BMT Recipients Research Articles

Eco-Friendly Product: The Role of Pounding Ecoprint Training on Green Economy

The problems found in PPR Kerinchi are the natural potential of the area that has not been utilized properly, the lack of skills in managing the potential of this community group, the majority of whom are housewives with minimal income. The International Community Service Program is carried out to provide solutions by utilizing the natural potential of PPR Kerinchi and increasing the ability of BMT recipient members to create an eco-friendly product by utilizing the natural potential in their area. This study aims to determine the differences between before-and-after the community service program by holding training in making ecoprints by utilizing natural materials found in the surrounding environment, thus providing direct benefits to people’s lives in the social and economic fields as well as improving the quality of resources through empowering BMT recipient mothers, in PPR Kerinchi also supports the creation of a green economy. The study revealed that the pounding ecoprint training can support the green economy by providing community members with skills to create eco-friendly products, thus increasing their income. Additionally, incorporating online market training empowers these communities to reach broader markets, further enhancing their income growth. The findings demonstrate the importance of increasing the community capacities in utilizing local natural potential and improving community welfare in supporting the green economy.

Association between non-European ancestry, low socioeconomic status, and receipt of HLA-disparate allografts in adult BMT recipients

Association between non-European ancestry, low socioeconomic status, and receipt of HLA-disparate allografts in adult BMT recipients

Inhibition of the Receptor Interactive Protein Kinase 1 (RIP1) Pathway Prevents Acute Gvhd

Acute graft-versus-host disease (GVHD) causes apoptosis of intestinal stem cells (ISCs) that are key targets in the gastrointestinal (GI) tract (Zeiser and Blazar, NEJM 2917; 377:2167). The receptor interactive protein kinase 1 (RIP1) is an important mediator of apoptosis and we investigated the role of the (RIP1) pathway in GI tract and ISC damage in both human and mouse GVHD models. Immunohistochemical staining of human GI biopsies from GVHD patients showed strong phospho-RIP1 staining in biopsies with Lerner grades 2/3 compared to grades 0/1 (12/17 v 0/8, p < 0.01). We then established in vitro models of ISC apoptosis (at least 50% loss) by adding 10 ng/ml of both IFNg and TNFa to organoid cultures from both ileum and colon (organoids cultures with either IFNg or TNFa alone showed no change). Addition to cultures of 0.1 mcg/ml GNE684, a well-characterized small molecule RIP inhibitor (Patel et al, Cell Death Diff 2020; 27:161), dramatically reversed ISC loss in both human and mouse ileal organoids as measured by cross sectional area (Fig 1). WE observed an identical pattern in colon cultures where GNE684 reversed loss of human ISCs (70.0 + 7.7 % v 29.3 + 2.5 %, p < 0.001) and mouse ISCs (100 + 6.6 % v 41 + 6.6 % p < 0.001). In vivo, 14 daily ip injections of GNE684 prevented mortality in a GVHD mouse model (C3H.SW → B6) in a dose dependent fashion (Fig 2). Systemic GNE administration also improved survival in a second model (Balb/c → B6: day 30 survival 7/10 v 0/10, p < 0.001). In the C3H.SW → B6 model there were dramatic reductions in CD4+ lamina propria effector lymphocytes secreting either IFNg (56 + 11 v 9 + 2, p < 0.001) or IL-17 ( 22 + 10 v 7 + 4, p < 0.01) and in CD8+ effector cells secreting IFNg (39 + 11 v 8 + 3, p < 0.001) or IL-17 ( 19 + 8 v 7 + 2, p < 0.001) on day 10 after HCT. Importantly, GVHD was dramatically reduced when administration of GNE684 was used as treatment for GVHD beginning on day +7 after GVHD was established (day 40 survival: 8/12 v 2/12, p = 0.02). Additionally, survival dramatically improved in RIP1 kinase-dead recipients compared to wild-type B6 controls in both the C3H.SW → B6 model (13/16 v 4/16, p < 0.001) and in the Balb/c →B6 model (13/20 v 1/20, p < 0.001). IFNg secreting CD4+ and CD8+ effector lymphocytes that infiltrated the lamina propria were significantly reduced in RIP1 kinase-dead recipients compared to wild-type controls (58 + 13 v 11 + 7 and 39 + 11 v 14 + 4, respectively [p < 0.001 for both]). Infiltrating lymphocytes secreting IL-17 were also reduced four fold. Importantly, administration of GNE684 preserved beneficial GVL effects when lethal doses of myeloid tumor C1498 cells that caused 100% mortality by day 28 in syngeneic BMT recipients were added to the donor inoculum (day 42 survival: 7/10 v 4/13, p = 0.03). We conclude that RIP1 is a key mediator of GI damage during GVHD. In vitro RIP1 inhibition prevents ISC apoptosis in both mouse and human models of GI GVHD. RIP1 inhibition, either through genetic alteration of the RIP1 kinase domain in transplant recipients or through systemic administration of the RIP1 inhibitor GNE684, protects the GI tract from acute GVHD damage as measured both clinically and histologically in preclinical models while preserving beneficial GVL effects. These data suggest that GNE684 a clinical trial of GNE684 is warranted in patients with gastrointestinal GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

THE INFLUENCE OF EARNING ZAKAT DISTRIBUTION LEVEL ON ZAKAT MUSTAHIK BUSINESS PROFIT

This research is motivated by the administration that has not been well organized and the absence of training for BMT and mustahik recipients of productive zakat funds by the National Amil Zakat Agency (BAZNAS) Ciamis Regency. Quantitative field research using a simple linear regression method uses a sample of 30 people. The results showed that (1) Most respondents agreed that the utilization of productive zakat funds at BAZNAS Ciamis Regency through BMT-BMT in Cijeunjing District was quite good. (2) Most respondents agree that the empowerment of mustahik at BAZNAS in Ciamis Regency through BMT-BMT in Cijeunjing District is quite good. (3) The variable utilization of productive zakat funds (X) does not affect the empowerment of mustahik (Y) BAZNAS Ciamis Regency through BMT-BMT in Cijeunjing District.

Donor Plasmacytoid Dendritic Cells Regulate GvHD in a VIP Dependent Manner in Allogeneic BMT Recipients

