Host Antigen-presenting Cells Research Articles

Antigen-Specific Priming is Dispensable in Depletion of Apoptosis-Sensitive T Cells for GvHD Prophylaxis.

Prophylactic approaches to graft versus host disease (GvHD) have employed both phenotypic reduction of T cells and selective elimination of host-primed donor T cells in vitro and in vivo. An additional approach to GvHD prophylaxis by functional depletion of apoptosis-sensitive donor T cells without host-specific sensitization ex vivo showed remarkable reduction in GHD incidence and severity. We address the role and significance of antigen-specific sensitization of donor T cells and discuss the mechanisms of functional T cell purging by apoptosis for GvHD prevention. Host-specific sensitization is dispensable because migration is antigen-independent and donor T cell sensitization is mediated by multiple and redundant mechanisms of presentation of major and minor histocompatibility complex and tissue antigens by donor and host antigen-presenting cells. Our data suggest that potential murine and human GvH effectors reside within subsets of preactivated T cells susceptible to negative regulation by apoptosis prior to encounter of and sensitization to specific antigens.

Indirect presentation of antigen-coupled cells mediates specific CD4+ T cell tolerance by PD-L1 for the prevention of type 1 diabetes (BA14P.207)

Abstract Antigen coupled to the surface of cells using ethylene carbodiimide is a potent inducer of tolerance in multiple models of autoimmunity including Type 1 Diabetes (T1D). Yet, the mechanisms contributing to the induction of T cell tolerance in vivo during T1D are unclear. We used the BDC2.5 T cell receptor transgenic T cell transfer model to study how p31-coupled cells inhibit T cell function in vivo. Antigen-coupled cells that were either deficient in MHC class II or MHC-mismatched induced tolerance similarly to MHC class II-sufficient matched cells, suggesting that indirect presentation of coupled cell antigens is required for tolerance induction in vivo. We also show that BDC2.5 T cells predominantly encounter coupled cell antigen that has been ingested by host antigen presenting cells, and that the inhibitory receptor PD-L1 is required on the host but not the coupled cells to induce tolerance, further highlighting an indirect mechanism of tolerance induction. These data add to our current understanding of how antigen-coupled cells induce tolerance and aid in the translation of this therapeutic into the clinic.

How Do CD4(+) T Cells Detect and Eliminate Tumor Cells That Either Lack or Express MHC Class II Molecules?

CD4+ T cells contribute to tumor eradication, even in the absence of CD8+ T cells. Cytotoxic CD4+ T cells can directly kill MHC class II positive tumor cells. More surprisingly, CD4+ T cells can indirectly eliminate tumor cells that lack MHC class II expression. Here, we review the mechanisms of direct and indirect CD4+ T cell-mediated elimination of tumor cells. An emphasis is put on T cell receptor (TCR) transgenic models, where anti-tumor responses of naïve CD4+ T cells of defined specificity can be tracked. Some generalizations can tentatively be made. For both MHCIIPOS and MHCIINEG tumors, presentation of tumor-specific antigen by host antigen-presenting cells (APCs) appears to be required for CD4+ T cell priming. This has been extensively studied in a myeloma model (MOPC315), where host APCs in tumor-draining lymph nodes are primed with secreted tumor antigen. Upon antigen recognition, naïve CD4+ T cells differentiate into Th1 cells and migrate to the tumor. At the tumor site, the mechanisms for elimination of MHCIIPOS and MHCIINEG tumor cells differ. In a TCR-transgenic B16 melanoma model, MHCIIPOS melanoma cells are directly killed by cytotoxic CD4+ T cells in a perforin/granzyme B-dependent manner. By contrast, MHCIINEG myeloma cells are killed by IFN-γ stimulated M1-like macrophages. In summary, while the priming phase of CD4+ T cells appears similar for MHCIIPOS and MHCIINEG tumors, the killing mechanisms are different. Unresolved issues and directions for future research are addressed.

