Onset Of Graft-versus-host Disease Research Articles

Abstract 4191: Delayed onset of GvHD in PBMC humanized NCG-B2m KO mice provides an enhanced model for oncology studies

Abstract Humanized mice are specialized animal models incorporating components of the human immune system. These models are valuable tools for assessing the efficacy of human immunotherapies on tumors. Humanized mice are created by injecting human CD34+ hematopoietic stem cells (HSCs) or human peripheral blood mononuclear cells (PBMCs) into an immunodeficient mouse. PBMC humanized (HuPBMC) mice engraft within a few weeks and can recapitulate a robust human T cell population. One challenge when using HuPBMC mice is the onset of graft versus host disease (GvHD), limiting their lifespan compared to hCD34+ HSC humanized mice. The onset of GvHD varies in the HuPBMC model depending on the donor cells. The development of xenogeneic GvHD is dependent on the expression of major histocompatibility complex (MHC) on host cells and lowering MHC expression can delay the onset of GvHD.NCG-B2m KO mice lack beta-2 microglobulin, a component of MHC I, and have lower cell surface expression of MHC I molecules. This leads to a decrease in recognition of host cells by human immune cells. Here we compared the onset of GvHD between HuPBMC NCG-B2m KO mice and HuPBMC NCG mice. PBMC donor variability was also compared between the two PBMC humanized models by evaluating multiple donors and assessing additional collections from the same donor. After the PBMC engraftment was characterized, HuPBMC NCG mice and HuPBMC NCG-B2m KO mice were enrolled in tumor studies comparing outcomes and tumor growth kinetics in mice treated with human immune checkpoint blockade therapy.NCG and NCG-B2m KO mice were injected with 1x107 PBMCs from individual donors. Animals were weighed three times a week to monitor for changes in body weight and general health status. Peripheral blood was collected at day 10, 20, 30, 40, 60 and 90 post injection for flow cytometry screening of human immune cell engraftment (hCD45+, hCD3+, hCD4+ and hCD8+). Human PBMCs isolated from additional collections from the same donors were also injected into both NCG-B2m KO mice and NCG mice and flow cytometry was performed. HuPBMC NCG-B2m KO mice survived longer than HuPBMC NCG mice allowing for the potential use in longer term tumor studies before the results are confounded by GvHD. PBMC humanized mice are an important model for translational research using immunotherapies and the PBMC humanized NCG-B2m KO model provides an expanded study window ideal for oncology studies. Citation Format: Steven Bronson, David Harris, Anya Avrutskaya, Christoph Eberle, Jenny Rowe. Delayed onset of GvHD in PBMC humanized NCG-B2m KO mice provides an enhanced model for oncology studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4191.

Graft-versus-host disease after autologous stem cell transplantation in a recipient who underwent allogeneic stem cell transplantation 20 years earlier.

A literature review does not provide information about the safety of autologous hematopoietic stem cell transplantation (HSCT) in a recipient who has previously received allogeneic HSCT. We treated a 69-year-old woman with diffuse large B cell lymphoma. The patient received autologous stem cell transplantation (ASCT) in the second complete remission of malignant lymphoma. The patient had undergone allogeneic hematopoietic SCT (allo-HSCT) for chronic myeloid leukemia 20 years ago. Chronic myeloid leukemia had been in complete remission for the previous 20 years. Thus, the patient received autologous and allogenic HSCT 20 years apart. ASCT involves the patient receiving "self" hematopoietic cells from an allogeneic donor. In other words, this is immunologically the second allo-HSCT. The allo-HSCT 20 years ago was undergone by a related healthy brother, a human leukocyte antigen (HLA) 8/8 full matched donor. The conditioning regimen was reduced-intensity consisting of fludarabine and busulfan. The patient did not experience acute or chronic graft-versus-host disease (GVHD) after allo-HSCT. The second transplantation, ASCT was performed to the MEAM conditioning regimen. Engraftment was uneventful, and complete donor chimerism had been achieved even after ASCT. She suffered from an acute gastric mucosal lesion 52 days after ASCT. Pathological finding of gastric mucosa was nonspecific acute gastritis with significant neutrophil infiltration. Sex chromosome analysis of gastric mucosa demonstrated that mucosal cells had XX signals, whereas infiltrating neutrophils had XY signals. We speculated the patient onset of an acute gastric GVHD in this recipient after the second transplantation. This case remarked infiltration of neutrophils triggered GVHD reaction by resetting allogeneic immune reaction after the second transplantation. We describe a rare occurrence of GVHD reaction in a recipient of ASCT following allo-HSCT.

A Validated Risk Stratification That Incorporates MAGIC Biomarkers Predicts Long-Term Outcomes in Pediatric Patients with Acute GVHD

Acute graft versus host disease (GVHD) is a common and serious complication of allogeneic hematopoietic cell transplantation (HCT) in children but overall clinical grade at onset only modestly predicts response to treatment and survival outcomes. Two tools to assess risk at initiation of treatment were recently developed. The Minnesota risk system stratifies children for risk of nonrelapse mortality (NRM) according to the pattern of GVHD target organ severity. The Mount Sinai Acute GVHD International Consortium (MAGIC) algorithm of 2 serum biomarkers (ST2 and REG3α) predicts NRM in adult patients but has not been validated in a pediatric population. We aimed to develop and validate a system that stratifies children at the onset of GVHD for risk of 6-month NRM. We determined the MAGIC algorithm probabilities (MAPs) and Minnesota risk for a multicenter cohort of 315 pediatric patients who developed GVHD requiring treatment with systemic corticosteroids. MAPs created 3 risk groups with distinct outcomes at the start of treatment and were more accurate than Minnesota risk stratification for prediction of NRM (area under the receiver operating curve (AUC), .79 versus .62, P = .001). A novel model that combined Minnesota risk and biomarker scores created from a training cohort was more accurate than either biomarkers or clinical systems in a validation cohort (AUC .87) and stratified patients into 2 groups with highly different 6-month NRM (5% versus 38%, P < .001). In summary, we validated the MAP as a prognostic biomarker in pediatric patients with GVHD, and a novel risk stratification that combines Minnesota risk and biomarker risk performed best. Biomarker-based risk stratification can be used in clinical trials to develop more tailored approaches for children who require treatment for GVHD.

