Graft-versus-host Disease Response Research Articles

AGvHDtrackR and cGvHDtrackR: shiny applications for graft versus host disease management and clinical data collection.

Graft-versus-host disease (GvHD) is one of the most common and troublesome complications after allogeneic hematopoietic stem cell transplantation (HSCT). Despite adequate GvHD prophylaxis, 30-50% of the patients still develop acute or chronic GvHD, often requiring multiple lines of therapy. Therefore, it is crucial to closely monitor the onset and the response of GvHD to therapies to identify the best available treatment for each patient. Currently, some applications (desktop or mobile) that allow to score GvHD severity at the bedside are available. However, none of the published systems is designed to record ongoing therapies and to upload data in a database, which can support both the clinical decision-making process as well as data collection. To this aim, we developed two Shiny apps: aGvHDtrackR for acute GvHD and cGvHDtrackR for chronic GvHD. These applications record GvHD grading alongside the therapies used for each patient and allow to export of the data in a longitudinal patient-specific database. This is of help for the clinical management of patients and for future multicentric studies on GvHD.

Chemokine receptors are required for effector T-cell trafficking to GVHD tissues but not to bone marrow

AbstractIn allogeneic hematopoietic stem cell transplantation (allo-SCT), alloreactive donor T cells mediate the graft-versus-leukemia effect but also attack nonhematopoietic tissues, causing graft-versus-host disease (GVHD). Reducing alloreactive T-cell trafficking to GVHD target tissues while allowing their access to bone marrow (BM) and spleen, major sites of malignant hematopoiesis, is a rational strategy for reducing the GVHD risk when using alloreactive T cells as a therapeutic. Here, we show that effector T-cell (Teff) entry into BM and spleen in unmanipulated mice and in mice that received transplantation without alloreactive T cells is augmented by pertussis toxin (PTX)-sensitive chemokine receptor signaling. However, unexpectedly, in the presence of a GVH response, chemokines no longer draw T cells into BM and spleen but remain critical for their recruitment to GVHD target tissues. Consistent with this, PTX-treated Teff cells were as efficacious as untreated T cells in killing leukemia cells in BM and spleen in mice with a concurrent GVHD response. These results suggest a strategy to improve the safety of alloreactive T-cell therapeutics in treating leukemias in the context of an allo-SCT.

Cord Blood-Derived Multipotent Stem Cells Ameliorate in Vitro/in Vivo Alloreactive Responses, and This Effect Is Associated with Exosomal Microvesicles in Vitro

Human cord blood derived-multipotent stem cells (CB-SCs) have been found to have immunomodulatory capabilities that can result in inhibition of immune activation. Clinically, when used to interact with apheresed peripheral blood mononuclear cells (PBMCs) before reinfusion, they can counteract inflammation and restore immune balance in patients with autoimmune diseases, including alopecia areata and type 1 diabetes. The present study aimed to explore the potential application of CB-SCs to control donor alloreactive responses involved in allogeneic hematopoietic cell transplantation, which often results in acute graft-versus-host disease (GVHD). Phenotypically, we demonstrated that CB-SCs express CD45, CD11b, and CD9 markers on the cell surface; express Oct3/4, a transcription factor for embryonic stem cells; are negative for CD3, CD14, and CD34 expression; and have low expression of HLA-DR. In an allogeneic mixed lymphocyte culture (MLC) using human CD4 T cell enriched PBMCs and allogeneic myeloid derived dendritic cells, direct coculture with CB-SCs decreased CD4 T cell proliferation and activation, as evidenced by a marked decrease in the expression of the late activation markers CD25 and HLA-DR and a reduction of the PKH26 cell proliferation membrane lipophilic marker. Cytokine profiling of MLC supernatants revealed decreased concentrations of inflammatory proteins, including IFN-γ, IL-17, IL-13, IL-2, IL-6, and MIP1-α, along with marked increases in IL-1RA, IP-10, and MCP-1 concentrations in the presence of CB-SCs. Furthermore, transwell MLC experiments revealed that a soluble component was partially responsible for the immunomodulatory effects of CB-SCs. In this regard, exosomal microvesicles (EVs) positive for CD9, CD63, and CD81 were found in CB-SC-derived, ultrafiltered, and ultracentrifuged culture supernatants. CB-SC-EVs inhibited T cell proliferation in allogeneic MLC, suggesting a potential mode of action in allogeneic responses. Finally, CB-SCs were evaluated for their cellular therapy potential in vivo and found to ameliorate the development of GVHD responses in a xenogeneic human PBMC-induced NSG mouse model. Taken together, our results indicate that CB-SCs can directly and indirectly attenuate alloreactive CD4 T cell activation and proliferation in vitro with a potentially related EV mode of action and may have potential as a cellular therapy to control donor T cell-mediated GVHD responses in vivo.

Placenta-Derived Decidua Stromal Cells: A New Frontier in the Therapy of Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (GVHD) is a frequent and potentially life-threatening complication following allogeneic hematopoietic cell transplantation (HCT). Mesenchymal stromal cells (MSCs), rare precursors found in all body tissues, possess immunosuppressive properties and can inhibit alloreactivity both in vitro and in vivo. Two decades ago, we introduced bone marrow-derived (BM) MSCs as a novel therapy for acute GVHD. While some patients responded to BM-MSCs, the response was not universal. Commercially available BM-MSCs are now used for acute GVHD treatment in Canada, Japan, and New Zealand. The fetus is protected from the mother's immune system by the placenta, and our research found that placenta-derived decidua stromal cells (DSCs) offer a stronger immunosuppressive effect than other sources of stromal cells. Safety studies in rabbits, rats, mice, and humans have shown negligible or no side effects from BM-MSCs or DSCs. In a phase I/II trial for severe acute GVHD, we treated 21 patients (median age, 49 years; range 1.6-72 years) with severe biopsy-proven gastrointestinal acute GVHD. The median cell dose of DSCs was 1.2 × 106 (range 0.9-2.9) cells/kg body weight, with a median of 2 (range 1-6) infusions given 1 week apart. The cell viability of DSCs was 93% (range, 69%-100%), and the median cell passage number was 4 (range, 2-4). All patients responded, with a complete response of acute GVHD in 11 patients and partial response in 10 and 1-year survival of 81%. Randomized trials are needed to prove the superiority of DSCs compared to ruxolitinib and/or other novel immunosuppressive therapies.