Origin of Langerhans cells in normal skin and chronic GVHD after hematopoietic stem‐cell transplantation

Chronic graft-versus-host disease (cGVHD) is a common complication following allogeneic stem-cell transplantation (SCT). Past studies have implicated the persistence of host antigen-presenting cells (APCs) in GVHD. Our objective was to determine the frequency of host Langerhans cells (LCs) in normal skin post-SCT and ask if their persistence could predict cGVHD. Biopsies of normal skin from 124 sex-mismatched T-cell-replete allogenic SCT recipients were taken 100 days post-transplant. Patients with acute GVHD and those with <9 months of follow-up were excluded and prospective follow-up information was collected from remaining 22 patients. CD1a staining and X and Y chromosome in-situ hybridization were performed to label LCs and to identify their host or donor origin. At 3 months, 59 ± 5% of LCs were host derived. The density of LCs and the proportion of host-derived LCs were similar between patients that did or did not develop cGVHD. Most LCs in the skin remained of host origin 3 months after SCT regardless of cGVHD status. This finding is in line with the redundant role of LCs in acute GVHD initiation uncovered in recent experimental models.

The Histone Methyltransferase Ezh2 Is a Crucial Epigenetic Regulator Of Allogeneic T Cell Responses and Graft-Versus-Host Disease In Mice

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for many hematological malignancies, however, it remains to be limited by morbidity and mortality related to graft-versus-host disease (GVHD). During GVHD, donor T cells are activated by host antigen-presenting cells (APCs) and differentiate into effector cells that mediate host tissue damage. Development of alloreactive effector T cells requires orchestrated expression of myriad genes initiated by host APCs. However, little is known about the key epigenetic factor that controls this process. Histone methylation, which is catalyzed by histone methyltransferanses (HMT), has been correlated with genes encoding effector cytokines and transcription factors critical for effector differentiation. We recently demonstrated that inhibition of histone methylation using 3-Deazaneplanocin A (DZNep) arrested ongoing GVHD through induction of apoptosis in alloreactive T cells. However, since DZNep reduced multiple histone methylation marks, including trimethylation of histone H3 at lysine 4 (H3K4me3), H3K27me3, H3K36me3 and H4K20me3, the key HMT that regulates the transcription program important for allogeneic T cell responses remains unclear. Using genetic approaches and mouse GVHD models, we identify that the HMT Ezh2, which specifically catalyzes H3K27me3 and acts primarily as a gene silencer, plays a central role in regulating allogeneic T cell proliferation, differentiation, and function during the GVHD process. Transfer of donor T cells derived from Ezh2 conditional knockout C57BL/6 (B6) mice did not induce GVHD in lethally irradiated allogeneic BALB/C mice, with all recipients surviving free of disease. In contrast, wild-type (WT) donor B6 T cells caused uniformly lethal GVHD in these recipients. We found that loss of Ezh2 selectively impaired alloantigen-specific T cell responses. Three days after allo-HSCT, both Ezh2-deficient and WT T cells were similarly activated to proliferate in vivo. However, 7 days after transplantation, there was a significant reduction in the number of highly proliferative Ezh2-deficient T cells compared to WT T cells, leading to a dramatic reduction of infiltrating Ezh2-deficient T cells in GVHD target organs. Interestingly, conditionally deleting Ezh2 did not affect proliferation and survival of B6 T cells that were transferred into lethally irradiated syngeneic B6/SJL mice by 7 days after transplantation. Thus, Ezh2 is critical for the continual proliferation of alloantigen-responding T cells during later stages of GVHD induction. In contrast, Ezh2 is dispensable for both the initial activation and proliferation of T cells upon alloantigen-priming and homeostatic expansion of T cells in response to lymphopenia. Previous studies suggest that IFN-g-producing alloreactive effector cells cause damage to the liver and gastrointestinal tract. We found that loss of Ezh2 led to selective reduction in frequency of IFN-γ-producing effector cells 7 days after allo-HSCT. This effect was accompanied with decreased expression of transcription factors TBX21 and STAT4, both of which are crucial for effector differentiation. These data suggest that in addition to regulating the transcription program critical for producing sufficient numbers of alloreactive effector T cells needed to mediate GVHD, Ezh2 is also important for orchestrating genes required to promote IFN-γ production in effector cells. Importantly, we found that Ezh2 ablation retained anti-leukemia activity in alloreactive T cells, leading to improved overall survival of transplant recipients. Collectively, these findings identify Ezh2 as a key epigenetic regulator that controls allogeneic T cell responses and GVHD. We propose that pharmacological modulation of Ezh2 using specific inhibitors should be investigated as a novel therapeutic strategy after allo-HSCT. Disclosures:No relevant conflicts of interest to declare.

