aGVHD Prophylaxis Research Articles

Toward optimization of cyclosporine concentration target to prevent acute graft‐versus‐host disease following myeloablative allogeneic stem cell transplant

Despite the common use of cyclosporine (CsA) for acute graft-versus-host disease (aGVHD) prophylaxis following allogeneic stem cell transplant, the optimal CsA trough target remains unknown. Here, we report on outcomes of adult patients following myeloablative conditioning to identify an optimal CsA trough target and characterize the most relevant timeframe post-transplant for CsA trough targeting to minimize aGVHD. We retrospectively reviewed 399 consecutive patients who underwent first peripheral blood allogeneic stem cell transplant for hematological malignancies between January 2009 and December 2018. In the unadjusted and adjusted analyses, the incidence of grades 2-4 aGVHD was significantly higher among patients with an average CsA trough concentration <250mcg/L compared to patients with an average CsA trough concentration ≥250mcg/L during days 15-28 post-transplant (31.5% versus 18.8%, P = 0.037), with an odds ratio (OR) of 1.97 (95% confidence interval 1.04-3.71). In contrast, no correlations between CsA trough concentration and relapse, non-relapse mortality and overall survival was found. In conclusion, early post-transplant CsA trough concentrations are an important factor in the prophylaxis against aGVHD. Our findings suggest that CsA trough concentrations should be maximized between days 15-28 post-myeloablative transplant.

Engineering T cells to prevent graft-versus-host disease and leukemia relapse following allogeneic stem cell transplantation

Abstract Acute graft-versus-host disease (aGvHD) and leukemia relapse remain major causes of mortality after allogeneic hematopoietic stem cell transplantation (alloHSCT). Prophylaxis and treatment of aGvHD rely on generalized immunosuppression, increasing the risk of tumor relapse and opportunistic infections and emphasizing the need for more targeted therapies. Alloreactive donor CD4+ T cells play a central role in aGvHD pathogenesis, we thus hypothesized that elimination of activated CD4+ T cells with engineered T cells would mitigate aGvHD while preserving protective CD8+ T cell immunity post alloHSCT. We developed an alloimmune defense receptor (ADR) targeting OX40, a surface marker predominantly upregulated on activated CD4+ T cells. OX40 ADR-expressing T cells eliminated activated CD4+ T cells during coculture but spared the majority of activated CD8+ T cells, including virus-specific T cells, and had no discernible activity against resting lymphocytes. A single infusion of ADR T cells fully protected mice from fatal xenogeneic aGvHD induced by intravenous injection of human PBMC, minimizing signs of aGvHD and maximizing survival. To enable simultaneous activity of engineered T cells against aGvHD and leukemia relapse, we further armed ADR T cells with a CD19-directed chimeric antigen receptor (CAR). In a mouse model of residual leukemia post alloHSCT, administration of T cells co-expressing OX40 ADR and CD19 CAR mediated dual protection against tumor relapse and aGvHD. These results support the feasibility of a bi-functional CAR.ADR T cell product to improve outcomes post alloHSCT and reduce transplant-related mortality. Supported by grant from Leukemia &amp; Lymphoma Society.

Low-Dose 5-Aza and DZnep Alleviate Acute Graft-Versus-Host Disease With Less Side Effects Through Altering T-Cell Differentiation.

ObjectivePrevious studies showed that hypomethylating agents (HMAs) could alleviate acute graft-versus-host disease (aGvHD), but affect engraftment after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The combination of two different HMAs in lower doses might overcome this problem. This study aimed to evaluate the treatment effect of the combination of two HMAs—azacitidine (5-Aza) and histone H3K27 methyltransferase inhibitor 3-deazaneplanocin (DZNep)—for the prophylaxis of aGvHD after allo-HSCT and to explore the possible mechanisms.MethodsWe first optimized the concentrations of individual and combinational 5-Aza and DZNep treatments to ensure no obvious toxicities on activated T cells by evaluating T-cell proliferation, viability, and differentiation. A mouse model of aGvHD was then established to assess the prophylactic efficacy of 5-Aza, DZNep, and their combination on aGvHD. The immunomodulatory effect on T cells and the hematopoietic reconstruction were assessed. Additionally, RNA sequencing (RNA-seq) was performed to identify the underlying molecular mechanisms.ResultsCompared with single treatments, the in vitro application of 5-Aza with DZNep could more powerfully reduce the production of T helper type 1 (Th1)/T cytotoxic type 1 (Tc1) cells and increase the production of regulatory T cells (Tregs). In an allo-HSCT mouse model, in vivo administration of 5-Aza with DZNep could enhance the prophylactic effect for aGvHD compared with single agents. The mechanism study demonstrated that the combination of 5-Aza and DZNep in vivo had an enhanced effect to inhibit the production of Th1/Tc1, increase the proportions of Th2/Tc2, and induce the differentiation of Tregs as in vitro. RNA-seq analysis revealed the cytokine and chemokine pathways as one mechanism for the alleviation of aGvHD with the combination of 5-Aza and DZNep.ConclusionThe combination of 5-Aza and DZNep could enhance the prophylactic effect for aGvHD by influencing donor T-cell differentiation through affecting cytokine and chemokine pathways. This study shed light on the effectively prophylactic measure for aGvHD using different epigenetic agent combinations.