Introduction: Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide known to induce differentiation of regulatory dendritic cells and regulatory T cells. Using allogeneic hematopoietic stem cell transplantation (allo-HSCT) models, we have shown that donor bone marrow (BM) plasmacytoid dendritic cells (pDCs) facilitate HSC engraftment and attenuate pathogenesis of graft vs. host disease (GvHD) through regulation of recipient T cells. However, the mechanism by which pDCs mitigate the GvHD activity of recipient T cells is not clearly understood. Here, we report that donor pDCs limit pathogenic T cell inflammation by VIP production.Methods: To study VIP production by pDCs, FACS-sorted pDCs from B6 mouse BM were cultured with or without PMA/ionomycin in-vitro. After activation and cytospin slide preparation, pDCs were labeled with anti-PDCA1 (pDC marker) and anti-VIP antibodies for confocal fluorescence microscopy. To investigate the effects of VIP production on T cell proliferation, an in-vitro co-culture assay was performed using R848 and CpG-activated WT or VIP-KO pDCs with anti-CD3-activated, CFSE-labeled syngeneic T cells. For GvHD experiments, irradiated B10.BR (H-2K k) mice received 5x10 3 HSCs, 5x10 4 pDCs and 1x10 6 T cells from WT B6 (H-2K b) or VIP-KO B6 (H-2K b) mice. H&E histology of intestine and colon was performed for GvHD scoring 7 days post-transplant. Graft vs. leukemia (GvL) effects were tested by inoculating recipient mice with 5x10 5 LBRM 33-5A4 cells in the same model. Recipient mice were monitored twice weekly using a 10-point GvHD scoring system. Gene expression analysis of FACS-sorted donor T-cells from recipient spleens was performed using the Nanostring Myeloid Innate Immunity Panel at days 8 and 15 post-transplant.Results: Confocal microscopic images of PMA/ionomycin stimulated or unstimulated sorted pDCs show that VIP is synthesized by pDCs (anti-VIP, green; anti-PCDA-1, red; DAPI counterstain, blue) (Fig 1). After in-vitro culture, VIP expression and frequencies of VIP + pDCs were similar in PMA/ionomycin treated or untreated cells (not shown). VIP-KO mice have significantly higher percentages of pDCs in BM compared to WT (Fig 2a). T cells co-cultured with VIP-KO pDCs showed higher proliferation than T cells co-cultured with WT pDCs, demonstrating that VIP secreted by pDCs reduces T cell proliferation (Fig 2b). Moreover, VIP-KO pDCs induce significantly greater proliferation of IFN-gamma + CD8 T cells compared to WT, indicating that pDCs lacking VIP promote Th1 polarization in-vitro (Fig 2c). The data are consistent with results from GvHD experiments showing increased frequencies of Th1 polarized T cells and fewer regulatory T cells in recipients of VIP-KO pDCs compared with recipients of WT pDCs. Intestinal GvHD scores and crypt apoptosis in the colon were higher in recipient groups transplanted without pDCs or with VIP-KO pDCs compared with recipients of WT pDCs (Fig 3a, b, c). These results indicate that VIP secreted from pDCs limits GvHD in the gut. In the GvL model, administration of pDCs lacking VIP did not alter the anti-tumor effect of donor T cells. Nanostring analysis of T cells recovered from VIP-KO pDC recipients had increased expression of the pro-inflammatory transcription factor Bhlhe40 during the first two weeks post-transplant, and higher transcription levels of the inflammatory mediator Cyclophilin A at day 15 post-transplant than T cells from recipients of WT pDCs.Conclusion: Data from in vitro and in vivo experiments suggest that VIP secreted by pDCs limits pathogenic T cell proliferation. In murine allo-BMT, increased gut GvHD scores and crypt apoptosis in recipients transplanted without pDCs or with VIP-KO pDCs indicates that VIP secreted by pDCs consolidates gut integrity without altering GvL. Gene expression analysis also supports a mechanism by which VIP-secreting donor pDCs reduce T cell inflammation through negative regulation of Bhlhe40. Our findings suggest paracrine VIP signaling is a novel immune checkpoint pathway by which donor pDCs limit T cell activation, Th1 polarization, and inflammation, and improve outcomes of allo-BMT by reducing GvHD activity. [Display omitted] DisclosuresWaller: Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Verastem Oncology: Consultancy, Research Funding.

Trends in Late Mortality and Life Expectancy after Autologous Blood or Marrow Transplantation (BMT) Performed over Three Decades - a BMT Survivor Study (BMTSS) Report

Background: Advances in therapeutic and supportive care strategies have led to increasing use of autologous BMT to ensure durable disease-free survival in patients with hematologic malignancies. However, autologous BMT recipients carry a high burden of morbidity. The cumulative effect of autologous BMT and the long-term morbidity on life expectancy remains unknown. Further, the impact of changes in transplant practice (older age at BMT, increasing proportion of patients undergoing autologous BMT for plasma cell dyscrasias [PCD], increasing use of peripheral blood stem cells [PBSCs] and decreasing use of total body irradiation [TBI]) on late mortality and life expectancy remains unknown. We determined trends in life expectancy and cause-specific late mortality after autologous BMT performed over a 33y period (1981-2014) using the resources offered by BMTSS - a multi-institutional collaborative designed to examine late morbidity and mortality after BMT.Methods: This retrospective cohort included 4,702 individuals who lived ≥2y after autologous BMT performed between 1981 and 2014 at three transplant centers. We examined trends in life expectancy and late mortality over four eras: 1981-1999; 2000-2005; 2006-2010; 2011-2014. Endpoints included all-cause, recurrence-related mortality (RRM) and non-recurrence-related mortality (NRM) and projected reduction in life expectancy. Information on vital status and cause of death was obtained from National Death Index (NDI) Plus program and Accurinct databases. End of follow-up was 04/19/2021.Results: Median age at BMT was 53y (range, 0-78); 58.7% were male, 67.8% non-Hispanic white. Median follow up after BMT was 9y (range, 2-36). PCD was the most common indication for autologous BMT (42.3%). PBSCs were used for 91.6% of the transplants and TBI was used for conditioning in 23.1% of the patients. Across the four eras, the median age at BMT increased (40y→58y), as did the proportion of patients receiving autologous BMT for PCD (13.7%→60.0%) and the proportion of patients receiving PBSCs as the stem cell source (66.6%→99.5%). There was a dramatic decline in the proportion of patients conditioned with TBI (56.4%→5.2%), and those transplanted for acute myeloid leukemia/ myelodysplastic syndrome (AML/MDS) (15.4%→0.2%). Conditional on surviving ≥2y after autologous BMT, patients experienced a 25.8% reduction in life expectancy, representing 7.0y of life lost. In an analysis adjusted for all relevant demographic and clinical predictors and restricted to 5y of follow-up, the years of life lost declined across the four eras from 5y to 1.6y (Fig 1) The adjusted hazard of all-cause mortality also declined over the four eras (reference: 1981-1999; hazard ratio [HR]2000-2005=0.77; 95%CI=0.7-0.9; HR 2006-2010=0.62; 95%CI=0.5-0.7; HR 2011-2014=0.5; 95%CI=0.4-0.6; P trend <0.0001) (Fig 2). Mediation analysis revealed that the reduction in late mortality was evident among those transplanted for Hodgkin lymphoma (HL) or PCD, and among those who did not receive TBI . The 30y cumulative incidence of NRM (29.2%) was higher than that RRM (23.8%) (Fig 3). The adjusted hazard of RRM declined over the four eras (reference: 1981-1999; HR 2011-2014=0.55; 95%CI=0.4-0.7, P trend: <0.0001). Cause-specific late mortality declined for mortality related to infection (P trend <0.0001), subsequent malignant neoplasms (P trend=0.01), cardiovascular disease (P trend=0.001), and renal disease (P trend=0.0002), but not for pulmonary disease (P trend=0.3).Conclusions and Relevance: Late mortality among autologous BMT recipients has declined over a 30y period, likely due to changes in transplant practice (reduction in use of TBI) and improvement in disease-specific therapeutic strategies (newer therapeutic options for PCD and reduction in dose/volume of radiation for HL). Continued efforts need to focus on mitigation strategies for disease recurrence, subsequent neoplasms, infections, and cardiovascular, renal and pulmonary disease in this population. [Display omitted] DisclosuresArora: Kadmom: Research Funding; Pharmacyclics: Research Funding; Syndax: Research Funding. Forman: Allogene: Consultancy; Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company; Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company. Weisdorf: Incyte: Research Funding; Fate Therapeutics: Research Funding.