Anandamide reduces the migration of lymphocytes to the intestine by CB2 activation and reduces TNF-α in the target organs, protecting mice from graft-versus-host disease

Graft-versus-host disease (GVHD) is a serious inflammatory illness that often occurs as a secondary complication of bone marrow transplantation. Current therapies have limited effectiveness and fail to achieve a balance between inflammation and the graft-versus-tumor effect. In this study, we investigate the effects of the endocannabinoid anandamide on the complex pathology of GVHD. We assess the effects of an irreversible inhibitor of fatty acid amine hydrolase or exogenous anandamide and find that they increase survival and reduce clinical signs in GVHD mice. In the intestine of GVHD mice, treatment with exogenous anandamide also leads to a reduction in the number of CD3+, CD3+CD4+, and CD3+CD8+ cells, which reduces the activation of CD3+CD4+ and CD3+CD8+ cells, as assessed by enhanced CD28 expression, a T cell co-stimulatory molecule. Exogenous AEA was also able to reduce TNF-α and increase IL-10 in the intestine of GVHD mice. In the liver, exogenous AEA reduces injury, TNF-α levels, and the number of CD3+CD8+ cells. Interestingly, anandamide reduces Mac-1α, which lowers the adhesion of transplanted cells in mesenteric veins. These effects are mimicked by JWH133—a CB2 selective agonist—and abolished by treatment with a CB2 antagonist. Furthermore, the effects caused by anandamide treatment on survival were related to the CB2 receptor, as the CB2 antagonist abolished it. This study shows the critical role of the CB2 receptor in the modulation of the inflammatory response of GVHD by treatment with anandamide, the most prominent endocannabinoid.

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.

Graft Versus Leukaemia (GvL): Identification & Characterisation of Alloreactive Antigens and Cognate T Cell Responses in Acute Myeloid Leukemia

Allogeneic stem cell transplantation (allo-SCT) is the most established, curative T cell cancer immunotherapy. The curative effect of allo-SCT relies on a graft-versus-leukemia (GvL) effect; but this can be difficult to dissociate from graft-versus-host disease (GvHD). For decades, the key unsolved challenge has been to identify common alloreactive peptide-human leukocyte antigen (HLA) complexes targeted by T cells that mediate GvL but not GvHD. We developed a workflow for unbiased identification of alloreactive antigens and analysis of cognate donor alloreactive T cells and their T cell receptors (TCRs; Fig 1). We studied sequential bone marrow and peripheral blood samples, collected over 15 years, from 12 high-risk AML patients who remain in continuous complete remission following allo-SCT (all >2 years). All patients had relapsed at least once pre-allo-SCT and/or were AML flow cytometry minimal residual disease (MRD)+ pre-allo-SCT. 7 had received donor lymphocyte infusions. No patients developed >1 grade GvHD. We performed whole exome sequencing of DNA from isolated patient T cells (patient germline), AML blasts and donor DNA to identify: (i) AML-specific coding variants; and (ii) germline coding single nucleotide polymorphisms (SNPs), mismatched between patient and donor. 98.7% of the mismatched variants were germline. More variants were identified in unrelated compared with sibling donors (mean: 5744 vs 3253 variants). To narrow the list of variants taken forward for functional studies, we applied two sequential filtering steps: (i) we restricted analysis to variants expressed in the patients' AML cells (determined by RNA-Seq). 28.7% of all mismatched coding variants were expressed in AML cells; and (ii) we used in silico prediction of HLA-binding affinities of peptides spanning variant amino acids to patient-specific HLA-I and -II using NetMHCpan 4.0 and NetMHCIIpan 3.0. To functionally identify alloreactive antigens, we then screened ~200-900 peptides per patient-donor pair (total 5300 peptides) for recognition by T cells present in patient peripheral blood collected 2-7 years after allo-SCT. This enriches for the identification of long-lived anti-AML responses at a time when patients were both infection- and GvHD- free. Response screening, using IFNγ ELISPOT assays, identified T cell responses to 24 germline, mismatched epitopes. In all cases, responses were only observed when the amino acid encoded by the patient SNP, but not the donor allele, was present in the peptide. The phenotype of the epitope-specific T cells was established by intracellular cytokine staining (ICS) and flow cytometry analysis, and the HLA restriction of each T cell response was determined using HLA-I, -DR, -DP and -DQ blocking antibodies. Of the 24 alloreactive peptides,19 elicited CD4+ HLA class II restricted responses and 3 were targeted by CD8+ class I restricted T cells. Two remain to be characterised (Fig 2). The population allele frequency of the germline variant SNPs targeted varies from 0.5% to 72% in the Caucasian population. Likewise, some of the restricting HLA alleles are very common (e.g. a HLA-DP allele presenting 3 epitope peptides is present in ~66% of the Caucasian population). ICS showed that epitope-specific CD4+ T cells produce Th1 cytokines (IFNg, TNFa) and degranulate (express CD107a) in response to peptide stimulation (Fig 2). Next, we performed single cell 5'RNA-seq with TCR-seq of epitope-restricted T cells (isolated via IFN-g catch assay) for all CD4+ epitope-specific T cells. Alloreactive CD4+ T cells have cytokine-producing and cytotoxic effector potential, expressing MIP1a and b, IFNg, granzyme B and FasL (Fig 2). Finally, lentiviral-based gene transfer of one of the candidate alloreactive epitope-specific TCRs into third party primary T cells confers the capacity to generate IFNg and TNFa in response to peptide stimulation, and to recognise primary AML cell-derived antigen in the presence of monocyte-derived dendritic cells (Fig 2). In vitro and in vivo killing assays are in progress. The high proportion of CD4+ T Cell responses identified by our pipeline is concordant with downregulation of HLA II expression on AML blasts at relapse post-allo-SCT, which is a common mechanism of immune evasion, found in one-third of patients. Our approach dissects the biology of alloreactive anti-AML GvL and GvHD responses and to manipulate them for therapeutic purposes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Extracorporeal photopheresis in paediatric patients: A retrospective comparison between different 'off-line' protocols.