The combination of four main components in Xuebijing injection improved the preventive effects of Cyclosporin A in acute graft-versus-host disease mice by protecting intestinal microenvironment

Acute graft-versus-host disease (aGVHD) is a major life-threatening complication after Allogeneic Hematopoietic Stem Cell Transplant (allo-HSCT). Although a series of immunosuppressant agents are routinely used as the first-line prevention, the morbidity and mortality rate remains high in allo-HSCT recipients. Our previous work indicated that combining Xuebijing (XBJ) with Cyclosporin A (CSA) is superior to CSA alone in preventing aGVHD. However, it was not clear which compounds in XBJ may prevent aGVHD. Whether the effective compounds in XBJ can be safely combined with CSA to prevent GVHD remain to be evaluated. Here, we accessed whether the combination of four main components in XBJ (C0127) had the same efficacy as XBJ in preventing aGVHD. In addition, the effectiveness of a novel combination therapy (C0127 + CSA) on aGVHD prophylaxis was evaluated using 16 s rRNA sequencing and RNA sequencing approaches in vitro and in vivo. In aGVHD mice, C0127 enhanced the preventive effects of CSA including decreasing mortality, maintaining weight, reducing GVHD score and reducing the expression of IL-6 and TNF-α in serum. Fatal GVHD is a frequent consequence of intestinal tract damage. We found combining C0127 with CSA alleviated the gut damage and maintained the normal physiological function of intestine by H&E staining, intestinal permeability and short chain fatty acid (SCFA) assays. Next, 16 S sequencing analysis of feces showed the combination treatment maintained the intestinal microbial diversity, normalized the intestinal microorganism and prevented flora disorder by reducing the relative abundances of Escherichia coli and Enterococcus. Further, RNA-seq analysis of colonic epithelium revealed C0127 combined with CSA chiefly regulated chemokines and cytokines in IL-17 signaling pathway. The combination treatment reduced the expression of G-CSF and its effector STAT3 (an axis that aggravated gut inflammation and flora disorder) in gut epithelium on mRNA and protein level. These findings indicated that C0127 improved the prevention of CSA in aGVHD mice partially by protecting the gut from damage through normalizing G-CSF signaling, which regulates the intestinal microbiota and the integrity of the epithelial barrier.

A Prospective, Multicenter Study of Closed-System Extracorporeal Photopheresis for Children with Steroid-Refractory Acute Graft-versus-Host Disease

Therapy for steroid-refractory (SR) acute graft-versus-host disease (aGVHD) involves intensive immunosuppression, which is associated with significant risk of infection. Extracorporeal photopheresis (ECP) is used to treat SR-aGVHD and is considered more immunomodulatory than immunosuppressive. However, pediatric data are mostly retrospective and often involve multistep ECP that includes apheresis followed by separate photosensitizing/reinfusion on another device. This study aimed to prospectively evaluate the efficacy and safety of a single-device ECP system in children with SR-aGVHD. In this open-label multicenter phase 3 study of the Therakos CellEx Photopheresis System in children/young adults age 1 to 21 years with SR-aGVHD. Patients were treated 3 times per week for 4 weeks, then twice weekly through week 12 while maintaining standard aGVHD prophylaxis. The primary efficacy endpoint was the proportion of patients achieving an overall response (OR) at day +28 without the addition of next-line systemic treatment. Secondary endpoints included the proportion of patients achieving OR at weeks 8 and 12; the mean weekly steroid dose at weeks 4, 8, and 12; and treatment-emergent adverse events (TEAEs). Twenty-nine children (median age, 8 years) were enrolled. OR was 55% by day 28, 74% by week 8, and 79% by week 12. Progressive improvements were observed in the skin and the gastrointestinal tract. The mean steroid dose was decreased from 1.54 mg/kg/day at baseline to 0.90 mg/kg/day at week 4; 35% of patients achieved a >50% steroid dose reduction by week 4, and 75% achieved a >50% steroid dose reduction by week 12. Of the 168 TEAEs reported among 25 patients (86%), 28 events (17%) were infections and 14 events (8%) were considered likely treatment related (all nonserious). Of 627 ECP treatments administered in children and young adults, 68% required blood priming. TEAEs related to Uvadex or ECP were rare, hypocalcemia was the most common (3 events total). Three deaths occurred and were deemed unrelated to ECP by the investigators. Use of the Therakos CellEx Photopheresis System was effective in children with SR-aGVHD, with more than one-half experiencing improvement by day 28 and further responses observed over 12 weeks. Very few TEAEs were attributable to ECP, and no new safety signals were observed.

Inhibition of the miR-155 and protein prenylation feedback loop alleviated acute graft-versus-host disease through regulating the balance between T helper 17 and Treg cells

MicroRNA-155(miR-155) and protein prenylation have been reported to participate in acute graft-versus-host disease (aGVHD) through modulating T lymphocyte differentiation, however the mechanism remains elusive. In this study, we found that the expression of miR-155 and protein prenyltransferases in peripheral blood T lymphocytes of aGVHD mice was significantly increased. Suppression of miR-155 by antagomir-155 could remarkably reduce prenyltransferases mRNA and protein expression in T lymphocytes of aGVHD mice. Conversely, prenyltransferase inhibitors significantly reduced the level of miR-155. Inhibition of this feedback loop of miR-155 and protein prenylation in aGVHD mice led to improved survival and lower aGVHD histopathology scores and significantly induced T cell deficient differentiation towards T helper 17 (Th17) cells and titled differentiation towards CD4+CD25hi regulatory T (Treg) cells. Furthermore, the immunoregulatory effects and protection from aGVHD of prenyltransferase inhibitors could be reversed by the addition of miR-155. The dual treatment of prenylation inhibitors and antagomir-155 showed synergistic effects on T polarization and protection from aGVHD. Consistent with the in vivo changes, inhibition of this feedback loop of miR-155 and protein prenylation affected Th17 and Treg cell polarization in vitro. Our data suggest that miR-155 and protein prenylation may constitute a feedback loop that amplifies immune and inflammatory responses in subjects with aGVHD, and they may serve as potential targets for aGVHD prophylaxis and treatment.

Transitioning tacrolimus to sirolimus in allogeneic hematopoietic cell transplantation.