Oxidative Stress Regulates T Cell Pathogenicity in Gvhd

Allogeneic hematopoietic transplantation (allo-HCT) is a classic immunotherapy for hematologic malignances, but graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality.Oxidative stress is considered as an unavoidable consequence of allo-HCT and could contribute to GVHD pathogenicity significantly. Owing to the contributions of pre-existing disease conditions and the requirement for conditioning regimens that increases cellular reactive oxygen species (ROS), oxidative stress is elevated in all BMT recipients. Thioredoxin (Trx) is ubiquitously expressed enzymes, counteracts oxidative stress by scavenging ROS and regulating other enzymes metabolizing H2O2. In addition, it has been shown that regulatory T cells (Tregs) express and secrete higher levels of Trx than conventional CD4 T cells. Trx-1 is critical for the resilience of Tregs to oxidative stress and sustained expression of surface thiols at a high density. We hypothesis that oxidative stress plays a critical role in the pathology of GVHD development, and thus the maintenance of a redox balance would be an effective strategy to control GVHD after allo-BMT.To test this hypothesis, we used a strain of mice that express transgenic (Tg) human Trx-1 ubiquitously, refer as Trx-Tg mice. We first verified that Trx-Tg T cells demonstrated significantly reduced ROS accumulation after activation in vitro and in vivo. We next sought to test whether Trx1 affects T cell activation and function. Upon alloantigen stimulation in vitro, Trx-Tg T cells have substantially reduced ability to produce IFN-γ compared with WT counterparts. These results prompted us to evaluate T cell responses in vivo . Upon transfer of T cells into irradiated allogeneic recipients, we observed that the Trx-Tg T cells significantly reduced proliferation and IFN-γ production compared with WT counterparts. Given that Trx-Tg T cells displayed a reduced allogeneic response in vitro and in vivo, we further hypothesized that Trx plays an essential role in regulating allogeneic T cells in GVHD. We performed a MHC-mismatched B6→BALB/c BMT model, and found that recipients of WT T cells developed severe and lethal GVHD, whereas the majority of the recipients of Trx-Tg T cells survived long term and had significantly less weight loss and lower clinical score. Development of GVHD requires donor T cell expansion in lymphoid organs and migration into target organs. Hence, we asked whether TRX-1 is required for T cell infiltration and expansion into target organs. Three weeks after BMT, we observed that recipients of Trx-Tg T cells had significantly fewer pathological injuries in all GVHD target organs, which was consistent with the GVHD outcome. While comparable numbers of Trx-Tg T and WT T cells were found in recipient spleens, a significantly lower number of Trx-Tg T cells were found in recipient livers as compared with that of WT T cells. In addition, T cells that migrated into the liver expressed significantly lower levels of intercellular IFN-γ in the Trx-Tg group. These data suggest that Trx-Tg T cells may reduce expansion and/or migration. For translational purpose, we evaluated therapeutic potential of recombinant human Trx-1 (rhTrx-1), and found that administration of recombinant human Trx-1 significantly reduced ability of T cells to produce IFN-γ compared with control treatment upon alloantigen stimulation in vivo . Furthermore, we performed a MHC-mismatched B6→BALB/c BMT model and found that administration of rhTrx-1had significantly reduced the mortality and mobility of BLAB/c recipients. Theraputic effect of rhTrx-1 in the prevention of GVHD was validated in a haploidentical BMT model (B6→BD2F1). More importantly, administration of rhTrx-1 did not impair the graft-versus-leukemia (GVL) against p815 tumor. Taken together, the current work provides a strong rationale and demonstrates the feasibility to target ROS pathway, which can be readily translated into clinic. DisclosuresNo relevant conflicts of interest to declare.

NKG2D Negatively Regulates CD4 T Cells in Allo-Response in Mice

NKG2D is a C-type lectin-like receptor which mainly expresses on mouse NK, activated CD8 and some γδ T cells. NKG2D is also found to express on human and mouse CD4 T cells, but the role of NKG2D in CD4 T cells is essentially unknown. We asked what subsets of CD4 T cells could express NKG2D; we activated and polarized CD4 T cells into Th0, Th1,Th2, Th17 and Foxp3+ Tregs in vitro and measured NKG2D expression on those subsets. We found that only a proportion of Th1 cells expressed NKG2D, whereas little or no NKG2D was expressed on other subsets of T cells. In a mixed lymphocyte reaction (MLR) assay, NKG2D was induced on a subset of CD4 T cells after stimulation with allogeneic antigen presenting cells (APCs) (Figure 1A). NKG2D+CD4+ T cells expressed higher levels of LAG-3, CD49b, CD80, FasL and PD-L1 (Figure 1B) and secreted more IL-10 and IFN-γ than NKG2D- CD4+ T cells (Figure 1C).Previous reports by others showed that NKG2D on NK and activated CD8 T cells contributing to graft versus host disease (GVHD) pathogenesis have been well-established, but the expression and function of NKG2D on CD4 T cells in GVHD development are unknown.To address these questions, we utilized a CD4-driven, MHC-mismatched aGVHD model of allogeneic bone marrow transplantation (allo-BMT), C57BL/6 to BALB/c. We monitored the development of NKG2D+CD4+ T cells in allogeneic recipients. We observed that NKG2D was continuously induced on CD4 T cells during the whole aGVHD process - gradually increased, peaked on day 14 and then slowly decreased after allo-BMT. By comparing the severity of aGVHD in recipients transferred with WT versus NKG2D-/- CD4 T cells. We observed that recipients transferred with NKG2D-/- CD4 T cells showed more severe GVHD demonstrated by a higher lethality (Figure 1D) and more severe diarrhea compared to WT counterpart. Mechanistically, NKG2D-/- CD4 T cells had a higher capacity of migrating to gut and liver than WT CD4 T cells reflected by the higher expression of α4β7 and CXCR3. Furthermore, NKG2D+CD4+ T cells secreted less proinflammatory cytokine IL-17 and more anti-inflammatory cytokine IL-10. Strikingly, NKG2D expression coincided with T-bet, but excluded RORγt or Foxp3 expression of CD4 T cells. Given NKG2D+CD4+ T cells expressed regulatory markers and secreted IL-10 and IFN-γ; we hypothesized that NKG2D+CD4+ T cells may serve as suppressive cells (e.g. Tr1 cells). To test this hypothesis, we sorted NKG2D+CD4+ and NKG2D-CD4+ T cells from recipients of allogeneic BMT, and performed in vitro suppressive assay. We observed that NKG2D+CD4+ T cells had a higher suppressive activity than NKG2D-CD4+ T cells. (Figure 1E)Taken together, the current work demonstrates that NKG2D may serve as a negative regulator on CD4 T cells, and NKG2D+CD4+ subset can function as a regulatory cell subset in the development of GVHD after allo-BMT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Long-Term Morbidity Associated with Chronic Graft Vs. Host Disease (cGvHD) - a Blood or Marrow Transplant Survivor Study (BMTSS)