Graft-Versus-Host-Disease Recruits Both Monocyte and Pre-Conventional Dendritic Cell-Derived Tissue Antigen Presenting Cells Independent Of CCR2 and CCR6

Graft-versus-host disease (GVHD) limits the broader application of allogeneic hematopoietic stem cell transplantation. In prior studies we defined roles for both host and donor-derived antigen presenting cells (APCs) in the activation of alloreactive donor T cells and in promotion of GVHD. While initial T cell activation in GVHD occurs predominantly in secondary lymphoid organs, we have consistently observed MHCII+ donor-derived APCs, including dendritic cells (DCs), in histopathologic GVHD lesions, frequently adjacent to infiltrating T cells, suggesting they have a role in local GVHD reactions. Donor-derived tissue APCs (t-APCs), including tissue-DCs (t-DCs) could activate donor T cells through indirect or cross-presentation of host antigens, produce chemokines that recruit other effectors, and elaborate inflammatory mediators or suppressors of inflammation. We first characterized t-DC subsets in the skin and bowel of GVHD-affected mice. 129 (H-2b) hosts were irradiated and reconstituted with B6 (H-2b) BM with or without CD4+ and CD8+ T cells to induce GVHD and analyzed mononuclear cells from skin and bowel approximately 4 weeks post transplant. In skin, both main dermal DC populations (CD11b+ and CD103+) were significantly increased in GVHD mice as compared to BM alone controls, though the ratios of CD11b+: CD103+ DCs were similar. In the bowel lamina propria, the ratios of CD11b+CD103- to CD11b+CD103+ were increased in GVHD mice in the colon but were similar to that in BM alone controls in the small bowel. We next studied the roles of CCR6 and CCR2 in the recruitment of donor-derived APCs to skin and bowel. We transplanted mice with CCR6-/- BM in competition with wild type (wt) BM and found that the contribution of each to skin and bowel APCs matched their contributions to myeloid hematopoiesis in BM, spleen and blood, indicating that CCR6 is not required. To study the role of CCR2 we first compared mice transplanted with either wt or CCR2-/- BM with wt T cells. Despite having a profound reduction in blood monocytes, all skin and bowel t-APC subsets were present in CCR2-/- recipients, indicating that CCR2 is not required for t-APC recruitment in contrast to its role in many other models of inflammation. However, CD103+ DCs were more prevalent relative to CD11b+ DCs, consistent with a pre-cDC origin. Despite monocytopenia, recipients of CCR2-/- BM developed clinical GVHD; histology data is being analyzed and will be presented. To better define the contributions of CCR2 to t-APC recruitment and to determine monocyte versus pre-cDC origin of t-DCs, we transplanted mice with CCR2-/- BM in competition with wt BM and compared ratios of BM and blood precursors (pre-cDCs and monocytes) to t-DC ratios. For CD103+ DCs, wt/KO ratios matched the ratios of general myeloid hematopoiesis and pre-cDCs, indicating a pre-cDC origin. For CD11b+CD103- DCs, the ratio of wt/KO matched that in blood monocytes. We further subsetted CD11b+ t-DCs based on the expression of Ly6C, MAR1, CD64 and CD24, used to differentiate pre-cDC from mono-derived DCs in other organs, and did not identify any population with wt/KO ratios that did not match that of the general CD11b+ DC population, suggesting that most if not all CD11b+ t-DCs are of monocyte origin. Experiments are underway examining the role of CX3CR1 in t-APC recruitment and these data will be presented. Disclosures: No relevant conflicts of interest to declare.