Calcineurin inhibitor (CNI) use for acute graft-versus-host disease (aGVHD) prophylaxis in allogeneic hematopoietic cell transplantation (allo-HCT) recipients has been associated with toxicities. Toxicities may be managed by converting CNI to sirolimus as often done in solid organ transplantation. This study aimed to characterize allo-HCT patients who completely transitioned from tacrolimus to sirolimus and evaluate the incidence of aGVHD within 100days post-transition, overall survival (OS), and incidence of relapse. Safety and efficacy data were collected at baseline and at day 30 and 90 post-transition from tacrolimus to sirolimus and at one-year post-HCT. Most patients who transitioned had acute leukemia, received a matched unrelated donor allo-HCT, and transitioned due to nephrotoxicity or neurotoxicity. The resolution rate was 83% and 48% in the nephrotoxicity group, 78% and 61% in the neurotoxicity group, 33% and 33% in the group that developed both nephrotoxicity and transplant-associated thrombotic microangiopathy at 30 and 90days of assessments, respectively. Patients who transitioned before day 55 post-allo-HCT were more likely to develop new or worsening aGVHD. The one-year OS and relapse rates were 37% and 20%, respectively. The conversion from tacrolimus to sirolimus demonstrates promising resolution of acute toxicities; however, overall mortality remains high.

Promising Results at 1 Year Follow-up Following Familial Haploidentical (FHI) T-Cell Depleted (TCD) with CD34 Enrichment and T-Cell (CD3) Addback Allogeneic Stem Cell Transplantation in Patients with High-Risk Sickle Cell Disease (SCD) (IND 14359)

AlloSCT from HLA-matched MSD has been successful for high-risk SCD, and is the only known curative therapy (Talano/Cairo et al EJH, 2014). We have recently demonstrated 100% EFS and absence of sickle cell symptoms following reduced toxicity conditioning in HLA matched sibling donor (MSD) or cord blood AlloSCT (Bhatia/Cairo et al BMT, 2014). However, 5 out of 6 children lack an HLA MSD without SCD. Only 19% of recipients of African descent will identify a well-matched unrelated donor (MUD) and results after unrelated UCBT are poor (Radhakrishnan/Cairo et al BBMT, 2013). Recent results utilizing matched unrelated donors in a multi-center trial showed unacceptable rates of cGVHD at 62% (95% CI 41-77) (Shenoy et al Blood, 2016). We demonstrated CD34+ selection followed by T-cell addback from MUD in pediatric recipients led to 100% engraftment with minimal aGVHD (Geyer/Cairo et al BJH, 2011). Limitations in the past of various T-cell depletion methods have included a higher incidence of graft failure, delayed immune reconstitution, opportunistic fungal infection and higher incidence of cGVHD. FHI TCD AlloSCT utilizing the approach of CD34+ enrichment and T-cell addback could expand the donor pool and improve outcomes for patients with high risk SCD and have similar outcomes to AlloSCT from HLA MSD.This SCD consortium trial (www.sicklecelltransplantconsortium.org) (NCT01461837) is investigating the safety, feasibility, EFS, donor chimerism, graft failure, aGVHD and cGVHD after FHI TCD AlloSCT in high-risk SCD patients.High risk included: ≥1 CVA, ≥2 ACS, ≥3 VOC in past 2 years, or 2 abnormal TCDs. Patients (2-<21 yrs) who have ≥1 high-risk SCD features were eligible. Patients received hydroxyurea and azathioprine, day -59 - day -11, fludarabine (150mg/m2), busulfan (12.8mg/kg) (targeted trough Css of 600-900), thiotepa (10 mg/kg), cyclophosphamide (200mg/kg), R-ATG (8mg/kg), and TLI (500cGy) followed by FHI T-cell depleted AlloSCT. AGVHD prophylaxis: tacrolimus. We utilized the CliniMACS to enrich for peripheral blood HPC's; target dose of 10 x 106 CD34+ cells/kg with a fixed dose of 2 x 105 CD3+ T cells/kg. The Child Health Rating Inventories (CHRIs) was used to serially collect parent proxy-rated health-related quality of life (HRQL).Twenty-one patients have been enrolled. Nineteen patients have received AlloSCT to date. Fifteen patients utilized maternal donors and 4 patients utilized paternal donors. Two donors encountered thrombocytopenia that resolved spontaneously. The mean ± SD CD34+ enrichment prior to cryopreservation was 12.66 ± 3.3 x 106/kg and log T-cell depletion was 4.8 ± 0.58. Evaluable pts had early neutrophil engraftment (median day +9), and ≥95% whole blood and RBC (CD71) donor chimerism at 1yr. Probability of aGVHD n=17, 7.1% (CI95: 0-70.0); cGVHD n=17, 20% (CI95: 0.7-59.6) (Figure 1A and 1B). Probability of 1-Year Overall Survival (OS) n=17; 88.2% (CI95: 60.6-96.9) (Figure 1C). Immune reconstitution was robust with 1 yr NK, CD3, CD4 and CD8 204±37 cells/μL, 795±168 cells/μL, 408±102 cells/μL and 375±90 cells/μL, respectively. Two-yr PFT results were also robust with % predicted FVC, FEV1, TLC and DLCO of 89%, 84%, 82%, and 68%, respectively. All survivors are free of SCD symptoms and without signs of progression of multi-organ toxicity. One patient developed late hepatic SOS and died at day +59; one patient died at day +390 due to complications of CGVHD, and one patient died at day +141 of steroid refractory aGVHD, the remainder are alive and free of SCD symptoms with a median follow-up of +711 days (range, +231 to +1684 days). Most importantly, mean + SD HRQL score (0-100) reported at 1 yr for emotional, physical functioning, role, and global scores were 82.9±9.7 points, 82.8±15.8 points, 89.6±13.3 points, and 86±8.2 points, respectively.Early results indicate promising results in 1 yr OS, stable donor chimerism, and low incidence of acute and/or cGVHD after FHI HCT utilizing CD34 enrichment and CD3/MNC addback in high-risk SCD patients who lack a MSD or URD. A longer term follow-up is needed to assess long-term safety and outcomes. This research was supported by RO1FD004090-01A1. [Display omitted] DisclosuresWalters:AllCells, Inc: Other: Medical Director; bluebird bio: Research Funding; Sangamo Therapeutics: Consultancy; ViaCord Processing Lab: Other: Medical Director. Cairo:Jazz Pharmaceuticals: Speakers Bureau.