▪Background: Patients with cGvHD are at risk for morbidity and compromised quality of life. However, the risk of specific morbidities observed long-term in patients with cGvHD is not clearly understood. Furthermore, whether patients with cGvHD are more likely to be frail, with added vulnerability to an already compromised functional status, remains unstudied. Methods: Study participants were drawn from BMTSS - a retrospective cohort of patients who had received a BMT at City of Hope or University of Minnesota between 1974 and 1998 for a hematologic malignancies or severe aplastic anemia (SAA) and survived at least 2y post-BMT. This study included 581 allogeneic BMT recipients ≥18y of age at study participation and alive at baseline survey (2000-2004) or follow-up survey (2013-2017). The survey included a self-report of sociodemographic characteristics and physical health conditions (Blood. 2010;116:3129-3) . The frailty phenotype was constructed from responses provided by BMT survivors using modified Fried criteria (JAMA Oncol. 2016;2:1277-86) . Details regarding cGvHD were abstracted from medical records. We conducted a systematic evaluation of the association between cGvHD and key chronic health conditions (xerostomia, dry eyes, cataract, glaucoma, diabetes, hypothyroidism, cardiomyopathy, coronary artery disease, stroke, tremors, problems with balance, pain, sensory loss, subsequent malignancies, esophageal or rectal/anal stricture, cirrhosis and pulmonary complications), using multivariable cox regression analyses adjusted for sociodemographics, primary diagnosis, smoking status, pre-existing comorbidities and TBI. We also examined the association between cGvHD and frailty using logistic regression and adjusting for the variables listed above, as well as chronic health conditions. Results: The 581 study participants underwent BMT at a median age of 30.9y (0.4-62.9) and participated in the study at a median age of 48.0y (18.2-73.7), after a median of 17.1y (2.0-40.5) from BMT; 55% were males and 77% were non-Hispanic whites. Primary diagnoses included CML (37%), AML (28%), ALL (14%), SAA (9%), other (12%). In this cohort of 581 allogeneic BMT recipients, 304 (52.3%) carried a history of cGvHD. All patients received myeloablative conditioning; TBI was used in 85%. The frequency of the following chronic health conditions was statistically-significantly higher in BMT patients with a history of cGvHD vs. those without cGvHD. (Fig 1): oral (difficulty in tasting/swallowing/chewing) and/or ocular (dry eyes) conditions (54% vs. 34%, p<0.0001), diabetes (17% vs. 11%, p=0.04), osteonecrosis (13% vs. 5%, p=0.002), neurological symptoms (tremors/decreased sense/prolonged pain: 33% vs. 23%, p=0.007), pulmonary complications (fibrosis/scarring/oxygen supplementation: 23% vs. 12%, p=0.0005), and GI complications (esophageal strictures + rectal/ anal strictures: 13% vs. 8%, p=0.05). The frequency of frailty was also higher in those with cGvHD vs. those without cGvHD (17.1% vs. 6.14%, p <0.0001). Overall, 45% of the allogeneic BMT recipients with cGvHD vs. 25% of those without cGvHD had 2 or more chronic health conditions (p<0.0001). As shown in Fig 1, multivariable analyses showed statistically significant associations between cGvHD and oral/ocular complications (HR=1.91, 95%CI, 1.4-2.5, p<0.0001), diabetes (HR=1.58, 95%CI, 0.96-2.6, p=0.07), osteonecrosis (HR=2.59, 95%CI, 1.4-5.0, p=0.004), neurological complications (HR=1.52, 95%CI, 1.1-2.2, p=0.02), pulmonary complications (HR=2.04, 95%CI, 1.3-3.2, p=0.002), and GI complications (HR=1.87, 95%CI, 1.1-3.3, p=0.03). In a multivariable model, adjusting for demographic and clinical variables and chronic health conditions, the odds of frailty were 2.66-fold higher among those with cGvHD (95%CI, 1.4-5.1, p=0.003) when compared with those without cGvHD. Conclusions: Allogeneic BMT recipients with a history of cGvHD carry a significantly higher burden of long-term morbidity, and are also at a significantly higher risk of frailty. The association between cGvHD and key chronic health conditions provides evidence for developing targeted surveillance and intervention strategies to reduce the burden of morbidity and frailty. [Display omitted] DisclosuresArora:Takeda Oncology: Consultancy.

Adverse Effects of Busulfan Plus Cyclophosphamide versus Busulfan Plus Fludarabine as Conditioning Regimens for Allogeneic Bone Marrow Transplantation

Background:The side effects of conditioning regimens on the success rate of allogeneic transplantation around the world have been challenging. In this study, we aimed to investigate the side effect of Bu/Cy and Bu/Flu regimens on our patients who underwent allogeneic bone marrow transplantation. Methods:We analyzed 180 patients receiving bone marrow transplantation in Taleghani Hospital, in Tehran, Iran between April 2016 and December 2019. Patients in group A received a combination of intravenous busulfan 0.8 mg/kg QID over two hours for 4 consecutive days (12.8 mg/kg in total)(Savani et al., 2006) and cyclophosphamide 60 mg/kg per day for two consecutive days. Patients in group B received busulfan the same as the first group in combination with fludarabine equal to 40 mg/m² per day. Patients were followed up at regular intervals up to two years after transplantation. Result:Various items were evaluated for patients, including cardiopulmonary function, psychological disorders, GVHD, and endocrine disorders such as hypothyroidism, fertility, or gonad dysfunction. Primary hypothyroidism developed in 13.3% and 11.1% of the Bu/Cy and Bu/Flu groups, respectively (p=0.230). None of the patients in either group experienced infertility or gonad dysfunction. In group A versus group B, pulmonary diseases were detected in 4.4% versus 6.6% of BMT recipients, respectively (p = 0.223). In both groups, mitral and tricuspid regurgitation were observed in patients (8.9% vs. 11.1%; p = 0.189). Incidence of Psychological disorders was no significant difference between the two groups. 32.2% of group A versus 34.45% of group B had skin and liver GVHD, respectively (p = 0.235). Conclusion:The therapeutic-related adverse effects of the two conditioning regimens in patients who underwent allogeneic bone marrow transplant were almost similar. To improve quality of life and overall survival among BMT patients, careful evaluation of treatment-related complications should be part of the regular follow-up of them.

Cytomegalovirus in Adult Allogeneic Blood and Marrow Transplant Patients Before or Around the Period of Neutrophil Recovery: A Single-Center, Retrospective, Descriptive Study.

BackgroundFew reports exist on pre-engraftment cytomegalovirus (CMV) DNAemia in allogeneic blood or marrow transplant (allo BMT) recipients. We describe this clinical entity, its management, and the potential effect of 3 different quantitative CMV deoxyribonucleic acid (DNA) tests used during the 6-year study period. MethodsWe performed a retrospective, single-center study of allo BMT recipients from 2010 to 2015 who developed CMV DNAemia before neutrophil recovery (absolute neutrophil count [ANC] <1000 cells/mm3, “pre-engraftment CMV”) or who became neutropenic concomitant with detectable CMV DNA (“peri-engraftment CMV”). Clinical data were collected from the electronic medical record.ResultsAmong 1151 adult allo BMT patients, 73 developed CMV DNAemia before engraftment or while neutropenic after initial engraftment. Most patients were eventually treated (valganciclovir or ganciclovir, N = 68; foscarnet, N = 1); 4 were not treated. First CMV detection occurred at median day +12 (range, 0–48), but treatment was not started until median day +33 (range, 4–105) at median ANC of 760 cells/mm3. Six patients had peak viral loads >5000 IU/mL; none had tissue-invasive disease. One developed ganciclovir resistance. No significant differences were observed upon stratification by quantitative CMV DNA test. ConclusionsCytomegalovirus DNA was detected in 6.3% of pre- and peri-engraftment allo-HSCT patients. Ganciclovir derivatives were commonly used for treatment despite risk of neutropenia. Treatment was typically deferred until CMV DNA and ANC rose. With rare exceptions, this treatment strategy did not appear to have adverse clinical consequences with respect to acute CMV. Different CMV DNA quantification tests used performed similarly from a clinical perspective despite different analytical performance characteristics.

