Marrow Recipients Research Articles

Deletion of Macrophage Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Accelerates Atherosclerosis Regression and Increases C-C Chemokine Receptor Type 7 (CCR7) Expression in Plaque Macrophages.

We previously showed that mice lacking MΦLRP1-/- (low-density lipoprotein receptor-related protein 1 in macrophages) undergo accelerated atherosclerotic plaque formation due to changes in macrophages including increased apoptosis, decreased efferocytosis, and exaggerated transition to the inflammatory M1 phenotype. Here we sought to explore the role of macrophage low-density lipoprotein receptor-related protein 1 during regression of atherosclerosis since regressing plaques are characterized by transitioning of macrophages to M2 status as inflammation resolves. Apolipoprotein E-/- mice on a high-fat diet for 12 weeks were reconstituted with bone marrow from apolipoprotein E-producing wild-type or MΦLRP1-/- mice, and then placed on a chow diet for 10 weeks (n=9 to 11 mice/group). A cohort of apolipoprotein E-/- mice reconstituted with apolipoprotein E-/- bone marrow served as baseline controls (n=9). Plaques of both wild-type and MΦLRP1-/- bone marrow recipients regressed compared with controls (11% and 22%, respectively; P<0.05), and plaques of MΦLRP1-/- recipients were 13% smaller than those of wild-type recipients ( P<0.05). Recipients of MΦLRP1-/- marrow had 36% fewer M1 macrophages ( P<0.01) and 2.5-fold more CCR7 (C-C chemokine receptor type 7)-positive macrophages in the plaque relative to wild-type mice ( P<0.01). Additionally, in vivo studies of cellular egress showed a 4.6-fold increase in 5-ethynyl-2´-deoxyuridine-labeled CCR7+ macrophages in mediastinal lymph nodes. Finally, in vivo studies of reverse cholesterol transport showed a 1.4-fold higher reverse cholesterol transport in MΦLRP1-/- recipient mice ( P<0.01). Absence of macrophage low-density lipoprotein receptor-related protein 1 unexpectedly accelerates atherosclerosis regression, enhances reverse cholesterol transport, and increases expression of the motility receptor CCR7, which drives macrophage egress from lesions.

Loss of ACAT1 Attenuates Atherosclerosis Aggravated by Loss of NCEH1 in Bone Marrow-Derived Cells.

Aim: Acyl-CoA cholesterol acyltransferase 1 (ACAT1) esterifies free cholesterol to cholesteryl esters (CE), which are subsequently hydrolyzed by neutral cholesterol ester hydrolase 1 (NCEH1). The elimination of ACAT1 in vitro reduces the amounts of CE accumulated in Nceh1-deficient macrophages. The present study aimed at examining whether the loss of ACAT1 attenuates atherosclerosis which is aggravated by the loss of NCEH1 in vivo.Methods: Low density lipoprotein receptor (Ldlr)-deficient mice were transplanted with bone marrow from wild-type mice and mice lacking ACAT1, NCEH1, or both. The four types of mice were fed a high-cholesterol diet and, then, were examined for atherosclerosis.Results: The cross-sectional lesion size of the recipients of Nceh1-deficient bone marrow was 1.6-fold larger than that of the wild-type bone marrow. The lesions of the recipients of Nceh1-deficient bone marrow were enriched with MOMA2-positive macrophages compared with the lesions of the recipients of the wild-type bone marrow. The size and the macrophage content of the lesions of the recipients of bone marrow lacking both ACAT1 and NCEH1 were significantly smaller than the recipients of the Nceh1-deficient bone marrow, indicating that the loss of ACAT1 decreases the excess CE in the Nceh1-deficient lesions. The collagen-rich and/or mucin-rich areas and en face lesion size were enlarged in the recipients of the Acat1−/− bone marrow compared with those of the recipients of the WT bone marrow.Conclusion: The loss of ACAT1 in bone marrow-derived cells attenuates atherosclerosis, which is aggravated by the loss of NCEH1, corroborating the in vitro functions of ACAT1 (formation of CE) and NCEH1 (hydrolysis of CE).

Summary of the Third International Workshop on Clinical Tolerance.

