Graft-versus-host Disease Symptoms Research Articles

Successful Immune Reconstitution in a Patient with a TYK2 Deficiency after Allogeneic Stem Cell Transplantation from Unrelated Donors.

A boy with primary immunodeficiency, caused by a tyrosine kinase 2 (TYK2) mutation, presented with immune defects and a lifelong history of severe infections. Our aim was to determine whether allogeneic hematopoietic stem cell transplantation (HSCT) could restore the patient's immune defenses and reduce susceptibility to infection. In the absence of a suitable HLA-matched blood relative to act as a donor, the patient received an allogeneic HSCT from unrelated donors. The patient's clinical data were analyzed in the Children's Hospital of Chongqing Medical University (Chongqing, China) before transplantation and during the 4-year follow-up period using a combination of western blotting (e.g., TYK2 and STAT levels), qRT-PCR (e.g., T cell receptor rearrangement excision circles, kappa deletion element recombination circles, and TYK2 transcript levels), and flow cytometry (e.g., lymphocyte subpopulations and CD107α secretion). We found that HSCT significantly reduced the incidence of severe infections, restored normal TKY2 levels, and reversed defects such as impaired JAK/STAT signaling in response to interferon-α or interleukin-10 treatment. Although the patient did not develop acute graft-versus-host disease (GVHD) after transplantation, he did experience chronic GVHD symptoms in a number of organs, which were effectively managed. Our findings suggest that HSCT is a feasible strategy for reconstituting the immune system in TYK2-deficient patients; however, the factors associated with GVHD and autoimmune thyroiditis development in TYK2-deficient patients undergoing HSCT warrant further investigation.

Mesenchymal stem cell-derived exosomes can alleviate GVHD and preserve the GVL effect in allogeneic stem cell transplantation animal models.

Mesenchymal stem cells (MSCs) can alleviate graft-versus-host disease (GVHD) in hematopoietic stem cell transplantation (HSCT). MSCs-derived exosomes (MEXs) can mirror the biological function of their parent cells. Whether MEXs can alleviate GVHD like their parent cells or not is unclear. In this study, we investigate the effects of MEXs on GVHD and graft-versus-leukemia (GVL) effect in vitro and in HSCT animal models. MSCs were produced using bone marrow mononuclear cells (MNCs), and MEXs were separated from the supernatants of MSCs. Electron microscopy, western blot, and nanoparticle tracking analysis (NTA) were used to determine the characteristics of MEXs. The immunomodulatory function of MEXs and their effects on GVHD and GVL were examined in vitro and in vivo. Like other cell-type derived exosomes, our data revealed that MEXs were also disc-shaped vesicles with a diameter of 100-200 nm under electron microscopy and were positive for the exosomal hallmark proteins. MEXs can notably inhibit the expression of costimulatory molecules and functional cytokine secretion of dendritic cells (DCs). Meanwhile, MEXs can exert suppressive effects on T lymphocyte proliferation and activation. Moreover, MEXs can also encourage the polarization of macrophages toward the M2 type. In animal HSCT models, MEXs can promote the differentiation of Treg cells in spleens, decrease the GVHD score, increase the survival rate of mice, and preserve the cytotoxic antileukemia effects of CD8+ T lymphocytes from recipient mice. These findings showed that MEXs exert their effects by inhibiting the immunomodulatory function of DCs, macrophages, and T lymphocytes. In the animal model, MEXs ameliorate the clinical symptoms of GVHD, while maintaining the antitumor effects of CD8+ T lymphocytes. Therefore, it can be inferred that MEXs can separate GVHD from GVL in HSCT. Our study suggests that MEXs have broad clinical application potential in the prevention and treatment of GVHD in HSCT in the near future.

Flares of Acute Graft-Versus-Host Disease: Mount Sinai Acute Gvhd International Consortium (MAGIC) Study