Effective and Ineffective Samples of Mesenchymal Multipotent Stromal Cells Used for Acute Graft-Versus-Host Disease Prophylaxis after Allogeneic Bone Marrow Transplantation Differ in the Expression Level of HLA-DR When Co-Cultured with Activated Lymphocytes

IntroductionMultipotent mesenchymal stromal cells (MSCs) are widely used for the prevention and treatment of acute graft versus host disease (aGVHD) after allogeneic bone marrow transplantation (allo-BMT). In some cases prophylaxis of aGVHD failed. The reasons of the failure could be either the result of particular qualities of donor-recipient interaction, patient status or differences in MSC samples. In order to discriminate effective and ineffective for aGVHD prophylaxis MSCs the growth and differentiation characteristics, relative expression levels of different genes and changes in the expression of surface MSC molecules during co-cultivation with activated lymphocytes were investigated in all MSC samples.MethodsMSCs from the bone marrow of corresponding hematopoietic stem cells donor were injected into 59 patients after allo-BMT at the moment of blood count recovery. MSCs from the bone marrow of those donors were analyzed by cultivation in standard conditions (aMEM, 10% fetal calf serum) for 3 passages. Relative expression level (REL) of 30 genes involved in proliferation, differentiation and immunomodulation was estimated by RT-qPCR in all MSC samples. MSCs were seeded 105 cells per T25 flask, and then 106 allogeneic lymphocytes from single donor were added to all MSCs cultures. For lymphocytes activation 5 mg/ml phytohemagglutinin (PHA) was added to half of these cultures. Lymphocytes were removed from MSCs on 1st and 4th days of co-cultivation. Than trypsinized MSCs were collected from the bottom of the flask and expression of HLA-DR, HLA-ABC, CD73, PD-L1, CD54, CD80, CD86, CD105, CD146 and FAS-L on their surface was measured by flow cytometry. p<0.05 was considered statistically significant, all data is present as medium ± SEM.ResultsThe injection of MSCs reduced the incidence of aGVHD 2 times and increased the 5 years overall survival of patients (p=0.047). Eight of 51 MSC samples had been ineffective for preventing aGVHD. Analysis of individual donor characteristics (gender, age, body mass index) found no significant differences between the MSCs, effective and ineffective for preventing aGVHD. REL analysis of 30 genes involved in proliferation, differentiation and immunomodulation in MSCs reviled only 4 genes differs significantly between effective and ineffective MSC samples (table). During 4 days of cultivation the MFI of HLA-DR did not change on MSCs. When MSCs co-cultivated with non-activated lymphocytes for 4 days MFI of HLA-DR increased only in 1.3 fold (p=0.03). Co-cultivation of MSCs with activated lymphocytes led to 7 fold increases in HLA-DR expression between 1st and 4th days (p≤0,001). On 4th day of co-cultivation MSCs with activated lymphocytes the MFI of HLA-DR was 3 fold lower in ineffective MSC samples than in effective (table). These data indicate that the effectiveness of MSCs used depends on the degree of their immunogenicity and, accordingly, on the immunomodulating potential. Among studied molecules only Fas L was significantly lower in effective MSC samples than in ineffective (table). It is possible that such MSC samples survived in the organism better than ineffective and their immunomodulating function lasts longer in the organism.ConclusionsIn summary, this study showed that MSC injections resulted in a significant 2-fold decrease in aGvHD development compared with the standard prophylaxis treatment. When compared with ineffective MSC, effective MSC samples are characterized by higher total cell production and REL of CFH, FGFR1, PDGFRa, and ICAM1. ExpressionofHLA-DR increased significantly in MSCs co-cultivated with lymphocytes, so these cells became immunogenic in organism. It is evident that the level of HLA-DR expression on MSCs important for MSCs efficiency in aGVHD prophylaxis. It is possible to predict efficiency of MSCs injection by simple test of co-cultivation of these cells with activated lymphocytes and measuring the expression level of HLA-DR on them.The materials are supported by grant from the Russian Science Foundation, Project № 16-15-00102 [Display omitted] DisclosuresShipounova:Russian Foundation for Basic Research, Project № 15-04-02514: Research Funding.

Treg in GVHD Prophylaxis: Unique Transcription Factor Regulation of Foxp3 Expression in Itreg Derived from Human Umbilical Cord Blood (UCB) Vs Adult (AB) Naive CD4 T Cells

