Host Antigen-presenting Cells Research Articles

Extracellular release of damaged mitochondria induced by prehematopoietic stem cell transplant conditioning exacerbates GVHD

AbstractDespite therapeutic advancements, graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HSCT). In current models of GVHD, tissue injury induced by cytotoxic conditioning regimens, along with translocation of microbes expressing pathogen-associated molecular patterns, result in activation of host antigen-presenting cells (APCs) to stimulate alloreactive donor T lymphocytes. Recent studies have demonstrated that in many pathologic states, tissue injury results in the release of mitochondria from the cytoplasm to the extracellular space. We hypothesized that extracellular mitochondria, which are related to archaebacteria, could also trigger GVHD by stimulation of host APCs. We found that clinically relevant doses of radiation or busulfan induced extracellular release of mitochondria by various cell types, including cultured intestinal epithelial cells. Conditioning-mediated mitochondrial release was associated with mitochondrial damage and impaired quality control but did not affect the viability of the cells. Extracellular mitochondria directly stimulated host APCs to express higher levels of major histocompatibility complex II (MHC-II), costimulatory CD86, and proinflammatory cytokines, resulting in increased donor T-cell activation, and proliferation in mixed lymphocyte reactions. Analyses of plasma from both experimental mice and a cohort of children undergoing HSCT demonstrated that conditioning induced extracellular mitochondrial release in vivo. In mice undergoing MHC-mismatched HSCT, administration of purified syngeneic extracellular mitochondria increased host APC activation and exacerbated GVHD. Our data suggest that pre-HSCT conditioning results in extracellular release of damaged mitochondria, which increase alloreactivity and exacerbate GVHD. Therefore, decreasing the extracellular release of damaged mitochondria after conditioning could serve as a novel strategy for GVHD prevention.

CD4+ T cell immunity against cutaneous melanoma encompasses multifaceted MHC II-dependent responses.

Whereas CD4+ T cells conventionally mediate antitumor immunity by providing help to CD8+ T cells, recent clinical studies have implied an important role for cytotoxic CD4+ T cells in cancer immunity. Using an orthotopic melanoma model, we provide a detailed account of antitumoral CD4+ T cell responses and their regulation by major histocompatibility complex class II (MHC II) in the skin. Intravital imaging revealed prominent interactions of CD4+ T cells with tumor debris-laden MHC II+ host antigen-presenting cells that accumulated around tumor cell nests, although direct recognition of MHC II+ melanoma cells alone could also promote CD4+ T cell control. CD4+ T cells stably suppressed or eradicated tumors even in the absence of other lymphocytes by using tumor necrosis factor-α and Fas ligand (FasL) but not perforin-mediated cytotoxicity. Interferon-γ was critical for protection, acting both directly on melanoma cells and via induction of nitric oxide synthase in myeloid cells. Our results illustrate multifaceted and context-specific aspects of MHC II-dependent CD4+ T cell immunity against cutaneous melanoma, emphasizing modulation of this axis as a potential avenue for immunotherapies.

Listeria monocytogenes: a promising vector for tumor immunotherapy.

Cancer receives enduring international attention due to its extremely high morbidity and mortality. Immunotherapy, which is generally expected to overcome the limits of traditional treatments, serves as a promising direction for patients with recurrent or metastatic malignancies. Bacteria-based vectors such as Listeria monocytogenes take advantage of their unique characteristics, including preferential infection of host antigen presenting cells, intracellular growth within immune cells, and intercellular dissemination, to further improve the efficacy and minimize off-target effects of tailed immune treatments. Listeria monocytogenes can reshape the tumor microenvironment to bolster the anti-tumor effects both through the enhancement of T cells activity and a decrease in the frequency and population of immunosuppressive cells. Modified Listeria monocytogenes has been employed as a tool to elicit immune responses against different tumor cells. Currently, Listeria monocytogenes vaccine alone is insufficient to treat all patients effectively, which can be addressed if combined with other treatments, such as immune checkpoint inhibitors, reactivated adoptive cell therapy, and radiotherapy. This review summarizes the recent advances in the molecular mechanisms underlying the involvement of Listeria monocytogenes vaccine in anti-tumor immunity, and discusses the most concerned issues for future research.

Adoptive T cell transfer and host antigen-presenting cell recruitment with cryogel scaffolds promotes long-term protection against solid tumors

Although adoptive T cell therapy provides the T cell pool needed for immediate tumor debulking, the infused T cells generally have a narrow repertoire for antigen recognition and limited ability for long-term protection. Here, we present a hydrogel that locally delivers adoptively transferred T cells to the tumor site while recruiting and activating host antigen-presenting cells with GMCSF or FLT3L and CpG, respectively. T cells alone loaded into these localized cell depots provided significantly better control of subcutaneous B16-F10 tumors than T cells delivered through direct peritumoral injection or intravenous infusion. T cell delivery combined with biomaterial-driven accumulation and activation of host immune cells prolonged the activation of the delivered T cells, minimized host T cell exhaustion, and enabled long-term tumor control. These findings highlight how this integrated approach provide both immediate tumor debulking and long-term protection against solid tumors, including against tumor antigen escape.

Impact of oral microbiota on pathophysiology of GVHD.