Human MAIT cells inhibit alloreactive T cell responses and protect against acute graft-versus-host disease.

Adoptive transfer of immunoregulatory cells can prevent or ameliorate graft-versus-host disease (GVHD), which remains the main cause of nonrelapse mortality after allogeneic hematopoietic stem cell transplantation. Mucosal-associated invariant T (MAIT) cells were recently associated with tissue repair capacities and with lower rates of GVHD in humans. Here, we analyzed the immunosuppressive effect of MAIT cells in an in vitro model of alloreactivity and explored their adoptive transfer in a preclinical xenogeneic GVHD model. We found that MAIT cells, whether freshly purified or short-term expanded, dose-dependently inhibited proliferation and activation of alloreactive T cells. In immunodeficient mice injected with human PBMCs, MAIT cells greatly delayed GVHD onset and decreased severity when transferred early after PBMC injection but could also control ongoing GVHD when transferred at delayed time points. This effect was associated with decreased proliferation and effector function of human T cells infiltrating tissues of diseased mice and was correlated with lower circulating IFN-γ and TNF-α levels and increased IL-10 levels. MAIT cells acted partly in a contact-dependent manner, which likely required direct interaction of their T cell receptor with MHC class I-related molecule (MR1) induced on host-reactive T cells. These results support the setup of clinical trials using MAIT cells as universal therapeutic tools to control severe GVHD or mucosal inflammatory disorders.

Acute graft versus host disease 1976-2020: reduced incidence and predictive factors.

We studied the incidence of acute graft versus host disease (GvHD) and its outcome in three consecutive time frames (year <2000; 2000-2010; >2010), in 3,120 patients allografted in two transplant Centers between 1976 and 2020. The median age increased over the three periods from 32 to 42 to 54 years (p < 0.00001). The median day of onset of GvHD in the three periods was day +14, day +16, and day +30, respectively (p < 0.0001). The cumulative incidence (CI) of GvHD grades II-IV in the three periods was 47, 24, and 16%, respectively (p < 0.00001). The CI of GvHD grades III-IV was 13, 5, and 4% (p < 0.001). In multivariate analysis, significant predictive factors for GvHD II-IV, on top of year of transplant, were anti-thymocyte globulin (ATG) (RR 0.67, p > 0.001); post-transplant cyclophosphamide (PTCY) (RR 0.41, p < 0.001), a family mismatched donor (RR 1.31, p = 0.03) a matched unrelated donor (RR 2.1, p < 0.001), an unrelated mismatched donor (RR1.8, p = 0.001), donor age above 40 years (RR 1.27, p < 0.001), hematological malignancy-as compared to aplastic anemia (RR 2.3, p < 0.001). When selecting only GvHD grade II, in a multivariate analysis, there was a significant reduction of transplant-related mortality (TRM) for patients grafted in 2001-2010 (RR 0.62, p < 0.0001) and for patients grafted in 2011-2020 (RR 0.35, p < 0.0001) as compared to grafts before the year 2000. A similar reduction in time was seen for patients with GvHD grades III-IV. The overall TRM in the three periods was 30, 22, and 16% (p < 0.0001) and survival was 47, 51, and 58% (p < 0.0001). Relapse risk was unchanged. In conclusion, we showed improved prevention of acute GvHD with time, together with a significant delay in the onset of the disease. Treatment of GvHD has also improved over time, as suggested by both reduced TRM and improved survival in more recent transplant periods.

Study protocol: Close Assessment and Testing for Chronic Graft-vs.-Host disease (CATCH).

Chronic graft-versus-host disease (GVHD) is an immune-mediated disorder that causes significant late morbidity and mortality following allogeneic hematopoietic cell transplantation. The "Close Assessment and Testing for Chronic GVHD (CATCH)" study is a multi-center Chronic GVHD Consortium prospective, longitudinal cohort study designed to enroll patients before hematopoietic cell transplantation and follow them closely to capture the development of chronic GVHD and to identify clinical and biologic biomarkers of chronic GVHD onset. Data are collected pre-transplant and every two months through one-year post-transplant with chart review thereafter. Evaluations include clinician assessment of chronic GVHD and its manifestations, patient-reported outcomes, multiple biospecimens (blood, saliva, tears, buccal mucosa and fecal samples, biopsies of skin and mouth), laboratory testing, and medical record abstraction. This report describes the rationale, design, and methods of the CATCH study, and invites collaboration with other investigators to leverage this resource. trial registration: This study is registered at www.clinicaltrials.gov as NCT04188912.