Two Photon Intravital Microscopy Reveals CD4+ T Cells Making Cognate Interactions With Tissue Dendritic Cells In Skin Graft-Versus-Host-Disease

Graft-versus-host disease (GVHD) limits the broader application of allogeneic hematopoietic stem cell transplantation. In prior studies we defined roles for both host and donor-derived antigen presenting cells (APCs) in the activation of alloreactive donor T cells and promotion of GVHD. While initial T cell activation in GVHD occurs predominantly in secondary lymphoid organs (SLO), we have consistently observed MHCII+ donor-derived tissue APCs (t-APCs), including tissue dendritic cells (t-DCs) in histopathologic GVHD lesions, frequently adjacent to infiltrating T cells. We hypothesize that productive interactions occur between donor APCs and T cells in situ in GVHD target tissues, which propagate disease locally. We could not use knockout or APC depletion approaches to study T cell: t-APCs interactions as they impact APCs systemically and might therefore affect T cell stimulation in SLO. We therefore utilized two-photon intravital microscopy to analyze interactions between fluorescent donor CD4+ T cells and t-DCs in skin. 129 (H-2b) hosts were irradiated and reconstituted with B6 (H-2b) CD11c-YFP transgenic (Tg) Bone Marrow (BM) with B6 RFP Tg CD4 cells and nonfluorescent B6 CD8 cells. We imaged ear skin in GVHD mice 4 weeks later. In general CD4 cells co-localized with DCs. We observed CD4+ T cells that were highly motile and only in transient contact with DCs and others that made stable contact with DCs. To determine how much TCR: MHCII interactions drive sustained CD4+ T cell: DC interactions and arrest CD4+ T cell motility, mice were imaged and then injected with an MHCII blocking antibody (Ab; Y3P) with continued imaging of the same regions. After injection, T cell mean speed significantly increased and the proportion of T cells in stable contact with DCs decreased, indicating that transient disruption of TCR: MHCII is sufficient to restore motility to some T cells. In a second approach to assess the specificity of CD4+ T cell: t-DC interactions we transplanted 129 mice with B6 RFP+ CD4 cells, nonfluorescent CD8 cells and a mix of CD11c-YFP MHCII-/- and RFP wt BM or a mix of CD11c-YFP wt and RFP Tg MHCII-/- BM. We are currently comparing the motility of CD4 cells that make contact with MHCII+ as compared to MHCII- DCs, with the prediction that contact times will be shorter with the latter. Our data suggest that CD4+ T cells make cognate interactions with t-DCs in skin and we hypothesize that these interactions promote GVHD locally. Because the graft-versus-leukemia effect occurs primarily in bone marrow and secondary lymphoid tissues, targeting of tissue-infiltrating APCs could represent a unique strategy to ameliorate GVHD while preserving GVL. Disclosures:No relevant conflicts of interest to declare.

Inhibiting retinoic acid signaling ameliorates graft-versus-host disease by modifying T-cell differentiation and intestinal migration

AbstractGraft-versus-host disease (GVHD) is a critical complication after allogeneic bone marrow transplantation. During GVHD, donor T cells are activated by host antigen-presenting cells and differentiate into T-effector cells (Teffs) that migrate to GVHD target organs. However, local environmental factors influencing Teff differentiation and migration are largely unknown. Vitamin A metabolism within the intestine produces retinoic acid, which contributes to intestinal homeostasis and tolerance induction. Here, we show that the expression and function of vitamin A–metabolizing enzymes were increased in the intestine and mesenteric lymph nodes in mice with active GVHD. Moreover, transgenic donor T cells expressing a retinoic acid receptor (RAR) response element luciferase reporter responded to increased vitamin A metabolites in GVHD-affected organs. Increasing RAR signaling accelerated GVHD lethality, whereas donor T cells expressing a dominant-negative RARα (dnRARα) showed markedly diminished lethality. The dnRARα transgenic T cells showed reduced Th1 differentiation and α4β7 and CCR9 expression associated with poor intestinal migration, low GVHD pathology, and reduced intestinal permeability, primarily via CD4+ T cells. The inhibition of RAR signaling augmented donor-induced Treg generation and expansion in vivo, while preserving graft-versus-leukemia effects. Together, these results suggested that reagents blunting donor T-cell RAR signaling may possess therapeutic anti-GVHD properties.

