all complete responses). Day-28 overall response was 50% (5/10) in Minnesota high-risk and 42% (5/12) in high-risk Ann Arbor patients, increasing to 58% (7/12) by Day-56. Non-relapse mortality at 6-months was 24% (95% CI 11-53). The most common treatment-related adverse event was infection (6/25, 24%). Neither baseline complement levels (except for C5), activity, nor inhibition of C5a with ALXN1007 correlated with GVHD severity or responses. Further studies are needed to evaluate the role of complement inhibition in GVHD treatment.

Acute graft-versus-host disease after allogeneic stem cell transplantation from a haploidentical donor with post-transplant cyclophosphamide administration

Allogeneic hematopoietic stem cell transplantation is the only curative treatment for many life-threatening diseases. Due to the difficulty of finding an HLA-identical donor, transplantation from a haploidentical donor has become a frequent alternative. This option is associated with a higher risk of graft-versus-host disease (GVHD). In this article, we present the current view on the pathogenesis, treatment and prevention of GVHD and report a clinical case of acute GVHD after allogeneic hematopoietic stem cell transplantation from a haploidentical donor with post-transplant cyclophosphamide prophylaxis. The patient's parents gave consent to the use of their child's data, including photographs, for research purposes and in publications.

Acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation

Acute graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic stem cell transplantation determining the prognosis of hematopoietic stem cell recipients because of the impairment of multiple organs and systems and subsequent secondary immunodeficiency caused by combination immunosuppressive therapy. Current strategies are focused on the prevention of this life-threatening complication and on the timely start of treatment with appropriate selection of medications based on the knowledge of acute GVHD pathogenesis and grading as well as of risk factors for its development / progression in different patients. Here we provide a brief overview of acute GVHD and the existing approaches to its prevention and treatment.

Severe graft‐versus‐host disease post allogeneic hematopoietic stem cell transplantation due to loss of HLA heterozygosity in recipient lymphocytes after full graft rejection

AbstractGerm cell tumors complicated by hematological malignancy (HM) are a rare clinical phenomenon. Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is a potentially effective therapy, but graft‐versus‐host disease (GVHD) is a life‐threatening complication. We report a case of a 13‐year‐old female patient diagnosed with germ cell tumors followed by acute lymphoblastic leukemia. After chemotherapy, she received allo‐HSCT and her chimerism rate decreased rapidly to near zero by 6 months without evidence of HM recurrence. However, she developed severe, multiorgan GVHD‐like manifestations. DNA analysis revealed the pathogenesis of GVHD to be loss of HLA heterozygosity in recipient hematopoietic cells.

Effects of immune system cells in GvHD and corresponding therapeutic strategies.

Allogeneic tissue transplantation is one of the most effective treatments for several diseases and injuries, in particular, malignant and non-malignant hematological conditions. Following this procedure, transplanted tissue encounters various complications, one of the most serious being graft-versus-host disease (GvHD). The management of GvHD directly affects the success of transplantation and the survival rate of the patient; therefore, many studies have focused on GvHD prevention and control. This review briefly explains the transplantation process, causes of graft rejection, and importance of the human leukocyte antigen system. Initially, we address the pathophysiology and immunobiology of GvHD, the cells involved in this complication, the differences between chronic and acute GvHD, and the importance of graft-versus-leukemia. Interestingly, various types of immune cells are involved in GvHD pathogenesis. After explaining how these cells affect the GvHD process, we discuss the studies conducted to control and reduce GvHD symptoms.

IFN-γR/STAT1 signaling in recipient hematopoietic antigen-presenting cells suppresses graft-versus-host disease.

The absence of IFN-γ receptor (IFN-γR) or STAT1 signaling in donor cells has been shown to result in reduced induction of acute graft-versus-host disease (GVHD). In this study, we unexpectedly observed increased activation and expansion of donor lymphocytes in both lymphohematopoietic organs and GVHD target tissues of IFN-γR/STAT1-deficient recipient mice, leading to rapid mortality following the induction of GVHD. LPS-matured, BM-derived Ifngr1-/- Stat1-/- DCs (BMDCs) were more potent allogeneic stimulators and expressed increased levels of MHC II and costimulatory molecules. Similar effects were observed in human antigen-presenting cells (APCs) with knockdown of Stat1 by CRISPR/Cas9 and treatment with a JAK1/2 inhibitor. Furthermore, we demonstrated that the absence of IFN-γR/STAT1 signaling in hematopoietic APCs impaired the presentation of exogenous antigens, while promoting the presentation of endogenous antigens. Thus, the indirect presentation of host antigens to donor lymphocytes was defective in IFN-γR/STAT1-deficient, donor-derived APCs in fully donor chimeric mice. The differential effects of IFN-γR/STAT1 signaling on endogenous and exogenous antigen presentation could provide further insight into the roles of the IFN-γ/STAT1 signaling pathway in the pathogenesis of GVHD, organ rejection, and autoimmune diseases.

Recent advances in ocular graft-versus-host disease.