Allogeneic transplantation of hematopoietic cells is the only curative therapy for several hematopoietic disease in which patients receive cytotoxic conditioning regimens followed by infusion of hematopoietic stem cells. Although the outcomes have improved over the past decades, graft-versus-host-disease (GVHD), the most common life-threatening complication, remains a major cause of non-relapse morbidity and mortality. Pathophysiology of acute GVHD characterized by host antigen-presenting cells after tissue damage and donor T-cells is well studied, and additionally the importance of recipient microbiota in the intestine is elucidated in the GVHD setting. Oral microbiota is the second most abundant bacterial flora in the body after the intestinal tract, and it is related to chronic inflammation and carcinogenesis. Recently, composition of the oral microbiome in GVHD related to transplantation has been characterized and several common patterns, dysbiosis and enrichment of the specific bacterial groups, have been reported. This review focuses on the role of the oral microbiota in the context of GVHD.

A Novel Human OX40L-CAR-Treg Specifically Targets Activated Antigen-Presenting Cells and Effectively Controls T Cell Alloreactivity and Xeno-GvHD

Background: Graft-versus-host disease (GvHD) remains the leading cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (HCT). Adoptive transfer of regulatory T cells (Tregs) has demonstrated promising results in controlling GvHD in both animal models and phase I/II clinical trials. However, strategies to increase the stability of ex-vivo expanded Tregs, and to optimize their potency, are needed. Recent efforts to accomplish these goals have focused on engineering Chimeric Antigen Receptor (CAR) Tregs, with the first attempts using CARs targeted against disparate HLA antigens. However, Tregs targeting HLA have several limitations, including the downregulation of these antigens that accompanies some diseases, as well as the lack of utility of this approach in the HLA-matched setting. In this study, we tested a novel GvHD prophylaxis approach consisting of infusing donor Tregs engineered to express an anti-OX40L CAR that, upon engagement with activated OX40L+ host antigen-presenting cells (APCs), increases their suppressor potency to dampen APC-mediated activation of alloreactive T cells. Methods and Results: We designed a CAR construct containing an scFv targeting OX40L, a CD28 costimulatory domain, along with CD3ζ, as well as the fluorescent reporter NeonGreen, all under the control of a synthetic FOXP3 promoter. This design enabled the selective and sustained expression of the anti-OX40L CAR on CD25+CD127- FoxP3+ Treg cells. We also created Tregs transduced with a non-targeting NeonGreen-only control construct (Neon-Tregs). Engineered OX40L CAR-Tregs maintained expression of the canonical Treg markers FOXP3, CD25, and CTLA-4, and demonstrated robust activation in the presence of OX40L-expressing cells. In co-culture assays of OX40L CAR-Tregs with OX40L-expressing K562 target cells, we demonstrate upregulation of Treg suppressive proteins CTLA-4, LAP, CD71, GARP and LAG3 without inducing expression of pro-inflammatory cytokines (no upregulation of IFNγ, TNFα, IL17a) (Figure 1A). Activation of Tregs via CAR recognition of OX40L+ cells resulted in 2.5-fold enhancement in their potency to suppress anti-CD3/CD28-driven T cell proliferation in vitro compared to Neon-Tregs at a 1:4 Treg-to-Teff ratio (p<0.05). Furthermore, OX40L-CAR-Tregs demonstrated a greater capacity to inhibit monocyte-derived dendritic cell activation, highlighting the superior suppressive activity of these OX40L-CAR-Tregs compared to non-targeting Neon-Tregs. Importantly, in a human xenograft model of GvHD, induced by transplantation of human PBMC into sublethally irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, OX40L CAR-Treg demonstrated superior GvHD disease control compared to both PBMC alone and Neon-Tregs. Clinical scores were assessed by a combinatorial system including five parameters: weight loss, posture, mobility, skin, and fur conditions, and curves were compared using a 2-way ANOVA mixed-effects analysis, which demonstrated improvement in clinical xeno-GvHD with the OX40L CAR-Treg > Neon-Treg > PBMC alone over the entire length of analysis (p <0.001). An example of the substantial improvement in the clinical score on Day +14 is as follows: combined GvHD clinical score = 6.5 for PBMC vs 2.5 for Neon-Treg vs 0.15 for OX40L CAR-Treg, p <0.0001 for all comparisons. This control of clinical GvHD resulted in prolonged recipient survival when compared to both PBMC alone and to control Neon-Tregs (MST = 34 days for OX40L-CAR-Treg, 20 days for Neon-Treg, 14 days for PBMC alone, p<0.001, Figure 1B). Conclusions: Collectively, these results demonstrate a novel and efficacious approach using OX40L-CAR Tregs to enhance Treg suppressive function and to control GvHD. Because OX40L is upregulated on APCs in inflammatory environments, and its expression is limited to APCs and activated endothelial cells, this approach provides a unique strategy for the control of allo-immunity after HCT. Moreover, these OX40L-CAR Treg may be broadly applicable beyond HCT, to control allo-immunity after solid organ transplant and to suppress T cell activation in autoimmune diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lacticaseibacillus rhamnosus R0011 secretome attenuates Salmonella enterica serovar Typhimurium secretome-induced intestinal epithelial cell monolayer damage and pro-inflammatory mediator production in intestinal epithelial cell and antigen-presenting cell co-cultures.