Stroke Risk in Blood or Marrow Transplant (BMT) Survivors - a Report from the BMT Survivor Study (BMTSS)

BACKGROUND: BMT recipients are vulnerable to accelerated atherosclerosis due to prior exposure to radiation with or without chemotherapy, and consequent long-term cardiovascular morbidity, such as stroke. A comprehensive evaluation of the risk of late-occurring stroke in adult BMT survivors and the associated risk factors has not been performed. We addressed this gap using the resources offered by the BMTSS. METHODS: BMTSS includes patients transplanted between 1974 and 2014 at 3 US sites who survived ≥2y after BMT, were alive and ≥18y at BMTSS survey completion. The survey asked participants to report if a healthcare provider had diagnosed specific chronic health conditions (including stroke), or relapse of primary cancer or development of new cancer, along with age at diagnosis. The participants provided information on sociodemographics, health behaviors and medication use. Medical record abstraction was used for information regarding primary cancer diagnosis, therapeutic exposures (pre-BMT chemotherapy/radiation, transplant preparative regimens), stem cell source (autologous, allogeneic), graft type (bone marrow, cord blood or peripheral blood stem cells), and history of chronic graft vs. host disease (GvHD). A cohort of 908 siblings also completed the BMTSS survey and served as a comparison group. Informed consent was obtained from all participants. RESULTS: The study included 3,479 BMT survivors; 50.3% had received an allogeneic BMT, 54.8% were males; 71.4% were non-Hispanic whites. Median age at study participation was 59y (range: 18-89y) for BMT survivors and 57y (range: 18-90y) for siblings. Patient characteristics are shown in Table 1. BMT survivors were followed for a median of 9y (range: 2-41 y) from BMT. Stroke was reported by 136 BMT survivors (67 allogeneic, 69 autologous); of these, 75 (55%) patients developed stroke ≥2y after BMT. Conditional on surviving ≥2y after BMT, the 10y cumulative incidence of stroke was 3.8% (Fig 1), and was comparable for allogeneic (3.4±0.5%) and autologous (4.2±0.6%) BMT recipients, p=0.3. Stroke in BMT recipients compared with siblings: Using logistic regression, and after adjusting for sociodemographics, physical activity and relevant comorbidities, we found that allogeneic BMT survivors were at a 2.1-fold higher odds of reporting stroke as compared to siblings (95%CI: 1.2-3.7, p=0.01), and autologous BMT recipients were at a 1.7-fold higher odds of reporting stroke compared to siblings (95%CI: 0.9-3.0, p=0.09). Stroke after Allogeneic BMT: History of hypertension (HR=2.2, 95%CI: 1.2-3.9, p=0.007), venous thromboembolism (HR=3.4, 95%CI: 1.6-7.1, p=0.002), diagnosis of acute lymphoblastic leukemia (HR=4.9, 95%CI: 1.6-15.0, p=0.006), acute myeloid leukemia/ myelodysplastic syndrome (HR=5.2, 95%CI: 1.4-19.0, p=0.013) (ref: non Hodgkin lymphoma), pre-BMT exposure to alkylating agents (HR=3.3, 95%CI: 1.3-8.5, p=0.01) and pre-BMT neck radiation (HR=5.4, 95%CI: 1.2-23.8, p=0.03) were associated with increased stroke risk. Exercise was associated with lower stroke risk (HR: 0.5, 95%CI: 0.3-0.9, p=0.01). Stroke after Autologous BMT: The risk factors for stroke in autologous BMT survivors included: increasing age at BMT (HR=1.02/y, 95%CI: 1.0-1.1, p=0.05), history of hypertension (HR=1.8, 95%CI: 1.1-3.2, p=0.03), coronary heart disease (HR=2.8, 95%CI: 1.3-6.4, p=0.01) and venous thromboembolism (HR=2.3, 95%CI: 1.1-4.7, p=0.02). Relapse of primary disease or development of new cancer were not associated with increased stroke risk in either autologous or allogeneic BMT recipients. CONCLUSION: In this large study, we found that the incidence of stroke was 4% among BMT survivors, and that they are at an increased risk of developing stroke when compared to an unaffected comparison group. The study also identified subgroups among BMT survivors at increased risk of stroke such as those who received neck radiation and those with cardiovascular comorbidity. These findings suggest a need for increased awareness of stroke as a late complication of BMT, such that aggressive management of cardiovascular risk factors can be instituted among those at highest risk. Disclosures Weisdorf: Incyte: Research Funding; Fate Therapeutics: Consultancy; Pharmacyclics: Consultancy.

PD-1 Is Expressed at Low Levels on All Peripheral Blood Natural Killer Cells but Is a Significant Suppressor of NK Function Against PD-1 Ligand Expressing Tumor Targets

PD-1 Is Expressed at Low Levels on All Peripheral Blood Natural Killer Cells but Is a Significant Suppressor of NK Function Against PD-1 Ligand Expressing Tumor Targets