Inflammatory Change of FoxP3 + Regulatory T Cells in Human Acute Graft-Versus-Host Disease: Implication in Immunopathogenesis

FoxP3 + regulatory T (Treg) cells are a subset of CD4 + T cells that are requisite for control of immune responses in various settings, thereby preventing excessive inflammation and maintaining homeostasis of the immune system. Paradoxically, in several autoimmune disorders, it has been suggested that Treg cells undergo inflammatory conversion to produce effector cytokines promoting tissue inflammation. Although Treg cells are known to play an important role in the pathogenesis of acute graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT), such inflammatory changes have not been focused on so far especially in the setting of human acute GvHD. Herein, in a prospective cohort of patients with hematologic malignancies who were treated with allogeneic SCT, we investigated the quantitative and qualitative changes of Treg cells in subjects with acute GvHD, including production of inflammatory cytokines from Treg cells. We also studied a cellular mechanism for production of effector cytokines by Treg cells and clinical significance of inflammatory changes of Treg cells. We first determined the frequency of Treg cells in the peripheral blood of patients with acute GvHD and compared it with that of patients who did not develop acute GvHD. Percentage of CD25 hiCD127 loFoxP3 + Treg cells among CD4 + T cells was significantly lower in patients with acute GvHD. When we analyzed for the composition of Treg cell subsets in more detail, we observed that the proportion of CD45RA -FoxP3 hi (actively suppressive) Treg cells was decreased and the proportion of CD45RA -FoxP3 lo (cytokine-producing non-suppressive) Treg cells was increased in patients with acute GvHD. Next, we assessed the production of inflammatory cytokines from Treg cells following T cell receptor (TCR) stimulation of peripheral blood lymphocytes with anti-CD3/CD28 antibody, using intracellular cytokine staining. As a result, we found that a remarkable proportion of CD4 +CD25 hiFoxP3 + Treg cells of acute GvHD patients produced TNF-α upon TCR stimulation. Interestingly, a significant proportion of TNF-α-producing Treg cells also secreted IL-17A simultaneously. These findings are consistent with the increased CD45RA -FoxP3 lo fraction among the Treg cell subsets. To explore a potential mechanism underlying inflammatory change of Treg cells, we quantified a variety of soluble factors in systemic circulation before and after onset of acute GvHD. The concentration of IL-6 and IL-21 was significantly higher in the serum drawn after onset of acute GvHD compared to baseline, suggesting that these Th17-polarizing cytokines might be responsible for the inflammatory conversion of Treg cells during acute GvHD. We further checked the suppressive activity of TNF-α-producing CD4 +CD25 hiFoxP3 + Treg cells of acute GvHD patients. TNF-α + Treg cells of acute GvHD patients expressed lower level of FoxP3 than the TNF-α - counterpart. Similarly, the percentage of CD39 + cells was lower in TNF-α + Treg cells than in the TNF-α - counterpart. In fact, Treg cell suppressive activity during acute GvHD was significantly reduced compared with that of pre-SCT control when non-Treg CD4 + T cells or CD8 + T cells were used as responder cells. Finally, we examined the clinical significance of TNF-α-producing CD4 +CD25 hiFoxP3 + Treg cells in acute GvHD patients. The frequency of TNF-α + cells among circulating Treg cells significantly correlated with the severity of acute GvHD, with higher proportion of TNF-α + Treg cells in patients with the higher grade of acute GvHD. This result indicates that the pathologic conversion of Treg cells to produce TNF-α contributes to immune-mediated tissue injury during acute GvHD. Taken together, during human acute GvHD, Treg cells undergo functional alteration to produce proinflammatory cytokines including TNF-α and IL-17A and to exhibit the reduced suppressive activity. TNF-α production by Treg cells is closely associated with the clinical severity of acute GvHD.

Bacterial and Bacteriophage Consortia Are Associated with Protective Intestinal Immuno-Modulatory Metabolites in Allogeneic Stem Cell Transplantation Patients

The human microbiome is a predictor of clinical outcome in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-SCT). Besides bacteria, fungi and viruses as well as intestinal microbiota-derived metabolites are involved. However, it is still unclear how dynamic shifts in these three kingdoms contribute to the production of intestinal metabolites, how metabolites are impacted by graft-versus-host disease (GvHD) or antibiotics and whether they are associated with clinical outcome. We report the two-year follow-up of a prospective, observational, longitudinal cohort of allo-SCT patients (n=78) at two German transplantation centers that combined three-kingdom (bacteria, fungi, viruses) analysis of intestinal microbial communities with targeted metabolomics ( Figure 1). Using Multi-omics factor analysis (MOFA), we uncovered a functional microbiome signature of bacteria from the Lachnospiraceae and Oscillospiraceae families and their associated bacteriophages, which correlated with the production of immuno-modulatory metabolites (IMMs) including short-chain fatty acids (SCFAs), branched-chain fatty acids (BCFA), metabolites associated with induction of type-I IFN signaling (IIMs) and immuno-modulatory secondary bile acids ( Figure 2). We established an Immuno-modulatory Metabolite Risk Index (IMM-RI) consisting of five index IMMs, which was associated with improved survival, less transplant-related mortality and reduced relapse rate. Onset of gastrointestinal GvHD and exposure to antibiotics significantly impacted intestinal levels of protective IMMs. Using whole shotgun metagenomic sequencing, we observed that in IMM-RI low-risk patients, sustained production of protective IMMs was associated with a high abundance of microbial SCFA biosynthesis pathways, specifically butyric acid via butyryl-CoA:acetate CoA-transferase (BCoAT). Through genome assembly from viral metagenomic sequencing data, we detected two distinct bacteriophages which encoded BCoAT as an auxiliary metabolic gene. They were more abundant in IMM-RI low-risk patients and positively correlated with intestinal butyric acid levels, suggesting that these bacteriophages may modulate bacterial metabolite biosynthesis. Our study identifies a specific microbiome signature associated with protective IMMs that could improve fecal microbiota transplantation (FMT) donor selection and provides a rationale for the development of engineered metabolite-producing consortia and defined metabolite combination drugs as novel microbiome-based therapies for cancer patients.