Certain lactic acid bacteria (LAB) are associated with immune modulatory activities including down-regulation of pro-inflammatory gene transcription and expression. While host antigen-presenting cells (APCs) and intestinal epithelial cells (IEC) can interact directly with both pathogenic and commensal bacteria through innate immune pattern recognition receptors, recent evidence indicates indirect communication through secreted molecules is an important inter-domain communication mechanism. This communication route may be especially important in the context of IEC and APC interactions which shape host immune responses within the gut environment. We have previously shown that the Lacticaseibacillus rhamnosus R0011 secretome (LrS) dampens pro-inflammatory gene transcription and mediator production from Tumor Necrosis Factor-α and Salmonella enterica serovar Typhimurium secretome (STS)-challenged HT-29 IECs through the induction of negative regulators of innate immunity. However, many questions remain about interactions mediated through these bacterial-derived soluble components and the resulting host immune outcomes in the context of IEC and APC interactions. In the present study, we examined the ability of the LrS to down-regulate pro-inflammatory gene transcription and cytokine production from STS-challenged T84 human IEC and THP-1 human monocyte co-cultures. Cytokine and chemokine profiling revealed that apically delivered LrS induces apical secretion of macrophage inhibitory factor (MIF) and down-regulates STS-induced pro-inflammatory mediator secretion into the apical and basolateral chambers of the T84/THP-1 co-culture. Transcriptional profiling confirmed these results, as the LrS attenuated STS challenge-induced CXCL8 and NFκB1 expression in T84 IECs and THP-1 APCs. Interestingly, the LrS also reversed STS-induced damage to monolayer transepithelial resistance (TER) and permeability, results which were confirmed by ZO-1 gene expression and immunofluorescence visualization of ZO-1 expression in T84 IEC monolayers. The addition of a MIF-neutralizing antibody abrogated the ability of the LrS to reverse STS-induced damage to T84 IEC monolayer integrity, suggesting a novel role for MIF in maintaining IEC barrier function and integrity in response to soluble components derived from LAB. The results presented here provide mechanistic evidence for indirect communication mechanisms used by LAB to modulate immune responses to pathogen challenge, using in vitro approaches which allow for IEC and APC cell communication in a context which more closely mimics that which occurs in vivo.

Profiles of interferon-gamma and interleukin-2 in patients after allogeneic hematopoietic stem cell transplantation.

BACKGROUNDAllogeneic hematopoietic stem cell transplantation (allo-HSCT) may be related to the occurrence of complications, including graft-versus-host disease (GvHD) and infections. The pathogenesis of acute GvHD is connected with T lymphocytes, which identify alloantigens on host's antigen-presenting cells, activate production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), and act on the immune effector cells and damage tissues and organs.AIMThe aim of the study was to investigate and distinguish serum concentration profiles of IFN-gamma and IL-2 within a 30-d period after allo-HSCT.METHODSWe enrolled 62 patients, i.e., 30 (48%) male and 32 (52%) female subjects [median age 49.5 (19-68) years], after allo-HSCT from siblings (n = 12) or unrelated donors (n = 50) due to acute myeloid leukemia with myeloablative conditioning (n = 26; 42%) and with non-myeloablative conditioning (n = 36; 58%). All patients were given standard immunosuppressive therapy with cyclosporin-A and methotrexate and pre-transplant antithymocyte globulin in the unrelated setting. Blood samples were collected pre-transplant before and after (on day -1) the conditioning therapy and on days +2,+4, +6, +10, +20, and +30 after allo-HSCT. Serum levels of IL-2 and IFN-gamma were determined using ELISA. RESULTSPatients were divided into four groups depending on the presence of acute GvHD and clinical manifestations of infection. Group I included patients with neither acute GvHD nor infections [n = 15 (24%)], group II consisted of patients with infections without acute GvHD [n = 17 (27%)], group III was comprised of patients with acute GvHD without infections [n = 9 (15%)], and group IV included patients with both acute GvHD and infections [n = 21 (34%)]. IFN-gamma concentrations were higher in Group II than in other groups on days +20 (P = 0.014) and +30 (P = 0.008). Post-hoc tests showed lower concentrations of IFN-gamma on day +30 in groups I (P = 0.039) and IV (P = 0.017) compared to group II. The levels of IL-2 were mostly undetectable.CONCLUSIONSerum levels of IFN-gamma following allo-HSCT progressively escalate. High serum levels of IFN-gamma are related to infectious complications rather than acute GvHD. Serum concentrations of IL-2 in most patients are undetectable.