Loss of Asxl1 Cooperates with Oncogenic Nras to Drive CMML Progression

Chronic myelomonocytic leukemia (CMML) belongs to the group of "mixed myelodysplastic/myeloproliferative neoplasm". There is currently no effective chemotherapy to treat CMML. Approximately 30% of CMML cases evolve to acute myeloid leukemia (AML) soon after their initial diagnosis, contributing to the poor prognosis of CMML patients. Ras pathway genes are frequently mutated in CMML patients (NRAS:11%; CBL:10%; KRAS:8%). Our whole exome sequencing of CMML patient samples and targeted sequencing of CMML patient-derived myeloid colonies showed that oncogenic NRAS could serve as an initiating or progression mutation. Consistent with human genetics, all the oncogenic Nras mice we and others characterized (NrasG12D/+, NrasG12D/G12D, and NrasQ61R/+), develop myeloproliferative (MP)-CMML like phenotypes.However, oncogenic NRAS alone is insufficient to drive CMML progression or transformation to AML. Additional sex combs-like 1 (ASXL1) is a human homolog of fly Asx. Mutations in ASXL1are predominantly nonsense and frequently identified in all myeloid malignancies (e.g. 40% in CMML). ASXL1mutations predict inferior overall survival in univariable analysis in multiple large CMML cohort studies and are significantly associated with NRASmutations in CMML patients. Therefore, we hypothesize that loss of Asxl1 cooperates with oncogenic Nrasto drive CMML progression. To test our hypothesis, we used Vav-Cre to drive oncogenic Nras expression and/or Asxl1 deletion in hematopoietic system. We refer these mice as NrasG12D/+, Asxl1-/-and NrasG12D/+;Asxl1-/-(NA) mice. Consistent with the literature, Asxl1-/-mice are normal at 6-week old. NrasG12D/+ mice showed mild CMML-like phenotypes, including enlarged spleen, increased white blood cell and monocyte counts, and expanded LSKs (Lin-Sca-1+c-Kit+) and myeloid progenitors (MPs). Compared to NrasG12D/+ mice, NA mice showed more sever phenotypes that were associated with hyperactivated ERK signaling in MPs at both basal level and upon GM-CSF stimulation. Our new NrasG12D/+mice developed a fully penetrant MP-CMML with the median survival of ~430 days. Loss of Asxl1 significantly shortened the median survival of NrasG12D/+ mice to 220 days. Half of NA mice displayed CMML transformation to AML. The transformed AML was serially transplantable to recipients. To investigate the mechanism(s) underlying NA-driven CMML/AML, we performed RNA-Seq analysis in Lin- c-Kit+BM cells from age-matched control, Asxl1-/-, NrasG12D/+, and moribund NA mice with CMML or AML phenotypes. We found 844 differentially expressed genes (DEG) in AML cells compared to control cells (FDR&lt;0.05 and fold change&gt;2). Among these genes, 341 are common between NA and published Nf1+/-;Asxl1+/-AML cells, including AP-1 complex genes (Jun, Junb, Jund and Fosb) that are crucial for malignant propagation in several AML subtypes. The remaining 503 genes are unique in NA AML, including Flt3, one of the mostly mutated genes in AML. These data suggest that our NA AML model demonstrates some distinct mechanisms from Nf1+/-;Asxl1+/-model. In-depth analysis of RNA-seq data is still ongoing. We also examined multiple histone marks using Western blot in BM cells of 6-week old mice. Our data showed that levels of H3K4me3, H3K4me1, and H3K27me3 were downregulated in Asxl1-/-cells, whereas H3K27Ac level was comparable to that of control cells. In contrast, H3K27Ac level was upregulated in NrasG12D/+and NA cells, while levels of H3K4me3, H3K4me1, and H3K27me3 were indistinguishable from those of control cells. BRD4 is a member of the BET family of bromodomain-containing proteins that binds to acetylated histones to activate gene transcription. Thus, we hypothesized that Ras/MEK/ERK signaling and BRD/BET are potential targets to inhibit AML progression. To test this hypothesis, we treated the AML cells and BMT recipients using trametinib (a MEK inhibitor approved by FDA to treat melanoma) and GSK525762 (a pan BET inhibitor under clinical development). We demonstrated thatcombined treatment significantly inhibited the growth of leukemia cells in vitro and prolonged the survival of recipients in vivo. It was more effective than single agent alone. Overall, we established a novel CMML/AML mouse model, which represents a significant group of CMML patients with poor prognosis. Our study provides a strong rational to treat these patients with combined MEK and BET inhibition. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Longitudinal Trajectory of Frailty in Blood or Marrow Transplant (BMT) Survivors - a BMT Survivor Study (BMTSS)

Introduction: Frailty is a state of increased vulnerability resulting in a decline in reserve and function across multiple physiologic systems such that the ability to cope with every day or acute stressors is compromised. We previously reported that BMT recipients with frailty were at a 2.8-fold higher risk of subsequent mortality (JAMA Oncol. 2016;2:1277-86) when compared with non-frail survivors. However, longitudinal trends in frailty several years after BMT are not known, and could possibly help in identifying the vulnerable sub-populations at highest risk of subsequent adverse events. We used BMTSS to report transitions in the frailty state over time. Methods: We included 484 patients who were i) transplanted for a hematologic malignancy between 1974 and 1998 at 2 sites and had survived ≥2y post-BMT; ii) were ≥18y of age at study participation; and iii) were alive both at completion of the baseline survey (t1: median of 7.3 y after BMT) and at follow-up survey (t2: median of 20.6y after BMT); median interval between t1 and t2 was 13.2y. Frailty phenotype used the previous definition (JAMA Oncol. 2016;2:1277-86), and included the presence of ≥3 of the following indices: 1) clinically underweight (body mass index [BMI] &amp;lt;18.5); (2) exhaustion; (3) low energy expenditure; (4) slowness; and (5) weakness. Patients reporting the presence of 1-2 of the 5 indices were classified as pre-frail. We created the following categories of change in frailty status over time; A) Worsening of frailty status/ stable frail status : pre-frailty [t1] → frailty [t2]; no-frailty [t1] → pre-frailty/frailty [t2]; and frailty [t1] → frailty [t2]; B) Improvement of frailty status: frailty [t1] → pre-frailty [t2]; pre-frailty [t1] → no frailty [t2]; C) Stable non-frail/pre-frail status: non-frail [t1]→ non-frail [t2]; pre-frail [t1] → pre-frail [t2]. We used logistic regression to evaluate predictors of A) worsening of frailty status/stable frail status, and B) frailty at t2, controlling for age at questionnaire, sex, diagnosis, smoking status, socio-economic, and BMT related variables (conditioning, donor type, presence of cGvHD), grade 3-4 chronic health conditions, and pre-BMT treatments therapeutic exposures. Results: Median age (range) at t1 was 42.9y (18.5-67.4) and at t2 was 56.5y (31.0-80.0); 49.4% were females; 85.4% non-Hispanic whites; 15% had &amp;lt;high school education, and 50% had annual household income of &amp;lt;$60,000. Primary diagnoses included acute leukemia (31%), lymphoma (31%), and chronic myelogenous leukemia (25%); 57% had received an allogeneic BMT; 77% received TBI; 40.3% had cGvHD. Prevalence of frailty was 4.8% at t1 and 9.6% at t2, and that of pre-frailty was 13.9% at t1 and 16.1% at t2 (Figure). Worsening of frailty status was seen in 18.8% of the patients, improvement in 9.7%; 68.7% of the patients remained non-frail or pre-frail. Predictors of worsening of frailty status: Pre-BMT exposure to vincristine (OR=5.1, 95%CI: 2.3-11.4) and female sex (OR=1.5, 95%CI, 0.9-2.6, p=0.08) were associated with worsening. Predictors of frailty at t2: Pre-BMT exposure to vincristine (OR=4.6, 95%CI, 1.7-12.5, p=0.003), history of cGvHD at t1 (OR=2.3, 95%CI, 1.0-5.2, p=0.04) and grades 3-4 chronic health conditions at t1 (OR=2.1, 95%CI, 1.0-4.5, p=0.04) were associated with frailty at t2. Age was not a significant predictor in either analysis. Conclusions: In a cohort of patients followed longitudinally for a median of 20.6y from BMT, with measurement of frailty status 13.2y apart, we observed that the frailty status worsened for ~20% of the patients. Pre-transplant exposure to vincristine was associated with worsening of frailty status. Patients with cGvHD or morbidity at t1, as well as pre-BMT vincristine exposure placed BMT survivors at high risk for subsequent frailty, identifying sub-populations that could benefit from targeted interventions. Disclosures Weisdorf: Incyte: Research Funding; Fate Therapeutics: Consultancy; Pharmacyclics: Consultancy.