The Third International Workshop on Clinical Tolerance was held in Stanford, California, September 8-9, 2017. This is a summary of Workshop presentations of clinical trials designed to withdraw or minimize immunosuppressive (IS) drugs in kidney and liver transplant patients without subsequent evidence of rejection. All clinical protocols had in common the use of donor or recipient cell therapy combined with organ transplantation. Tolerance to HLA matched and mismatched living donor kidney transplants with complete withdrawal of IS drugs without subsequent rejection for up to 14 years of observation was achieved in more than 50 patients enrolled in trials in four medical centers after the establishment of transient or persistent chimerism. Complete IS drug withdrawal without chimerism was reported in a prospective trial of liver transplantation combined with injection of regulatory T cells. IS drug minimization without rejection was reported in recipients of living donor kidney transplants enrolled in the One Study consortium after injection of recipient regulatory T cells, or injection of donor regulatory monocytes or dendritic cells. In conclusion, considerable progress has been made in achieving IS drug withdrawal after cell therapy in recipients of organ transplants.

Abstract A81: Early TLR-mediated killing of leukemia in bone marrow is correlated with durable protection against B cell precursor acute lymphoblastic leukemia

Abstract The ability of Toll-like receptor (TLR)-induced innate immune responses to activate adaptive immune responses offers considerable therapeutic potential for cancer treatment, most likely as part of combination therapies. The potent immunostimulatory capacity of TLR agonists provides means to generate antigen-specific antitumor T cell responses from antigen-nonspecific innate immune stimulation. B cell precursor (BCP) acute lymphoblastic leukemia (ALL) exhibits the very low mutational burden common to other pediatric cancers. Despite this limited neo-antigenicity, we previously reported the ability of CpG oligodioxynucleotides (ODN), a ligand for TLR9, to mediate anti-leukemia activities that establish long-term protection against ALL outgrowth in a transplantable syngeneic model using cell lines. CpG ODN also reduced the burden of primary human ALL in mouse xenografts, primarily via induction of NK cell-mediated cytotoxicity. However, our recent demonstration that endosomal TLR agonists generated distinct effects on pre-leukemic BCP cells, coupled with the divergent effects of TLR ligands on the immunogenicity of primary human ALL cells suggest that there are significant variables that contribute to the overall outcome of TLR stimulation in the context of BCP cell malignancy. In this study, we compared the ligands for TLR3 (polyI:C), TLR7/8(R848) and TLR9 (CpG ODN) for their ability to elicit control of primary ALL cells and correlated protection with early innate immune activation to identify the necessary component of a long-term protective anti-leukemic response. We adoptively transferred syngeneic primary ALL cells to wild-type mice then administered TLR ligands starting at day 7 for 3 doses every 4 days. 21 days after leukemia challenge, we evaluated the disease burden in the peripheral blood, spleen, and bone marrow of recipients. While a significantly lower disease burden was observed in all three anatomical compartments of mice treated with polyI:C, CpG ODN- and R848-treated mice failed to control the early outgrowth of transferred cells in the spleen and bone marrow, respectively. While R848-treated mice had a slightly delayed disease progression with median survival (MS) of 40 days compared to that of 28 days of PBS-treated control group, CpG ODN treatment conferred a significant survival advantage where over 57% of treated mice achieved a prolonged disease-free status. Despite successfully inducing systemic anti-ALL responses, poly I:C treatment failed to confer durable protection, achieving only a modest increase in disease-free survival in treated mice (MS=42 days). Moreover, CpG ODN-induced long-term survival in the absence of effective early immune-mediated control of ALL in spleen implies that bone marrow is not only an important homing niche supporting the survival and proliferation of leukemia blasts prior to migration into the circulation, but a critical target site for controlling disease progression. Furthermore, to investigate the early innate immune responses associated with the differential overall effects induced by TLR agonists, we evaluated the activation status of innate immune system using RAG-knockout mice. A single administration of CpG ODN, but not polyI:C nor R848, was sufficient to rapidly induce anti-ALL immune responses involving activated natural killer and antigen-presenting cells in the bone marrow of the mice bearing ALL in 4 days. This suggests that CpG ODN has superior capacity to initiate innate immune-mediated anti-ALL immune responses and activate subsequent antigen-specific T cell responses. TLR-mediate innate immune responses induced early in life are distinct from that induced in adult life. Given the peak incidence of pediatric ALL between 3-5 years of age, we are currently examining the influence of age on the TLR-induced anti-ALL immune responses to further evaluate the therapeutic benefits of TLR ligands. Our results demonstrate the differential magnitude of tissue-specific innate immune-mediated control of leukemia outgrowth induced by TLR stimulations correlating with durable disease-free survival. These findings provide mechanistic explanation for TLR-mediated protective immune responses and highlight the potential target site necessary for effective elimination of leukemia cells. Citation Format: Sumin Jo, Peter van den Elzen, Gregor S.D Reid. Early TLR-mediated killing of leukemia in bone marrow is correlated with durable protection against B cell precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A81.