Early clinical experience identifies third party allogeneic natural T regulatory cells (nTreg) to be safe and effective in acute GVHD (aGVHD) prophylaxis (CG Brunstein et al. 2016). However the low proliferative potential of nTreg remains a challenge to broader clinical application. Inducible Treg (iTreg) can reestablish tolerance in settings where Tregs are decreased or defective. However instability of expression of Transcription factor Fork head box P3 (Foxp3), essential for CD4+ iTreg induction and function, also poses a significant barrier to successful clinical application. Further understanding of Foxp3 transcription factor partners and their function is emerging. Foxp3 is known to regulate Treg function through cooperation with Nuclear Factor of Activated T-Cells (NFAT1). More recently, broad complex-Tramtrack-Bric-a-brac domain (BTB) and Cap'n'collar (CNC) homology 1, basic leucine zipper transcription factor 2 (BACH2) has been shown to repress effector programs to stabilize Treg-mediated immune homeostasis in murine KO studies (Roychoudhuri R. 2013). However, little is known in normal human CD4 T cells what role, if any, BACH2 may play in Foxp3+ regulation. Previous work by this group identified higher BACH2 expression in UCB vs. AB CD4+ T cells, and further that BACH2 regulates expression of IL-2 in UCB CD4+ T-cells lacking normal expression of NFAT1 protein (M. Lesniewski et al. 2008). In our current studies, we compared BACH2 function in Foxp3+ iTreg derived from UCB vs. AB naïve CD4+CD45RA+ cells. We observe that: i.) naïve UCB CD4+ T cells highly express BACH2, 21-fold higher compared to AB CD4+ T cells (including protein and mRNA expression (n=6; p<0.01), ii.) Foxp3+ expression is 2.5-fold higher in iTreg derived from UCB CD4+CD45RA+ T cells vs. AB (86.8 ± 3.13 vs. 34.3 ± 4.2, n=12; p<0.001), iii) absolute number of Foxp3+ iTreg generated from UCB vs. AB CD4+CD45RA+ T cells is 4-fold higher (3.8 x 106 vs. 1.0 x 106), in standard iTreg differentiation conditions, e.g. CD2/3/2/28 stimulation in X-VIVO15 media with 5 ng/ml transforming growth factor # (TGF#, iv.) suppressive function of UCB Foxp3+ iTreg is more potent in mixed lymphocyte culture compared to AB Foxp3+ iTreg (89% vs. 58% inhibition of T cell proliferation in 1:1 ratio, n=9; p<0.001), v.) UCB iTreg exhibit higher surface PD-1 and CTLA-4 MFI expression (PD-1: UCB: 2835 vs. AB: 1398; p<0.01, CTLA-4: UCB: 1558 vs. AB: 653; p<0.001, n=9). Loss of BACH2 expression in UCB CD4+ T cells results in 42% loss of Foxp3 expression by FACS (Empty shRNA: 78.8% vs. Bach2 shRNA: 46.2%, n=7; p<0.001) in transient shRNA knock-down experiments. Cross-linking chromatin immunoprecipitation (ChIP) showed BACH2 binding to the Foxp3 promoter in UCB iTreg but not AB iTreg (n=2, *; p<0.01) (Figure 1A). BACH2 inhibition via shRNA knockdown notably results in 36-fold reduction of Foxp3 mRNA expression (n=4, *; p<0.01) and 16-fold reduction of Foxp3 down-stream CTLA-4 (n=4, *; p<0.01) by qPCR (Figure 1B). Collectively, these data support the hypothesis that BACH2 is an important transcription factor partner that serves to stabilize Foxp3+ inducible regulatory T cells derived from UCB CD4+ naïve T cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bortezomib Consolidation after Nonmyeloablative Allogeneic Transplant Is Safe and Leads to a High Incidence of Immunophenotypic Complete Response in High-Risk or Young Myeloma Patients

▪Introduction: Allogeneic stem cell transplantation (alloSCT) is the only curative treatment for newly diagnosed multiple myeloma (NDMM) patients but is hampered by a high incidence of relapse and chronic graft-versus-host disease (cGVHD). We hypothesized that bortezomib (BTZ) in consolidation after tandem autologous stem cell transplantation (ASCT) and nonmyeloablative (NMA) alloSCT could decrease the incidence of cGVHD while improving quality of response.Method: NDMM patients ≤65 years with high-risk (HR) disease (defined as HR cytogenetics [del17p, t(4;14), t(14;16), t(14;20), del1p or gain 1q], ISS 3 or plasma cell leukemia) or ≤ 50 year regardless of risk status with an 8/8 HLA identical sibling or unrelated donor are eligible to participate in this prospective trial. After triplet BTZ-based induction chemotherapy and ASCT, outpatient NMA alloSCT is performed with either fludarabine 30 mg/m2 and cyclophosphamide 300 mg/m2 for 5 days (sibling donor) or fludarabine 30 mg/m2 for 3 days and total body irradiation 2 Gy (unrelated donor) followed by peripheral blood stem cell infusion. Acute GVHD (aGVHD) prophylaxis consists of tacrolimus and mycophenolate mofetil (MMF). BTZ is started on day +120 post-transplant at 1.3 mg/m2 every 2 weeks for 1 year. Response evaluation is based on IMWG criteria. Bone marrow minimal residual disease (MRD) evaluation is performed with highly sensitive (≥10-5) next-generation flow cytometry using 8-color Euroflow protocol before alloSCT, before BTZ, every 3 months for 2 years and at relapse. Immunophenotypic complete response (iCR) is defined as stringent CR in addition to 2 consecutive negative MRD results. aGVHD and cGVHD are evaluated according to modified Glucksberg and NIH criteria, respectively.Results: As of 07/10/2017, 33 NDMM patients (16 males) have been enrolled; median age is 55 (range: 35-64) years. ISS 3 is found in 39%, Durie-Salmon stage 3A or 3B in 85%, HR cytogenetics in 58% (12% del17p, 15% t(4;14), 33% gain 1q21, 9% del1p and 12% with >1 HR cytogenetics). Induction consisted of CyBorD (79%) or VTD (21%) for a median of 4 (range: 4-7) cycles followed by ASCT. Median times from induction to ASCT and from ASCT to alloSCT are 5.7 and 4.4 months, respectively. Sibling donor transplants were performed in 42%. With a median follow-up of 15 months after alloSCT, 23 patients have started BTZ and received 96% of planned doses. No dose reduction was needed for toxicity. Consolidation BTZ improve depth of response, increasing ≥CR rate from 55% to 80% and iCR rate from 17% to 67% (Table 1), regardless of cytogenetic abnormalities. Median progression-free survival (PFS) is not reached; 12-month PFS is 87% (CI: 66-96) (Fig. 1). The cumulative incidence of progression at 12 months is 8%. Median overall survival (OS) is also not reached and 12-month OS is 91% (CI: 68-98). In 23 patients followed ≥6 months, cumulative incidences of grade II-IV and grade III-IV aGVHD are 30% and 15%, respectively. In 18 patients followed ≥12 months, the cumulative incidence of all grade and moderate-severe cGVHD at 12 months are 58% and 40%, respectively, with mostly mouth, skin and liver involvement. There is no clear association between time of occurrence of cGVHD and improvement of response. Three patients died, one from myeloma progression and 2 from grade III aGVHD, with a non-relapse mortality at 12 months of only 5%. Observed grade ≥3 non-hematologic toxicities possibly/related to BTZ include diarrhea (n=1), viral hemorrhagic cystitis (2 adenovirus, 1BK virus) and subclinical EBV reactivations (n=4).Conclusion: Tandem ASCT-NMA alloSCT and BTZ consolidation result in a remarkably high rate of CR or better including iCR. Despite a HR NDMM population, a low relapse rate is observed. BTZ following alloSCT is safe and may contribute to decrease both the incidence of cGVHD or its severity. [Display omitted] DisclosuresSebag:Celgene, Janssen: Consultancy. Cohen:University of Montreal: Patents & Royalties: royalties.