P1312: FORODESINE AMPLIFIES HOST INNATE IMMUNE RESPONSE THROUGH TOLL-LIKE RECEPTOR 7 ACTIVATION WHILE PREVENTING EXPERIMENTAL GRAFT-VERSUS-HOST DISEASE

Background: Forodesine, a novel inhibitor of purine nucleoside phosphorylase (PNP), causes nucleotide pool imbalances by accumulating intracellular deoxyguanosine, thereby resulting in selective apoptosis of T-cells. Recent evidence indicates that specific types of nuclear acids including guanosine and its derivatives act as natural ligands for toll-like receptor 7 (TLR7). So far, it is unclear whether and how forodesine can modulate host innate immunity, including antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs), which express TLR7 abundantly. Aims: The aim of this study is to investigate the role of forodesine in host APC function as well as donor T-cell activation both in vivo graft-versus-host disease (GVHD) models and in vitro cell assays. Methods: NOG mice were subjected to 250 cGy total body irradiation followed by intravenous injection of human isolated T-cells. Mice were treated with forodesine (20 mg/kg) or vehicle daily after transplantation. Results: Despite the mice without treatment developing severe GVHD after transplantation, guanosine and deoxyguanosine were hardly detected in the plasma, suggesting that purine nucleosides released from damaged tissues were rapidly degraded by PNP. In fact, inhibition of PNP activity by forodesine markedly increased plasma guanosine levels, whereas deoxyguanosine was still undetectable. This was also the case in GVHD target organs, including liver and lungs. At the same level of guanosine in the liver, forodesine promoted TNF-a production from in vitro alveolar macrophage cultures. Preincubation with a TLR7 antagonist, ODN20958, attenuated the TNF-a production from macrophages stimulated by guanosine and forodesine. Nuclease treatment did not reduce macrophage TNF-a production, suggesting that single-strand RNA, a well-known TLR7 ligand, is not involved in this response. Similar results were observed in Flt3-ligand-induced plasmacytoid DCs. Not only TNF-a but also IL-12 production was induced by the combination of guanosine and forodesine, and was comparable to that in pDCs pulsed with R848, a potent agonist of TLR7/8. These observations suggest that a sustained high level of guanosine caused by forodesine treatment lead to continuous activation of TLR7 signaling, resulting in cytokine production from APCs. Meanwhile, as reported previously, forodesine inhibited the proliferation of human T-cells activated with CD3 and CD28 antibodies in the presence of deoxyguanosine. Of note, however, this combination did not impair but rather stimulated IFN-g production from activated T-cells cocultured with pDCs. This suggests that T-cell priming capacity mediated by the interaction with pDCs is conserved through TLR7 activation. Finally, we investigated the in vivo effects of forodesine on survival, body weight change, and disease severity of GVHD mice. Forodesine treatment significantly prolonged survival of the mice compared with control mice, with a median survival of 17.5 days vs 14 days, respectively (p = 0.029). The mean body weight loss percentage at day 9 was 11.5% vs 19.0% in the forodesine group and control, respectively (p = 0.003). Furthermore, the clinical GVHD scores were also lower in the forodesine group vs control (4.9 vs 0.6 points, p = 0.001). Image:Summary/Conclusion: This study demonstrates that forodesine amplifies host innate immune response through TLR7 signaling, while ameliorating T-cell GVH reaction. Further studies are needed to investigate whether forodesine can provide host protection against infections in a transplant setting.

Is IDO1 an adequate target for treatment in glioblastoma?

e14039 Background: Glioblastoma (GB) is the most prevalent primary brain tumor in adults. The first-line treatment is based on standard brain surgery and adjuvant radio-chemotherapy. Indoleamine 2,3-dioxygenase 1 (IDO1) is a catabolic enzyme that plays a role in the metabolism of tryptophan (Trp), thereby promoting a state of immunosuppression. New treatments inhibiting this pathway are being tested in GB. This study aims to assess the expression of IDO in GB patients to determine whether this enzyme may be an actionable target for the treatment of GB. Methods: This is a retrospective transversal study whose database comes from the GLIOCAT project. Expression of IDO protein and mRNA was evaluated in tumor specimens of newly diagnosed GB patients by immunohistochemistry (IHC) (percentage of positive tumor cells) with an antibody anti-IDO1 (Sigma-Aldrich Cat#HPA027772) and by RNA sequencing (RNA-seq). RNA-seq (IIlumina HiSeq2000) in paired-end mode with a read length of 2x76bp using TruSeq SBS Kit v4). Reads were mapped to the human reference genome version hg38 with STAR. Genes were quantified with RSEM using gencode annotation version 24. Normalization of gene expression was performed with the trimmed-mean of M values (TMM) method. IDO gene expression values of -6 logCPM were considered as not expressed. Results: From the 255 samples assessed by IHC, 199 patients (78%) did not express IDO1 protein (mean 0.8±2.73, range 0-21%). From the 139 samples assessed by RNA-seq, TMM values, were negative for 49.6%, while low expression was detected for the other samples (mean -3.36 +/- 3.15 logCPM, range -6.12-4.32). Conclusions: Although IDO1 is barely expressed in normal brain tissue, certain studies show that its expression is upregulated in GB, either by tumor cells or indirectly induced in host antigen presenting cells, leading to immune evasion. Therefore, the metabolism of Trp is an attractive pathway to be targeted for the treatment of GB; however, our results show that IDO may not be the main target to be inhibited due to its undetectable or very low levels of expression.