OR35 A novel mixed lymphocyte culture method to evaluate donor HLA specific immunity in different HLA type mismatched donors and recipients

Aim A novel flow cytometry based mixed lymphocyte culture assay (Flow Cellular Crossmatch: Flow-CXM) was developed for detecting antigen or donor specific T cell immunity. The preliminary study demonstrated that the donor specific T cell response in recipient was correlated with HLA mismatch (MM) numbers between donors & recipients. The aim of this study is to further evaluate the significance of Flow-CXM in different HLA type MM donors and recipients. Methods CXM (Fig. 1): 0.1 × 106 irradiated donor PBMCs were incubated with 0.1 × 106 recipient’s PBMCs and Th1 (IFNγ & IL-2) or Th2 (IL-4, 5, 10) cytokine capture beads for 16 h. The cells were lysed and the captured cytokines were detected by corresponding PE-labeled 2nd antibodies. The beads with different fluorescence were gated and PE stained beads (Flowspot %) were counted to represent the relative number of donor specific responding T cells. HLA Typing High-resolution HLA typing of all donor and recipient samples were performed by NGS or SSO. Results A total 109 paired bone marrow donor and recipient samples were tested by Flow-CXM. Compared to the HLA identical group, IL-2 but not IFNγ response significantly increased in HLA-class I (CI) (P Conclusions The newly developed Flow-CXM is able to detect earlier T cell activation of donor HLA specific immunity in recipient within 24 h. The multiple T cell activation markers (Th1/Th2 cytokines) can be measured in a single reaction. The methodology of Flow-CXM is simple, sensitive, and HLA specific. It hold a potential to be used in clinical transplantation for predicting and monitoring GVHD/GVL in BMT and cellular rejection in solid organ transplantation. Download : Download high-res image (404KB) Download : Download full-size image

Bone Marrow versus Peripheral Blood from Unrelated Donors for Children and Adolescents with Acute Leukemia

Graft-versus-host disease (GVHD) rates are higher after unrelated donor transplantation; thus, we examined whether there would be differences in transplant outcomes by graft type in children and adolescents with acute leukemia. The primary endpoint was overall survival. We studied 872 patients <18 years old who were transplanted with bone marrow (n = 650) or peripheral blood (n = 222) from unrelated donors. The characteristics of the 2 groups were comparable, except recipients of bone marrow were younger than recipients of peripheral blood (median age, 10 versus 12 years). Grades 2 to 4 (hazard ratio [HR], 1.48; P < .001) and grades 3 and 4 acute (HR, 1.69; P < .001) and chronic GVHD (HR, 1.92; P < .001) were higher with transplantation of peripheral blood than with bone marrow. Although relapse risks were lower after peripheral blood transplants (HR, 0.76; P = .05), transplant-related mortality (HR, 1.91; P = .003) and overall mortality (HR, 1.34; P = .032) were higher than with bone marrow transplants. The 8-year probability of overall survival after transplantation of bone marrow was 47% compared with 42% after peripheral blood. The 8-year probability of leukemia-free survival was 40% after transplantation of bone marrow and peripheral blood. Lower relapse after transplantation of peripheral blood negated the survival advantage after transplantation of bone marrow. The observed higher acute and chronic GVHD seen with peripheral blood suggest cautious use of this graft in children and adolescents with acute leukemia.

3135 - Self-Repopulating Recipient Bone Marrow Recipient Macrophages Promote Hematopoietic Stem Cell Engraftment Post Autologous Transplantation

3135 - Self-Repopulating Recipient Bone Marrow Recipient Macrophages Promote Hematopoietic Stem Cell Engraftment Post Autologous Transplantation

Allograft and patient survival after sequential HSCT and kidney transplantation from the same donor-A multicenter analysis.

Tolerance induction through simultaneous hematopoietic stem cell and renal transplantation has shown promising results, but it is hampered by the toxicity of preconditioning therapies and graft‐versus‐host disease (GVHD). Moreover, renal function has never been compared to conventionally transplanted patients, thus, whether donor‐specific tolerance results in improved outcomes remains unanswered. We collected follow‐up data of published cases of renal transplantations after hematopoietic stem cell transplantation from the same donor and compared patient and transplant kidney survival as well as function with caliper‐matched living‐donor renal transplantations from the Austrian dialysis and transplant registry. Overall, 22 tolerant and 20 control patients were included (median observation period 10 years [range 11 months to 26 years]). In the tolerant group, no renal allograft loss was reported, whereas 3 were lost in the control group. Median creatinine levels were 85 μmol/l (interquartile range [IQR] 72‐99) in the tolerant cohort and 118 μmol/l (IQR 99‐143) in the control group. Mixed linear‐model showed around 29% lower average creatinine levels throughout follow‐up in the tolerant group (P < .01). Our data clearly show stable renal graft function without long‐term immunosuppression for many years, suggesting permanent donor‐specific tolerance. Thus sequential transplantation might be an alternative approach for future studies targeting tolerance induction in renal allograft recipients.