Clinical Outcomes of Patients Receiving 3 Versus 4 Doses of Methotrexate with Concomitant Anti-Thymocyte Globulin; A Single Center Experience

Background: Four doses of methotrexate (MTX) given on day +1, +3, +6, and +11 after allogeneic stem cell transplant (HSCT) is considered one of the most common graft-versus-host disease (GVHD) prophylaxis regimen. However, mucositis and occasionally transaminitis prohibit safe delivery of the fourth dose of MTX. Prior single institution studies showed conflicting results comparing the outcomes of patients who received 3 vs 4 doses of MTX but to our knowledge, the effect of concomitant anti-thymocyte globulin (ATG) use has not been reported. As both ATG and MTX synergistically prevent GVHD via suppressive effects on T-cells. Our institution uniquely incorporates ATG as part of the conditioning regimen for patients receiving HSCT from 10/10 loci match unrelated donors (MUD) and here we report the clinical outcomes between patients who received 3 versus (vs) 4 doses of MTX. Methods: We retrospectively reviewed the charts of patients who underwent HSCT from MUD between 2009 to 2023 at the University of Iowa Hospital and Clinics. We use rabbit ATG (Thymoglobulin R), given at 0.5 mg/kg on day -3, 2 mg/kg on day -2, and 2.5 mg/kg on day -1. MTX is given at 15 mg/m 2 on day +1 and 10 mg/m 2 on day +3, +6 and +11 per institutional protocol. Statistical Analysis: Time was calculated from transplant to recurrence or death for recurrence-free survival (RFS) and overall survival (OS), respectively. For acute and chronic GVHD (aGVHD and cGVHD), time was calculated from transplant to onset of acute and chronic GVHD. Relapse and death were considered as competing events. For non-relapse mortality (NRM), time was calculated from transplant to death; relapse was considered a competing event. Cox regression and Fine-Gray competing risk models were used to estimate the effect of patient, disease, and treatment characteristics on outcomes Results: We identified 296 patients with various indications of HSCT; the most common was acute myeloid leukemia (45.3%). There were 117 and 179 patients who received 3 doses and 4 doses of MTX respectively. Patients characteristics reported in table 1. Median follow-up was 21.9 months (range 0.6 to 150.8). Patients who received 4 doses of MTX were more frequently male (68.2% vs 49.6%, p&amp;lt;0.01), received a reduced intensity (compared to myeloablative) preparative regimen (38.0% vs 23.1%, p&amp;lt;0.01), were older (median 58 vs 54 years, p=0.01), and underwent transplant at an earlier year (median year of transplant 2014 vs 2018, p&amp;lt;0.01). Severe mucositis was the most common reason for omission of the 4 th dose of MTX. Maximum grade of aGVHD was similar between cohorts (p=0.83). A statistically significant difference was not evidenced between patients receiving 4 versus 3 doses of MTX for the following outcomes: aGVHD (HR 1.11, 95% CI 0.84-1.46), grade 3-4 aGVHD (HR 1.41, 95% CI 0.77-2.60), RFS (HR 1.01, 95% CI 0.66-1.56), NRM (HR 1.33, 95% CI 0.84-2.10) and OS (HR 1.21, 95% CI 0.87-1.69). While significant on univariate analysis, MTX dose was not significantly associated with extensive cGVHD (HR 1.44, 95% CI 0.92-2.26) after adjustment for transplant year. Additionally, the median time to neutrophil engraftment was 14 days in both cohorts. Conclusion: When ATG is used concomitantly, omission of the 4 th dose of MTX does not significantly impact the rate of neutrophil engraftment, cumulative incidence of aGVHD, cGVHD, RFS, NRM, and OS after HSCT from MUD. However, a small sample size and non-randomized nature of this study prompts further confirmation study from a large prospective multicenter randomized control trial.