[Background] The current standard first-line treatment for acute graft-versus-host disease (GVHD), systemic high-dose steroids, induces clinical responses in a majority of patients. Flares of GVHD after initial improvement often occur during tapering or discontinuation of immunosuppression. There is no consensus regarding the definition of GVHD flare and little has been published regarding its natural history. [Methods] We retrospectively evaluated clinical data and blood samples from 968 allogeneic hematopoietic cell transplantation (HCT) recipients from 23 Mount Sinai Acute GVHD International Consortium (MAGIC) transplant centers who achieved complete response (CR) or very good partial response (VGPR) within 4 weeks of acute GVHD treatment between 2014 and 2021. VGPR was defined as the complete resolution of acute GVHD manifestations except residual stage 1 skin disease. Flares were defined as a recurrence of acute GVHD after achievement of CR/VGPR if (1) patients had an increased symptom severity of at least 1 organ stage; (2) received intensified treatment (increase in steroid dose ≥ 0.25 mg/kg or initiation of additional systemic immunosuppression); and (3) had no prior history of primary disease relapse or donor lymphocyte infusion preceding the flare. Serum samples obtained at the time of CR/VGPR and at the time of flares were analyzed for ST2 and REG3α concentrations that generated MAGIC algorithm probabilities (MAPs) resulting in previously validated Ann Arbor (AA) scores (1, 2, and 3). [Results] The median recipient age at HCT was 55 years (range: 0 to 79); 18.6% of patients had grades III-IV acute GVHD before CR/VGPR. The median maximum daily dose of corticosteroids before CR/VGPR was 1 mg/kg methylprednisolone equivalent (range, 0.1 to 3.2 mg/kg). Flares developed within 6 months following CR/VGPR in 210/968 (21.7%) patients with a median onset of 28 days (range: 2 to 448). Symptom severity at the onset of flare symptoms varied widely (grade I: 22%; grade II: 38%; grade III-IV: 41%) and the development of a flare was associated with a five-fold increase in NRM (hazard ratio [HR], 4.84 [95% CI, 3.19-7.36], P < 0.001) when considered as a time-dependent covariate in a multivariate regression model. The large majority of non-relapse deaths after flares (54/71, 76%) were due to acute GVHD or complications from its treatment. The AA scores at the time of first CR/VGPR successfully stratified patients for risk of six-month NRM (AA1: 5%, AA2: 11%, AA3: 34%, P < 0.001) (Figure A). Increasing AA score at CR/VGPR was also associated with significantly increased risk of GVHD flares (Figure B). Not only did the risk of flare increase with each rising AA score, but the number of patients with severe (grade III/IV) symptoms at flare onset also increased (AA1: 34%, AA2: 43%, AA3: 54%). We next generated a multivariate regression model of risk factors for the development of flares. High AA scores at the time of CR/VGPR (AA2: HR, 1.81 [95% CI, 1.32-2.48], P = 0.001; AA3: HR, 3.14 [95% CI, 1.98-4.98], P < 0.001), HCT from HLA mismatched unrelated donor (HR, 1.74 [95% CI, 1.00-3.02], P = 0.049), and interestingly, rapid achievement of CR/VGPR from the time of initial treatment (≤14 days) (HR, 1.84 [95% CI, 1.21-2.80], P = 0.004) were all identified as significant risk factors. Meanwhile, no other transplant, GVHD, or treatment characteristics associated with the development of flares including maximum GVHD severity, maximum steroid dose, and use of additional immunosuppressive agents other than steroids before CR/VGPR. AA scores measured at the onset of flare symptoms in 98 patients also predicted six-month NRM (AA1, 6%; AA2, 19%; AA3 42%, P = 0.01). [Conclusion] Despite advances in treatment, flares of GVHD symptoms following excellent (CR/VGPR) responses still occur in over one fifth of patients in current practice and are associated with a five-fold increase in six-month NRM. AA scores from serum biomarkers predict outcomes despite the absence of symptoms, suggesting that MAGIC biomarkers detect subclinical damage to GI crypts that can drive later recrudescence of clinical GVHD. Measurement of the MAP at the time of response to GVHD treatment may thus help to risk-stratify tapering strategies of immunosuppressive therapy.

Combined Haploidentical Hematopoetic Stem Cell Transplantation and Liver Transplantation in a Pediatric Patient

Solid organ transplantation from the same donor is an established procedure for end-stage organ failure that developed after a previous hematopoietic stem cell transplantation (HSCT); however, it is rarely done in patients transplanted with unmanipulated haplo-HSCT. There are no pediatric reports regarding the long-term performance of organ transplantation after haplo-HSCT with post-transplant cyclophosphamide (PTCY).
 A juvenile myelomonocytic leukemia patient, who underwent unmanipulated haplo-HSCT with PTCY from her mother at the age of 3 years, developed chronic liver graft versus host disease (GvHD) which was refractory to specific GvHD treatment. Liver transplantation (LT) from her mother (the donor of her haplo-HSCT) was decided as the next line of treatment.
 LT was performed on day 540 post-HSCT, and the donor's left lateral segment was appropriately removed and attached to the recipient. The symptoms of GvHD completely regressed in a month. The patient died on day 121 after LT, because of a possible hepato-pulmonary syndrome.
 Organ failure can develop after allo-HSCT secondary to GvHD and therefore performing HSCT from a haplo-donor may be superior to a matched unrelated donor in terms of subsequent organ transplantation for organ failure.

Drug exporter expression correlates with group 3 innate lymphoid cell resistance to immunosuppressive agents