Extracorporeal photopheresis (ECP) has been shown to be an effective treatment for graft-versus-host disease (GvHD). However, information regarding lymphocyte collection for ECP in children is limited. The aim of this study was to analyse and compare lymphocyte collection for ECP in children using different devices and protocols. Moreover, we have studied both safety and variables of the infused product related to treatment efficacy. This was a retrospective study of 91 patients who underwent 1524 apheresis procedures with either the COBE Spectra or Spectra Optia system. The comparison study between the Optia protocols (MNC and CMNC) was prioritized. We analysed 578 procedures using the Optia blood cell separator: 204 and 374 using the MNC and the CMNC protocol, respectively. The Optia CMNC protocol showed better collection efficiency, with increased lymphocyte collection per kg of body weight (p< 0.001). On multivariate analysis, the type of protocol showed no relationship with haematocrit or platelet loss. Most procedures were well-tolerated, with the most frequent adverse events related to venous access (21.7%). Seventy-one percent of patients had either partial or complete clinical GvHD response. In the multivariate model, only two variables were associated with a better response to ECP, younger age and a greater increase of B lymphocytes after treatment. Lymphocyte collection for ECP is well-tolerated in most children, achieving complete or partial response in more than half of GvHD patients. CMNC is the optimal software to perform lymphocyte collection in children.

Naive T Cells in Graft Versus Host Disease and Graft Versus Leukemia: Innocent or Guilty?

The outcome of allogeneic hematopoietic cell transplantation (allo-HCT) largely depends on the development and management of graft-versus-host disease (GvHD), infections, and the occurrence of relapse of malignancies. Recent studies showed a lower incidence of chronic GvHD and severe acute GvHD in patients receiving naive T cell depleted grafts compared to patients receiving complete T cell depleted grafts. On the other hand, the incidence of acute GvHD in patients receiving cord blood grafts containing only naive T cells is rather low, while potent graft-versus-leukemia (GvL) responses have been observed. These data suggest the significance of naive T cells as both drivers and regulators of allogeneic reactions. The naive T cell pool was previously thought to be a quiescent, homogenous pool of antigen-inexperienced cells. However, recent studies showed important differences in phenotype, differentiation status, location, and function within the naive T cell population. Therefore, the adequate recovery of these seemingly innocent T cells might be relevant in the imminent allogeneic reactions after allo-HCT. Here, an extensive review on naive T cells and their contribution to the development of GvHD and GvL responses after allo-HCT is provided. In addition, strategies specifically directed to stimulate adequate reconstitution of naive T cells while reducing the risk of GvHD are discussed. A better understanding of the relation between naive T cells and alloreactivity after allo-HCT could provide opportunities to improve GvHD prevention, while maintaining GvL effects to lower relapse risk.

The Effects of Interferons on Allogeneic T Cell Response in GVHD: The Multifaced Biology and Epigenetic Regulations.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. This beneficial effect is derived mainly from graft-versus-leukemia (GVL) effects mediated by alloreactive T cells. However, these alloreactive T cells can also induce graft-versus-host disease (GVHD), a life-threatening complication after allo-HSCT. Significant progress has been made in the dissociation of GVL effects from GVHD by modulating alloreactive T cell immunity. However, many factors may influence alloreactive T cell responses in the host undergoing allo-HSCT, including the interaction of alloreactive T cells with both donor and recipient hematopoietic cells and host non-hematopoietic tissues, cytokines, chemokines and inflammatory mediators. Interferons (IFNs), including type I IFNs and IFN-γ, primarily produced by monocytes, dendritic cells and T cells, play essential roles in regulating alloreactive T cell differentiation and function. Many studies have shown pleiotropic effects of IFNs on allogeneic T cell responses during GVH reaction. Epigenetic mechanisms, such as DNA methylation and histone modifications, are important to regulate IFNs’ production and function during GVHD. In this review, we discuss recent findings from preclinical models and clinical studies that characterize T cell responses regulated by IFNs and epigenetic mechanisms, and further discuss pharmacological approaches that modulate epigenetic effects in the setting of allo-HSCT.