A Day 14 Endpoint for Acute Gvhd Clinical Trials

The overall response rate (ORR) 28 days (D28) after treatment has been adopted as the primary endpoint for clinical trials of acute graft versus host disease (GVHD). ORR combines complete response (CR) and partial response (PR) because CR and PR have very similar non relapse mortality (NRM). D28 ORR has some disadvantages: first, physicians usually decide to modify immunosuppression much earlier than D28, and second, NRM does not always correlate well with ORR at D28, particularly for patients who present with intermediate risk or low risk disease. MAGIC serum biomarkers at day 14 (D14) of GVHD treatment have been shown to correlate better with NRM than clinical symptom response (Srinagesh et al, Blood Advances, 2019). We therefore hypothesized that a combination of clinical symptom severity and biomarkers at D14 of treatment would predict NRM at least as well and possibly better than clinical response at D28. We studied 1144 patients from 23 MAGIC sites who were systemically treated with at least 0.25 mg/kg/day of systemic steroids (prednisone equivalents) and had clinical data and serum samples available in the MAGIC database and biorepository. We divided the patients into a training set (n=764) and a validation set (n=380). We used 12-month NRM from the time of acute GVHD onset as the primary outcome of interest. We first trained a recursive partitioning algorithm in the training set to create a response model (MAGIC D14 clinical response) according to overall GVHD grade at both onset and D14 that predicted 12-month NRM and then applied it to the validation set. Patients that either progressed to grade III/IV GVHD, continued having grade III/IV GVHD, or only improved to grade II at D14 from grade III/IV at D0 had high NRM and were classified as non-responders. Patients with grade I/II GVHD at day 0 who had grade 0-II GVHD at day 14 and patients who improved to grade 0/I at day 14 from grade III/IV at day 0 had low NRM and were classified as responders. Although counterintuitive, responders included patients whose GVHD remained at grade II because the 12 month NRM of this group was only 20%. The new clinical model predicted NRM as well as the D28 standard response model in the validation set. When we integrated the D14 MAP biomarker score (low: ≤ 0.290 vs high: &amp;gt;0.290) with the new D14 clinical response (D14 MAGIC integrated response), we observed three response groups of integrated response with strikingly different 12 month NRM (8%, 35%, 76%, p&amp;lt;0.001), in contrast to the D28 standard response (Figure 1A, 1B). D14 complete response required MAGIC clinical response with low MAP; D14 partial response required either MAGIC clinical response with high MAP or MAGIC clinical non-response with low MAP; and D14 non-response required MAGIC clinical non-response with high MAP. The D14 response groups also displayed significant differences in 12 month OS (82%, 58%, 14% respectively, p&amp;lt;0.001) We then compared the D14 integrated response with the D28 standard response for 12 month NRM prediction. This D14 integrated response model was superior for time-dependent AUC (0.78 vs 0.71, P=0.03), sensitivity (0.71 vs 0.54), positive predictive value (0.52 vs 0.48) and negative predictive value (0.92 vs 0.89), with minimal loss in specificity (0.85 vs 0.87) in the prediction of 12-month NRM compared to the D28 standard response model. Using decision curve analysis, the D14 integrated response displayed higher net benefit (correct identification of patients that will experience NRM by 12 months) over the whole range of threshold probabilities/preferences for changing immunosuppression when 12-month NRM was used as the outcome. We conclude first, that the definition of response that uses onset and D14 grades is as accurate as D28 ORR model; and second, that a model using clinical grades and biomarkers on D14 more accurately predicts long term NRM than the standard D28 definition and may be useful as a clinical trial endpoint.

Improved Gvhd-Relapse Free Survival of Ex Vivo αβtcr/CD19 Depleted Allo-Sct Combined with In Vivo T Cell Depletion with ATG When Compared to T Cell Replete Transplantations with or without the Addition of ATG

Introduction The aim of allogeneic stem cell transplantation (allo-SCT) is to reach graft-versus-host disease, relapse free survival (GRFS). To reduce the incidence of graft-versus-host disease (GVHD), we have developed an allo-SCT platform combining ex vivo αβTCR/CD19 depletion with in vivo T-cell depletion through administration of antithymoglobulin (ATG). Here we compare GRFS to historical cohorts of T-cell replete allo-SCT. Methods Adults with hematological malignancies and transplanted between 2011 and 2022 were included in this retrospective analysis. Written informed consent was obtained in accordance with the JACIE guidelines. Clinical data was extracted from the EBMT registry. 3 cohorts were identified. Cohort A: Allo-SCT of unmanipulated peripheral blood derived mononuclear cells (PBMCs) of matched related donors (MRD) after non-myeloablative (NMA) or myeloablative (MA) conditioning without ATG. Cohort B: Allo-SCT of unmanipulated PBMCs of 10/10 or 9/10 matched unrelated donors (MUD) after NMA or MA conditioning with ATG. Patients in cohorts A &amp; B received dual immunosuppression with cyclosporin (CsA) and mycophenolic acid (MMF). Cohort C: Allo-SCT of αβTCR/CD19 depleted PBMCs of MRD and MUD (10/10 and 9/10) after ATG and a myeloablative conditioning as previously described[1]. Patients in cohort C received 28 days of MMF. Cumulative incidence (CI) of GVHD was defined as time to onset of GVHD, with relapse and death as competing events. Overall survival (OS) was defined as time to death from any cause. CI of relapse was defined as time to relapse, with death as a competing event. Non-relapse mortality (NRM) was defined as time to death, without relapse or progression. Event free survival (EFS) was defined as the time to relapse, graft failure or death. GRFS was defined as the time to relapse, aGVHD 3-4 or extensive cGVHD, graft failure or death. CI of CMV and EBV reactivations were calculated with relapse and death as competing events. Results 341 patients were included (cohort A: N=63; cohort B: N=150; cohort C: N=128) (table 1). In T cell replete allo-SCT (A&amp;B) ATG was administered to recipients of MUD and MA conditioning was administered to patients &amp;lt; 40 years with acute leukemia. In cohort C, all patients received ATG and an MA conditioning, regardless of age or underlying malignancy. This explains differences donor types and intensity of conditioning between the cohorts (table 1). Other clinical characteristics were comparable. Two year OS (A=66.7%; B=58.7%; C=63.8%) and EFS (A=57.1%; B=52%; C=55.7%) was comparable between the cohorts. Two year CI of relapse was also comparable (A=30%; B=23%; C=28%). Two year NRM appeared higher in B, but this difference did not reach significance (A= 9.8%; B=24%; C=15%). The CI of aGVHD grade 2-4 and 3-4 at day 100 was significantly higher in T cell replete allo-SCT with MUD (2-4; A=23%; B=37%; C=20% (p=0.002); 3-4: A=5%; B=16%; C=4.7% (p=0.043)). The incidence of extensive cGVHD was significantly higher in T cell replete allo-SCT of MRD. (A= 25%; B=13%; C= 2.3% (p=&amp;lt; 0.001)). The low incidence of grade 3-4 aGVHD and extensive cGVHD without an increase in relapse or NRM, translated into a superior 2 year GRFS in patients receiving ATG combined with an αβTCR/CD19 allograft (C=55.6%) as compared to T cell replete allo-HSCT of MRD (A=38.1%) and MUD (B=40.8%) (Figure 1, p=0.04). This difference remained significant in a multivariate analysis. Conclusion We demonstrate that αβTCR/CD19 depletion combined with ATG, with a myeloablative conditioning and a very short course of immunosuppression results in very low incidences of life-threatening GVHD and an improved GRFS as compared to T cell replete allo-SCT for adult patients up to the age of 70. This is likely to positively impact the long term quality of life in survivors of allo-SCT[2]. In addition, this platform provides a window for additional early post allo-interventions to further improve the control of underlying hematological malignancies. 1. de Witte, M.A., et al., alphabeta T-cell graft depletion for allogeneic HSCT in adults with hematological malignancies. Blood Adv, 2021. 5(1): p. 240-249. 2. Oerlemans, S., et al. Relapse and severe Graft-versus-Host Disease have a negative impact on long-term symptoms and quality of life of patients three years after allogeneic haematopoietic stem cell transplantation. Annual meeting EBMT 2022.