Role of Graft-derived Graft-versus-Host T cells in Facilitating Multilineage Blood Chimerism after Human Intestinal Transplantation

Long-term multilineage chimerism often appears in blood following human intestinal transplantation (ITx), especially in multivisceral transplant (MVTx) recipients. Patients with donor T cell macrochimerism (>4%) in blood, frequently without graft-versus-host disease (GVHD), displayed less graft rejection and donor-specific antibody (DSA) production and slower graft T cell replacement by the recipient. To address the hypothesis that the balance of graft-versus-host (GvH) and host-versus-graft (HvG) alloreactivity might underly these relationships, we analyzed the phenotype, repertoire and alloreactivity of donor- and recipient-derived T cells in the intestinal allograft, peripheral blood and bone marrow (BM) of ITx recipients. We identified GvH and HvG clones on the basis of high throughput TCRβ CDR3 sequencing from pre-Tx donor and recipient mixed lymphocyte reactions as we have described1. Enrichment of GvH compared to HvG clones in early intestinal biopsies and absence of Class I DSA were associated with donor T cell macrochimerism. Local expansion of GvH clones in the graft was likely driven by uniformly rapid replacement of recipient antigen presenting cells (APCs) within allografts early post-Tx. Cumulative frequency of GvH clones in blood early post-Tx was positively correlated with the peak level of donor T cell chimerism (Fig.1).Unlike donor ileal lymphocytes, which display a tissue-resident memory phenotype, long-term circulating donor T cells were markedly enriched for the recent thymic emigrant phenotype compared with recipient cells, and GvH clones were undetectable among these late circulating naïve donor-derived T cells. Enrichment of donor circulating naïve B cells and T cells and recurrent myeloid chimerism late post-Tx are consistent with our observation that donor-derived hematopoietic stem and progenitor cells are present in intestinal allografts and are detectable in the circulation of patients with macrochimerism. Multilineage chimerism might reflect a lymphohematopoietic GvH response (LGVHR) migrating from recipient circulation to the BM, making “space” for engraftment of progenitor cells from the graft. Indeed, 0.0356% of CD45+ cells in the BM of an MVTx patient on day 105 post-Tx were donor-derived, among which 72.9% were T cells and 1.37% were CD34+ progenitors. TCR sequencing of sorted donor cells in recipient BM identified donor-mappable clones, among CD4s of which 60% by frequency were GvH-reactive. GvH CD4 cells did not show marked enrichment in donor-mappable PBMCs on the same day. (Fig.2)Together, these data suggest that donor GvH-reactive T cells expanding within the graft in response to recipient APCs entering the graft migrate into the recipient circulation and BM, playing a key role in promoting and maintaining mixed chimerism. LGVHR without GVHD in ITx recipients suggests a novel approach to achieving intestinal allograft tolerance. 1. Zuber et al., 2016 Science Immunology. The study was funded by the National Institute of Allergy and Infectious Diseases grant P01 AI106697. Research reported in this study was performed in the CCTI Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056.

Mechanisms of Vascularized Bone Marrow Graft Protection of Vascularized Composite Allografts