PD-1H/VISTA expressed on host myeloid cells regulates acute graft-versus-host disease (aGVHD)

Abstract Programmed Death-1 Homolog (PD-1H, also known as VISTA) is a co-inhibitory molecule of the immunoglobulin superfamily and broadly found in hematopoietic cells. We previously demonstrated that PD-1H agonists prevent aGVHD in a murine MHC mismatched bone marrow (BM) transplantation model. aGVHD occurs when donor T cells are primed and activated by host antigen-presenting cells (APCs). PD-1H agonism suppressed donor allogeneic T cell activation leading to the suppression of aGVHD. However, it remains unknown whether PD-1H agonists also suppress host myeloid APCs, in which PD-1H is highly expressed. To test this, we adoptively transferred wildtype (WT) C57B/L6 (B6) BM into either PD-1H knock-out (KO) or WT BALB/c mice as hosts. PD-1H KO host mice had more severe aGVHD clinical scores and a worse survival rate when compared to WT host mice. To determine whether PD-1H agonism impacts host immune cells, we transferred B6 PD-1H KO BM into BALB/c WT host mice and treated host recipients with or without PD-1H agonists. Consistent with prior data, host mice treated with PD-1H agonists suppressed aGVHD and extended survival compared with isotype control treated mice. To test which cell subsets expressing PD-1H in host mice regulate aGVHD, we transferred WT BALB/c BM into myeloid lineage specific KO of PD-1H or WT littermate hosts. Interestingly, the myeloid lineage specific KO mice host had significantly worse survival rates and worse aGVHD symptoms compared with WT littermates. Our studies indicate that PD-1H expression on host myeloid cells regulates aGVHD in addition to donor T cell PD-1H. Finding new mechanisms of PD-1H agonism on aGVHD enables development of future therapeutic approaches for aGVHD treatment, such as myeloid APC suppression. Supported by an Edward P. Evans Foundation Young Investigator Award, a Conquer Cancer Foundation American Society of Clinical Oncology Career Development Grant, and an American Cancer Society Clinician Scientist Development Grant

Transfer of Cellular Content from the Allogeneic Cell-Based Cancer Vaccine DCP-001 to Host Dendritic Cells Hinges on Phosphatidylserine and Is Enhanced by CD47 Blockade.

DCP-001 is a cell-based cancer vaccine generated by differentiation and maturation of cells from the human DCOne myeloid leukemic cell line. This results in a vaccine comprising a broad array of endogenous tumor antigens combined with a mature dendritic cell (mDC) costimulatory profile, functioning as a local inflammatory adjuvant when injected into an allogeneic recipient. Intradermal DCP-001 vaccination has been shown to be safe and feasible as a post-remission therapy in acute myeloid leukemia. In the current study, the mode of action of DCP-001 was further characterized by static and dynamic analysis of the interaction between labelled DCP-001 and host antigen-presenting cells (APCs). Direct cell–cell interactions and uptake of DCP-001 cellular content by APCs were shown to depend on DCP-001 cell surface expression of calreticulin and phosphatidylserine, while blockade of CD47 enhanced the process. Injection of DCP-001 in an ex vivo human skin model led to its uptake by activated skin-emigrating DCs. These data suggest that, following intradermal DCP-001 vaccination, local and recruited host APCs capture tumor-associated antigens from the vaccine, become activated and migrate to the draining lymph nodes to subsequently (re)activate tumor-reactive T-cells. The improved uptake of DCP-001 by blocking CD47 rationalizes the possible combination of DCP-001 vaccination with CD47 blocking therapies.

219 CD47 and phosphatidylserine contribute to the interaction between antigen presenting cells and the allogeneic cell-based relapse vaccine DCP-001

BackgroundDCP-001 is a cancer relapse vaccine derived from the DCOne® human leukemic cell line. During manufacturing, DCOne® cells are shifted towards a mature dendritic cell (mDC) phenotype, combining an endogenous tumor antigen repertoire (e.g. WT-1, RHAMM and PRAME) with a mDC costimulatory profile and providing the basis for the highly immunogenic vaccine DCP-001. In a phase I clinical study in acute myeloid leukemia (AML), DCP-001 demonstrated to be safe and to induce multifunctional antitumor immune responses.1 It has also been reported that DCP-001 induces antitumor immunity against multiple myeloma cells in peripheral blood mononuclear cells (PBMC) from multiple myeloma patients and that DCP-001 antigenic material is transferred to host antigen presenting cells (APC), possibly via extracellular vesicles.2 However, the possibility of direct interactions between DCP-001 and host APC has not yet been investigated.MethodsTo further elucidate the mode of action of DCP-001, we studied the interactions of DCP-001 with human PBMC and isolated immature monocyte-derived DCs (iMoDC) in in vitro co-culture studies. A human skin explant model was used to determine uptake of DCP-001 by migrating skin DCs after intradermal injection.ResultsWe found that DCP-001 stimulates the secretion of various proinflammatory cytokines (IL-1β, GM-CSF, IFN-γ, IL-2, TNF-α, and IL-6) and chemokines (IL-8 and RANTES) in PBMC. In addition, we demonstrate that DCP-001 is efficiently taken up by iMoDC via direct cell-cell interactions and that this phagocytic process is influenced by ”eat-me” and ”don’t eat me” signaling pathways. Blocking of the ”eat-me” signals calreticulin and phosphatidylserine inhibited the uptake of DCP-001, whereas blockade of the ”don’t eat me” signal CD47 enhanced DCP-001 uptake. After intradermal injection of DCP-001 in an ex-vivo human skin model, its uptake by skin-emigrating DCs was demonstrated as well as simultaneous activation of these DCs.ConclusionsOur data suggest a key role for host antigen presenting cells in the triggering of immune responses upon DCP-001 vaccination. In addition, the data provide rationale for potential combination therapies based on DCP-001 and inhibitors of the CD47 pathway.Referencesvan de Loosdrecht AA, et al. A novel allogeneic off-the-shelf dendritic cell vaccine for post-remission treatment of elderly patients with acute myeloid leukemia. Cancer Immunol Immunother 2018;67(10):1505–1518.Leaf RK, et al. DCOne as an allogeneic cell-based vaccine for multiple myeloma. J Immunother 2017;40(9):315–322.