Immune Profiling To Predict Treatment Response From Extracorporeal Photopheresis In Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients receiving stem cell transplant. Extracorporeal photopheresis has demonstrated efficacy in a proportion of GVHD patients that are steroid refractory. However, given the expense, the challenging logistics, and unpredictable GVHD response to ECP, a predictive immune biomarker to assist with the selection of patients to treat with ECP can make this treatment modality more efficacious and cost-effective. One of the challenges of using leukocyte as biomarkers is that any single leukocyte population does not adequately describe the immune system. We have recently described an approach to characterize the immune system in a comprehensive manner. We have used a combination of whole blood quantitative flow cytometry and bioinformatics to generate immune profiles among patients with GVHD who were prescribed ECP. GVHD grading and clinical responses were confirmed by serial physical exams and review of medical records over a 6 month period. Response to ECP was defined as a decrease in clinical grade of GVHD (IBMTR and NIH consensus criteria) at the end of treatment. Peripheral blood drawn prior to initiation of ECP was used to generate a patient's immune phenotype by analyzing 33 lymphoid and myeloid surface marker combinations by flow cytometry. Immune phenotypes from 27 age-matched healthy volunteers were used as controls.From July 2011 to April 2013, 20 patients were enrolled (3 aGVHD, 17 cGVHD). At the time of this abstract, 15 patients were evaluable for treatment response, with 6 responders and 9 non-responders (median time on treatment 5.28 months, range 0.25 – 6 months). Using multivariate analysis by principal component analysis and unsupervised hierarchical clustering, we identified clustering patterns (immune profiles) in the composite immune phenotypes of the GVHD patients and controls. The healthy controls clustered together into a single immune profile. Four of the six responders clustered closely together and away from the 9 non-responders and healthy controls. Anova analysis identified 21 individual markers that were different among the clusters. Using non-parametric Wilcoxon comparison, we found that patients who responded to ECP had lower numbers of lymphocytes (p=2.5x10-12), CD4 T cells (p=0.002), CD4+CD69+ T cells (p=0.02), and CD8+CTLA4+ (p=1.8x10-5) than healthy controls and non-responders.To our knowledge, this is the first report of a bioinformatics approach to comprehensively examine systemic immune profiles of GVHD patients. Our results provide the proof-of-concept for using this method to identify an immune profile as a predictive biomarker in the treatment of GVHD. Our data supports the need for a larger study to definitively define the immune profiles and validate this approach. Disclosures:No relevant conflicts of interest to declare.

Abstract 2945: Novel engineered B-NDG B2M/KO mice plus and steroid treated B-NDG Mice shows delayed GvHD response when engrafted with hPBMC

Abstract Severely immunodeficient mouse strains, such as NOD.Cg-Prkdcscid Il2rg-/-, have been used to study the immune response in humans. These mice lack T, B and natural killer (NK) cells and allow for engraftment of human peripheral blood mononuclear cells (PBMCs). However, hPBMC engraftment may lead to severe acute xenogeneic graft versus host disease (GvHD) which arises due to a response from T cells against human leukocyte antigens (HLA) that are encoded by major histocompatibility complex (MHC) class I and II. GvHD shortens animal life span and results in only a brief window of evaluation of human immune cell functions in such mouse models. Efforts have been undertaken to reduce human PBMC-induced GvHD and to subsequently extend the window of human immunity assessment by eliminating mouse MHC I and MHC II. While these beta-2-microglobulin (B2M) knockout mice which are MHC deficient showed fewer symptoms of GvHD, an unintended result was the shorter half life of the antibodies which lead to its rapid clearance. This occurred because B2M is also associated with neonatal Fc receptor (FcRn) and is important to FcRn-mediated in vivo antibody recycling and retention. This is a pharmacokinetic limitation because it renders antibody efficacy assessment and makes these mice unsuitable for studying immune responses. To overcome this limitation, we engineered B-NDG B2M knockout mice plus (NOD.Cg-Prkdcscid Il2rg-/-/Bcgen) that is deficient in MHC I expression but expresses the B2M gene fused in the FcRn gene. In the present study we demonstrate the phenotypic changes in the immune system of the engineered mice using flow cytometry and validate immune tolerance developed over the several weeks of post engraftment by looking at the homeostatic balance between CD8/Treg ratio in the partially reconstituted humanized immune systems. In a parallel study, we used corticosteroid treatment in hPBMC induced B-NDG mice GvHD model. Corticosteroids are the fine line of therapy for GvHD and the phenotypic response generated in B-NDG mice confirms this finding in our GvHD model. Citation Format: Lan Gao, Shruti Sharma, Eugene Lin, Xiaoen Wang, Chengzhang Shang, Yuting Hu, W. Frank An, Mehma Kaur Chawla, Kaiqi Li, Qingcong Lin, Zhaoxue (Luke) Yu, Yujie Liu. Novel engineered B-NDG B2M/KO mice plus and steroid treated B-NDG Mice shows delayed GvHD response when engrafted with hPBMC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2945.

Complement Promotes Gvhd through Suppressing Autophagy in Recipient Dendritic Cells after Allogeneic Hematopoietic Cell Transplantation in Mice

Graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic stem cell transplantation (allo-HCT). Traditional therapies have targeted T cells, yet immunostimulatory dendritic cells (DCs) are critical in the pathogenesis of GVHD. Autophagy is a self-degradative process for cytosolic components and is required for maintaining homeostasis after allo-HCT. Complement receptors C3aR/C5aR can regulate immune responses by translating information gathered by complement fluid phase sensors into cellular responses. C3aR/C5aR have been reported to activate the autophagy inhibitor mTOR in cells. While depletion of autophagy in recipient DCs aggravates GVHD severity, and enhancing autophagy represents as a promising approach for treating immunological diseases, it is not known how escalation of autophagy regulates DC-functionality in GVHD pathogenesis. In the current study, we found that deficiency of C3aR/C5aR signaling significantly upregulated mTOR-independent autophagy in DCs (Figure 1B), which is associated with increased apoptosis, and reduced activation and antigen presenting capacity of C3aR/C5aR-/-DCs (Figure 1, A-C).To evaluate the role of C3aR/C5aR in modulating DC function in GVHD pathogenesis, we compared the severity of GVHD in WT versus C3aR/C5aR-/- recipients using murine models of allo-HCT. C3aR/C5aR-/- recipients developed less severe GVHD than their WT counterparts, reflected by a significantly higher rate of survival and lower clinical scores (Figure 1D). Donor T-cell survival and Th1-differentiation were significantly decreased, whereas Treg generation was increased in C3aR/C5aR-/- recipients. Glycolytic activity of donor T cells was significantly abrogated in the absence of recipient C3aR/C5ar (Figure 1E). Donor T-cells expressed lower levels of chemokine receptors, CXCR3 and CCR6, which were likely contributed to the decreased migration of the effector T cells into GVHD target organs of C3aR/C5aR-/- recipients. Using chimeric recipients in which either the hematopoietic or non-hematopoietic compartment is deficient for C3aR/C5aR, we found that C3aR/C5aR in the recipient hematopoietic cells was primarily responsible for donor T-cell response and pathogenicity in GVHD. Transfusion of C3aR/C5aR-/- DCs along with allo-HCT mitigated GVHD severity, indicating that C3aR/5aR signaling in DCs plays a predominant role in the pathogenesis of GVHD. Elevated autophagy was required for the suppressive effect of C3aR/C5aR-/- DCs on GVHD as autophagy inhibitor chloroquine (CQ) reverted the GVHD-suppressive effect of C3aR/C5aR-/-DCs (Figure 1F). For translational purpose, we evaluated the therapeutic potential of a combination of C3aR and C5aR antagonists, and found that pharmacological blockade of C3aR/C5aR could effectively reduce GVHD severity while sparing GVL effect through persevering CD8 cytolytic activity.The current study reveals for the first time that escalation of autophagy in DCs is an effective therapeutic approach for the control of GVHD, and establishes and interaction between complement and autophagy in allo-HCT. We also provide a novel strategy of enhancing autophagy as a DC-based vaccination strategy for the control of GVHD after transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pseudo-Allogeneic CAR-T Therapy after Allogeneic Stem Cell Transplantation in Relapsed/Refractory B-Cell NHL

Introduction Chimeric antigen receptor T-cell (CAR-T) therapy has emerged as a powerful therapy for relapsed and refractory (R/R) B-cell malignancies. A growing body of research suggests that CAR-T therapy can be safe and effective in patients who have previously received allogeneic hematopoietic stem cell transplantation (alloHSCT). "Pseudo-allogeneic" CAR-T therapy refers to manufacture of engineered T-cells collected from an alloHSCT recipient exhibiting 100% donor chimerism. This CAR-T approach poses theoretical risks due to alloreactivity and increased CAR-T toxicity. Most studies to date in this area have enrolled patients with leukemia. We present here a single-center experience with pseudo-allogeneic CAR-T following alloHSCT in 6 patients with large B-cell lymphoma (LBCL). Patients and Methods Patients with R/R LBCL who underwent treatment with axicabtagene ciloleucel (axi-cel) CAR-T after prior alloHSCT were identified. All patients underwent lymphodepletion with cyclophosphamide and fludarabine prior to CAR-T infusion. CAR-T toxicities, namely cytokine release syndrome (CRS) and immune effector-cell associated neurotoxicity syndrome (ICANS) were graded in accordance with the 2019 American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading. Results Six patients who underwent axi-cel CAR-T due to post-alloHSCT R/R LBCL were identified. All patients had stage ≥II disease. At the time of CAR-T apheresis, all patients had ≥95% donor chimerism and were off immunosuppression, without evidence of GVHD. Manufacturing failure occurred in only one patient (case 2) due to low viability and upon second apheresis, manufacture was successful. All 6 pseudo-allogeneic CAR-T therapy patients tolerated infusion and did not show increased incidence or severity of CRS or ICANS compared to our center's experience with standard CAR-T patients, with only one patient experiencing greater than grade 2 toxicity. Three of six patients developed graft-versus-host disease (GVHD); however, one case was in the setting of PD-1 blockade and two were isolated cutaneous manifestations. There was no incidence of severe GVHD. All three cases of GVHD underwent CAR-T within ten months after alloHSCT, whereas the three patients without GVHD were &amp;gt;1 year post-HSCT at time of CAR-T. Two cases who had donor lymphocyte infusions prior to CAR-T did not develop GVHD. In terms of efficacy, three patients achieved a complete remission. There was no perceptible relationship between the development of GVHD and the response to CAR-T. Four of six had cytopenias at day 30 after CAR-T, with only two exhibiting persistent cytopenias at day 90. Lastly, in this small sample size, no differences were seen in incidence of GVHD or treatment response between haploidentical, matched-related, and matched-unrelated alloHSCT donors. Conclusion In our experience, pseudo-allogeneic CAR-T therapy following alloHSCT appears to be a safe and well-tolerated treatment modality for R/R B-cell lymphoma. The efficacy of this modality will need to be borne out in further studies. Disclosures No relevant conflicts of interest to declare.

Fecal microbiota transplantation for graft-versus-host disease in children and adults: methods, clinical effects, safety

Was to evaluate clinical efficacy, adverse events and changes in the gut microbiome after fecal microbiota transplantation (FMT) in patients with gastrointestinal (GI) form of graft-versus-host disease (GVHD). The prospective single-center study in R.M. Gorbacheva institute included 27 patients with GI GVHD after allogeneic stem cell transplantation. 19 patients received FMT, 8 patients received placebo. Clinical scales for GI autoimmune diseases were used to evaluate response. Microbiome alterations were assessed with multiplex PCR. After FMT higher overall bacterial mass (р=0.00088), higher bacterial numbers ofBifidobacteriumspp. (р=0.021),Escherichia coli(р=0.049) andBacteroides fragilisgr. (р=0.000043) compared to placebo group. Also higher bacterial mass was observed in patients with clinical response (р=0.0057). The bacterial mass after procedure in non-responders was compared to the placebo group (р=0.31). Partial response of GVHD was achieved faster in the FMT group compared to placebo (median 4 days vs 48 days,p=0.014). Complete response was observed in 8 (42%), 14 (74%) and 16 (84%) at 30, 60 and 90 days respectively, while in the placebo group only 0%, 1 (13%) and 4 (50%) achieved complete response at the same time points. The incidence and severity of adverse events was comparable between FMT and the placebo group. FMT in patients with refractory GI GVHD was associated with favorable clinical outcomes and recovery in certain marker bacterial populations. Multiplex PCR can be used to assess an engraftment of a donor microbiota. FMT in GI GVHD was not associated with life-threatening adverse events, but further studies are required to validate clinical efficacy.