Allogeneic hematopoietic cell transplantation (HCT) is often complicated by graft versus host disease (GvHD), an alloreactive immune response triggered by tissue damage. Interleukin (IL)-22 producing type 3 innate lymphoid cells (ILC3) protect epithelial tissues against chemo(radio)therapy-induced damage, suppress alloreactive T cells and mitigate acute GvHD symptoms after allogeneic HCT. Relatively high numbers of ILC before and after allogeneic HCT has been associated with significantly reduced tissue damage and less acute GvHD. While most transplantation conditioning and GvHD prophylaxis regimens are aimed at eliminating host and alloreactive donor lymphocytes, the effect of these regimens on ILC remain elusive. Here, we studied the effect of conditioning chemotherapy and immunosuppressive agents on the survival, proliferation, activation and function of human ILC3 in vitro. Tonsil-derived ILC3 were activated and incubated with agents commonly used to prevent and treat GvHD. While fludarabine, rapamycin, mycophenolic acid and prednisolone suppressed ILC3 to a similar degree as T cells, the effect of other agents, including cyclosporine A, methotrexate, imatinib, ibrutinib and ruxolitinib, was milder on ILC3 than on T cells. ILC3 are less sensitive to immunosuppressants potentially because of their expression of functionally active ATP Binding Cassette Subfamily B Member 1 (ABCB1) drug exporter proteins. This suggests less intracellular accumulation of immunosuppressive agents, which renders ILC3 resistant to these compounds. The present findings may help to develop strategies to simultaneously maintain the tissue protective properties of ILC3 and at the same time suppress alloreactive lymphocytes, which is important in the prevention and treatment of acute GvHD.

Independent human mesenchymal stromal cell–derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model

Background aimsExtracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow–derived MSCs, this study focused on improving the MSC-EV production for clinical application. MethodsIndependent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. ResultsThe functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. ConclusionsStandardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.

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.

Cellular senescence marker p16INK4a and NFKB1 gene polymorphisms in lower gastro-intestinal acute graft versus host disease

BackgroundLower gastrointestinal (GI) graft versus host disease (GVHD) represents a severe complication in allogeneic hematopoietic stem cell transplant (HSCT) recipients with high rates of transplant-related mortality. Deregulated innate immunity reactions are the features of its pathogenesis. Cellular senescence has been considered a program of the innate immunity. We focused on lower GI GVHD from the perspective of cellular senescence. ObjectiveWe analyzed the impact of p16INK4a expression, a hallmark of cellular senescence, in intestinal biopsies of patients with lower GI GVHD symptoms and NFKB1 gene polymorphisms (rs3774937 C/T and rs3774959 A/G) on HSCT outcome. Study designFifty-two single-center patients who presented with symptoms of lower GI GVHD were analyzed in a retrospective manner. Two SNPs located in the NFKB1 gene regions (rs3774937 C/T and rs3774959 A/G) were genotyped from the peripheral blood samples collected before the start of the conditioning. All patients underwent proctosigmoidoscopy with biopsy of the mucosa. The expression of p16INK4a was analyzed in normal intestinal crypts and stroma. ResultsFifty-two patients (50% male) received HSCT for hematological diseases (acute leukemias in 67%) and developed lower GI symptoms. Patients with p16INK4a expression in the intestinal stroma were in lower risk of developing histological grade 3–4 aGVHD (RR 0.18 [95% CI 0.05–0.65]; p = 0.009). The multivariate linear regression confirmed the independent effect of p16INK4a expression on time of the lower GI aGVHD symptoms onset (Coef. 38.9 [95% CI 12.7–65.1]; p = 0.005). The NFKB1 rs3774937 CC and TT/TC genotype were present in 40 and 80% of patients with p16INK4a expression, respectively (p = 0.04). The rs3774959 AA and GG/AG genotype were present among 43 and 82% of patients with p16INK4a expression, respectively (p = 0.02). Expression of p16INK4a was associated with no clinical variable but NFKB1 genotype. ConclusionsOur results address possible new mechanisms that may lead to better understanding of HSCT-related immune complications. Cellular senescence may bring novel approaches in GVHD diagnostics and therapy.

Patient-reported symptom burden and impact on daily activities in chronic graft-versus-host disease.

Chronic graft-versus-host disease (GVHD) is a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT) treatment for hematologic malignancies. There are limited patient-reported data concerning symptom burden and effects on activities of daily living (ADL). The cross-sectional Living With Chronic GVHD Patient Survey was administered online in the United States (May-August 2020) to participants aged ≥18 years who underwent allogeneic HSCT, were diagnosed with chronic GVHD by a healthcare provider, and self-reported active chronic GVHD (within past 5 years). Information on patient demographics, disease characteristics, symptom burden, and ability to perform ADL was collected. Symptom burden was assessed using the validated Lee Symptom Scale (range from 0-100 with higher scores indicating greater burden). All data were summarized using descriptive statistics; no formal statistical comparisons were conducted. Out of 580 participants who entered the survey screener, 165 participants (28.4%) across 33 states fulfilled all study eligibility criteria, completed the entire survey, and were included (age: mean [SD], 53.7 (13.8) years; median [range], 57.0 [18-78] years; female, n= 105 [63.6%]; White, n= 137 [83.0%]). Respondents described their chronic GVHD severity primarily as moderate (n= 54 [32.7%]) or severe (n= 102 [61.8%]) at the time when symptoms were at their worst. One-third of respondents (33.9%) indicated that their chronic GVHD symptoms were at their worst for ≥1year in duration. Mean (SD; range) Lee Symptom Scale score was 44.8 (19.4; 2-100); 44% of respondents considered "dry eye" the most burdensome symptom. Almost half of respondents (n= 73 [44.2%]) rated their overall quality of life (QoL) as poor. Participants reported a detrimental impact of symptoms on ADL, including basic activities (eg, eating, personal hygiene, dressing). Survey respondents self-reported high chronic GVHD symptom burden and felt that their symptoms severely interfered with physical function and ADL. Effective strategies to mitigate chronic GVHD symptoms are needed to improve QoL among HSCT survivors.