Background Co-transplantation of vascularized bone marrow (VBM) promotes vascularized composite allograft (VCA) survival with standard immunosuppression in a non-human primate (NHP) model. We investigated mechanisms of immunomodulation by comparing VCA survival following donor bone marrow cell (BMC) infusion, donor and recipient conditioning strategies, and heterotopic VBM allografts to historical groups. Methods Groups of 3–4 NHP received facial subunit VCA and immunosuppression of tacrolimus (Tac) and mycophenolate mofetil (MMF). Comparative groups, VCA+VBM (n=4) and VCA (no VBM, n=3) were from historical cohorts (Group 1 and 2, respectively). Group 3 received VCA without VBM and transfused donor BMC at 20x 106 cells/kg on d 1. Group 4 received VCA+VBM following donor mandible irradiation (1.5 Gy) on d −1, while Group 5 received irradiated donor mandible followed by BMC transfusion on d 1. Group 6 underwent co-transplantation of heterotopic VBM and VCA without VBM. Group 7 received VCA+VBM after donor T cell depletion with anti-thymocyte globulin (ATGAM, 50 mg/kg) on days −2, and −1. An additional dose of ATGAM was given on d 0 if CD3+ cells remained >50/mm3. Group 8 recipients received ATGAM depletion as above, VCA+VBM, and a 21-d steroid taper. Banff classification was determined by histopathologic analysis. Chimerism was assessed by flow cytometry from peripheral blood and sampled tissues. Results VCA + VBM treated with Tac/MMF demonstrated prolonged graft survival and rejected only after immunosuppression was withdrawn compared to Groups 2–8 (mean survival 348d vs 36d). This effect was statistically significant (p=0.05). Endpoints were met secondary to Banff IV rejection and malignancy. Macrochimerism was present only in Group 1; remaining groups demonstrated only transient microchimerism. Discussion The immunologic advantage of VBM prolonging VCA graft survival is contingent on proximity of the VBM to the VCA allograft. Donor conditioning nullifies this benefit and it is not reconstituted by transfusion of donor BMC. Recipient T cell depletion mirrors the early graft loss observed with donor T cell depletion. Conclusion Our data elucidate a mechanism for VCA graft survival that is mediated by locally acting, radiosensitive T cell populations in both donor and recipient and is dependent on the unique immunologic and structural niche of VBM. Astellas Pharma, Inc.

Parasitic Infections Associated with Unfavourable Outcomes in Transplant Recipients.

Introduction. The immunosuppression used after transplantation (Tx) is associated with an increased risk of opportunistic infections. In Europe, parasitic infections after Tx are much less common than viral, bacterial and fungal ones. However, diseases caused by parasites are very common in tropical countries. In the last years the number of travellers with immunosuppression visiting tropical countries has increased. Methods. We performed a literature review to evaluate a risk of parasitic infections after Tx in Europe. Results. There is a real risk of parasitic infection in patients after Tx travelling to tropical countries. Malaria, leishmaniasis, strongyloidiasis and schistosomiasis are the most dangerous and relatively common. Although the incidence of these tropical infections after Tx has not increased, the course of disease could be fatal. There are also some cosmopolitan parasitic infections dangerous for patients after Tx. The greatest threat in Europe is toxoplasmosis, especially in heart and bone marrow recipients. The most severe manifestations of toxoplasmosis are myocarditis, encephalitis and disseminated disease. Diarrhoea is one of the most common symptoms of parasitic infection. In Europe the most prevalent pathogens causing diarrhoea are Giardia duodenalis and Cryptosporidium. Conclusions. Solid organ and bone marrow transplantations, blood transfusions and immunosuppressive treatment are associated with a small but real risk of parasitic infections in European citizens. In patients with severe parasitic infection, i.e., those with lung or brain involvement or a disseminated disease, the progression is very rapid and the prognosis is bad. Establishing a diagnosis before the patient’s death is challenging.

The gp130 Cytokine Interleukin-11 Regulates Engraftment of Vav1-/- Hematopoietic Stem and Progenitor Cells in Lethally Irradiated Recipients.

During bone marrow transplantation, hematopoietic stem and progenitor cells (HSPCs) respond to signals from the hematopoietic microenvironment by coordinately activating molecular pathways through Rho GTPases, including Rac. We have previously shown that deletion of Vav1, a hematopoietic-specific activator of Rac, compromises engraftment of transplanted adult HSPCs without affecting steady-state hematopoiesis in adult animals. Here, we show that Vav1-/- fetal HSPCs can appropriately seed hematopoietic tissues during ontogeny but cannot engraft into lethally irradiated recipients. We demonstrate that the engraftment defect of Vav1-/- HSPCs is abrogated in the absence of irradiation and demonstrate that Vav1 is critical for the response of HSPCs to the proinflammatory cytokine interleukin-11 (IL-11) that is upregulated in the marrow of irradiated recipients. Vav1-/- HSPCs display abnormal proliferative responses to IL-11 in vitro and dysregulated activation of pathways critical to engraftment of HSPCs. The engraftment of Vav1-/- HSPCs can be partially rescued in irradiated recipients treated with an anti-IL-11 antibody. These data suggest that HSPCs may respond to different functional demands by selective usage of the IL-11-Vav-Rac pathway, contextualizing further the recent view that HSPCs capable of reconstituting the blood system following transplantation might be distinct from those supporting hematopoiesis during homeostatic conditions. Stem Cells 2018; 36:446-457.

Distinct NG2 proteoglycan-dependent roles of resident microglia and bone marrow-derived macrophages during myelin damage and repair.