JAK2/mTOR Inhibition Fails to Prevent Acute Gvhd Despite Reduced Th1/Th17 Cells: Final Phase II Trial Results

Introduction: Our phase I graft-versus-host disease (GVHD) prevention trial of pacritinib (recommended phase II dose: 100mg po BID day 0 to +70, dose level 2) plus sirolimus (8-14ng/ml) and tacrolimus (3-7ng/ml) (PAC/SIR/TAC) demonstrated the regimen was safe and free of pan-JAK myelosuppression after allogeneic hematopoietic cell transplantation (alloHCT). PAC inhibits JAK2 with no activity against JAK1, avoiding off-target suppression of IL-2 required by Tregs. JAK2/STAT3 activity mediates IL-6, IL-12, and IL-23 receptor signaling and subsequent pathogenic Th1 and Th17 differentiation. JAK2/mTOR blockade supports Treg potency, providing further rationale for the PAC/SIR/TAC combination. Herein we report on our completed phase II trial of PAC/SIR/TAC after 8/8-HLA matched alloHCT. Methods: This single-arm phase II trial (NCT02891603) was powered to determine if PAC/SIR/TAC suppressed %pSTAT3 + CD4 + T cells at day +21 (primary endpoint: %pSTAT3 + CD4 + T cells ≤ 35%) and determine the cumulative incidence of grade II-IV acute GVHD by day +100. We also evaluated the impact of PAC/SIR/TAC on CD4 T cell differentiation (Treg, Th1, Th17) and related CD28 (pS6 and pH3ser10) and IL-2 receptor (pSTAT5) signal transduction after alloHCT. Eligible patients (n=28) received alloHCT for AML, MDS, ALL, and MF. Reduced (n=21) or myeloablative (n=7) intensity conditioning was permitted. HLA-A, -B, -C, and -DRB1 matched-related (n=7) or unrelated donors (n=21) were allowed. Adequate vital organ function and Karnofsky performance status (KPS ≥ 80%) were required. Results: PAC/SIR/TAC (PAC 100mg BID) met the primary endpoint of the phase II study, reducing %pSTAT3 + CD4 + T cells to 9.62% at day +21 ( Figure 1A). PAC/SIR/TAC significantly improved CD4 + T cell STAT5 phosphorylation at day +21, increasing the ratio of pSTAT5 + to pSTAT3 + CD4 + T cells (ratio 80.7 v 1.71 P&amp;lt;0.0001) compared to dose level 1 PAC/SIR/TAC of the phase I trial that lacked effective JAK2 blockade (PAC 100mg daily, day +21 %pSTAT3 + CD4 + T cells 59.5%). Partial CD28 signaling blockade was achieved by PAC/SIR/TAC compared to dose level 1, with suppression of mTOR (%pS6 + CD4 + T cells 7.67 v 22.5% P=0.0009) but only modest inhibition of Aurora kinase A activity (%pH3ser10 + CD4 + T cells 71.3 v 98.6% P&amp;lt;0.0001), a pathway for escape alloreactivity. Th1 (0.01 v 0.03 k/µl P=0.026) and Th17 (0.016 v 0.032 P=0.031) cells were reduced at day +21, increasing the ratio of Tregs to Th1 and Th17 cells (0.84 v 0.21 P=0.043) with PAC/SIR/TAC compared to dose level 1. Like JAK2 KO murine T cells, Th2 cells at day +21 were increased with PAC/SIR/TAC (0.056 v 0.001 k/µl P=0.036), compared to dose level 1. T, B, and NK cell engraftment at day +21 was comparable to dose level 1. Despite suppression of Th1 and Th17 cells, the cumulative incidence of grade II-IV acute GVHD by day +100 with PAC/SIR/TAC was similar to historic SIR/TAC values (46.4 v 43%) ( Figure 1B). Acute GVHD onset did not correlate with duration of PAC therapy, depth of pSTAT3 inhibition, or burden of circulating Th1, Th17, or Th2 cells. Conclusions: While PAC/SIR/TAC successfully reduced pSTAT3, increased pSTAT5, and suppressed Th1 and Th17 cells, the regimen did not reduce acute GVHD risk. Completed phase II and III trials testing tocilizumab (anti-IL-6 monoclonal antibody, ACTRN12612000726853, ACTRN12614000266662) and now PAC reveal a biologic disconnect between effective IL-6/JAK2/pSTAT3 axis blockade and a disappointing lack of clinical improvement in acute GVHD prevention. We surmise uncontrolled T cell Aurora kinase A activity contributed to acute GVHD via CD28 costimulation in this trial. As PAC polarizes the pSTAT5:pSTAT3 CD4 + T cell ratio and targets IRAK1 and CSFR1, it may have activity in refractory chronic GVHD. This concept is now being tested by others (NCT05531786).