Effective Treatment of Low Risk Acute Gvhd with Itacitinib Monotherapy

Introduction The primary treatment of acute graft-versus-host disease (GVHD), prolonged courses of high dose systemic corticosteroids (SCS), cause significant morbidities, including an increased risk of serious infections. We used validated clinical (Minnesota standard [Minn std] risk) and biomarker (Ann Arbor [AA] 1) criteria to identify a group of patients with GVHD that is low risk (LR) for treatment failure and non-relapse mortality (NRM) and who might benefit from less toxic alternatives to SCS. Itacitinib (ita), a novel JAK1 inhibitor, blocks the inflammatory cytokine pathways key to target organ damage without the myelosuppression of JAK1/2 inhibitors such as ruxolitinib. We tested the hypothesis that ita monotherapy would be a safe and effective primary treatment of low risk GVHD in a multicenter phase II trial and compared outcomes to a matched control cohort of MAGIC pts with LR GVHD treated with SCS. Methods From February 2019 to April 2021, 70 pts with LR GVHD (Minn std risk/AA1) GVHD were treated with ita 200mg/day for 28 days; responders were allowed a second 28 day cycle. Ita was discontinued without tapering. Biomarker scores were available within 30 hours from sample collections, and treatment could be delayed up to 4 days after screening to accommodate a mostly outpatient population. A contemporaneous cohort of 140 control pts, matched for key characteristics, including target organ severity, donor type, HLA-match, and GVHD prophylaxis, was created from the Mount Sinai Acute GVHD International Consortium (MAGIC) database and biorepository. Control pts were treated with standard doses of SCS (median starting dose 1.1 mg/kg). ORR at 28 days of treatment was defined as the proportion of pts achieving complete or partial response of GVHD symptoms without additional systemic therapy. Infections required laboratory/imaging proof and were considered serious if they required systemic treatment. Results Both ita and SCS treatment produced excellent ORR (ita: 89%; SCS: 86%; p=0.67) including in pts with lower GI GVHD (86% vs 86%, p=1). Similarly high ORR was observed for both cohorts for all subgroup comparisons including by GVHD grade, target organ involvement, age, donor type, HLA-match, conditioning regimen, GVHD prophylaxis, and number of days post-BMT to treatment initiation. ORR was unaffected by the time from screening to initiation of ita (≤48 hours: 90%; >48 hours: 87%; p=1). Significantly more patients treated with itacitinib achieved a response by day 7 compared to those treated with SCS (81% vs 66%, p=0.02) and among responders, ORR were equally durable up to day 90 in both groups (89% vs 88%; p=1). There were no significant differences between ita and SCS pts for 1-year relapse (18% vs 21%, p=0.64), chronic GVHD (28% vs 32%, p=0.33), NRM (4% vs 11%, p=0.21), or survival (88% vs 80%, p=0.11). Regarding the safety data of ita monotherapy, the incidence of grade ≥3 non-hematologic and non-infectious TEAEs was similar to the SCS cohort with no event occurring in >10% of pts. The incidence of new or worsening grade ≥3 neutropenia, anemia, or thrombocytopenia was similar between ita and SCS cohorts. In fact, grade ≥3 leukopenia occurred less frequently in the ita group (16% vs 31%, p=0.02). Ita treatment resulted in an 91% lower cumulative steroid dose through day 28 compared to SCS (1.9 vs 22.0 mg/kg, p<0.001) and fewer pts developed at least one serious infection within 90 days (27% vs 42%, p=0.02; Fig 1). The mean number of infections per pt was also lower (0.43 vs 0.66; rate ratio 0.65 [0.43-0.97]), as was the likelihood of developing a severe (BMT CTN grade 2 or 3) infection (odds ratio 0.50 [95% CI: 0.27, 0.93]). The reduction in serious infections was driven by fewer viral infections in the ita group (Fig 2), particularly in the first 28 days and not explained by differences in letermovir use. Conclusion A short course of itacitinib monotherapy produced very high response rates that occurred faster than treatment with SCS, were equally durable and with a similar risk for cGVHD in patients treated for LR GVHD. Importantly, ita treatment resulted in significantly less severe leukopenia and a significantly lower incidence of serious infections than SCS, likely a result of dramatically less exposure to systemic steroids. Ita monotherapy should be further studied as primary treatment for LR GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CpG-Activated Regulatory B-Cell Progenitors Alleviate Murine Graft-Versus-Host-Disease.