Combined CCR5 and CXCR3 Blockade Attenuates Murine Agvhd through Alternating Donor-Derived T Cell Distribution and Function

Acute graft-versus-host disease (aGVHD) is one of the major complications and a leading cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Chemokines and their cognate receptors play an important role in directing the migration of donor derived T cells to target organs. Chief among these chemokine receptors are CXCR3 and CCR5. Our previous study found that CCR5 blockade combined with cyclosporine A in a murine model could attenuate the severity of aGVHD in a less extent. This might be due to multiple expression of chemokine receptors on immune cell, only one kind receptor blockade might not completely block the migration of T cells to aGVHD target organs. In this study, we investigated whether combined CCR5 and CXCR3 blockade could further attenuate murine aGVHD, and also explored its immunologic mechanism.BALB/c(H-2Kd)recipients received 8.0 Gy of total body irradiation (60Coγsource) prior to transplantation followed by the infusion of bone marrow cells (BMCs) and splenic cells (SCs) from C57BL/6J(H-2Kb)or CCR5 knockout mice(H-2Kb)to establish an allogeneic bone marrow transplantation model. Body weight change, clinical scores of aGVHD, degree of histopathologic damage and survival rate were dynamically recorded to assess the efficacy of CCR5 and CXCR3 blockade in this transplantation model. Mechanically, donor derived lymphocytes were labeled with fluorescence dye to detect their distribution in vivo. Immunohistochemical analysis for CD4+ and CD8+ were performed on target organs of recipient mice at 7 days post-transplantation. Flow cytometry was used to calculate the absolute number of T cells in target organs, and to detect its proliferation, activation and differentiation in the spleen. ELISA was used to determine the concentration of T cells related cytokines.The results showed that both CCR5 and CXCR3 are enhanced during the aGVHD process. Although transplantation of allogeneic BMCs and SCs induced severe clinical aGVHD with high aGVHD scores (4.67±0.33) and a lethality of 90%, combined CCR5 and CXCR3 blockade attenuated clinical signs of aGVHD, reduced aGVHD scores (1.25±0.25, P< 0.001), reduced aGVHD-induced death (a lethality of 40%, P< 0.05). Compared with aGVHD group, combined CCR5 and CXCR3 blockade reduced liver histologic score (3.3±0.3 versus 1.3±0.3, P< 0.05), lung histologic score (3.6±0.3 versus 1.3±0.3, P< 0.05) and intestine histologic score (3.7±0.3 versus 1.3±0.3, P< 0.05). On days 7 after transplantation, more fluorescent signal emanated from the spleen and from lymph nodes after the use of CCR5 and CXCR3 blockade. Also combined CCR5 and CXCR3 blockade reduced the infiltration of CD4+ and CD8+ T cells into the liver and intestine, and retained part of CD4+ and CD8+ T cells within SLOs. As for the effector function of donor T cells, the results showed that combined CCR5 and CXCR3 blockade dampened T-cell activation, suppressed the percentage of Th1 and Tc1, and induced Treg induction. Additionally, combined CCR5 and CXCR3 blockade altered the concentrations of IFN-γ and IL10. Furthermore, the transplantation of CCR5-/- splenic cells with CXCR3 blockade showed a stronger protection effect on aGVHD.Combined CCR5 and CXCR3 blockade further attenuated aGVHD. aGVHD inhibition was associated with alternated donor T-cell distribution, limited donor T-cell activation and negatively impacted T-cell effector function in vivo. These data suggest that the combined use of CCR5 and CXCR3 blockade might be applicable for aGVHD prophylaxis in clinical settings. DisclosuresNo relevant conflicts of interest to declare.

Impact of the combination of donor age and HLA disparity on the outcomes of unrelated bone marrow transplantation.