Ocular graft-versus-host-disease (GVHD) remains a significant clinical complication after allogeneic hematopoietic stem cell transplantation. Impaired visual function, pain, and other symptoms severely affect affected individuals' quality of life. However, the diagnosis of and therapy for ocular GVHD involve a multidisciplinary approach and remain challenging for both hematologists and ophthalmologists, as there are no unified international criteria. Through an exploration of the complex pathogenesis of ocular GVHD, this review comprehensively summarizes the pathogenic mechanism, related tear biomarkers, and clinical characteristics of this disease. Novel therapies based on the mechanisms are also discussed to provide insights into the ocular GVHD treatment.

Pre-transplant CD69+ extracellular vesicles are negatively correlated with active ATLG serum levels and associate with the onset of GVHD in allogeneic HSCT patients.

Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Rabbit anti-T lymphocyte globulin (ATLG) in addition to calcineurin inhibitors and antimetabolites is a suitable strategy to prevent GVHD in several transplant settings. Randomized studies already demonstrated its efficacy in terms of GVHD prevention, although the effect on relapse remains the major concern for a wider use. Tailoring of ATLG dose on host characteristics is expected to minimize its side effects (immunological reconstitution, relapse, and infections). Here, day -6 to day +15 pharmacokinetics of active ATLG serum level was first assayed in an explorative cohort of 23 patients by testing the ability of the polyclonal serum to bind antigens on human leukocytes. Significantly lower levels of serum active ATLG were found in the patients who developed GVHD (ATLG_AUCCD45: 241.52 ± 152.16 vs. 766.63 +/- 283.52 (μg*day)/ml, p = 1.46e-5). Consistent results were obtained when the ATLG binding capacity was assessed on CD3+ and CD3+/CD4+ T lymphocytes (ATLG_AUCCD3: 335.83 ± 208.15 vs. 903.54 ± 378.78 (μg*day)/ml, p = 1.92e-4; ATLG_AUCCD4: 317.75 ± 170.70 vs. 910.54 ± 353.35 (μg*day)/ml, p = 3.78e-5. Concomitantly, at pre-infusion time points, increased concentrations of CD69+ extracellular vesicles (EVs) were found in patients who developed GVHD (mean fold 9.01 ± 1.33; p = 2.12e-5). Consistent results were obtained in a validation cohort of 12 additional ATLG-treated HSCT patients. Serum CD69+ EVs were mainly represented in the nano (i.e. 100 nm in diameter) EV compartment and expressed the leukocyte marker CD45, the EV markers CD9 and CD63, and CD103, a marker of tissue-resident memory T cells. The latter are expected to set up a host pro-inflammatory cell compartment that can survive in the recipient for years after conditioning regimen and contribute to GVHD pathogenesis. In summary, high levels of CD69+ EVs are significantly correlated with an increased risk of GVHD, and they may be proposed as a tool to tailor ATLG dose for personalized GVHD prevention.

1281: EXTRACELLULAR MITOCHONDRIA EXACERBATE GRAFT-VERSUS-HOST DISEASE MORTALITY IN A MOUSE MODEL

Introduction: Graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HSCT). Previous studies have shown an association between pre-transplant conditioning mediated tissue damage and incidence of GVHD, albeit the pathobiology is not clearly delineated. Recent studies have shown that tissue damage mediates release of extracellular mitochondria (exMito) and associated byproducts, which have immunomodulatory functions due to their archaebacterial origin. However, the role of exMito in the pathogenesis of GVHD has not been explored. Methods: Intestinal epithelial cells (Caco-2) were subjected to doses of irradiation used as pre-transplant conditioning. Subsequently, changes in intracellular and extracellular mitochondrial quantity and function were evaluated. BALB/c mice were subjected to total body irradiation (TBI) and plasma was analyzed to evaluate the quantity and function of circulating exMito. Finally, BALB/c mice undergoing a MHC-mismatched allogeneic HSCT were co-transplanted with exogenous isolated syngeneic mitochondria to study the impact of exMito on GVHD mortality. Results: Seahorse XF flux assays revealed that irradiation impaired intracellular mitochondrial function with a decrease in basal, ATP-linked and maximal respiratory capacity in Caco-2 cells. Analyses of the cell-culture supernatant showed elevated exMito and increased oxidative mitochondrial protein modification (carbonylation), following irradiation. BALB/c mice which received TBI demonstrated morphological signs of mitochondrial fission and damage in intestinal epithelium along with an increase in the amount of exMito in the plasma. In addition, exMito isolated from the plasma of irradiated animals had a markedly diminished response to pyruvate/malate-dependent-induction of mitochondrial membrane potential suggestive of damage. Finally, recipient mice co-transplanted with damaged cell-free mitochondria in a MHC-mismatched allogeneic HSCT demonstrated dose-dependent increased GVHD-induced mortality. Conclusions: The results indicate that conditioning regimen-mediated extracellular release of damaged mitochondria plays a role in GVHD mortality. Future studies targeting therapies to preserve mitochondrial function and decrease extracellular mitochondrial load for GVHD management is warranted.