We used a bone marrow transplantation approach to distinguish the activities of bone marrow-derived macrophages from the activities of central nervous system-resident microglia in phenomena associated with axon demyelination and remyelination. We transplanted wild type or germline NG2 null beta-actin-EGFP expressing bone marrow into irradiated wild type or NG2 null recipient mice, followed by analysis of lysolecithin-induced spinal cord demyelination and remyelination and quantification of Iba-1+/ F4/80+/ EGFP+ macrophages and Iba-1+/ F4/80+/ EGFP- microglia. One week after microinjection of 1% lysolecithin into the spinal cord, wild type recipients receiving NG2 null bone marrow exhibit greatly reduced infiltration of macrophages into lesions, compared to wild type recipients receiving wild type bone marrow. Wild type bone marrow recipients also exhibit larger numbers of demyelinated axons than NG2 null recipients, indicative of macrophage participation in the initial myelin damage. However, wild type bone marrow recipients also exhibit superior myelin repair at 6 weeks post-injury, compared to NG2 null bone marrow recipients, demonstrating the additional importance of macrophages in remyelination. Incompletely repaired lesions in NG2 null bone marrow recipients at 6 weeks post-injury retain elevated numbers of macrophages, in contrast to lower numbers of macrophages in more completely repaired lesions in wild type bone marrow recipients. This suggests that NG2 expression renders macrophages more effective in myelin repair and less likely to promote chronic inflammation. Effective macrophage involvement in myelin repair is due in part to effects on the proliferation and/or recruitment of oligodendrocyte progenitor cells. Reduced numbers of oligodendrocyte progenitors are seen in lesions in NG2 null bone marrow recipients, likely due to deficits in macrophage production of oligodendrocyte progenitor-relevant mitogens and in phagocytosis of inhibitory myelin debris. Microglia also appear to be important for clearance of myelin debris, as indicated by reduced phagocytosis in NG2 null recipients receiving wild type bone marrow.

PD-1 and CTLA-4 up regulation on donor T cells is insufficient to prevent GvHD in allo-HSCT recipients.

The expression of checkpoint blockade molecules PD-1, PD-L1, CTLA-4, and foxp3+CD25+CD4+ T cells (Tregs) regulate donor T cell activation and graft-vs-host disease (GvHD) in allogeneic hematopoietic stem cell transplant (allo-HSCT). Detailed kinetics of PD-1-, CTLA-4-, and PD-L1 expression on donor and host cells in GvHD target organs have not been well studied. Using an established GvHD model of allo-HSCT (B6 → CB6F1), we noted transient increases of PD-1- and CTLA-4-expressing donor CD4+ and CD8+ T cells on day 10 post transplant in spleens of allo-HSCT recipients compared with syngeneic HSCT (syn-HSCT) recipients. In contrast, expression of PD-1- and CTLA-4 on donor T cells was persistently increased in bone marrow (BM) of allo-HSCT recipients compared with syn-HSCT recipients. Similar differential patterns of donor T cell immune response were observed in a minor histocompatibility (miHA) mismatched transplant model of GvHD. Despite higher PD-1 and CTLA-4 expression in BM, numbers of foxp3+ T cells and Tregs were much lower in allo-HSCT recipients compared with syn-HSCT recipients. PD-L1-expressing host cells were markedly decreased concomitant with elimination of residual host hematopoietic elements in spleens of allo-HSCT recipients. Allo-HSCT recipients lacking PD-L1 rapidly developed increased serum inflammatory cytokines and lethal acute GvHD compared with wild-type (WT) B6 allo-HSCT recipients. These data suggest that increased expression of checkpoint blockade molecules PD-1 and CTLA-4 on donor T cells is not sufficient to prevent GvHD, and that cooperation between checkpoint blockade signaling by host cells and donor Tregs is necessary to limit GvHD in allo-HSCT recipients.

OR46 A novel cellular crossmatch to detect donor HLA specific immunity

Aim To develop a cellular crossmatch method (CXM) for evaluating donor HLA specific cellular immunity in HLA mismatched recipients. Methods HLA Typing: HLA types of all samples were obtained by high resolution typing. CXM: Irradiated donor cells were used as the donor HLA specific stimulant to co-incubate with recipient (responder) cells and Th1 (IFNγ & IL-2) or Th2 (IL-4, 5, 10) cytokine capture beads for overnight in fixed cell:bead ratio. The cells were lysed and the captured cytokines were detected by corresponding fluorescent 2nd step antibodies. The percentage of positive beads (Flowspot %) were calculated which proportionately correlated with numbers of donor specific responding cells. The MFI level correlated with the cytokine secretion capability of responding cells. Results A total 147 paired bone marrow donor and recipient samples were tested by CXM. Compared to the HLA identical group, IL-2 but not IFNγ was significantly increased in HLA-class I (CI) (P Conclusions Unlike mixed lymphocyte reaction (MLR) which detects DNA synthesis by radioisotope, the newly developed CXM is able to detect donor HLA specific immunity in responder within 24 h using standard flow cytometry. CXM detects earlier T cell activation (e.g. cytokine secretions) in a uni-or bi-directional multiplex detecting system. The method of CXM is simple, sensitive, and donor HLA specific. It potentially has wide application in both the research and clinical fields for predicting GVH/GVL in BM transplantation, cellular rejection in solid organ transplantation, and in other applications where cell–cell responses are of interest. Download : Download high-res image (207KB) Download : Download full-size image