Absence of Vasoactive Intestinal Peptide Synthesis in Recipients in Allogeneic Bone Marrow Transplant Increases Graft Versus Host Diseases

Introduction: Vasoactive intestinal polypeptide (VIP) is an immunosuppressive neuropeptide. Activation of donor T cells leading to graft-versus-host disease (GvHD) in allogeneic bone marrow transplant (allo-BMT) occurs via early post transplant interactions between donor T cells and host antigen presenting cells and leads to upregulation of immune co-inhibitory pathways, including expression of VIP in T cells and dendritic cells. We have previously shown VIP signaling favorably regulates the activation status and enhances the graft-versus-leukemia (GvL) activities of donor T cells (Li et al., Cancer Research 2016). The role of the VIP produced by host antigen presenting cells in regulating GvHD in allo-BMT is unknown. We tested the hypothesis that absence of VIP expression by host cells would augment donor T cell activation and increase GvHD.Methods and Results: We used the B10BR->B6 MHC fully mismatched murine model of allogeneic BMT. Recipient wildtype (WT) and VIP knockout B6 (VIP-KO) mice were lethally irradiated with 11Gy and injected i.v. with 5×106 T cell depleted (TCD) bone marrow cells (BM) plus 0, 1 or 3 ×106 splenocytes from WT B10BR donors. Fifteen mice (males and females) were assigned to each splenocytes dosage group. The survival of recipients after BMT was recorded daily and the average clinical GvHD scores were obtained by combining weight-loss, activity, posture, fur texture and skin integrity data following standard published procedures. All three groups of WT recipients had better survival than VIP-KO recipients. VIP-KO mice transplanted with TCD BM alone had only 33.3% survival at day 75 compared with 73.3% survival among the WT recipients. Chimerism studies on day 10 post-BMT showed equivalent level of donor chimerism: 80% donor in WT recipients vs 72% donor in VIP-KO recipients, indicating increased mortality in the VIP-KO recipients was not due to early graft rejection. The addition of donor splenocytes to the graft resulted in significantly more GvHD-related mortality in VIP-KO recipients compared with WT recipients (Figure 1), with 80% vs 100% day 75 survival among recipients of 1x106 splenocytes (p=NS) and 0% vs 40% survival (p<0.01) among recipients of 3x106 donor splenocytes, respectively.To determine how the absence of VIP production in KO recipients modulates immune effector mechanisms that contributes to the differential survival rate post allo-BMT, we analyzed the recipients' splenocytes on day 30 post transplant with flow cytometry and staining with CD3, CD4, CD8, CD62L, CD44 and PD1 antibodies. VIP-KO recipients transplanted with 5M TCD BM plus 1M splenocytes had more CD4+ central memory and effector T cells, and CD8+ central memory and effector T cells. VIP-KO recipients transplanted with 5×106 TCD BM alone in addition had significantly more CD4 and CD8 effector T cells (p<0.01). VIP-KO recipients transplanted with 5×106 TCD BM plus 1x106 splenocytes had more PD1 expression on donor CD4+ central memory and effector T cells, and CD8+ central memory effector T cells, consistent with enhanced antigen-mediated activation.To visualize VIP produced by the host cells, we transplanted C57/BL6 recipients expressing a GFP transgene under the control of the VIP promoter (n=10) with 5×106 TCD BM plus 3 ×106 splenocytes from WT B10BR donors, and visualized VIP gene promoter activity as GFP expression in recipient lymphoid tissues and GvHD target organs. Only small number of CD45+ VIP-GFP+ cells were detected from in host liver, spleen and lymph nodes on day 10 post transplant (Figure 2), suggesting residual host leukocytes, particularly dendritic cells, expressed VIP.Conclusion: VIP production by recipient antigen presenting cells is a key pathway in limiting the activation of donor T cells. Physiological expression of VIP by host leukocytes regulate the GvHD activity of donor T cells. Novel methods to enhance VIP production in transplant recipients or the parenteral administration of VIP receptor agonists may limit GvHD, and may be particularly important in allo-BMT where GvL is not necessarily critical for success, such as transplants for hemoglobinopathy. [Display omitted] DisclosuresWaller:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; PRA: Consultancy; AMGEN: Consultancy; National Institutes of Health: Research Funding; Coulter Foundation: Research Funding; Helocyte: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Katz Foundation: Research Funding; Chimerix: Equity Ownership; Cerus: Equity Ownership; Celldex: Consultancy.