Gut microbiota trajectory in β-thalassemia major children who underwent allogeneic hematopoietic stem cell transplantation.

The gut microbiota of patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) changes, leading to complications such as acute graft-versus-host disease (GVHD). This study aimed to evaluate the human microbiota composition before and after HSCT in β-thalassemia major (β-TM) children. Twenty-two β-TM children who received allo-HSCT between December 2018 and March 2020 were enrolled. They were followed up for more than 100 days after HSCT, and their gut microbiota information and disease data were recorded at five-time points. The dominant bacteria were Bacteroidetes and Firmicutes at the phylum level and Lachnospiraceae at the family level before and after HSCT. In the differential analysis, Ruminococcaceae constantly decreased after HSCT. Besides, Rothia mucilaginosa was the most abundant 2 months after HSCT compared to before it. Additionally, GVHD patients presented decreased levels of Bacteroidetes compared to those without GVHD. Moreover, Blautia levels significantly decreased in critically ill GVHD patients. The gut microbiota of the 22 β-TM children showed a clear trend of destruction and reconstruction within 100 days after HSCT. The extra-oral infections and inflammations of Rothia mucilaginosa, a Gram-positive bacterium of the normal oropharyngeal tract microbiota, might play an important role in the recovery process of HSCT. Finally, decreased Bacteroidetes levels were associated with GVHD onset.

Tocilizumab increases regulatory T cells, reduces natural killer cells and delays graft-versus-host disease development in humanized mice treated with post-transplant cyclophosphamide.

Graft-versus-host disease (GVHD) is a life-threatening complication following donor hematopoietic stem cell transplantation, where donor T cells damage host tissues. This study investigated the effect of tocilizumab (TOC) combined with post-transplant cyclophosphamide (PTCy) on immune cell engraftment and GVHD development in a humanized mouse model. NOD-scid-IL2Rγnull (NSG) mice were injected intraperitoneally with 2 × 107 human (h) peripheral blood mononuclear cells and cyclophosphamide (33 mg kg-1 ) or saline on days 3 and 4, then TOC or control antibody (0.5 mg mouse-1 ) twice weekly for 28 days. Mice were monitored for clinical signs of GVHD for either 28 or 70 days. Spleens and livers were assessed for human leukocyte subsets, and serum cytokines and tissue histology were analyzed. In the short-term model (day 28), liver and lung damage were reduced in PTCy + TOC compared with control mice. All groups showed similar splenic hCD45+ leukocyte engraftment (55-60%); however, PTCy + TOC mice demonstrated significantly increased (1.5-2-fold) splenic regulatory T cells. Serum human interferon gamma was significantly reduced in PTCy + TOC compared with control mice. Long-term (day 70), prolonged survival was similar in PTCy + TOC (median survival time, > 70 days) and PTCy mice (median survival time, 56 days). GVHD onset was significantly delayed in PTCy + TOC, compared with TOC or control mice. Notably, natural killer cells were reduced (77.5%) in TOC and PTCy + TOC mice. Overall, combining PTCy with TOC increases regulatory T cells and reduces clinical signs of early GVHD, but does not improve long-term survival compared with PTCy alone.

Abstract 38: PBMC donor selection affects engraftment rates and onset of graft versus host disease in humanized NCG mice for use in cancer studies

Abstract Preclinical studies examining tumor biology and immunity are limited by differences between the mouse and human immune systems. Humanized mouse models offer a unique tool to assess the anti-tumor response by recapitulating aspects of human tumor biology. Humanized mice are created by injecting either human CD34+ hematopoietic stem cells or peripheral blood mononuclear cells (PBMCs) into an immunodeficient mouse strain. Here we utilized a PBMC humanized NCG mouse model to investigate human T-cell mediated anti-tumor response (&amp;gt;95% CD3+ T-cells by day 21). The challenges encountered with this model include the onset of graft versus host disease (GvHD) and donor variability leading to donor specific engraftment rates. Consistency is required with humanized PBMC models to ensure their use as an appropriate research model. Therefore, we examined the relationship between donor selection and engraftment rates as well as the onset of GvHD. Inter-donor variability was assessed by collecting PBMCs from multiple healthy donors. Intra-donor consistency was assessed by collecting PBMCs from the same donors at subsequent collections and comparing to initial human immune cell profiling by flow cytometry. NCG mice were injected with 1 × 107 PBMCs from individual donors. Animals were weighed three times a week to monitor for changes in body weight and general health status. Peripheral blood was collected at day 9, 21 and 42 for flow cytometry screening of human immune cell engraftment (hCD45+, hCD3+, hCD4+ and hCD8+) at the initial collection. Human PBMCs isolated from additional collections from the same donors were also injected into NCG mice and flow cytometry was performed on the new cell lots at day 21. Donors were grouped into categories based on engraftment rates, which were donor dependent. Donor variability is an important aspect to consider when humanizing mice with human PBMCs. PBMC humanized models are important for the study of pre-clinical therapeutics, but donor engraftment rates must be considered when engrafting the mice to create a consistently humanized mouse. Citation Format: Steven Bronson, Jenny Rowe, Christoph Eberle, Stephen Festin. PBMC donor selection affects engraftment rates and onset of graft versus host disease in humanized NCG mice for use in cancer studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 38.

Genetic engineering strategies to enhance antitumor reactivity and reduce alloreactivity for allogeneic cell-based cancer therapy.

The realm of cell-based immunotherapy holds untapped potential for the development of next-generation cancer treatment through genetic engineering of chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapies for targeted eradication of cancerous malignancies. Such allogeneic "off-the-shelf" cell products can be advantageously manufactured in large quantities, stored for extended periods, and easily distributed to treat an exponential number of cancer patients. At current, patient risk of graft-versus-host disease (GvHD) and host-versus-graft (HvG) allorejection severely restrict the development of allogeneic CAR-T cell products. To address these limitations, a variety of genetic engineering strategies have been implemented to enhance antitumor efficacy, reduce GvHD and HvG onset, and improve the overall safety profile of T-cell based immunotherapies. In this review, we summarize these genetic engineering strategies and discuss the challenges and prospects these approaches provide to expedite progression of translational and clinical studies for adoption of a universal cell-based cancer immunotherapy.

EASIX score predicts inferior survival after allogeneic hematopoietic cell transplantation.

The Endothelial Activation and Stress Index (EASIX) is a prognostic tool that uses common clinical laboratory values and has been shown to predict non-relapse mortality (NRM) and overall survival (OS) at the onset of acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). We hypothesized that EASIX calculated at different time points pre- and post- HCT may predict NRM and OS, and that EASIX calculated at onset of GVHD may predict response to steroids. We evaluated the EASIX score pre- and post-HCT in 152 patients with lymphoid malignancies undergoing unmodified reduced intensity conditioning (RIC) alloHCT with uniform GVHD prophylaxis. In multivariate analysis, EASIX calculated pre-HCT was significantly associated with higher NRM (HR = 1.64, p = 0.009) and lower OS (HR = 1.33, p = 0.046). Furthermore, EASIX calculated at day 30 and at day 100 was associated with increased NRM (HR = 1.65, p < 0.001; and HR = 1.65, p < 0.001) and decreased OS (HR = 1.27, p = 0.018; and HR = 1.49, p < 0.001), independent of HCT-CI, disease and conditioning regimen. Our study shows that high EASIX scores at various time points pre- and post-HCT are significantly associated with poorer overall outcomes. EASIX provides an independent and easily accessible tool to predict outcomes that can be complementary to other measures of risk stratification for patients undergoing HCT.

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

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

Lenalidomide treatment for recurrent adult T-cell leukemia/lymphoma after allogeneic hematopoietic cell transplantation.

Patients with recurrent adult T-cell leukemia/lymphoma (ATL) after allogeneic hematopoietic cell transplantation (allo-HCT) have a dismal prognosis. We retrospectively evaluated the safety and efficacy of lenalidomide (LEN)in 11 consecutive patients with recurrent ATL after allo-HCT. The median time from allo-HCT to ATL recurrence was 111days (range, 20-1476), and that from allo-HCT to the initiation of LEN was 162days (range, 43-1560). The median initial daily dose of LEN was 10mg (range, 5-25), and the median duration of LEN treatment was 37days (range, 3-1078). Three patients (27%) achieved complete response and two (18%) achieved partial response (PR). The rates of complete or PR according to the involved site were 57% for skin and 50% for nodal or extranodal lesions. With a median follow-up of 1033days (range, 601-1465) among survivors, the 1-year probability of overall survival (OS) after ATL recurrence was 55%. Grade ≥3 toxicities included cytopenia (n=4), superficial vein thrombosis (n=1), and deep vein thrombosis (n=1). Graft-versus-host disease (GVHD) newly developed in five patients (45%) and worsened in four patients (36%). The median duration from the initiation of LEN to GVHD onset or worsening was 5days (range, 1-9). GVHD was manageable in all patients. Seven patients received mogamulizumab (MOG) for recurrent ATL before LEN treatment. The overall response rates to LEN were 57% in patients who had previously received MOG and 25% in those who had not. The 1-year probabilities of OS after recurrent ATL were 71% in patients who had previously received MOG and 25% in those who had not. Although cytopenia and GVHD are common among patients with recurrent ATL after allo-HCT, LEN may improve survival. Administering MOG before LEN may augment treatment efficacy in the allo-HCT population.

EASIX predicts non-relapse mortality after haploidentical transplantation with post-transplant cyclophosphamide.

Endothelial Activation and Stress Index (EASIX) is a prognostic score reflecting endothelial damage. It can identify cohorts of patients at higher risk of non-relapse mortality (NRM) after allogeneic stem cell transplantation (SCT) from a matched-related or -unrelated donor. No data are available in the setting of haploidentical-SCT with post-transplant cyclophosphamide (PT-Cy). We retrospectively analyzed the role of EASIX score in a cohort of 266 patients receiving Haplo-SCT with PT-Cy at our center. By a decision-tree model, 1-year NRM was 16% vs. 29% and overall survival was 59% vs. 32%, respectively, for patients with a pre-transplant EASIX (EASIX-PRE) <0.8 vs. ≥0.8 (p < 0.001). By multivariable analysis, EASIX-PRE was an independent predictor of NRM (hazard ratio [HR] 2.43, p < 0.001) and overall survival (HR: 1.64, p = 0.011). EASIX-PRE did not predict patients at higher risk of developing acute graft-versus-host disease (GVHD) but was an independent predictor of 1-year NRM (3.2 cutoff, HR 6.61, p = 0.002; <3.2 vs. ≥3.2: 10% vs. 56%, p < 0.001) in patients developing acute GVHD. EASIX score can also represent an important tool to predict mortality in the setting of Haplo-SCT with PT-Cy. It may help to make therapeutic decisions both before the transplant and at the onset of acute GVHD.