A real life use of ruxolitinib in patients with acute and chronic graft versus host disease refractory to corticosteroid treatment in Latin American patients.

IntroductionGraft-versus-host disease (GVHD) is a serious complication in allogeneic transplantation. The first-line treatment is high doses of corticosteroids. In the absence of response to corticosteroids, several immunosuppressive drugs can be used, but they entail an elevated risk of severe infections. Added to this, there are patients who do not improve on any immunosuppressive treatment, with subsequent deteriorated quality of life and high mortality. Ruxolitinib has been shown to induce responses in refractory patients. In this study we have presented our real-life experience.MethodsA retrospective analysis was performed on patients with severe GVHD refractory to corticosteroids. Demographic, previous treatment, response and mortality data were collected.ResultsSince 2014, seventeen patients with GVHD were treated with ruxolitinib due to refractoriness to corticosteroids and immunosuppressants and a few to extracorporeal photopheresis, 8 with acute GVHD (1 pulmonary, 4 cutaneous grade IV and 3 digestive grade IV) and 9 with chronic GHVD (5 cutaneous sclerodermiform, 2 pulmonary and 1 multisystemic). The overall response to ruxolitinib treatment for acute GVHD was 80%, 40% with partial response and 40% with complete remission. Global response in chronic GVHD was 79%. The GVHD mortality was only seen in acute disease and was 40%. Causes of mortality in those patients were severe viral pneumonia, post-transplantation hemophagocytic syndrome and meningeal GVHD refractory to ruxolitinib.ConclusionsIn our series, the use of ruxolitinib as a rescue strategy in acute or chronic GVHD was satisfactory. Ruxolitinib treatment in patients with a very poor prognosis showed encouraging results. However, the GVHD mortality remains high in refractory patients, showing that better therapeutic strategies are needed.

Successful and Safe Treatment of Intestinal Graft-Versus-Host Disease (GvHD) with Pooled-Donor Full Ecosystem Microbiota Biotherapeutics

Introduction Intestinal Graft-versus-Host Disease (GvHD), following allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT), comes with a high mortality rate and a reduced life-expectancy. In this context, failure to respond to steroid therapy is associated with an absence of further therapeutic options, thereby representing an unmet medical need. A marked reduction in gut microbiota diversity leading to loss of functions was strongly associated with reduced overall survival in GvHD, while a high gut microbiota diversity appears to be protective. Aiming at restoring microbiome functions, Fecal Microbiota Transfer proved to be a promising treatment modality in this challenging clinical situation, with recent studies reporting favorable results in steroid refractory-acute GVHD (SR-aGvHD) patients. Here we report on the use of "MaaT013", a standardized, pooled-donor, high-richness microbiota biotherapeutic, used to treat 8 patients with intestinal-predominant aGvHD. Patients and methods Eight allo-HSCT recipients with steroid-dependent or steroid-refractory gastrointestinal GvHD (classical aGVHD n=3, late-onset aGVHD n=2; aGvHD with overlap syndrome n=3) were treated with the MaaT013 biotherapeutic as part of a compassionate use program. These patients had previously received and failed 1 to 5 lines (median 2.5) of GvHD systemic treatments. MaaT013 microbiota biotherapeutics were provided as a pharmaceutical preparation to hospitals by the developer, "MaaT Pharma". Each patient received 1 to 3 doses (median: 3; total doses administered: 21) of MaaT013, a 150 mL bag, by enema (n=7) or nasogastric tube (n=1). GvHD response was evaluated 7 days after each administration and 28 days after the first dose. Prepared under Good Manufacturing Practices, MaaT013 biotherapeutics are characterized by a highly consistent richness of 455 +/- 3% Operational Taxonomic Units (OTUs) and an Inverse Simpson index greater than 20. Batch release specifications are based on potency (viability), identity (diversity), and purity (microbiological safety testing following regulatory guidelines and proportion of proinflammatory species), ensuring the desired consistency between batches. Results We observed 6/8 positive responses at D28 after first dosing, including 3 Complete Responses (CR), one Very Good Partial Response (VGPR), and 2 Partial Responses (PR). Considering the best GI response achieved, all (8/8) patients experienced at least a PR, with 3 CRs, 2 VGPRs and 3 PRs. The 3 patients with CR were still alive at last follow-up (60 to 192 days after last dosing; median: 115 days) and were able to taper and stop steroids and immunosuppressants without relapse of GI symptoms. Of note, among these 3 patients, mild mouth chronic GvHD symptoms persisted in one patient, and relapse of hematologic malignancy was observed in another patient. Among the 8 treated patients, 5 were still alive at last follow-up (60 to 232 days after last dosing; median: 125 days). The safety of the MaaT013 microbiota biotherapeutic was satisfactory in all patients. One patient developed a possibly related sepsis one day after the third MaaT013 administration. In the latter case, no pathogen was identified in blood cultures, and the patient recovered after a course of antibiotics. Conclusions We herein report for the first time the treatment of 8 patients with steroid-dependent or steroid-refractory intestinal aGvHD using a full ecosystem, standardized, pooled-donor, high-richness biotherapeutic. Overall, 3/8 patients attained a complete response following treatment with the off-the-shelf MaaT013 product, shown to be safe and effective in these immunocompromised patients with severe conditions, warranting further exploration of the full ecosystem microbiota restoration approach. Disclosures Malard: Sanofi: Honoraria; JAZZ pharmaceutical: Honoraria; Astellas: Honoraria; Therakos/Mallinckrodt: Honoraria; Keocyte: Honoraria; Janssen: Honoraria. Plantamura:MaaT Pharma: Employment. Carter:MaaT Pharma: Employment. Chevallier:Jazz Pharmaceuticals: Honoraria; Incyte: Consultancy, Honoraria; Daiichi Sankyo: Honoraria. Blaise:Pierre Fabre medicaments: Honoraria; Molmed: Consultancy, Honoraria; Jazz Pharmaceuticals: Honoraria; Sanofi: Honoraria. Mohty:Jazz Pharmaceuticals: Honoraria, Research Funding.