Tpl2 Signaling Regulates Dendritic Cell Activation and Responding T Cell Differentiation Following Mycobacterium tuberculosis Infection

Abstract Mycobacterium tuberculosis (Mtb) utilizes a number of immune evasion mechanisms in order to persist inside of host antigen-presenting cells. Dendritic cells (DCs) are important in restricting Mtb growth by migrating to draining lymph nodes and activating antigen-specific T cell responses, but the roles of DCs in Mtb infection require further study. This study investigated DC activation and functional outcomes following Mtb H37Ra-driven tumor progression locus 2 (Tpl2) signaling. The role of Tpl2 was interrogated genetically, utilizing bone marrow-derived DCs from Tpl2−/− mice. We assessed cytokine production via ELISA, mRNA levels via qRT-PCR, and expression of cell surface molecules via flow cytometry. In Mtb-treated DCs, genetic depletion of Tpl2 increased production of pro-inflammatory cytokines such as IL-12p40 and IL-6. Loss of Tpl2 in Mtb-treated DCs also led to decreased E-cadherin expression, and increased expression of Icam-1 (Cd54) and Mmp2, which are molecules involved in DC transmigration. Expression of Ccr7 and Ccr4 was also enhanced in Mtb-treated Tpl2−/− DCs, which correlated with improved migration of Tpl2−/− DCs towards CCL19 and CCL21 in vitro (assessed using a trans-well assay). When antigen-specific CD4+ T cells were co-cultured with Mtb-infected DCs, deletion of Tpl2−/− in the DCs resulted in increased T cell production of IFNγ and IL-2, as well as increased Tbet expression, indicative of enhanced Th1 polarization. Together, these data indicate that Mtb-induced Tpl2 signaling suppresses certain aspects of DC activation, leading to blunted Th1 responses against the pathogen.

Recent developments in prophylaxis and treatment of graft-versus-host disease

Acute graft-versus-host disease (GVHD) is a systemic immune reaction in which mature T cells in the donor inoculum are abnormally activated by the host and donor antigen-presenting cells. These activated cells then attack normal host tissue after allogeneic hematopoietic stem cell transplantation (HSCT). Clinical GVHD develops when the damage exceeds the threshold for tissue tolerance. Furthermore, functional damage of hematopoietic and non-hematopoietic organs due to this acute immune response triggers the subsequent development of chronic GVHD. The endpoint of HSCT is shifting from just surviving the acute phase to having an enriched life until later years; therefore, seamless and detailed immune management is required from the acute to chronic phase after HSCT. Acute GVHD prophylaxis consisted of calcineurin inhibitors and short-term methotrexate for a long time. However, various novel immune-modulating strategies have been developed against the background of recent diversification in donor sources and changes in treatment goals. In this review, we discuss recent clinical developments in basic and clinical research regarding acute or chronic GVHD.

Donor-derived regulatory dendritic cell infusion results in host cell cross-dressing and T cell subset changes in prospective living donor liver transplant recipients.

Regulatory dendritic cells (DCreg) promote transplant tolerance following their adoptive transfer in experimental animals. We investigated the feasibility, safety, fate, and impact on host T cells of donor monocyte-derived DCreg infused into prospective, living donor liver transplant patients, 7days before transplantation. The DCreg expressed a tolerogenic gene transcriptional profile, high cell surface programed death ligand-1 (PD-L1):CD86 ratios, high IL-10/no IL-12 productivity and poor ability to stimulate allogeneic T cell proliferation. Target DCreg doses (range 2.5-10×106 cells/kg) were achieved in all but 1 of 15 recipients, with no infusion reactions. Following DCreg infusion, transiently elevated levels of donor HLA and immunoregulatory PD-L1, CD39, and CD73 were detected in circulating small extracellular vesicles. At the same time, flow and advanced image stream analysis revealed intact DCreg and "cross-dressing" of host DCs in blood and lymph nodes. PD-L1 co-localization with donor HLA was observed at higher levels than with recipient HLA. Between DCreg infusion and transplantation, T-bethi Eomeshi memory CD8+ T cells decreased, whereas regulatory (CD25hi CD127- Foxp3+ ): T-bethi Eomeshi CD8+ T cell ratios increased. Thus, donor-derived DCreg infusion may induce systemic changes in host antigen-presenting cells and T cells potentially conducive to modulated anti-donor immune reactivity at the time of transplant.

AllerScreener – A Server for Allergenicity and Cross-Reactivity Prediction

Abstract Allergenicity of proteins is a subtle property encoded in their structures. The prediction of allergenicity of novel proteins saves time and resources for subsequent experimental work. In the host antigen-presenting cells, the allergens are processed as antigens by the means of Human Leukocyte Antigens (HLA) class II proteins. Sometimes, people allergic to a given protein show allergic reaction to a different protein, even when the two proteins have different routes of exposure. This phenomenon is termed cross-reactivity. Here, we describe a server for allergenicity and cross-reactivity prediction based on the abilities of allergenic proteins to generate binders to HLA class II proteins. The generated peptides are compared to HLA binders originating from known allergens. As a result, the server returns a list of common binders, origin proteins, and species. Different species generate common HLA binders and this determines their cross-reactivity. The server is named AllerScreener and is freely accessible at: http://www.ddg-pharmfac.net/AllerScreener .

MHC Class II on Ccl19+ Reticular Cells Mitigates Graft-Versus-Host Disease Via Maintenance of Regulatory T Cells

Allogeneic T cell priming is considered as an essential event determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HCT), ideally triggering anti-leukemic responses (GvL effect) or, at worst, causing life-threatening acute graft-versus-host disease (aGvHD). During aGvHD initiation, alloreactive T cells are activated by host antigen presenting cells (APCs), rapidly expand and subsequently exert tissue damage. Recently, it was discovered that in absence of host hematopoietic APCs, aGvHD cannot be prevented, suggesting a crucial role of non-hematopoietic APCs for priming alloreactive T cells (Toubai et al., Blood 2012, Li et al., J Immunol. 2012, Koyama et al., Nat Med 2012). However, the exact location and identity of host non-hematopoietic APCs triggering alloreactive T cell responses remains controversial and needs to be proven in vivo. Fibroblastic reticular cells (FRCs) have shown to provide the crucial delta-like notch ligand to alloreactive T cells (Chung et al., JCI 2017) in aGvHD, therefore we investigated the role of FRCs MHC class II in aGvHD and their potential role as non-hematopoietic APCs in MHC class II dependent manner. In vitro cultured FRCs cell line as well as FRCs from lethally irradiated mice upregulate MHCII and co-stimulatory molecules. Moreover, FACS sorted FRCs (CD45-CD24-CD31-gp38+) were able to process DQ-OVA via MHC class II machinery, indicating that FRCs have the potential to activate CD4+ T cells. Employing allo-HCT mouse models in combination with flow cytometry and advanced microscopy techniques, we explored early alloreactive T cells activation initially in a myeloablatively conditioned MHC major mismatch allo-HCT setting (FVB/NàC57Bl/6). We generated MHCIIΔCcl19 mice with a Ccl19-intrinsic deletion of MHC class II on all Ccl19 expressing reticular lineage cells by crossing mice with floxed H2-Ab1 gene (H2-Ab1fl) with a mouse expressing Cre recombinase under the control of the Ccl19 promoter (Ccl19Cre). On day+3 after allo-HCT, CD4+ T cells activation (CD44 and CD25 expression) and proliferation (Ki67 expression and CFSE dilution) did not differ in the MHCIIΔCcl19 mice from H2-Ab1fl wildtype littermates. To further elucidate FRCs MHC class II in aGvHD milieu, we utilized iFABP-tOVA transgenic model in which OVA is expressed by intestinal epithelial cells as well as ectopically by FRCs of the lymphoid organs. OT-II cells transferred from RagΔ background mice failed to proliferate in the mLNs of lethally irradiated iFABP-tOVA, whereas excessive proliferation was observed in CD11c.DOG mice (where OVA is presented by CD11c-expressing cells). Taken together these results indicate that MHCII on FRCs does not play a role in direct antigen presentation and CD4+ T cell activation. Next, we asked whether MHCII on FRCs influences alloreactivity of CD4+ T cells in the symptomatic phase of aGvHD. Indeed, in MHCIIΔCcl19 mice, CD4+ T cells expressed higher levels of effector molecules: CD44 and CD127 as well as the proliferation marker Ki67 on day +30 of allo-HCT. Furthermore, the proportion of donor CD90.1+CD4+FoxP3+ regulatory T cells (Tregs) were reduced in MHCIIΔCcl19 mice as compared to H2-Ab1fl wild-type littermates. This led to overall poor survival of MHCIIΔCcl19 mice by day+60 after allo-HCT. At this time point in MHCIIΔCcl19 mice CD4+ T cells displayed higher levels of CD44, CD127 and Ki67 and down-regulated PD-1 and Lag3. To further elucidate the effect of FRCs MHC class II on CD4+FoxP3+ donor Tregs, we transplanted CD90.1+CD4+CD25hi Tregs, TCD BM from FVB mice along with naïve luc+ CD90.1+CD4+ T cells from FVB.L2G85 mice. Tregs protected against aGvHD in H2-Ab1fl littermate controls whereas Tregs could not protect MHCIIΔCcl19 recipients rendering them susceptible to aGvHD and to poor overall survival. Conclusively, these results indicate for the first time that MHC class II on FRCs assists to maintain donor Tregs in the SLOs after allo-HCT. Conclusively, we propose a model in which FRCs promote T cell alloreactivity by providing notch ligands (Chung et al., JCI 2017) in the initiation phase and mitigate aGvHD by maintenance of Tregs via MHC class II in the aGvHD-effector phase. Disclosures Einsele: Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Research Funding, Speakers Bureau.

Alternative mechanisms that mediate graft-versus-host disease in allogeneic hematopoietic cell transplants.

Allogeneic hematopoietic cell transplantation (alloHCT) benefits increasing numbers of patients with otherwise lethal diseases. Graft-versus-host disease (GVHD), however, remains one of the most potentially life-threatening complications due to its own comorbidities and the side effects of its treatment. In this issue of the JCI, two groups have turned dogma on its head by providing evidence for alternative mechanisms of acute GVHD (aGVHD) in humans. The principle of donor T cell reactivity elicited by host antigen-presenting cells (APCs) expressing MHC-encoded major HLA disparities or expressing minor histocompatibility antigen (miHA) differences presented by identical HLA molecules remains intact. These reports, however, demonstrate that GVHD can additionally result from peripheral host T cells resident in skin and gut being stimulated against donor APCs in the form of monocyte-derived macrophages. Moreover, these donor monocyte-derived macrophages can themselves mediate cytopathic effects against resident host T cells in skin explants and against a keratinocyte-derived cell line.