Impact of donor age considering HLA disparity on hematopoietic cell transplantation (HCT) outcomes has not been fully evaluated. We evaluated 8486 patients who received unrelated bone marrow transplantation (UR-BMT) from 8/8 or 7/8 HLA-matched donors. Compared to 8/8 HLA-matched younger donors (<40 years), 8/8 HLA-matched older donors (subdistribution hazard ratio [SHR], 1.16; 95% CI, 0.97-1.38) and 7/8 HLA-matched younger donors (SHR, 1.33; 95% CI, 1.11-1.58) were associated with increased risk of grade III-IV acute graft-versus-host disease (aGVHD). 7/8 HLA-matched older donors had further increased risk (SHR, 2.00; 95% CI, 1.68-2.38) due to interaction between donor age and HLA disparity (p for interaction = 0.038). Progression-free survival (PFS) after UR-BMT with 8/8 HLA-matched younger donors was comparable to that after UR-BMT with 8/8 HLA-matched older donors, whereas UR-BMT with 7/8 HLA-matched younger or older donors was significantly associated with lower PFS than UR-BMT with 8/8 HLA-matched younger donors (younger donor; HR, 1.12; 95% CI, 1.04-1.21, older donor; HR, 1.28; 95% CI, 1.17-1.40; p for interaction = 0.079). In conclusion, adverse effect of increased donor age requires attention, especially in HLA-mismatched UR-BMT due to interaction between donor age and HLA disparity. Intensive aGVHD prophylaxis may be required to improve outcomes after HCT with mismatched older donors.

Ruxolitinib early administration reduces acute GVHD after alternative donor hematopoietic stem cell transplantation in acute leukemia

This study aimed to observe the safety and clinical efficacy of early application of ruxolitinib to prevent acute graft-versus-host disease (aGVHD) after alternative donor transplantation in acute leukemia. There were 57 patients undergoing allo-HSCT at the Affiliated Cancer Hospital of Zhengzhou University from July 2017 to October 2019. They were divided into control(16 patients) and ruxolitinib (41 patients) groups. For aGVHD prophylaxis, the control group received post-transplantation cyclophosphamide, antithymocyte globulin-Fresenius, cyclosporine A, and mycophenolate mofetil, while in the ruxolitinib group, ruxolitinib 5 mg/d in adults or 0.07–0.1 mg/(kg d) in children was administered from the day of neutrophil engraftment to 100 days post-transplantation based on control group. We found 55 patients had successful reconstitution of hematopoiesis; No significant difference was found in cGVHD, hemorrhagic cystitis, pulmonary infection, intestinal infection, Epstein-Barr virus infection, cytomegalovirus infection, relapse, death, and nonrelapse mortality. The incidences of aGVHD (50 vs. 22%, P = 0.046) and grade II–IV aGVHD (42.9 vs. 12.2%, P = 0.013) were significantly higher in the control group than in the ruxolitinib group. No significant differences were observed in overall survival (P = 0.514), disease-free survival (P = 0.691), and cumulative platelet transfusion within 100 days post-transplantation between two groups. This suggests early application of ruxolitinib can reduce the incidence and severity of aGVHD and patients are well tolerated.

Immune Suppression in Allogeneic Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for high-risk hematologic disorders. There are multiple immune-mediated complications following allo-HSCT that are prevented and/or treated by immunosuppressive agents. Principal among these immune-mediated complications is acute graft-versus-host disease (aGVHD), which occurs when the new donor immune system targets host tissue antigens. The immunobiology of aGVHD is complex and involves all aspects of the immune system. Due to the risk of aGVHD, immunosuppressive aGVHD prophylaxis is required for nearly all allogeneic HSCT recipients. Despite prophylaxis, aGVHD remains a major cause of nonrelapse mortality. Here, we discuss the clinical features of aGVHD, the immunobiology of aGVHD, the immunosuppressive therapies used to prevent and treat aGVHD, how to mitigate the side effects of these immunosuppressive therapies, and what additional immune-mediated post-allo-HSCT complications are also treated with immunosuppression.

581. Risks versus Benefits of Metronidazole Use for the Prevention of Acute GVHD in Allogeneic Stem Cell Transplant Recipients

BackgroundCurrently, acute graft versus host disease (aGVHD) prophylaxis in hematopoietic stem cell transplants (HSCT) varies amongst different institutions. There is a lack of data supporting the use of metronidazole for aGVHD prophylaxis in HSCT. To further investigate if metronidazole has an effect on aGVHD, allogeneic HSCT recipients will be examined to determine if metronidazole post-transplantation decreases the incidence of aGVHD and the risks of adverse drug events (ADE) associated with this practice.MethodsThis retrospective study included 120 adult patients who received an allogeneic HSCT between January 1, 2010 to December 31, 2013. The primary endpoint is the incidence of aGVHD, defined as within 100 days post-transplant. Secondary endpoints include the rate of metronidazole discontinuation due to intolerance, frequency of metronidazole-related adverse effects, incidence of Clostridioides difficile infection, mortality, and overall survival.ResultsOne hundred six patients met the inclusion criteria. The majority of patients received metronidazole (88 vs. 18). Less patients in the metronidazole arm developed aGHVD (51.1% vs 61.1%, p=0.44). In the subcategories of liver, skin, and gastrointestinal aGHVD, patients who received metronidazole developed less gastrointestinal aGVHD (26.1% vs 50.0%, p=0.045). Gastrointestinal ADEs were the most common metronidazole-related ADEs (19.3%, Table 1). There were no significant differences in the incidence of C. difficile infection, mortality, and overall survival between the two arms (Table 2).Table 1. Adverse Drug Events and Discontinuation of Therapy Table 2. Additional Secondary Outcomes ConclusionDespite a reduction in gastrointestinal aGVHD in the metronidazole arm, approximately one in four patients experienced an ADE to the medication, likely due to the prolonged use of the medication (33 days). The utilization of post-transplant cyclophosphamide for GVHD prophylaxis likely eliminates the need for metronidazole; however our findings suggest a benefit in preventing gastrointestinal aGVHD with metronidazole; albeit, caution is warranted given the high incidence of ADE associated with prolonged use.Disclosures All Authors: No reported disclosures

Fluvastatin-Pretreated Donor Cells Attenuated Murine aGVHD by Balancing Effector T Cell Distribution and Function under the Regulation of KLF2.