P2y12 Receptor Promotes Pressure Overload-Induced Cardiac Remodeling via Platelet-Driven Inflammation in Mice.

Inflammation plays a critical role in adverse cardiac remodeling and heart failure. The P2y12 receptor is one of the predominant activating receptors for platelets, thus initiating inflammatory responses under various diseases. In this study, we investigated the functional significance of P2y12-mediated platelet activation in pressure overload-induced cardiac remodeling. Notably, P2y12 knockout (P2y12-/-) mice exhibited suppressed transverse aortic constriction-induced changes in cardiac hypertrophy, collagen synthesis, inflammatory cell recruitment, and cardiac dysfunction. Activated platelets and platelet-leukocyte aggregates were markedly downregulated in P2y12 knockout mice compared with wild-type counterparts after transverse aortic constriction. Moreover, bone marrow chimera experiments revealed that wild-type recipients of P2y12 knockout bone marrow markedly improved cardiac function and attenuated cardiac remodeling, reversed by wild-type platelets reinjection. Platelet depletion and P-selectin inhibition mimicked these protective effects by limiting the interaction between activated platelets and leukocytes. Furthermore, activated wild-type platelets directly induced cardiomyocyte hypertrophy and collagen synthesis via α-granule exocytosis, vanished in P2y12 knockout platelets or those administered anti-NSF (N-ethlymalimide-sensitive factor) antibodies. The results suggest that P2y12-mediated platelet activation promotes cardiac remodeling by triggering a series of inflammatory changes and interacting with leukocytes and endotheliocytes.

Clinical characteristics and outcome of human herpesvirus-6 encephalitis after allogeneic hematopoietic stem cell transplantation

In this retrospective analysis using the Transplant Registry Unified Management Program, we identified 145 patients with human herpesvirus (HHV)-6 encephalitis among 6593 recipients. The cumulative incidences of HHV-6 encephalitis at 100 days after transplantation in all patients, recipients of bone marrow or PBSCs and recipients of cord blood were 2.3%, 1.6% and 5.0%, respectively. Risk factors identified in multivariate analysis were male sex, type of transplanted cells (relative risk in cord blood transplantation, 11.09, P<0.001; relative risk in transplantation from HLA-mismatched unrelated donor, 9.48, P<0.001; vs transplantation from HLA-matched related donor) and GvHD prophylaxis by calcineurin inhibitor alone. At 100 days after transplantation, the overall survival rate was 58.3% and 80.5% among patients with and without HHV-6 encephalitis, respectively (P<0.001). Neuropsychological sequelae remained in 57% of 121 evaluated patients. With both foscarnet and ganciclovir, full-dose therapy (foscarnet ⩾180 mg/kg, ganciclovir ⩾10 mg/kg) was associated with better response rate (foscarnet, 93% vs 74%, P=0.044; ganciclovir, 84% vs 58%, P=0.047). HHV-6 encephalitis is not rare not only in cord blood transplant recipients but also in recipients of HLA-mismatched unrelated donors. In this study, development of HHV-6 encephalitis was associated with a poor survival rate, and neurological sequelae remained in many patients.

Abstract 1126: Potent inhibition of HCMV by modulating the cellular SNARE syntaxin 5