T-Cell Derived Interferon Gamma Directly Targets Intestinal Epithelium to Induce Stem Cell Apoptosis in GI Gvhd

Crypt base intestinal stem cells (ISCs) marked by Lgr5 and Olfm4 maintain the intestinal epithelium, and Paneth cells (PCs) provide an epithelial niche for ISCs in the small bowel. ISCs are reduced during gastrointestinal (GI) GVHD, but the precise mechanisms including the role of niche injury are unknown. Additionally, the specific effects of Interferon-γ (IFNγ) on intestinal epithelium in GVHD remain ill-defined. We evaluated kinetics of ISC loss by histology after allogeneic (allo) BMT using Lgr5-LacZ reporter mice. In both MHC- and miHA-mismatched models (LP&amp;gt;B6 and B6&amp;gt;BDF1), ISC numbers quickly recovered from pretransplant TBI conditioning in recipients of T-cell-depleted (TCD) BMT by day +10, but ISCs failed to recover in recipients of allo T cells. T-cell-induced ISC reduction was functionally validated by genetic marking of stem cell progeny and by culturing intestinal organoids from crypts isolated post-BMT. Similar to the kinetics of ISC loss, ISC-dependent organoid-forming capacity was impaired in recipients of allo T cells compared with TCD BMT recipients on day +10 (p&amp;lt;0.05). Likewise, BMT into Olfm4 reporter mice showed significantly reduced lineage tracing from ISCs in recipients of allo T cells (Fig. 1). To better understand the potential for T cells to interact with the ISC compartment, we performed whole-mount 3-D microscopy to distinguish T cell localization within the intraepithelial and lamina propria compartments post-transplant. We found that donor T cells invading the small bowel after BMT were mostly in the crypt region, and infiltration within the lamina propria adjacent to the ISC compartment was much greater than invasion within the epithelium itself (Fig. 2). We next established an ex vivo co-culture system, to model interactions between intestinal epithelium and donor T cells and investigate mechanisms of T-cell-mediated ISC injury. Screening of effector pathways revealed no impact of perforin or FasL, but identified IFNγ as a principal mediator of ISC injury. Culture with allo T cells significantly reduced viable human and mouse intestinal organoid numbers, and this was inhibited by IFNγ neutralization. IFNγ receptor knockout (IFNγR-/-) organoids were resistant to T cells. IFNγ increased expression of Bak1 and decreased expression of Bcl2 in organoids, and induced ISC apoptosis defined by Annexin+Dapi-Lgr5+ phenotype. ISC killing was mediated by intraepithelial JAK/STAT signaling, as JAK1- and STAT1-deficient organoids were resistant, and it was inhibited by Ruxolitinib. Investigating the role of IFNγ in vivo, FACS analysis confirmed donor T cells to be the primary producers of IFNγ in crypt lamina propria, and BMT with IFNγ-/- donor T cells reduced crypt apoptosis, and preserved ISC frequencies. Moreover, BMT with recipients lacking IFNγR specifically in the intestinal epithelium significantly protected ISCs, reduced crypt apoptosis, and ameliorated GI GVHD pathology (Fig.3). Furthermore, ISCs were also protected by epithelial deletion of STAT1 and by Ruxolitinib treatment. As specific genetic manipulation of ISCs in vivo is not possible because genetic targeting of ISCs results in the same changes in their progeny, we utilized ex vivo models to determine if IFNγ kills ISCs by directly inducing their apoptosis or by damaging the PC niche. Manipulation of the niche by culturing wild-type ISCs with IFNγR-/- PCs was not protective to allo T cells. Using a niche-independent high-purity Lgr5+ ISC culture system based on combined GSK3β and HDAC inhibition, IFNγ directly induced cleaved-caspase-3+ ISC apoptosis and substantial ISC colony death, which were inhibited by Bak/Bax deficiency and by the pan-caspase inhibitor QVD. These results confirmed that IFNγ can directly induce ISC apoptosis independent of other cytotoxic effector molecules and independent of injury to the PC niche. In summary, T cells migrating to the GI tract primarily infiltrated the lamina propria adjacent to the ISC compartment, and T-cell-derived IFNγ directly targeted intestinal epithelium via JAK1/STAT1 signaling to induce ISC apoptosis in a PC-independent manner. ISC reduction and GI GVHD pathology were prevented by inhibiting the IFNγR/JAK1/STAT1 axis within the intestinal epithelium, indicating that in addition to their effects on T cells, JAK inhibitors may treat GVHD by inhibiting pathologic cytokine signaling within target organs and shielding them from allo T cells. Disclosures Hanash: Nexus Global Group LLC: Consultancy.

Long-term Kinetics of Intragraft Gene Signatures in Renal Allograft Tolerance Induced by Transient Mixed Chimerism.

Renal allograft tolerance (TOL) has been successfully induced in nonhuman primates (NHPs) and humans through the induction of transient mixed chimerism. To elucidate the mechanisms of TOL, we compared local immunologic responses in renal allografts with those in T-cell-mediated rejection (TCMR) and chronic antibody-mediated rejection (CAMR) in NHPs. Using the NanoString nCounter platform, we retrospectively studied 52 mRNAs in 256 kidney allograft samples taken from NHP kidney recipients of donor BMT. No immunosuppression was given after 1-month post-donor BMT. Recipients who achieved TOL (n = 13) survived for >1840 ± 1724 days with normal kidney function, while recipients with CAMR (n = 13) survived for 899 ± 550 days with compromised graft function, and recipients with TCMR (n = 15) achieved only short-term survival (132 ± 69 days). The most prominent difference between the groups was FOXP3, which was significantly higher in TOL than in CAMR and TCMR, both early (<1 y, P < 0.01) and late (≥1 y, P < 0.05) after transplant. Other mRNAs related to regulatory T cells (Treg), such as IL10, TGFB, and GATA3, were also high in TOL. In contrast, transcripts of inflammatory cytokines were higher in TCMR, while activated endothelium-associated transcripts were higher in CAMR than in TOL. The receiver operating characteristic analyses revealed that intragraft FOXP3 and CAV1 can reliably distinguish TOL from CAMR. High FOXP3 and other Treg-related mRNAs together with suppressed inflammatory responses and endothelial activation in renal allografts suggest that intragraft enrichment of Treg is a critical mechanism of renal allograft TOL induced by transient mixed chimerism.