Graft Versus Host Disease (GVHD) after HLA-Mismatched Haploidentical Transplantation with Post-Transplant Cyclophosphamide Compared to Matched Unrelated Donor: Incidence, Distribution and Response to Therapy

Unmanipulated T-cell replete HLA-mismatched haploidentical transplantation (haplo) with post-transplant cyclophosphamide (PTCY) is now widely used for patients lacking a suitably matched donor. The use of PTCY post haplo results in lower chronic GVHD rates compared to match unrelated donors (MUD) without PTCY. GVHD after PTCY in the haplo setting may have different presentation, response to treatment and impact on survival endpoints compared to a standard GVHD prophylaxis of calcineurin inhibitor plus MMF or methotrexate for MUD patients. To check for differences in GVHD presentation and response to treatment between MUD and haplo with PTCY, we assessed 394 consecutive patients who developed acute or chronic GVHD after receiving their first allogeneic transplantation (HCT) from a 10/10 HLA MUD (n=179) or a haplo (n=215) at our center between 2008 and 2017. Median follow up for survivors was 52.5 months. Our institution has prospectively documented onset, grading and therapy of patients with GVHD using a single dedicated practitioner since 2005. All haplo patients received standard GVHD prophylaxis of tacrolimus (days 5-180), MMF (stop day 35) and PTCY at 50mg/kg on days 3 and 4. Most commonly used GVHD prophylaxis for MUD patients were tacrolimus/methotrexate (68%) and tacrolimus/MMF (26%). MUD recipients were older (median age 56 vs 53 years, p=0.007), were more likely to be white (93% vs 58%, p&lt;0.001), received mainly PBSC as graft source (76% vs 63%, p0.013) and had a higher HCT-comorbidity index (HCT-CI≥3, 64% vs 47%, p&lt;0.001) compared to haplo recipients. The cumulative incidences for grade II-IV and III-IV aGVHD at day 180 post HCT were similar between both donor sources at 35% and 11% for haplo compared to 44% and 16% for MUD (p=NS). Haplo patients had lower cumulative incidence of mod-severe cGVHD at 22% versus 31% for MUD (p=0.026).The median times to onset of grade II-IV acute GVHD and moderate-severe chronic GVHD were: acute, 56 vs 49 days (p=0.19) and chronic 213 vs 280 days (p=0.011) for haplo versus MUD patients respectively. Among patients with grade II-IV acute GVHD, there was no significant difference in organ involvement between Haplo (n=101) and MUD (n=96) with skin being most commonly affected (74% haplo vs 71% MUD), Gut (70% haplo vs 69% MUD) and liver (14% haplo vs 17% MUD). For patients who developed chronic GVHD, organ involvement distribution is shown in table 1. Haplo patients had less involvement of the eyes (46% vs 75% for MUD, p&lt;0.001) and of the joints/fascia (12% vs 36%, p=0.001). Among patients who developed grade II-IV acute GVHD, there was no difference in all-cause mortality (28% vs 19%, p=0.34) and being off immunosuppression at one 1 year (49% vs 51% p=0.80) between haplo and MUD recipients. Among patients who developed cGVHD, haplo recipients had similar all-cause mortality (22% vs 18%, p=0.89) but were more likely to be off immunosuppression at 2 years post HCT (63% vs 43% p=0.03) compared to MUD (figure 1). A cox model was conducted on survival and relapse endpoints where donor type was retained in all models. The fixed covariates tested in Cox models included age (&lt;55, &gt;=55), gender, race, diagnosis, regimen intensity, graft source (BM, PBSC), disease risk index (low/intermediate, high/very high), CIBMTR risk (low, intermediate, high), HCT-CI (0-2, &gt;=3), CMV status, year of transplantation (2008-2012, 2013-2015, 2016-2017). The following variables were evaluated as time-dependent covariates in Cox models: all-grade cGVHD, moderate-severe cGVHD, severe cGVHD, grade 2-4 aGVHD, grade 3-4 aGVHD. Variables were selected if p values were less than 0.05. Developing grade 3-4aGVHD and severe chronic GVHD were both associated with worse OS, DFS and TRM (table 2). Both aGVHD and cGVHD were not significant factors for relapse. Our analysis reveals that compared to MUD, haplo transplant results in significantly lower incidence of moderate-severe chronic GVHD, faster cGVHD onset, different organ distribution and a higher chance of coming off immunosuppression. This data, added to prior publications from our center and others showing similar OS and DFS between MUD and haplo, enforces the notion that haplo transplant with PTCY is at least equivalent to MUD transplant. An ongoing BMTCTN 1702(CTRL-ALT-D) study will help answer assess this in a prospective fashion. figure 1: Chronic GVHD patients who are Immunosuppression free at 2 years Table 1: Organ Distribution of Chronic GVHD by Donor Type Disclosures No relevant conflicts of interest to declare.