Development of Graft Versus Host Disease (GVHD) represents a major impediment in allogeneic hematopoietic stem cell transplantation (HSCT). The observation that the presence of bone marrow and circulating hematogones correlated with reduced GVHD risks prompted us to evaluate whether B-cell progenitors, which provide protection in various autoimmune disease models following activation with the TLR-9 agonist CpG (CpG-proBs), could likewise reduce this allogeneic disorder. In a murine model of GVHD that recapitulates an initial phase of acute GVHD followed by a phase of chronic sclerodermatous GVHD, we found that CpG-proBs, adoptively transferred during the initial phase of disease, reduced the diarrhea score and mostly prevented cutaneous fibrosis. Progenitors migrated to the draining lymph nodes and to the skin where they mainly differentiated into follicular B cells. CpG activation and IFN-γ expression were required for the protective effect, which resulted in reduced CD4+ T-cell-derived production of critical cytokines such as TGF-β, IL-13 and IL-21. Adoptive transfer of CpG-proBs increased the T follicular regulatory to T follicular helper (Tfr/Tfh) ratio. Moreover, CpG-proBs privileged the accumulation of IL-10-positive CD8+ T cells, B cells and dendritic cells in the skin. However, CpG-proBs did not improve survival. Altogether, our findings support the notion that adoptively transferred CpG-proBs exert immunomodulating effect that alleviates symptoms of GVHD but require additional anti-inflammatory strategy to improve survival.

Antinuclear antibodies produced in HLA-DR transgenic humanized mice developed chronic graft-versus-host disease

BackgroundChronic graft versus host disease (GVHD) has been reported in humanized mice after the implantation of human hematopoietic stem cells (hu-HSC). As such, humanized mice have been applied to a mouse model of chronic GVHD; however, B-cell activation and autoantibody production did not occur, and the clinical features of chronic GVHD were not sufficiently reproduced. The purpose of this study was to establish an improved humanized mouse model with chronic GVHD using HLA-DR transgenic NOD/Shi-scid, IL-2RγKO (NOG) mice. MethodsCD34-positive cells were isolated from blood extracted from HLA-DRB1∗0405-positive umbilical cords using magnetic cell isolation. Then these were transplanted into NOG-Iab KO, HLA-DR 0405 Tg mice aged 8–16 weeks. GVHD symptoms were observed 26 weeks after transplantation. Histological findings of the skin, lung, liver, and spleen were compared with those of non-humanized mice. Antinuclear antibodies (ANA) were measured by indirect immunofluorescence using sera isolated 26 weeks after transplantation. ResultsAlthough GVHD symptoms were not observed in humanized (hu-HSC) NOG-Iab KO, HLA-DR 0405 Tg mice during the observation period, histological findings of human T-cell infiltration were observed in the skin, liver, and lung, suggesting that GVDH was present; human tingible body macrophages or clusters of BCL-6-positive human B-cells were observed in the spleen. Furthermore, human IgG ANA with peripheral or homogeneous staining patterns were also detected in the sera. ConclusionHu-HSC NOG-Iab KO, HLA-DR 0405 Tg mice differed from conventional models in terms of B-cell activation and ANA production. This study is the first to report on B-cell activation and autoantibody production in humanized mice with chronic GVHD, suggesting that hu-HSC NOG-Iab KO, HLA-DR 0405 Tg mice could be applied to a new humanized mouse model of chronic GVHD.

Ruxolitinib for Treating Steroid-Refractory Acute Graft-Versus-Host Disease in an Infant with Malignant Osteopetrosis Who Received Double-Unit Umbilical Cord Blood Transplantation

A 3-month-old male infant was transferred to our hospital due to bicytopenia. His bone marrow biopsy showed irregular bony trabeculae with cartilaginous core, which was consistent with osteopetrosis. In the genetic test, c.242del (p.Pro81Argfs*85) in TCIRG1 was found to be homozygotic, thus he was diagnosed with malignant infantile osteopetrosis. At 6 months of age, he received double-unit umbilical cord blood transplantation (UCBT) with the conditioning regimen including busulfan, cyclophosphamide, and rabbit anti-thymocyte globulin. Initially, single UCB was infused to the patient, but the post infusion viability of the UCB was unexpectedly low. Thus, another UCB was additionally infused. Cyclosporine and mycophenolate mofetil were used for graft-versus-host disease (GVHD) prophylaxis. Neutrophils and platelets were engrafted on day +13 and +33, respectively. With engraftment, he showed overall grade 4 acute GVHD involving the skin and gut, which was refractory to corticosteroids. Despite treating with low-dose weekly methotrexate (10 mg/m2) and oral beclomethasone, his symptoms persisted. After treating with ruxolitinib 2.5 mg/day for 2 weeks, and 5 mg/day thereafter, his diarrhea stopped in 2 weeks and his skin symptoms gradually improved over 3 months. The short tandem repeats showed 100% donor chimerism at 1 and 3 months after UCBT. Currently, 4 months after UCBT, he is 10 months old. The oral prednisolone has been tapered to 0.6 mg/kg/day, and the dose of ruxolitinib was decreased to 2.5 mg/day without recurrence of GVHD. We plan to taper off the immunosuppressive agents if his GVHD symptoms do not recur. Keywords: Child, Cord blood stem cell transplantation, Graft vs host disease, Infant, Osteopetrosis

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.