Graft-Versus-Host Disease Leads to Systemic Elimination of Innate Lymphoid Cells and Abolishes Their Development

Innate lymphoid cells (ILCs) are associated with protective mucosal response after experimental allogeneic BMT. However, the specific in vivo roles of ILCs and their capacity for reconstitution post-transplant remain unclear. We therefore sought to characterize the significance and tissue distribution of ILCs after allo-BMT. To study the role of ILCs after allo-BMT, we established a clinically relevant acute GVHD model based on H-Y antigen mismatch. Lethally irradiated Rag2−/− male mice (Thy1.2, B6) were transplanted with T cell depleted (TCD) bone marrow and purified T cells obtained from wild-type female mice (Thy1.1, B6). To deplete host-derived ILCs, recipient mice were treated with an anti-Thy1.2 antibody or an anti-isotype antibody prior to BMT. Pre-transplant depletion of ILCs induced significantly worse systemic signs of GVHD, as well as increased thymic injury and intestinal GVHD pathology, indicating an important role for ILCs after BMT (Fig. 1). To better understand the systemic roles of ILCs, we evaluated ILC subsets in the intestine, liver, and lungs after allo-BMT. Two weeks after MHC-mismatched (C57BL6 into BALB/c) TCD-BMT, both donor- and host-derived ILCs could be detected in all tissues. Liver was characterized by a high number of NK cells and ILC1s, while the lungs contained a high proportion of ILC2s, and small intestine contained relatively greater ILC3s. For all tissues analyzed, the majority of ILCs were host-derived. During T cell replete BMT resulting in GVHD, both donor and host-derived ILCs were significantly reduced in all tissues compared to TCD-BMT mice (Fig. 2). To better understand the loss of donor-derived ILCs caused by donor T cells, we investigated ILC precursors in recipient marrow post-BMT, and found a significant loss of CLPs, αLPs, CHILPs, and ILC2 precursors in mice with GVHD. These data suggested that bone marrow involvement with GVHD induced a loss of ILC precursors impairing their reconstitution post-BMT. Given the loss of ILCs occurring post-BMT and the increased GVHD severity in BMT recipients lacking ILCs, we hypothesized that expansion of the ILC compartment pre-transplant could help protect BMT recipients from GVHD. Aryl hydrocarbon receptor (AhR) signaling has been associated with ILC3 development and maintenance during intestinal homeostasis. We thus evaluated the effect of diet-derived AhR ligands (AhRL) on intestinal ILCs and BMT outcomes. Prior to transplantation, mice were given diets containing or lacking AhRL. Mice on an AhRL-poor diet showed reduced intestinal ILC3s, increased intestinal GVHD pathology, and increased mortality post-BMT. In conclusion, ILCs contribute to tissue protection following allo-BMT, but GVHD leads to elimination of pre-existing ILCs and abolishes their development. Dietary supplementation with AhRL-rich foods may represent an approach to enhance mucosal immunity and intestinal recovery post-BMT.

Molecular detection of Toxoplasma gondii in a sample of Iraqi patients with acute leukemia and stem cell transplantation

BACKGROUND: Acute leukemia and allogenic bone marrow transplantation BMT are immunocompromised conditions which may be susceptible for many opportunistic infections or reactivation of latent infections like Toxoplasma gondii (T.gondii). OBJECTIVES: The aims of study were to detect T.gondii in both acute leukemia patients and allogenic BMT recipients and to determine copy number of T.gondii in these groups in comparison to healthy individual as control group. METHODS: Sixty one acute leukemia patients enrolled in a prospective study from 1st December 2016 to 1st June 2017. Forty eight of them evaluated while induction chemotherapy (group I), while the other 13 within 1 year post bone marrow transplantation-BMT-(group II). In addition to 30 apparently healthy individuals as (control group), blood samples were collected from all groups. T.gondii DNA was extracted and then measured by Taqman quantitative real-time PCR. Measurement IgG and IgM antibody specific to T. gondii was investigated also in the control group by an enzyme-linked immune assay (ELISA). RESULTS: T.gondii parasitemia was detected in (8.3%) 4 out of 48 group I patient. While negative in group II and control group. The range of T.gondii load was (6.285×103-17.915×103) copy/ml, the mean of the copy numbers 11458.75± 5120.85. CONCLUSION: T.gondii should be looked for Leukemic patients at least by routine serological test for early diagnosis and early treatment if indicated. Quantitative PCR is used to monitor post BMT patients at risk for T.gondii disease and for a timely start of preemptive therapy.

Self-Endorsed Cognitive Problems Vs. Objectively-Assessed Cognitive Impairment in Blood or Marrow Transplantation (BMT) Recipients — a Longitudinal Study

Background: Cognitive impairment in BMT survivors is well-documented, and may include deficits in memory, attention, executive functioning and processing speed. However, the clinical relevance of self-endorsed cognitive problems and their relation to objectively-assessed cognitive impairment is not established.Methods: We assessed cognitive impairment in 379 BMT recipients (median age = 52.2y; 40% females; 68% non-Hispanic whites) at 5 pre-determined timepoints: baseline (pre-BMT), 6mo, 1y, 2y and 3y after BMT. Self-endorsed cognitive problems (0: not at all to 4: extremely) were evaluated using the Neuropsychological Impairment Scale (NIS) - a 95-item survey. Responses were used to derive a Global Measure of Impairment (GMI) T-score; higher scores indicated greater cognitive problems. A standardized 2h battery of objective cognitive testing was also administered at each timepoint to derive a Global Deficit Score (GDS); individuals with GDS >0.5 were considered impaired. Healthy controls (HC: n = 98; median age = 55.1y; 46% females; 62% non-Hispanic whites) also completed the NIS and underwent cognitive testing at the 5 corresponding timepoints. At each timepoint, a multivariable analysis of covariance was performed to examine the magnitude of difference in GMI between BMT recipients and HC. Multilevel modeling was employed to examine participants' rates of change in GMI across time. Among BMT recipients, we used multivariable analyses to identify clinical/demographic variables associated with greater self-endorsed cognitive problems; variables examined included age at BMT, sex, race/ethnicity, education, pre-BMT IQ, fatigue, type of BMT (autologous, allogeneic [myeloablative, reduced-intensity]) and use of total body irradiation. To evaluate the relation between NIS subscales and corresponding objective cognitive domains, within-group first-order partial correlation analyses were conducted at each timepoint. We also examined the association between cognitive impairment and return to work at 3y.Results: Significant differences (p<.001) were observed in mean GMI scores with BMT recipients having consistently higher scores than HC at all timepoints. Multilevel modeling revealed that the rate of change in NIS scores was significantly greater in BMT recipients when compared with HC, after adjusting for age, sex, education level, fatigue and baseline IQ (Fig 1). Among BMT recipients, predictors of endorsement of cognitive problems across all timepoints (p ≤.003) included younger age at study (Wilks' λ = .797), higher pre-BMT IQ (Wilks' λ = .858) and greater fatigue (Wilks' λ = .811). Overall, there was a statistically-significant, modest correlation between GMI and GDS scores (range: .401 to .445, p ≤ .01), with mean GMI scores being generally higher (indicating greater endorsement of cognitive impairment) among those who were found to be cognitively impaired (GDS) via objective testing (Fig 2); these relationships were statistically significant at 6mo, 1y and 2y. Multivariable analysis adjusting for age, sex, education level, fatigue and IQ revealed that higher self-reported cognitive impairment (GMI) and higher objectively assessed cognitive impairment (GDS) were associated with a 3.7-fold (p = .02) and 4.3-fold (p = .02) higher odds of not returning to work at 3y, respectively.Conclusions: BMT recipients are more likely to report cognitive problems as compared with HC. Further, these problems increase with time from BMT. Younger age at BMT, higher pre-BMT IQ and greater fatigue are associated with greater self-endorsed cognitive problems. Further, there is a modest-to-high correlation between self-endorsed cognitive problems and objectively determined cognitive impairment. Finally, both self-endorsed cognitive problems and objectively assessed cognitive impairment are associated with a higher likelihood of not returning to work. Overall, this study demonstrates that self-endorsed cognitive problems can serve as a good screen for identifying those with cognitive impairment. DisclosuresForman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding.