Graft-versus-host Reaction Research Articles

Pre-Engraftment Syndrome (PES) Is Frequent in Double Unit Cord Blood Transplantation (dCBT) Recipients and Is Potentiated By the Addition of Haplo-Identical CD34+ Cells

Introduction PES is common after dCBT. Manifestations have been described but severity & risk factors are not fully established. Methods We analyzed the incidence, manifestations, severity & risk factors of PES in adult dCBT recipients transplanted 8/2012-12/2017 for hematologic malignancies/ severe aplastic anemia with Cy 50/ Flu 150/ Thio 5-10/ TBI 400 & CSA/ MMF. Standard PES therapy was IV methylprednisolone (IV MP) 1 mg/kg/day x 3 days. Results 133 1st allograft dCBT recipients (median 49 years, range 21-65) were analyzed. Patients (pts) most commonly had acute leukemia (n = 98, 74%). The median TNC cell doses were 2.4 × 107/kg/unit (range 1.2-6.0). The median unit-recipient HLA-match was 5/8 (range 3-7); the median unit-unit HLA-match was 4/8 (range 0-8). 71 (53%) pts also received haploidentical CD34+ cells. 96% of pts engrafted with CB. Overall, 98 pts developed non-infectious sustained fevers ≥ 38.5°C consistent with PES for a cumulative incidence of 74% (95%CI: 65-80) by 21 days post-dCBT (Table). The median onset was 10 days (range 6-21) post-dCBT with a median temperature at onset of 38.9°C degrees (range 38-39.8). Nearly all (94, 95%) of PES pts required IV MP therapy. PES associated rash within the 1st 72 hrs only occurred in a minority of pts (incidence 24%; 95%CI: 17-32). Weight gain > 5% from the baseline 24 hrs prior to PES onset (incidence 18%; 95%CI: 12-25) was correlated with the development of hypoxia requiring oxygen (incidence 20%; 95%CI: 14-28). Incidence of PES associated renal impairment (gr 2-3 AKI) was low overall at 2% (95%CI: 0-8). No pt had hypotension. Concurrent bacteremias were treated in 2 pts but clinical manifestations/ therapy response was consistent with PES. Median time to MP therapy was 1 day (range 0-12). Therapy response was rapid (median 1 day, range 1-9). The median MP duration was 3 days (range 3-14). 7/94 (7%) PES pts [7/133, 5% of entire cohort) required ICU care; this correlated with delayed therapy [median MP start 4 days (range 0-5)]. 30 (32%) pts required a 2nd MP course. Addition of haploidentical cells was associated with a higher PES incidence & severity as measured by weight gain & hypoxia (Table). There was no association with age, disease (acute leukemia vs other), dominant unit-recipient or unit-unit HLA-match or total TNC infused dose (split at median). Conclusions PES is common after dCBT. It should be promptly treated with IV MP without waiting for results of infectious work-up as delay can be associated with severe capillary leak syndrome & multi-organ failure. The worse manifestations in haplo-dCBT recipients supports PES being mediated by GVG/ GVH reactions & an associated cytokine storm. This is highly relevant to CBT platforms that involve infusion of multiple donors including 3rd-party products. We are now investigating day -1 tocilizumab 8 mg/kg in dCBT recipients with the aim to abrogate PES & acute GVHD (Politikos et al, ASBMT 2019).

Experimental Modeling of Acute- and Chronic-GvHD By Xenotransplanting Human Donor PBMCs or Cord Blood CD34+ Cells (HSC) into NSG Mice

▪Graft vs. host diseases (GvHD) is a life-threatening complication accounting for 15-30% of deaths following allergenic hematopoietic stem cell transplantation (allo-HSCT) for treatment of malignant diseases. There are two types of GvHD: acute (aGvHD) and chronic (cGvHD), which have different parthenogenesis and phenotype. aGvHD typically involves skin, gastrointestinal and hepatic inflammation, and occurs within 100 days of transplantation; and cGvHD involves multiple organs and occurs beyond 100 days. The former is largely due to the rapid activation of donor T cells (Th1, CD8+ biased), causing tissue damage (via cell killing) and often leading to mortality; in contrast, the latter (Th2, CD4+ biased) typically display autoimmune-like syndrome, involving both T- and B-cell, as well as auto antibody production and systemic fibrosis. The current understanding of the parthenogenesis, as well as developing treatment strategy, is largely based on experimental animal models, many aspects of which remain to be investigated. At present, the most commonly used GvHD models are allo-transplantation between mice, the result of which many may not all adequately translate into clinic for the donor species difference. Xeno-transplantation of human donor into mice could potential represents a better alternative GvHD model in some respects.Methods. Human peripheral blood mononuclear cells (PBMC) derived from the consent normal donor were transplanted into NSG (other NSG-like strains) and B2M-NSG (MHC class I, b-2 micro-globulin, deficiency) mice for modeling aGvHD, while cord blood derived HSCs (hCD34+) (Jackson Lab, huNSG) were transplanted for modeling cGvHD. Following transplantation, the disease progress will be monitored twice weekly, including clinical observations (e.g. animal postures, activity, fur texture, and skin integrity), body weight changes, gross pathology and histo-pathology upon termination, along with human-immunological phenotype of peripheral blood, spleen, lung, and liver by flow (e.g. lymphocyte populations), multiplex, complete blood count, histo-pathology and immunohistochemistry at different time-points.Results. NSG mice engrafted with human PBMC, or purified T-cells, from normal donors rapidly developed typical symptoms of aGvHD, onset 2~3 weeks post transplantation, including severe body loss, reduced activity, hunched posture, loss of fur in combination with severe ruffling and overall poor grooming. Mortality is observed typically from 4th or 5th weeks post transplantation. In correlation, we have observed significant engraftment of human CD45+ human leukocytes (0.5~1.5% on Day 6, 10~30% on Day 16, 30~50% on Day 26 post transplantation, with variations depending on donors, route of transplantation, IP vs. IV, etc), including dominant CD3+ human T-cells of single positive of either CD4+ or CD8+ T-cells. This correlated kinetics between clinical symptoms and degree of engraftment of human leukocytes, particularly T-lymphocytes, suggests xenografting human T-cells are responsible for the observed aGvHD. Further comprehensive analysis on the pathology and immunophenotypes of relevant organs are ongoing. In parallel to NSG, we assessed aGvHD in the absence of mouse MHC-I using the PBMC engrafted B2M mice. Additionally, NSG-mice were engrafted with CD34+ cells derived from cord blood with cGvHD high- risk and low-risk HLA haplotypes. We observed cGvHD development between 18 weeks to 39 weeks post-engraftment, in contrast to the short duration seen aGvHD; symptoms include severe weight loss, impaired movement, severe ruffling, facial/full body alopecia, and scaly skin. Interestingly, in correlation of this, human engraftment of CD45+, particularly T-cells including CD4+, CD8+ and their CD30+ subsets, are kinetically increased in blood and spleen with similar timeline, suggesting their roles in the observed cGvHD.Conclusions. The xenograft murine model using adult human PBMC and cord blood derived CD34+ HSCs could be alternative experimental systems to model human aGvHD and cGvHD for investigating disease mechanisms and evaluating treatment strategy. DisclosuresNo relevant conflicts of interest to declare.

Graft-Vs-Host Reactivity Against the Bone Marrow Is Directed Against the Hematopoietic and Non-Hematopoietic Compartments in Mice

Hematopoietic dysfunction is frequently observed following allogeneic hematopoietic cell transplantation (allo-HCT) and contributes to substantial morbidity. Various factors, e.g. viral infections, poor graft-function, and certain drugs can cause cytopenias, but usually, graft-vs-host disease (GVHD) of the bone marrow (BM) is not taken into account as a major cause of hematopoietic dysfunction, particularly when no other signs of GVHD in classical target organs are present.Here, we studied in an MHC-matched, minor antigen mismatched HCT model (C3H.SWàC57BL/6 (B6)) the effects of lethal irradiation and allo-HCT of HSC (cKIT+Sca1+Lin-) alone or in combination with T cells (TC) on the hematopoietic compartment (HC) and non-hematopoietic compartment (NHC) of the BM. FACS analysis was used to assess relative and absolute cell counts of hematopoietic and non-hematopoietic populations. Confocal microscopy was used to establish 3D images of structural integrity vs. disruption, and qRT-PCR was used to examine functional alterations of gene expression within NHC populations.In this HCT model mice did not develop overt signs of acute GVHD. In recipients of HSC+TC recovery of hemoglobin (Hb) and neutrophils (Nph) was significantly delayed beyond d+56, while Hb and Nph were normalized by d+21 in recipients of pure HSC. Recovery of BM cell counts was also faster in HSC recipients, whereas addition of TC to the graft delayed reconstitution of total BM counts, BM B cells and BM neutrophils significantly. Not only the HC was affected by alloreactive TC, but also non-hematopoietic populations, including CXCL12-abundant reticular (CAR) cells (CD45-Ter119-CD31-CD140b+) and endothelial cells (EC; CD45-Ter119-CD31+) declined post-HCT, and showed a significantly slower recovery in mice given allogeneic TC within their HCT. 3D-confocal microscopy confirmed the alloreactive damage to the NHC, as imaging revealed rapid recovery of CAR cells in B6.CXCL12-GFP recipients of pure HSC, whereas regeneration of CAR cells was impaired in mice given HSC+TC. Likewise, the extracellular matrix and sinusoidal vascular structures recovered promptly in the HSC group, whereas HSC+TC recipients displayed severe disruption of the structural integrity with impaired recovery of the BM microvessels. Increased microvascular permeability was shown in all transplanted (and irradiated) mice on d+7 post-HCT by Evans blue dye extravasation assay. Recovery from leakiness was faster in mice transplanted with HSC alone compared with those given alloreactive TC. Of note, confocal microscopy revealed that in recipients of alloreactive TC hematopoietic cell content was replaced by an abundance of adipocytes for a prolonged period of time (Figure 1A). To further elucidate functional alterations of the NHC in the context of HCT with/without TC we FACS-isolated CAR cells and EC from HSC and HSC+TC recipients on d+14 and d+28 post-HCT and performed qRT-PCR of candidate genes in these populations. In HSC+TC recipients both CAR cells and EC showed significantly lower relative expression levels of SCF (stem cell factor, ligand to cKIT), CXCL12 (C-X-C-motif chemokine 12, = SDF-1, a cytokine with various functions in hematopoiesis and lymphopoiesis), and IL-7 (a growth factor that stimulates the differentiation of pluripotent HSC into lymphoid progenitors) compared with mice transplanted with HSC only. Rather, in CAR cells of HSC+TC recipients adiponectin and PPARγ (peroxisome proliferator-activated receptor γ, stimulates lipid uptake and adipogenesis) were significantly higher compared with HSC only recipients (Figure 1B). These findings suggest that alloreactive TC promote adipogenesis while at the same time suppressing hematopoiesis via deprivation of essential cytokines and growth factors required for HSC function and maturation.Delayed hematopoietic regeneration post allo-HCT represents a major problem in clinical patient care. GVH reactions of the BM have been poorly characterized. Our experiments help to further characterize “marrow GVHD” and its impact on hematopoietic output. We illustrate the damage that alloreactive TC can have on both hematopoiesis directly but also on the nonhematopoietic compartment and its critical functions in stimulating and supporting hematopoietic regeneration. DisclosuresNo relevant conflicts of interest to declare.

Generation of alloreactivity-reduced donor lymphocyte products retaining memory function by fully automatic depletion of CD45RA-positive cells

Background aimsFor patients needing allogeneic stem cell transplantation but lacking a major histocompatibility complex (MHC)-matched donor, haplo-identical (family) donors may be an alternative. Stringent T-cell depletion required in these cases to avoid lethal graft-versus-host disease (GVHD) can delay immune reconstitution, thus impairing defense against virus reactivation and attenuating graft-versus-leukemia (GVL) activity. Several groups reported that GVHD is caused by cells residing within the naive (CD45RA+) T-cell compartment and proposed use of CD45RA-depleted donor lymphocyte infusion (DLI) to accelerate immune reconstitution. We developed and tested the performance of a CD45RA depletion module for the automatic cell-processing device CliniMACS Prodigy and investigated quality attributes of the generated products. MethodsUnstimulated apheresis products from random volunteer donors were depleted of CD45RA+ cells on CliniMACS Prodigy, using Good Manufacturing Practice (GMP)-compliant reagents and methods throughout. Using phenotypic and functional in vitro assays, we assessed the cellular constitution of CD45RA-depleted products, including T-cell subset analyses, immunological memory function and allo-reactivity. ResultsSelections were technically uneventful and proceeded automatically with minimal hands-on time beyond tubing set installation. Products were near-qualitatively CD45RA+ depleted, that is, largely devoid of CD45RA+ T cells but also of almost all B and natural killer cells. Naive and effector as well as γ/δ T cells were greatly reduced. The CD4:CD8 ratio was fivefold increased. Mixed lymphocyte reaction assays of the product against third-party leukocytes revealed reduced allo-reactivity compared to starting material. Anti-pathogen responses were retained. DiscussionThe novel, closed, fully GMP-compatible process on Prodigy generates highly CD45RA-depleted cellular products predicted to be clinically meaningfully depleted of GvH reactivity.

Systemic Treatment with a miR-146a Mimic Suppresses Endotoxin Sensitivity and Partially Protects Mice from the Progression of Acute Graft-versus-Host Disease.

Acute GVHD (aGVHD) is driven by interactions between the allogenic T cell response, inflammation, tissue injury and microbial products that enter the circulation when protective barriers such as the intestinal epithelium become compromised. Mice with aGVHD become hypersensitive to LPS, secreting large quantities of inflammatory mediators that exacerbate tissue injury. We hypothesized that microRNA (miR) modulators could be used in vivo to mitigate LPS hypersensitivity, altering the course of aGVHD. Using the C57BL/6 → (C57BL/6 × DBA/2)F1 -hybrid model of aGVHD, we measured intestinal permeability over time and used a qPCR array to detect concomitant changes in the expression levels of certain microRNAs (miRs) in the intestine. Large increases in permeability were seen on day 15, when endotoxemia becomes detectable and GVHD-associated histopathological lesions develop. Amongst the miRs with altered expression levels were some that regulate sensitivity to endotoxin. We chose to focus on miR-146a and treated recipient mice systemically with a miR-146a mimic early in the GVH reaction. This led to a reduction in the burst of IFNγ that likely plays a priming role in the mechanism underlying heightened sensitivity to endotoxin. LPS-induced TNFα release and GVHD-associated weight loss were also diminished and survival was prolonged. In summary, systemic treatment with a miR-146a mimic dampens the heightened sensitivity to LPS that occurs concomitantly with increased intestinal permeability and provides partial protection from the progression of acute GVHD.

Differing Mechanisms for Early Versus Persistent Donor T cell Chimerism in Peripheral Blood of Human Intestinal Transplant Recipients

Donor T cell macrochimerism (>4%) often appears in blood following intestinal transplantation (ITx), usually without graft-versus-host disease[1]. Patients with donor T cell macrochimerism displayed less graft rejection, slower recipient T cell replacement within the graft and less donor-specific antibody (DSA) production[2]. However, the reasons for the development and persistence of donor T cell chimerism are unknown. We analyzed the repertoire, phenotype and origin of circulating donor T cells in ITx recipients using flow cytometry, mixed lymphocyte reaction (MLR) and high-throughput TCR sequencing. Expanded graft-versus-host (GvH) clones triggered by rapidly replaced recipient antigen-presenting cells were detected early in ileum biopsies (Figure 1A). Enrichment of GvH clones in the graft and absence of Class I DSA in the circulation were associated with donor T cell macrochimerism. Cumulative frequencies of GvH clones in blood early post-Tx correlated with the peak level of donor T cell chimerism (Figure 1B). Frequencies of host-versus-graft (HvG) clones subsequently declined in the blood of a patient with a high level of T cell chimerism (>38%), suggesting that lymphohematopoietic GvH responses may attenuate HvG responses (Figure 1C). Long-term donor T cells were markedly enriched for the naïve recent thymic emigrant phenotype compared with recipient cells regardless of donor age (Figure 2A). GvH clones were absent among long-term circulating naïve donor-derived T cells, which were enriched for CD31 expression and T-cell receptor excision circles (TRECs), suggesting they developed de novo in the recipient thymus. Long-lasting (POD > 200) circulating donor cells lacked GvH reactivity yet were immunocompetent in MLR, partially due to the presence of recipient-specific Tregs (Figure 2B). We also detected donor-derived hematopoietic stem cells (HSCs) and/or progenitors (HPs) in blood and stoma specimens post-Tx and in donor liver, ileum and perfusates pre-Tx. HSCs/HPs from ileum and bone marrow of each organ donor showed similar properties on CyTOF (mass cytometry) analysis, suggesting they may have similar functional phenotypes. Together, donor GvH-reactive T cells and HSCs/HPs carried within the graft promote and maintain donor T cell chimerism in blood, which correlates with better clinical outcomes and suggests an approach to reduce graft rejection and achieve tolerance post-ITx.FIGURE 1: (A) Enrichment of GvH clones in intestinal biopsies and absence of Class I DSA (POD <90) in the circulation were associated with donor T cell macrochimerismin blood. (B) Correlation between the cumulative frequency of GvH clones in blood early post-Tx and the peak level of donor T cell chimerism. (C) Cumulative frequency of HvG clones in blood post-Rx.FIGURE 2: (A) Circulating donor-derived naïve T cells display a recent thymic emigrant (RTE) phenotype. (B) Long-lasting (POD>200) circulating donor cells lacked GvH reactivity yet were immunocompetent in MLR, partially due to the presence of recipient-specific Tregs.Research reported in this publication was performed in the Columbia Center for Translational Immunology (CCTI) Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Sequential monitoring of lymphocyte subsets and of T-and-B cell neogenesis indexes to identify time-varying immunologic profiles in relation to graft-versus-host disease and relapse after allogeneic stem cell transplantation.

T and B lymphocyte subsets have been not univocally associated to Graft-versus-host disease (GVHD) and relapse of hematological malignancies after stem cell transplantation (SCT). Their sequential assessment together with B and T cell neogenesis indexes has been not thoroughly analysed in relation to these changing and interrelated immunologic/clinic events yet.Lymphocyte subsets in peripheral blood (PB) and B and T cell neogenesis indexes were analysed together at different time points in a prospective study of 50 patients. Principal component analysis (PCA) was used as first step of multivariate analysis to address issues related to a high number of variables versus a relatively low number of patients. Multivariate analysis was completed by Fine-Gray proportional hazard regression model. PCA identified 3 clusters of variables (PC1-3), which correlated with acute GVHD: PC1 (pre-SCT: KRECs≥6608/ml, unswitched memory B <2.4%, CD4+TCM cells <45%; HR 0.5, p = 0.001); PC2 (at aGVHD onset: CD4+>44%, CD8+TCM cells>4%; HR 1.9, p = 0.01), and PC3 (at aGVHD onset: CD4+TEMRA<1, total Treg<4, TregEM <2 cells/μl; HR 0.5, p = 0.002). Chronic GVHD was associated with one PC (TregEM <2 cells/μl at day+28, CD8+TEMRA<43% at day+90, immature B cells<6 cells/μl and KRECs<11710/ml at day+180; HR 0.4, P = 0.001). Two PC correlated with relapse: PC1 (pre-SCT: CD4+ <269, CD4+TCM <120, total Treg <18, TregCM <8 cells/μl; HR 4.0, p = 0.02); PC2 (pre-SCT mature CD19+ >69%, switched memory CD19+ = 0 cells and KRECs<6614/ml at +90; HR 0.1, p = 0.008). All these immunologic parameters were independent indicators of chronic GVHD and relapse, also considering the possible effect of previous steroid-therapy for acute GVHD. Specific time-varying immunologic profiles were associated to GVHD and relapse. Pre-SCT host immune-microenvironment and changes of B cell homeostasis could influence GVH- and Graft-versus-Tumor reactions. The paradoxical increase of EM Treg in PB of patients with GVHD could be explained by their compartmentalization outside lymphoid tissues, which are of critical relevance for regulation of GVH reactions.

An Early Biomarker Algorithm Predicts Lethal Graft-Versus-Host Disease and Survival after Allogeneic Hematopoietic Cell Transplantation

No laboratory test can predict non-relapse mortality (NRM) after hematopoietic cellular transplantation (HCT) prior to the onset graft-versus-host disease (GVHD). Recently, we have shown that a signature of three GVHD plasma biomarkers (TNFR1, ST2, and REG3α) can predict response to GVHD therapy and NRM at the onset of clinical GVHD (Levine, Lancet Haem, 2015). Our goal in the current study was to identify a blood biomarker signature that could predict lethal GVHD and six-month NRM well in advance of the onset of GVHD symptoms. Patient samples on day +7 after HCT were obtained from 1,287 patients from 11 HCT centers in the Mount Sinai Acute GVHD International Consortium (MAGIC). Samples from two large centers (n = 929) were combined and randomly assigned to a training set (n = 620) and test set (n = 309). 358 patients from nine others centers constituted an independent validation set. The overall cumulative incidences of 6-month NRM were 11%, 12%, and 13% for the training, test, and validation sets respectively. The incidence of lethal GVHD, defined as death without preceding relapse while under steroid treatment for acute GVHD, were 18%, 24%, and 14% in the same groups, respectively. The median day of GVHD onset was 28 days in the training set and 29 days in the test and validation sets. We measured four GVHD related biomarkers [ST2, REG3α, TNFR1, and IL2Rα] in all samples and used the training set alone to develop competing risks regression models that used all 13 possible combinations of one to four biomarkers to predict 6-month NRM. The best algorithm, which we rigorously confirmed through Monte Carlo cross-validation of 75 different combinations of training sets, included ST2 and REG3α. No combination of one, three, or four biomarkers was superior to the combination of these two biomarkers. The day 7 algorithm identified high risk (HR) and low risk (LR) groups with 6-month NRMs of 28% and 7%, respectively (p<0.001) (Fig 1A). The relapse rates did not differ between risk groups so that overall survival (OS) was 60% for HR and 84% for LR (p<0.001) (Fig 1B). When applied to the test set (Fig 1C/D), the algorithm identified 54/309 (17%) of the patients as HR with an NRM of 33% vs 7% for LR patients (p<0.001) and 6-month OS of 57% and 81% for HR and LR patients, respectively (p<0.001). In the independent validation set (Fig 1 E/F), the algorithm identified 72/358 (20%) of the patients as HR with an NRM of 26% vs 10% for LR patients (p<0.001) and OS of 68% and 85% for HR and LR patients, respectively (p<0.001). High risk patients were three times more likely to die from GVHD than LR patients in each cohort (p<0.001) (Fig 2). The GI tract is the GVHD target organ that is most resistant to treatment and represents a major cause of NRM, and we observed twice as much severe GI GVHD (stage 3 or 4) in HR patients as in LR patients (p<0.001, data not shown). The algorithm successfully separated HR and LR strata for 6 month NRM in several groups with differing risks for GVHD and NRM, including donor type, degree. of HLA-match, age group, and conditioning regimen intensity (Fig 3).In conclusion, we have developed a blood biomarker algorithm that predicts the development of lethal GVHD seven days after HCT, which performed successfully in large multicenter validation sets. The GVH reaction is already in progress by day +7, even though clinical symptoms may not occur until days or weeks later. We speculate that the blood biomarker concentrations at this early time point reflect subclinical GI pathology, a notion that is reinforced by the fact that ST2 and REG3α, the two biomarkers in the algorithm, are closely associated with GI GVHD. The algorithm identified HR and LR strata in several patient groups with different overall risk for lethal GVHD (donor, HLA match, conditioning regimen intensity, age). This day +7 algorithm should prove useful in clinical BMT research by identifying patients at high risk for lethal GVHD who might benefit from aggressive preemptive treatment strategies. [Display omitted] [Display omitted] [Display omitted] DisclosuresChen:Novartis: Research Funding; Incyte Corporation: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Jagasia:Therakos: Consultancy. Kitko:Therakos: Honoraria, Speakers Bureau. Kroeger:Novartis: Honoraria, Research Funding. Levine:Viracor: Patents & Royalties: GVHD biomarkers patent. Ferrara:Viracor: Patents & Royalties: GVHD biomarkers patent.

Adoptive Transfer of Purified Donor-B-Lymphocytes after Allogeneic Stem Cell Transplantation: Results from a Phase I/IIa Clinical Trial

Patients after allogeneic stem cell transplantation (alloSCT) show a long lasting immune deficiency involving both T- and B-lymphocytes. Antibody responses to vaccination, as a measure of functional B-cell immunity, are insufficient in the first months after alloSCT and usually recover within 1-2 years. To improve B-cell immune reconstitution after alloSCT, we developed a novel concept of adoptive transfer of memory B-cells from the original stem cell donor (Klenovsek et al., Blood 110:3472-79, 2007). To this end, we first established the technology for the manufactoring of a GMP-qualified B-cell product and initiated a first-in-man phase I/IIa clinical trial in patients after alloSCT to evaluate safety and tolerability of adoptively transferred donor B-cells.B-cells were manufactured under GMP conditions from 16 unstimulated leukapheresis products derived from the original stem cell donor. The separation protocol consists of two subsequent separation steps on the CliniMACS® System (Miltenyi Biotec) including depletion of CD3+ T-cells and subsequent positive separation of CD19+ B-cells by magnetic beads. In all of the 16 cell products high purities of CD20+ B-cells (average 96%, range 85-99%) with only low frequencies of contaminating T-cells (average 0.10 %, range 0.01-0.82%) were achieved. After manufacturing, B-cell products were cryopreserved for single administration to 4 groups of patients with escalating doses. To prevent GvH reactions the absolute number of CD3+ T lymphocytes in the B-cell product had to be below 4 x 104 CD3+ cells/kg BW.The clinical trial was designed as an open label dose escalating study using the traditional 3+3 design with 4 dose levels of 0.5-1-2-4x106 B-cells/kg BW. Patients were enrolled on day +140 ± 20 after alloSCT after tapering of immunosuppression. Patients with acute GVHD grade III/IV, chronic GVHD with intermediate to high risk, EBV reactivation (>10,000 EBV copies/ml) or after therapy with B-cell antibodies (i.e. rituximab) were not eligible. Up to now, 13 patients after alloSCT received donor B-cells in 4 different B-cell dosages (average day +147, range from day +130 to d +160). Three patients received 0.5x106 B-cells per kg BW, 3 patients 1.0x106 B-cells/kg BW, 5 patients 2.0x106 B-cells per kg BW, and 2 patients 4.0x106 B-cells/kg BW, respectively. The median number of infused T cells was 0.06x104 T-cells per kg BW (range 0.01-0.13 x104/kg BW). Regarding primary safety data, the B-cell transfer was well tolerated without any acute adverse reactions. Importantly, neither significant EBV-viremia nor acute or chronic GvHD reactions were observed during the observation period of 4 months after the transfer of B-cells.In addition to the analysis of the primary safety endpoints of the trial, the activity of infused donor memory B-cells in vivo were tested. A characteristic memory B-cell response is defined as a mobilization of plasmablasts (PBs) 7 days after booster vaccination into the peripheral blood (Odendahl et al., Blood 105: 1614-21, 2005). Preliminary results obtained from patients that received the donor derived B-cells demonstrated significant mobilization of PBs in some of the patients after re-vaccination with a pentavalent vaccine (Pentavac®), suggestive for a memory B-cell response. A detailed analysis of the vaccination data from the study patients in comparison to a control group of transplanted patients without B-cell transfer after routine re-vaccination will be presented. Furthermore, the analysis of immunoglobulin VH-sequences by next-generation-sequencing in memory B-cell populations of two individual patients after B-cell transfer revealed that at least 50% of all memory B-cell clones identified in the recipient are unambiguously contained within the memory B-cell population of the infused B-cell product.In summary, our data show that the manufacturing of a B-cell product from the original stem cell donor under GMP conditions without significant T-cell contamination is feasible. The transfer of donor-derived B-cells into patients after alloSCT is safe with regard to EBV reactivation and induction of acute or chronic GvHD. Future clinical trials will evaluate whether the adoptive transfer of memory B-cells from the donor can significantly reduce the frequency of infections after alloSCT.(Supported by the Deutsche Forschungsgemeinschaft through SFB 643 "Strategies of cellular immune intervention") DisclosuresNo relevant conflicts of interest to declare.

Preclinical Evaluation of Allogeneic Anti-Bcma Chimeric Antigen Receptor T Cells with Safety Switch Domains and Lymphodepletion Resistance for the Treatment of Multiple Myeloma

Multiple myeloma (MM) is a hematological disease of plasma B cells that remains incurable despite the availability of numerous therapies. The plasma cell-specific expression of the TNF superfamily receptor BCMA may allow targeting of normal and malignant plasma cells. Genetically engineered chimeric antigen-receptor T cells (CAR T) have shown tremendous promise in the treatment of several hematological diseases, including MM. However, conventional autologous CAR T therapies use patient-derived T cells and the logistics of on-demand CAR T manufacture limits their availability to a broad patient pool.Here we describe the preclinical evaluation of an allogeneic CAR T therapy targeting BCMA that has the potential for a readily available, off-the-shelf therapy for MM and other malignancies expressing BCMA. Human T cells were transduced with recombinant lentiviral vectors encoding three BCMA CAR candidates designed with fully human anti-BCMA scFvs, CD8a transmembrane domains and the intracellular signaling domains of 4-1BB and CD3zeta. All CAR T efficiently killed BCMA-expressing multiple myeloma cell lines (KMS12BM, MM1.S, Molp-8 and OPM-2), but not BCMA-negative REH cells in vitro and in vivo. Whereas 2 of the 3 candidates exhibited target-independent cytokine production, accelerated T cell differentiation and reduced target cell-induced expansion in vitro, the third candidate did not exhibit this scFv-induced autoactivation and was chosen as the lead molecule.Due to the allogeneic nature of this T cell therapy, the possibility of graft-versus-host (GvH) reactions can be a safety concern. We applied Cellectis’ know-how and TALEN® technology for the gene inactivation of the T cell receptor (TCR) alpha chain to significantly reduce the probability for TCR-mediated GvH reactions and found that TCR knockout did not affect CAR T activity in vitro or in vivo. Furthermore, we incorporated intra-CAR rituximab-recognition domains into the CAR molecule to enable depletion of CAR T cells from patients when necessary. We found that this modified CAR retained anti-BCMA CAR T activity and enabled CAR T depletion by rituximab.Another aspect of allogeneic CAR T therapies is the rejection of the CAR T by host-versus-graft (HvG) reactions. Lymphodepletion prior to CAR T infusion enhances CAR T efficacy in autologous CAR T trials and may also prevent anti-CAR HvG reactions in allogeneic therapy settings. Engineering lymphodepletion resistance into CAR T cells could therefore enable sustained lymphodepletion for enhanced allogeneic CAR T persistence and efficacy. CD52 is expressed on all lymphocytes and administration of the anti-CD52 antibody alemtuzumab for prolonged lymphodepletion is an approved treatment for multiple sclerosis. TALEN®-mediated knockout of CD52 protected BCMA CAR T from alemtuzumab-induced cytotoxicity and did not alter BCMA CAR T anti-tumor activity.Taken together these results support allogeneic BCMA CAR T as an off-the-shelf adoptive immunotherapy for the treatment of multiple myeloma and other BCMA-positive malignancies. DisclosuresBoldajipour:Pfizer: Employment. Galetto:Cellectis SA: Employment. Sommer:Pfizer Inc.: Employment. Pertel:Pfizer Inc.: Employment. Valton:Cellectis Inc.: Employment. Park:Pfizer Inc.: Employment. Gariboldi:Cellectis SA: Employment. Chen:Alexo Therapeutics: Employment. Geng:Kodiak Sciences: Employment. Dong:Pfizer Inc.: Employment. Boucher:Pfizer Inc.: Employment. Van Blarcom:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment. Rajpal:Pfizer Inc.: Employment. Smith:Cellectis SA: Employment. Kuo:Pfizer Inc.: Employment. Sasu:Pfizer Inc.: Employment.

Towards 'Off-the-Shelf ' Universal Chimeric Antigen Receptor (CAR) T Cells: Mouse Anti-3rd Party Central Memory CD8 Veto Cells Prolong Functional Engraftment of Allogeneic Genetically Modified T Cells

We recently demonstrated that mouse anti-3rd party central memory CD8 veto cells (Tcm) enable engraftment of fully mis-matched T cell depleted bone marrow (TDBM) in sublethally irradiated (5.5 Gy TBI) recipients (Eran et al. Blood 2013). Here we found that Tcm generated from (host x donor)F1 or fully allogeneic donors were able to engraft and sustain their presence in such sub-lethally irradiated mice in the absence of TDBM transplants. Limiting dilution analysis revealed that the reactivity of host T cells (HTC) against donor spleen cells was significantly diminished in the chimeric mice treated with Tcm, whereas reactivity against 3rd party was comparable betweenTcm treated and non-treated mice (Fig. 1). This specific immune tolerance indicated that vetoTcm could potentially enable the use of 'off-the-shelf' allogeneic CAR T cells or any other genetically modified T cells from the same donor.To further investigate this possibility, we tested the ability of Tcmveto cells to enable engraftment of CD8 T cells from OT1 mice, expressing a transgenic T cell receptor (TCR) directed againstovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. To that end, 1x106 OT1 CD8 T cells (H2b) were infused into Balb/c (H2Dd) recipients sublethally irradiated with 5.25 Gy TBI in the presence or absence of different doses of veto Tcm. As shown in Fig. 2, when tested at 2 months post-transplant, OT1 cells could not be detected in the peripheral blood of untreated recipient mice, while they were readily detectable in mice receiving 2x106 Tcm (0.78±0.36), reaching a higher level (1.18± 0.61) upon infusion of 5x106Tcm.To evaluate the functionality of the engrafted OT1 T cells in the chimeric mice, we developed and calibrated a new murine model, using a melanoma B16-cell line of C57BL background that expresses the ovalbumin peptide and the tdTomatomarker (B16-OVA-tdTomato) (Fig 3A). In this model, we tried to mimic a state of minimal residual disease by injecting a small number of B16-OVAtdTomato melanoma cells (0.25x106) into syngeneic C57BL mice; we then treated the mice with sublethal irradiation (6Gy TBI) to simulate treatment of the tumor, but also to clear out space for the T-cell adoptive transfer. Finally, we injected 1x106 naïve OT-1 cells generated on a (Balb x-OT1-CD45.1+RAG2-) F1 background in the presence or absence of Balb/c veto Tcm (5x106), and followed the development of the tumor. We found thatBalb/c vetoTcm prolonged the engraftment of the transgenic donor-derived F1 OT1 CD8 T cells inBalb/c recipients, thereby enabling significantly improved control of tumor growth when tested at day 20 post-transplant ( p<0.05). In contrast non OVA-expressing tumor cells grew at the same rate in all treatment groups, thereby demonstrating the specificity of the anti-tumor effect (Fig.3B).Previous studies demonstrated that Tcmare depleted of GVH reactivity by virtue of their culture with 3rd party stimulators under cytokine starvation. Our present results demonstrate that suchTcm veto cells can pave the way for functional engraftment of CD8 T cells with genetically modified specificity. Taken together, these results provide a proof of concept for the potential clinical use of such veto cells to enable therapy with 'off-the-shelf ' allogeneic CAR T cells. The relative ease of growing humanTcm in large numbers over a short period of time (9-12 days) suggests that it could be possible to harvest byleukapheresis from a single donor, a sufficient number of cells, to support CAR therapy for more than 50 allogeneic recipients.* N.O.G and R.G equally contributed [Display omitted] [Display omitted] [Display omitted] DisclosuresOr Geva:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

DETECTION OF ANTI-RETICULOENDOTHELIOSIS ANTIBODY BY ENZYME-LINKED IMMUNOSORBENT ASSAY USING ENVELOPE PROTEIN EXPRESSED IN BACULOVIRUS

Reticuloendotheliosis virus (REV) infects various animals including chickens, ducks, geese, pheasants, peafowl and other birds. REV causes tumor, runting disease syndrome and immunosuppression in infected birds. The purpose of this study was to develop a rapid, reliable and convenient method to detect anti-REV antibody. The REV envelope protein was expressed by baculovirus expression system and evaluated for the potential as an antigen in an enzyme linked-immunosorbent assay (ELISA). The env gene from a REV strain goose/3410/06 was cloned into bacmid vector and expressed in Sf9 insect cells. The expected recombinant envelope protein expressed in infected insect cells was demonstrated in western blot with size of 62 kDa, and purified for using as antigen in an ELISA. A total of 182 chicken serum samples were used to evaluate the suitability of the ELISA based on neutralization test as the gold standard. The results showed that the relative sensitivity and relative specificity of this ELISA were 88.5% and 97.7%, respectively. The present ELISA using baculovirus expression envelope protein can be used to detect anti-REV antibody in the field.

The role of danger signals and ectonucleotidases in acute graft-versus-host disease

Allogeneic hematopoietic cell transplantation (allo-HCT) represents the only curative treatment approach for many patients with benign or malignant diseases of the hematopoietic system. However, post-transplant morbidity and mortality are significantly increased by the development of acute graft-versus-host disease (GvHD). While alloreactive T cells act as the main cellular mediator of the GvH reaction, recent evidence suggests a critical role of the innate immune system in the early stages of GvHD initiation. Danger-associated molecular patterns released from the intracellular space as well as from the extracellular matrix activate antigen-presenting cells and set pro-inflammatory pathways in motion. This review gives an overview about danger signals representing therapeutic targets with a clinical perspective with a particular focus on extracellular nucleotides and ectonucleotidases.

Donor Leukocyte Infusion Mediated Graft-Versus-Host Responses in a Pre-Clinical Swine Model of Haploidentical Hematopoietic Cell Transplantation

The donor-anti-host cellular reactivity associated with allogeneic hematopoietic cell transplantation (HCT) is a powerful anti-leukemia treatment. However, graft-versus-host disease (GVHD) continues to limit HCT, especially across major histocompatibility (MHC) barriers. We previously described successful hematopoietic stem cell engraftment in MHC-mismatched swine without GVHD using novel reduced intensity conditioning (RIC) regimens consisting of partial, transient recipient T-cell depletion (with or without thymic or low-dose total body irradiation), and a short course of cyclosporine A (CyA). Here, we report that stable chimeric animals generated with these protocols are strongly resistant to donor leukocyte infusion (DLI) mediated GVH effects (n=17). Delayed DLI in tolerant chimeras at a dose consisting of 10-50X106 T cells /kg (Tc/kg) failed to induce conversion to full donor chimerism or cause GVHD. Sensitizing the donor with recipient skin prior to collecting donor cells for DLI did not have any demonstrable effect on DLI outcome (n=2). This resistance to DLI mediated GVH reactivity could be overcome in some cases (n=9) by either 1) increasing the dose of DLI, 2) removing chimeric host peripheral blood cell populations through extensive leukapheresis prior to DLI or 3) delivering lymphocytes fully mismatched to the host but not to the original HCT donor within the chimera. However, conversion to full donor chimerism in some recipients was associated with GVHD. Our studies suggest that delivery of non-manipulated HCT grafts under reduced intensity conditioning can induce a potent, GVHD free immune tolerant state that is strongly resistant to DLI. DLI induced conversion to full donor chimerism is often associated with GVHD despite full immune reconstitution of the host and lack of known inflammatory stimuli.

T Cells in Fish

Cartilaginous and bony fish are the most primitive vertebrates with a thymus, and possess T cells equivalent to those in mammals. There are a number of studies in fish demonstrating that the thymus is the essential organ for development of T lymphocytes from early thymocyte progenitors to functionally competent T cells. A high number of T cells in the intestine and gills has been reported in several fish species. Involvement of CD4+ and CD8α+ T cells in allograft rejection and graft-versus-host reaction (GVHR) has been demonstrated using monoclonal antibodies. Conservation of CD4+ helper T cell functions among teleost fishes has been suggested in a number studies employing mixed leukocyte culture (MLC) and hapten/carrier effect. Alloantigen- and virus-specific cytotoxicity has also been demonstrated in ginbuna and rainbow trout. Furthermore, the important role of cell-mediated immunity rather than humoral immunity has been reported in the protection against intracellular bacterial infection. Recently, the direct antibacterial activity of CD8α+, CD4+ T-cells and sIgM+ cells in fish has been reported. In this review, we summarize the recent progress in T cell research focusing on the tissue distribution and function of fish T cells.

Skin manifestations and treatment of graft-versus-host disease

Graft-versus-host disease (GVHD) is an autoimmune disease caused by graft-versus-host reaction, occurring most frequently in patients receiving allogeneic hematopoietic stem cell transplantation and occasionally in patients receiving organ transplantation. It often involves multiple organ systems, of which, skin and mucous membranes are the most frequently affected system with various clinical manifestations. Acute GVHD often appears as measles- or scarlet fever-like skin rashes with severe systemic symptoms such as fever and poor appetite, and may rapidly progress into toxic epidermal necrolysis (TEN) or Stevens-Johnson syndrome (SJS) in a few days. Lichen planus and sclerodermoid lesions are the most common clinical manifestations of chronic GVHD. With further understanding of this disease in recent years, some new manifestations have been observed, such as psoriasis- and atopic dermatitis-like skin lesions, as well as nail changes in some cases. It is especially important to select appropriate treatment protocols according to clinical manifestations. Glucocorticoids combined with immunosuppressive agents are a conventional treatment for GVHD, and innovative treatments have emerged with the development of therapeutics. Key words: Graft vs host disease; Skin manifestations; Clinical protocols; Biological agents; Mesenchymal stem cell transplantation

Current and future status of haploidentical hematopoietic stem cell transplantation

Stem cell transplantation from HLA-haploidentical related donors (haploSCT) has been highlighted as an alternative donor source. The regimen consisting of post-transplant cyclophosphamide (PTCY) has been highly prevalent in the US, Europe, and Japan. Considering the status overseas and the current status in Japan, we aim to show our efforts in haploSCT. We initially established the "haplo-full (original)" regimen, which was found to be excessively toxic for general use. Thus, we added ATG to diminish the GVH reaction (haplo-full with ATG). Unfortunately, "haplo-mini (original)" was found to be relatively weak against refractory diseases. Thus, we intensified the preconditioning regimen, which has enabled us to deal with refractory and post-transplant relapse (FAMC-T). One of the characteristics in haploSCT in Hyogo is the use of steroids from the beginning of SCT. The aim of this strategy is to diminish the inflammation affecting GVHD-target organs, and to suppress chemokine release. Since chemokines produced by the preconditioning regimen are well known to induce GVHD, chemokine suppression should effectively suppress GVHD development. The dependency of the GVL effect on chemokines is unclear, possibly serving as a therapeutic window to separate GVHD from the GVL effect.

Immunomodulatory effects of transforming growth factor-β in the liver.

Members of the transforming growth factor-β (TGF-β) family are potent regulatory cytokines that affect multiple cell types of the immune system mediating pro-inflammatory or anti-inflammatory responses. In the liver, TGF-β is produced by a multitude of non-parenchymal liver cells including hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), and dendritic cells (DCs) as well as natural killer (NK) T cells among other hepatic lymphocytes. The effect of TGF-β on other cells is highly versatile. In concert with other soluble factors, it controls the maturation, differentiation and activity of various T cell subsets that either prevent or actuate infections, graft-versus-host reactions, immune diseases, and cancer formation. During the last decades, it became evident that some TGFB1 polymorphisms are associated with the pathogenesis of hepatic disease and that plasma TGF-β is a suitable biomarker to detect liver lesions. Moreover, since TGF-β has capacity to influence the quantity and quality of T cell subsets as well as their activity, it is obvious that a well-balanced TGF-β activity is essential for liver homeostasis. In the present review, we highlight some pivotal functions of TGF-β in hepatic immunobiology. We discuss its regulatory function on adaptive immunity, the impact on differentiation of various T cell subsets, its crosstalk with Toll like receptor signaling, and its contribution to functional impairment of the liver.

Treg Induced Impairment of DC Function and Its Impact on GvH Reactions

Promising results from murine models and early stage clinical trials have shown that adoptive transfer of regulatory T cells (Treg) prevents graft-versus-host disease (GvHD). However, the primary target of Treg mediated protection against GvHD is yet to be fully defined. We have previously shown that the presence of Treg during effector T cell priming is able to ameliorate cutaneous GvH reactions in vitro by blocking effector cell migration. This has led to the hypothesis that Treg modulation of dendritic cells (DC) could be a key mechanism by which Treg exert their protective role in GvHD. DC are fundamental for the initiation of allo-reactive immune responses and are critical in GvHD pathogenesis. We investigated the effect of Treg on the phenotypic profile and allo-reactive functions of DCs. Furthermore, the impact of Treg treatment on the ability of DCs to induce GvH target tissue damage was examined for the first time using an in vitro human GvHD skin explant model.Immature, mature and Treg treated DCs were generated from immuno-magnetic isolated monocytes (im-DC, mat-DC and Treg-DC respectively). The three moDC populations were generated using the well-established 6 day culture with GM-CSF and IL-4 followed by 24 hour LPS maturation. Treg were added on day 3 of moDC culture. Im-DC, mat-DC and Treg-DC were harvested on day 7 and tested in parallel as stated below. Prior to functional assays Treg-moDC were isolated by FACS sorting via FSC/SSC/CD3neg gating to remove Treg present in the co-culture.Our results revealed that Treg-DC displayed a semi-mature phenotype with CD83, CD80 and CD86 expression significantly lower than mat-moDC (p<0.005) but significantly higher than im-moDC (P<0.05) whilst HLA-DR levels were comparable to mat-moDC but significantly higher than imDC (p<0.005). Treg-DC also expressed CCR7 comparable to that of im-DC but markedly higher than mat-DC (p=0.052). Distinct morphology of Treg-DC, defined by Giemsa staining, corroborated their semi-mature status. Data from FITC-dextran uptake showed a significant reduction in antigen-capture capacity of Treg treated im-DC compared to untreated im-DC (p=0.047). The Treg mediated functional impairment of DCs was also associated with significantly higher expression of LAP-TGFβ1 on Treg-DC when compared to that of mat-DC (P<0.05). Treg-DC were also markedly defective in stimulating activation and proliferation of allo-reactive CD8 T cells, detected by CD25 expression and CFSE dilution respectively (p=0.009, p=0.046). Interestingly the presence of Treg throughout the entire allo-response induction resulted in a more potent reduction in activation and proliferation than when only the DC were treated with Treg (p=0.009, p=0.0085). Furthermore, allo-reactive CD8 T cells primed with Treg treated moDC were less able to mediate cutaneous GvH tissue damage (Figure 1).In conclusion, attenuation of DC signature and function is key for Treg mediated protection against GvHD. However, isolated DC modulation by Treg was less effective in suppressing CD8 T cell allo-responses compared to the continued presence of Treg during the CD8 T cell priming, activation and proliferation, suggesting that Treg exert most effective function via multidimensional modulation on the DC, T cells and DC-T cell interactions simultaneously. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effect of isopropyl methylphosphonofluoridate (IMPF) poisoning on selected immunological parameters of angiogenesis.

Acetylcholinesterase (AChE) and cholinergic receptors play an important role in the immune system, including lymphocyte-induced angiogenesis. However, their exact role is not fully understood. The presented work tests the influence of isopropyl methylphosphonofluoridate (IMPF), an irreversible inhibitor of AChE, on selected immune parameters associated with angiogenesis in mice. The levels of VEGF, bFGF, TNF-α, and IFN-γ production were measured, together with the ability of lymphoid spleen cells to induce local GvH reaction after a single dose of IMPF. Experiments were performed in male BALB/c mice. Acetylcholinesterase activity in erythrocytes was determined by the Ellman`s procedure. Levels of cytokines were measured in serum using standard commercial ELISA kits. Influence of IMPF intoxication upon angiogenesis was examined by the LIA test, according to the Sidky and Auerbach procedure. The results showed a 6- and 8-fold increase in VEGF at days 1 and 7 of the experiment, respectively, as well as a decrease (at days 14 and 21 after administration), followed by a significant increase (day 1) in bFGF levels. A statistically significant decrease in the concentration of IFN-γ was observed throughout all experiments. The maximum decrease in the level of TNF-α was found at days 1 and 7 after administration of IMPF. Additionally, a significant decrease was found in the ability to form new blood vessels following IMPF administration. This study revealed that IMPF has a significant effect on the regulation of lymphocyte-induced angiogenesis, which is related with the modulation of angiogenic and pro-inflammatory cytokines secretion. The observed differences suggest a possible derangement of certain elements of the neuronal and/or non-neuronal cholinergic system.