Prevention of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is still to be explored. Statins are potent immunomodulatory agents that hold promise as novel and safe agents for aGVHD prophylaxis, yet the controversial effect and regulatory mechanism are incompletely understood. Here, in an MHC mismatched murine model, we found that Fluvastatin-pretreated donor cells could attenuate aGVHD severity by remission tissue pathological injury. Fluvastatin served to restrain effector T cells entry into aGVHD target organs from secondary lymphoid organs (SLOs). The potential mechanism of correcting the effector T cell biased distribution was that Fluvastatin elevated CD62L and CCR7 expression while decreased CXCR3 and CD44 levels, which were correlated with Kruppel-like factor 2 (KLF2) sustention in donor-derived cells. In addition, Fluvastatin was contributed to reducing cytokines IFN-γ, TNF-α, and granzyme-B production in allogeneic effector CD4+ and CD8+ T cells. Furthermore, evidence confirmed that Fluvastatin had a long-lasting effect to sustain KLF2 expression both in vitro and in vivo even under the stimulated circumstance. In conclusion, administration of Fluvastatin to donor mice showed protective effects against recipient aGVHD when compared to untreated mice due to the retention of effector T cells in lymphoid organs accompanying with reduction of nonlymphatic infiltration and related inflammatory cytokines.

How to Make an Immune System and a Foreign Host Quickly Cohabit in Peace? The Challenge of Acute Graft-Versus-Host Disease Prevention After Allogeneic Hematopoietic Cell Transplantation.

Allogeneic hematopoietic cell transplantation (alloHCT) has been used as cellular immunotherapy against hematological cancers for more than six decades. Its therapeutic efficacy relies on the cytoreductive effects of the conditioning regimen but also on potent graft-versus-tumor (GVT) reactions mediated by donor-derived immune cells. However, beneficial GVT effects may be counterbalanced by acute GVHD (aGVHD), a systemic syndrome in which donor immune cells attack healthy tissues of the recipient, resulting in severe inflammatory lesions mainly of the skin, gut, and liver. Despite standard prophylaxis regimens, aGVHD still occurs in approximately 20–50% of alloHCT recipients and remains a leading cause of transplant-related mortality. Over the past two decades, advances in the understanding its pathophysiology have helped to redefine aGVHD reactions and clinical presentations as well as developing novel strategies to optimize its prevention. In this review, we provide a brief overview of current knowledge on aGVHD immunopathology and discuss current approaches and novel strategies being developed and evaluated in clinical trials for aGVHD prevention. Optimal prophylaxis of aGVHD would prevent the development of clinically significant aGVHD, while preserving sufficient immune responsiveness to maintain beneficial GVT effects and immune defenses against pathogens.

Defibrotide inhibits donor leucocyte-endothelial interactions and protects against acute graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (allo‐HCT) is an effective therapy for the treatment of high‐risk haematological malignant disorders and other life‐threatening haematological and genetic diseases. Acute graft‐versus‐host disease (aGvHD) remains the most frequent cause of non‐relapse mortality following allo‐HCT and limits its extensive clinical application. Current pharmacologic agents used for prophylaxis and treatment of aGvHD are not uniformly successful and have serious secondary side effects. Therefore, more effective and safe prophylaxis and therapy for aGvHD are an unmet clinical need. Defibrotide is a multi‐target drug successfully employed for prophylaxis and treatment of veno‐occlusive disease/sinusoidal obstruction syndrome. Recent preliminary clinical data have suggested some efficacy of defibrotide in the prevention of aGvHD after allo‐HCT. Using a fully MHC‐mismatched murine model of allo‐HCT, we report here that defibrotide, either in prophylaxis or treatment, is effective in preventing T cell and neutrophil infiltration and aGvHD‐associated tissue injury, thus reducing aGvHD incidence and severity, with significantly improved survival after allo‐HCT. Moreover, we performed in vitro mechanistic studies using human cells revealing that defibrotide inhibits leucocyte‐endothelial interactions by down‐regulating expression of key endothelial adhesion molecules involved in leucocyte trafficking. Together, these findings provide evidence that defibrotide may represent an effective and safe clinical alternative for both prophylaxis and treatment of aGvHD after allo‐HCT, paving the way for new therapeutic approaches.

Influence of Germline Genetics on Tacrolimus Pharmacokinetics and Pharmacodynamics in Allogeneic Hematopoietic Stem Cell Transplant Patients.

Tacrolimus exhibits high inter-patient pharmacokinetics (PK) variability, as well as a narrow therapeutic index, and therefore requires therapeutic drug monitoring. Germline mutations in cytochrome P450 isoforms 4 and 5 genes (CYP3A4/5) and the ATP-binding cassette B1 gene (ABCB1) may contribute to interindividual tacrolimus PK variability, which may impact clinical outcomes among allogeneic hematopoietic stem cell transplantation (HSCT) patients. In this study, 252 adult patients who received tacrolimus for acute graft versus host disease (aGVHD) prophylaxis after allogeneic HSCT were genotyped to evaluate if germline genetic variants associated with tacrolimus PK and pharmacodynamic (PD) variability. Significant associations were detected between germline variants in CYP3A4/5 and ABCB1 and PK endpoints (e.g., median steady-state tacrolimus concentrations and time to goal tacrolimus concentration). However, significant associations were not observed between CYP3A4/5 or ABCB1 germline variants and PD endpoints (e.g., aGVHD and treatment-emergent nephrotoxicity). Decreased age and CYP3A5*1/*1 genotype were independently associated with subtherapeutic tacrolimus trough concentrations while CYP3A5*1*3 or CYP3A5*3/*3 genotypes, myeloablative allogeneic HSCT conditioning regimen (MAC) and increased weight were independently associated with supratherapeutic tacrolimus trough concentrations. Future lines of prospective research inquiry are warranted to use both germline genetic and clinical data to develop precision dosing tools that will optimize both tacrolimus dosing and clinical outcomes among adult HSCT patients.