Id3 Is Essential for Mediating Ezh2-Regulated Persistence of Alloreactive T Cells in Gvhd Target Tissues in Mice

Abstract: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is potentially a curative treatment for hematologic malignancies and non-malignant diseases. However, its application is limited by graft-versus-host disease (GVHD), which is induced by donor T cells that recognize and react to histocompatibilty differences. Despite pharmacological prophylaxis, acute and chronic GVHD still develop in 40-60% of allo-HSCT recipients. Data from our studies and others suggested critical roles of alloantigen-sensitized memory T cells in sustaining host tissue injury during GVHD. However, the molecular pathway(s) that are crucial for the development and maintenance of alloreactive memory T cells mediating persistent GVHD remain largely unknown. Id3 (Inhibitor of DNA Binding 3) is a transcription factor that negatively regulates the DNA-binding activity of E-protein. During response to acute viral infection, Id3 has been shown to regulate the generation of memory T cells, but is dispensable for primary effector response. Whether Id3 is responsible for persistent allo-immunity and tissue damage during GVHD has not been previously examined. Using experimental GVHD models, we demonstrate that Id3 functions as a key molecular gatekeeper for persistent generation of alloreactive T cells mediating tissue damage and GVHD. Infusion of donor T cells from wild-type (WT) B6 mice caused uniformly lethal GVHD in allogeneic BALB/C recipient mice. In contrast, transfer of donor T cells derived from Id3 conditional knockout (Id3-/-) B6 mice did not induce lethal GVHD. Notably, Id3-/- T cell recipients developed similar symptoms of GVHD (e.g. weight loss, hunched back and reduced mobility) as compared to WT T cell recipients within 20 days after allo-HSCT. However, while GVHD continually progressed in WT T cell recipients, which was evidenced by hair loss, severe diarrhea, skin ulceration, weight loss and death, it was gradually diminished in mice receiving allogeneic Id3-/- T-cells without apparent skin inflammation and tissue damage. As a result, all Id3-/- T-cell recipient mice survived over 70 days after allo-HSCT. We found that by 7 days and 14 days after transplantation, there were similar numbers of donor-derived CD4+ and CD8+ T cells in BALB/c recipients (e.g. spleen, liver, skin and gut) of Id3-/- T cells compared with WT T cells. Furthermore, during this acute alloreactive response phase both Id3-/- and WT T cells expressed high levels of activation markers (e.g. CD25, CD44, CD69, CD122) and had high IFN-g production. By 35 days after allo-HSCT, Id3-/- T cell recipient mice showed significantly increased number of donor T cells in the spleen compared to WT T cell recipients, but had 10-fold fewer numbers of alloreactve T cells in GVHD target organs including gut, skin and liver. Analysis of samples from human patients undergoing allo-HSCT revealed that GVHD patients (n=8) had significantly higher frequency of T cells expressing high levels of ID3 compared to time-matched non-aGVHD patients (n=6). These findings suggest that Id3 plays critical roles in mediating the persistence and accumulation of tissue-infiltrating alloreactive T cells, while being dispensable for early activation, proliferation and effector differentiation of alloreactive T cells. Our recent studies have discovered a central role of the epigenetic regulator Ezh2 in controlling allogeneic T cell proliferation, differentiation and function (Blood 2012, Blood 2013 and Blood 2017). Using genome-wide profiling analysis and functional assessment, we found that Ezh2 directly activates Id3 transcription, independent of its canonical role in repressing gene transcription. Retroviral engineering of Ezh2-deficient T cells to express Id3 significantly enhanced the persistence of antigen-activated T cells in vivo, suggesting a critical role of Id3 in mediating Ezh2 regulation of activated T cell survival and maintenance. In summary, our findings identify the importance of Id3 in regulating persistent allo-immunity and tissue damage, and illuminate a novel molecular pathway of Ezh2-Id3 that may be potentially targeted for GVHD therapy. DisclosuresNo relevant conflicts of interest to declare.

Human Bone Marrow Mesenchymal Stromal/Stem Cell-Derived Extracellular Vesicles Ameliorate Graft-Versus-Host Disease-Related Manifestations