Outcome of Repeated Vaccination to Hepatitis B Virus in Patients Failing to Respond to Vaccination Following Allogeneic Hematopoietic Stem Cell Transplantation (HSCT): If at First You Don't Succeed, Try Try Again

Background: Recipient immunity is compromised after HSCT, obligating patients (pts) to take prophylactic antimicrobial and antiviral agents and to be reimmunized to viral and bacterial pathogens. Hepatitis B virus (HBV) infection is a major public health problem, with about 30% of the world population having serological evidence of current or past HBV infection. HBV vaccination post-HSCT is imperative in these pts, as most lose protective HBV surface antibodies (anti-HBs) following conditioning, placing them at high risk for HBV reactivation. Guidelines recommend delaying vaccination (including HBV) for 6-12 months following transplantation to allow for cellular and humoral immune recovery. Even with delaying vaccination, immunosuppression, graft-versus-host disease (GVHD), and delayed immune reconstitution hinder the effectiveness of vaccines. The efficacy of HBV vaccination is not well defined in pts on immunosuppressive therapy (IST) and/or in those with GVHD. Further, little data exists on the efficacy of HBV revaccination in pts failing to respond to the 1st vaccination series. We studied factors impacting the success of vaccination in pts undergoing one to four HBV vaccination series after HSCT. Methods: This single-center, retrospective study evaluated the effectiveness of HBV vaccine in HSCT pts by assessing protective antibody generation after vaccination. Fifty-two pts (25 female, 27 male) who received at least one 3-dose HBV vaccination series post-HSCT and who had evaluable post-vaccine anti-HBs titers were included in the analysis. Pts with negative or indeterminate anti-HBs titers following the first vaccine series were eligible to receive one or more additional series of HBV vaccinations. All pts were treated with cyclophosphamide and fludarabine based conditioning (± anti-thymocyte globulin) and received GVHD prophylaxis with either cyclosporine/tacrolimus with or without mycophenolate mofetil. The vaccine response rate over a series of vaccinations was estimated by Kaplan-Meier methods. The development of response after the first vaccination was correlated with patient baseline and post-HSCT factors including pretransplant HBV titers, vaccination time post-transplant, use of rituximab and IST and absolute lymphocyte count (ALC), CD4, and CD8 cell counts and history of acute or chronic GVHD. Results: The studied cohort included 52 HSCT pts with a median age of 22 years (range 7-62) and a variety of diagnoses (38 aplastic anemia, 6 myelodysplastic syndrome and 8 hematological malignancies). Thirty-five pts underwent HSCT from an HLA-matched donor and 17 pts received a combined haploidentical and umbilical cord blood transplant. The median time to first HBV vaccination was 12 months (8-37) post-HSCT. Following the 1st vaccination series; 19, 4 and 2 pts received a 2nd, 3rd and 4th vaccination series. The estimated cumulative anti-HBs response rates were 51.9%, 82.3%, 91.1% and 100% for the four vaccination series, respectively (Figure). A logistic regression analysis revealed: a) Pts who achieved a response after the initial vaccination series had higher CD4 counts compared to those who failed to mount a response (median CD4 count 450 vs. 300/μL, P= 0.024, Figure); b) Pts without a history of acute GVHD (n=23) were significantly more likely to respond to the 1st vaccination series compared to those with acute GVHD (n=29) (response: 69.6% vs 37.9%, P= 0.029). Other factors included in this analysis were not found to be correlated with the anti-HBs response after the initial vaccination series. Conclusions: Multiple rounds of HBV vaccination may be required before a protective antibody response is achieved. After the first vaccination series, only 51.9% of pts achieved a response, with lower pre-vaccination CD4 counts and a prior history of acute GVHD being negatively associated with vaccine success. Remarkably, with continued vaccination attempts (up to four vaccination series), all evaluable pts ultimately developed a protective anti-HBs response. Figure Disclosures Shalabi: GlaxoSmithKline: Other: Spouse is employed by GSK Pharma.

Evaluation of treatment response in oral chronic graft-versus-host patients by power doppler ultrasound: a prospective study.

To date, imaging and non-invasive examination methods for evaluating the oral tissue of patients who with oral chronic graft-versus-host disease (GVHD) are still lacking. Herein, we assessed the ultrasonography imaging characteristics of chronic GVHD patients' buccal soft tissue and evaluated the response to treatment using gray-scale and power Doppler (PD) ultrasound. The overall objective of the study was to determine the potential of ultrasonography to provide an early and objective indication of a therapeutic response to treatment intervention in oral chronic GVHD. Buccal mucosa in 21 patients with clinically diagnosis of oral chronic GVHD were assessed before and after 14 day treatment between 2016 and 2018. Ultrasonography assessment included thickness and echogenicity evaluation, and PD. Echogenicity and PD were measured and scored according to a semi-quantitative method. Ultrasonography parameters were compared with clinical activity assessments of disease activity and patient-reported measures. Following 14 day therapy, a significant decrease in PD scores and patient-reported measures was observed (p < 0.01), and clinical assessment scores also decreased (p < 0.05). Changes from baseline PD scores correlated significantly with patient-reported measures (r = 0.85, p < 0.01). Changes in PD scores correlated moderately with the changes in the clinical assessments from baseline (r = 0.46, p < 0.05). A significant decrease in PD scores was observed in the buccal soft tissue of patients that responded clinically to the treatment. A significant decrease in PD scores was also observed in patients that were failed to show clinical improvement (p < 0.05). Ultrasonography is a novel and feasible imaging technique for measurement of the response of oral chronic GVHD to therapy. Ultrasonography yields additional information about buccal mucosa and is a complementaty to clinical examination.