Abstract Human cytomegalovirus (HCMV), also referred to as human herpesvirus-5 (HHV-5), can cause serious and even fatal disease in immunocompromised individuals and newborns, namely individuals with AIDS, solid organ transplant recipients, chemotherapy patients and recipients of bone marrow and stem cell transplants. HCMV is a ubiquitous virus found throughout all geographic regions and socioeconomic groups, infecting greater than 50% of adults in industrialized countries and as many as 100% in developing countries. Although current therapeutics improves clinical outcomes, they are limited by toxicity, intravenous infusion and the development of resistance by the virus. Thus, there is a pressing need to develop novel therapeutics to prevent HCMV infections with concomitant organ toxicity. Formation of the cytoplasmic viral assembly compartment (cVAC) is an important step for efficient HCMV assembly. To do this, the virus must alter and repurpose the normal cellular balance of membrane and protein flux, a process that is not well understood. We have identified a compound Retro94, which potently inhibits production of infectious HCMV virions in cells by operating against a host cell process, rather than directly targeting the virus. The presence of Retro94 results in the severely impaired production of infectious virions, as great as 5 logs (99-99.99%). Here we discuss in vitro, in vivo, stability, and binding study of Retro94. Overall, our findings have identified Retro94, as novel agent that affects key cellular trafficking factors important for supporting HCMV infection. Citation Format: Dhimant H. Desai, Linda Cruz, Nicholas T. Streck, Trisha D. Desai, Aron Lukacher, Shantu G. Amin, Nicholas J. Buchkovich. Potent inhibition of HCMV by modulating the cellular SNARE syntaxin 5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1126. doi:10.1158/1538-7445.AM2017-1126

Haemolysis, pure red cell aplasia and red cell antibody formation associated with major and bidirectional ABO incompatible haematopoietic stem cell transplantation.

Acute and delayed haemolysis, alloimmunisation and pure red cell aplasia (PRCA) are potential complications after ABO incompatible haematopoietic stem cell transplantation (HSCT). The aims of this study were to investigate acute and delayed red blood cell (RBC) antibody-associated complications, including haemolysis, PRCA and alloimmunisation in major and bidirectional ABO incompatible HSCT. We retrospectively examined the transplant courses of 36 recipients of bone marrow or peripheral blood stem cells from ABO incompatible donors and evaluated the current practice of performing plasmapheresis in patients with higher isoagglutinin titres. We investigated the role of ABO incompatibility in haematopoietic recovery, transfusion requirements, alloimmunisation and PRCA. Laboratory signs of acute haemolysis were noted in five (14%) patients, one (3%) of whom had clinically overt haemolysis. Patients with haemolysis had IgM titres ≥1:8 and received >16 mL of RBC in the HSCT. In patients with higher titres, plasmapheresis performed prior to the transplant prevented acute haemolysis. Delayed haemolysis was not recorded in the follow up. Haematopoietic recovery and transfusion requirements did not differ notably between patients with and without haemolysis. De novo RBC antibodies were detected in two (5.5%) patients after HSCT, and PRCA was noted in one (3%) patient. Carried out with adequate graft processing, plasmapheresis and blood component support, haemolysis is not a common complication after HSCT. Our results confirm that the occurrence of haemolysis depends on larger RBC volumes and higher isoagglutinin titres. Despite the reduction of patients' isoagglutinin titres by plasmapheresis, we still noted a critical combination for the development of laboratory signs of haemolysis (IgM titre ≥1:8 and RBC volume >16 mL). De novo immunisation to RBC antigens and PRCA are rare events following ABO incompatible HSCT.

Epithelial-specific Toll-like Receptor (TLR)5 Activation Mediates Barrier Dysfunction in Experimental Ileitis.

A large body of evidence supports a central role of TLR5 and its natural ligand, flagellin, in Crohn's disease (CD), with the precise mechanism(s) still unresolved. We investigated the role of flagellin/TLR5 in SAMP1/YitFc (SAMP) mice, a spontaneous model of Crohn's disease-like ileitis. Ileal Tlr5 and serum antiflagellin IgG antibodies were increased in SAMP before the onset of inflammation and during established disease; these trends were abrogated in the absence of colonizing commensal bacteria. Irradiated SAMP receiving either wild-type (AKR) or SAMP bone marrow (BM) developed severe ileitis and displayed increased ileal Tlr5 compared with AKR recipients of either SAMP or AKR bone marrow, neither of which conferred ileitis, suggesting that elevated TLR5 in native SAMP is derived primarily from a nonhematopoietic (e.g., epithelial) source. Indeed, ileal epithelial TLR5 in preinflamed SAMP was increased compared with age-matched AKR and germ-free SAMP. TLR5-specific ex vivo activation of SAMP ileal tissues decreased epithelial barrier resistance, indicative of increased permeability, and was accompanied by altered expression of the tight junction proteins, claudin-3, occludin, and zonula occludens-1. Our results provide evidence that aberrant, elevated TLR5 expression is present in the ileal epithelium of SAMP mice, is augmented in the presence of the gut microbiome, and that TLR5 activation in response to bacterial flagellin results in a deficiency to maintain appropriate epithelial barrier integrity. Together, these findings represent a potential mechanistic pathway leading to the exacerbation and perpetuation of chronic gut inflammation in experimental ileitis and possibly, in patients with Crohn's disease.