A substantial proportion of patients who receive allogeneic hematopoietic stem cell transplantation develop an immunologic adverse event of acute graft-versus-host disease (GVHD). Cell therapy with bone marrow mesenchymal stromal/stem cells (BM-MSC) is one of the effective treatments for such patients with GVHD. Lines of evidence have suggested that BM-MSC inhibit the function of immune cells mainly through secretion of soluble immune modulators and partially through cell-to-cell contact-dependent mechanism. However, further understanding of how they work has been an important issue. Recently, extracellular vesicles (EV) gain interests as mediators based on their capacity of transmitting signals to sites of action regardless of physiological barriers to reach. In this study, we examined the effects of human BM-MSC-derived EV on clinical, histological and immunologic manifestations of the acute GVHD model mice. We also analyzed whether biological processes mediated by microRNA (miRNA) in human BM-MSC-derived EV are involved in such immunologic effects.Human MSC were isolated from BM samples purchased from AllCells (Alameda, CA) based on our previously published method (Biochem Biophys Res Commun 2016;469:823). EV were isolated from the culture supernatant of BM-MSC by using the Total Exosome Isolation kit (Invitrogen, Carlsbad, CA). We confirmed that the isolated EV expressed CD63 and CD81 by immunoblot analysis and showed morphology of saucer-like shape by transmission electron microscopy. For in vitro experiments, peripheral blood mononuclear cells (PBMC) were activated by anti-CD3/CD28 antibodies with a Dynabeads Human T-Activator (Life Technologies AS, Oslo, Norway). For in vivo experiments, we used a mouse model where GVHD was induced by intravenously injecting spleen cells (1 x 108 per mouse) from C57BL/6 mice (H-2b) into irradiated (8 Gy) BDF1 mice (H-2b x d). The severity of GVHD was assessed histologically and clinically as previously described method (Blood 1996;88:3230, J Clin Invest 1998;102:115).We first tested the effects of BM-MSC-derived EV on T cell subsets in the expansion culture of anti-CD3/CD28-stimulated PBMC. In culture with human BM-MSC-derived EV, the expansion of PBMC was suppressed. Flow cytometric analysis showed that the expansion of CD3+ T cells including CD4+ and CD8+ T cells was suppressed. The frequency of CD45RA+Foxp3low cells (naive Treg cells) was higher and that of CD45RA-Foxp3high cells (effector Treg cells) was lower in culture with EV than those in a single culture of PBMC. In addition, the ratio of CD8/naive Treg cells was lower in culture with EV than that in a single culture of PBMC. Thus, BM-MSC-derived EV suppressed the T cell expansion and preserved naive Treg cells.We then investigated the effects of BM-MSC-derived EV on acute GVHD in mouse model. Systemic infusion of human BM-MSC-derived EV significantly prolonged the survival of GVHD mice with the median survival period of 16 days while that was 10 days in control untreated GVHD mice. The clinical scores of BM-MSC-derived EV-treated mice were lower than those of the control mice, indicating that EV ameliorated systemic symptoms of GVHD. In the histological assessment of GVHD-targeted organs, EV treatment significantly mitigated GVHD-associated abnormalities in specimens particularly from large bowel. Flow cytometric analysis of PB from the BM-MSC-derived EV-treated GVHD mice indicated that both CD4+ and CD8+ T cells were suppressed, and CD62L-CD44+ T cells was decreased and CD62L+CD44- T cells was increased compared with the control untreated GVHD mice. In addition, EV-treated mice still contained substantial Treg population. Thus, EV suppressed functional differentiation of peripheral T cells from naïve phenotype to effector phenotype and did not enhance depletion of Treg cells. Collectively, BM-MSC-derived EV showed immunomodulatory effects in murine GVHD model.Finally, we performed microarray analysis of microRNAs in BM-MSC-derived EV and found down-regulated cell proliferative processes as identified by Gene Ontology enrichment analysis.This study revealed that BM-MSC-derived EV captured the therapeutic effects of cell-of-origin against GVHD in association with immunologic mechanism of T cell suppression and peripheral naïve T cell preservation. Our findings provide the basis of BM-MSC-mediated anti-GVHD effect with the EV-mediated mechanism. DisclosuresTakaori-Kondo:celgene: Honoraria, Research Funding; Bristol-Myers Squibb, Novartis, Janssen pharma, Pfizer: Honoraria.

Pretransplant Short-Term Exposure of Donor Graft Cells to ITK Selective Inhibitor Ameliorates Acute Graft-versus-Host Disease by Inhibiting Effector T Cell Differentiation while Sparing Regulatory T Cells.

Graft-versus-host disease (GVHD) remains to be a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). IL-2-inducible T cell kinase (ITK), a TEC cytoplasmic tyrosine kinase, has an essential role in T cell development and receptor signaling. The ITK/Bruton tyrosine kinase inhibitor ibrutinib has been shown to improve chronic GVHD symptoms; however, the effect of ITK selective inhibition on acute GVHD remains unclear. In this study, we evaluated the pharmacological effects of an ITK selective inhibitor (ITKsi) on acute GVHD using murine bone marrow transplantation models. First, we found that CD4+ T cell differentiation toward Th1, Th2, or Th17 was inhibited following ITKsi treatment in a dose-dependent manner while maintaining regulatory T cells in the presence of alloantigens both invitro and invivo. ITKsi preferentially inhibited inflammatory cytokine production and invivo proliferation of alloreactive T cells. We then demonstrated that short-term exposure of donor graft cells to ITKsi significantly delayed the onset of GVHD-associated mortality without compromising the donor cell engraftment and the graft-versus-tumor effect, indicating the potential of ITK selective inhibition in the setting of clinical allogeneic HSCT. These findings suggest that ITK is a potential therapeutic target against GVHD, and the pharmacological ITK inhibitor may serve as a novel strategy for immune regulation after HSCT.

Removal of CD276+ cells from haploidentical memory T-cell grafts significantly lowers the risk of GVHD

Detrimental graft-versus-host disease (GVHD) still remains a major cause of death in hematopoietic stem cell transplantation (HSCT). The recently explored depletion of naive cells from mobilized grafts (CD45RA depletion) has shown considerable promise, yet is unable to eliminate the incidence of GVHD. Analysis of CD45RA-depleted haploidentical mixed lymphocytes culture (haplo-MLC) revealed insufficient suppression of alloresponses in the CD4+ compartment and identified CD276 as a marker for alloreactive memory Th1 T cells. Conclusively, depleting CD276+ cells from CD45RA-depleted haplo-MLC significantly attenuated alloreactivity to recipient cells while increasing antiviral reactivity and maintaining anti-third party reactivity in vitro. To evaluate these findings in vivo, bulk, CD45RA-depleted, or CD45RA/CD276-depleted CD4+ T cells from HLA-DR4negative healthy humans were transplanted into NSG-Ab°DR4 mice, a sensitive human allo-GVHD model. Compellingly, CD45RA/CD276-depleted grafts from HLA-DR4negative donors or in vivo depletion of CD276+ cells after transplant of HLA-DR4negative memory CD4 T cells significantly delay the onset of GVHD symptoms and significantly alleviate its severity in NSG-Ab°DR4 mice. The clinical courses correlated with diminished Th1-cytokine secretion and downregulated CXCR6 expression of engrafted peripheral T cells. Collectively, mismatched HLA-mediated GVHD can be controlled by depleting recipient-specific CD276+ alloreacting T cells from the graft, highlighting its application in haplo-HSCT.

National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: IIb. The 2020 Preemptive Therapy Working Group Report

Chronic graft-versus-host disease (GVHD) commonly occurs after allogeneic hematopoietic cell transplantation (HCT) despite standard prophylactic immune suppression. Intensified universal prophylaxis approaches are effective but risk possible overtreatment and may interfere with the graft-versus-malignancy immune response. Here we summarize conceptual and practical considerations regarding preemptive therapy of chronic GVHD, namely interventions applied after HCT based on evidence that the risk of developing chronic GVHD is higher than previously appreciated. This risk may be anticipated by clinical factors or risk assignment biomarkers or may be indicated by early signs and symptoms of chronic GVHD that do not fully meet National Institutes of Health diagnostic criteria. However, truly preemptive, individualized, and targeted chronic GVHD therapies currently do not exist. In this report, we (1) review current knowledge regarding clinical risk factors for chronic GVHD, (2) review what is known about chronic GVHD risk assignment biomarkers, (3) examine how chronic GVHD pathogenesis intersects with available targeted therapeutic agents, and (4) summarize considerations for preemptive therapy for chronic GVHD, emphasizing trial development, including trial design and statistical considerations. We conclude that robust risk assignment models that accurately predict chronic GVHD after HCT and early-phase preemptive therapy trials represent the most urgent priorities for advancing this novel area of research.

Current practice in nutrition after allogeneic hematopoietic stem cell transplantation – Results from a survey among hematopoietic stem cell transplant centers

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is frequently associated with impaired oral intake and malnutrition, which potentially increases morbidity and mortality. Therefore, nutrition is one of the major challenges in the post-transplant period. To document the current clinical approach in nutritional treatment, we designed a questionnaire concerning the current practice in nutrition after alloHSCT and distributed it to German speaking centers performing alloHSCT in Germany, Austria and Switzerland between November 2018 and March 2020. Twenty-eight (39%) of 72 contacted centers completed the survey, 23 from Germany, two from Austria and three from Switzerland, representing 50% of alloHSCT activity within the participating countries in 2018. All centers reported having nutritional guidelines for patients undergoing alloHSCT, whereby 86% (n=24) provided a low-microbial diet during the neutropenic phase. The criteria to start parenteral nutrition (PN) directly after alloHSCT seemed to be consistent, 75% (n=21) of the corresponding centers started PN if the oral nutritional intake or the bodyweight dropped below a certain limit. In the setting of intestinal graft-versus-host disease (GvHD) the current practice appeared to be more heterogenous. About 64% (n=18) of the centers followed a special diet, added food stepwise modulated by GvHD symptoms, while only four centers regularly stopped oral intake completely (intestinal GvHD grade >1). Half of the centers (54%, n=15) applied a lactose-free diet, followed by 43% (n=12) which provided fat- and 18% (n=5) gluten-free food in patients with intestinal GvHD. Supplementation of micronutrients in acute intestinal GvHD patients was performed by 54% (n=15) of the centers, whereas vitamin D (89%, n=25) and vitamin B12 (68%, n=19) was added regularly independently of the presence of GvHD. Only 5 (18%) participating centers ever observed a food-associated infection during hospitalization, whereas food-associated infections were reported to occur more often in the outpatient setting (64%, n=18). The survey documented a general consensus about the need for nutritional guidelines for patients undergoing alloHSCT. However, the nutritional treatment in clinical practice (i.e. lactose-, gluten- or fat-free in intestinal GvHD) as well as the use of food supplements was very heterogeneous. In line with current general recommendations the centers seemed to focus on safe food handling practice rather than providing a strict neutropenic diet. More high-quality data are required to provide evidence-based nutrition to patients during and after alloHSCT.