Advances in the treatment of acute graft‐versus‐host disease

Allogeneic hematopoietic stem cell transplantation (HSCT) has been widely used for the treatment of hematologic malignant and non-malignant hematologic diseases and other diseases. However, acute graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic transplantation. Acute GVHD may occur in 30% of transplant recipients, which is a syndrome of erythematous skin eruption, cholestatic liver disease and intestinal dysfunction, resulting from the activation of donor T lymphocytes by host antigen-presenting cells, resulting in an immune-mediated inflammatory response. Recent scientific advances in the understanding of the pathogenesis involved in the development of acute GVHD and clinical investigation have provided more effective therapeutic strategies for acute GVHD. This review focuses on major scientific and clinical advances in the treatment of acute GVHD.

Immunologie der Keratoplastik: Macht HLA-Matching bei lamellären Verfahren Sinn?

The immunological mechanisms of graft rejections after penetrating keratoplasty are largely investigated in rodent models. Here, antigens are predominantly processed by host antigen presenting cells (APCs). For this reason, graft rejections are not primarily triggered by mismatches in the major histocompatibility complex (MHC). Consequently, MHC matching (equivalent of HLA matching) does not robustly prevent immunological graft rejections in mice. This, however, may not apply to humans because anatomy and the clinical picture of immune reactions differ vastly. Immunological experiments are not feasible in humans for ethical reasons. However, the recent surgical modifications in keratoplasty inadvertently gave rise to several interesting immunological field experiments. We herein discuss the potential insight into human graft rejections from selected clinical observations. On this basis, we have evaluated HLA matching for keratoplasty techniques. Several clinical observations hint towards an active role of donor-derived APCs in graft rejections after human keratoplasty. Additionally, donor-specific anti-HLA antibodies may play a significant role. On this basis we suggest that HLA matching is potentially beneficial in human keratoplasty in contrast to the situation in mice. Graft rejections are rarely observed after Descemet membrane keratoplasty (DMEK). For this reason, we do not recommend HLA matching here. The same is true for deep anterior lamellar keratoplasty, where graft rejections can usually be treated well. However, HLA matching is a viable option in penetrating keratoplasty. This is especially true for high-risk eyes.

Brain microvessel cross‐presentation is a hallmark of experimental cerebral malaria

Cerebral malaria is a devastating complication of Plasmodium falciparum infection. Its pathogenesis is complex, involving both parasite- and immune-mediated events. CD8+ T cells play an effector role in murine experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA (PbA) infection. We have identified a highly immunogenic CD8 epitope in glideosome-associated protein 50 that is conserved across rodent malaria species. Epitope-specific CD8+ T cells are induced during PbA infection, migrating to the brain just before neurological signs manifest. They are functional, cytotoxic and can damage the blood–brain barrier in vivo. Such CD8+ T cells are also found in the brain during infection with parasite strains/species that do not induce neuropathology. We demonstrate here that PbA infection causes brain microvessels to cross-present parasite antigen, while non-ECM-causing parasites do not. Further, treatment with fast-acting anti-malarial drugs before the onset of ECM reduces parasite load and thus antigen presentation in the brain, preventing ECM death. Thus our data suggest that combined therapies targeting both the parasite and host antigen-presenting cells may improve the outcome of CM patients.

Optimization of methodology for production of CD25/CD71 allodepleted donor T cells for clinical use

Immunotherapy with allodepleted donor T cells improves immunity after T cell-depleted hematopoietic stem cell transplantation. We developed a methodology for selective depletion of alloreactive T cells after activation with host antigen-presenting cells by targeting T cells up-regulating CD25 and CD71. Combined depletion of these cells yields a pool of allodepleted donor T cells with antiviral properties with minimal capacity to cause graft-versus-host disease. Mature dendritic cells were irradiated and used to stimulate donor peripheral blood mononuclear cells for 4 days. The co-culture was stained with anti-CD71-biotin followed by CliniMACS CD25 and Anti-Biotin Reagents (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany) before depletion on the CliniMACS Plus (Miltenyi Biotec GmbH). Residual alloreactivity was tested by flow cytometry, a secondary mixed lymphocyte reaction and limiting dilution analysis, and specific anti-viral immunity with pentamer staining. The large-scale protocol was tested under current good manufacturing practice conditions in five donor-recipient pairs of human leukocyte antigen-matched volunteer donors. We developed a closed-system methodology using cell differentiation bags for cell culture and the COBE2991 Cell Processor (CaridianBCT, Lakewood, CO, USA). We also validated an anti-CD71-biotin generated for ex vivo clinical use. In five large-scale runs, the depleted fraction demonstrated excellent viability (99.9%), minimal residual expression of CD3/CD25 and CD3/CD71 (<0.2%) and passed tests for Mycoplasma, endotoxin, bacterial and fungal sterility. In secondary mixed lymphocyte reaction assays, the median response to host after allodepletion was 0%, whereas responses to third-party peripheral blood mononuclear cells were preserved (median, 105%; range 37%-350%). Limiting dilution analysis assays also demonstrated a reduction in response to host (median,-1.11 log) with preservation of third-party responses, and testing with human leukocyte antigen-restricted pentamers showed that populations of Epstein-Barr virus-specific and cytomegalovirus-specific CD8(+) T cells were retained after depletion. We optimized a protocol for the combined immunomagnetic depletion of alloreactive CD25/CD71 T cells under current good manufacturing practice conditions and tested the efficacy in five donor-recipient pairs.

CD24-Siglec-G Interaction Plays an Important in Reducing Experimental Graft-Versus-Host Disease (GVHD)

Abstract 453The role of host antigen presenting cells (APCs) on negatively regulating GVHD is not well understood. Members of the sialic acid binding Ig–like lectin-G (Siglec-G) is an immunoreceptor tyrosine-based inhibitory motifs (ITIM) or ITIM-like regions in its intracellular domain that negatively regulates immune activation induced by non-infectious damage associated molecules (DAMPs). Following conditioning for allogeneic BMT, several DAMPs are released which stimulate host APCs and enhance GVHD. But the role of negative regulators of DAMP associated immune activation, such as Siglecs, in regulating allo-reactivity is not known. We therefore utilized well defined clinically relevant murine models of allogeneic BMT to test the hypothesis that deficiency of a negative regulator of responses to DAMPs in the hosts, namely Siglec-G, will increase GVHD. B6 wild type (WT) and Siglec-G−/− animals were lethally irradiated (13Gy) and transplanted on day 0 with 5×106 bone marrow and 3×106 splenic CD90+T cells from either syngeneic WT-B6 or MHC mismatched BALB/c donors. The Siglec-G−/− animals showed significantly worse survival than the allo-WT animals (p=0.0045). The increased mortality was associated with an increase in GVHD specific clinical severity (p<0.05), donor T cell expansion (p<0.03), and serum levels of pro-inflammatory cytokines (TNFα, IFN-γ, p<0.05) on day +7 after BMT. We next evaluated whether this was because of Siglec-G deficiency only on the radiosensitive host APCs. To this end we generated [B6àB6Ly5.2] and [Siglec-G−/−àB6Ly5.2] BM chimeras and utilized them as recipients following lethal radiation. They were injected with 5×106 BM and 3×106 CD90+ T cells from either syngeneic WT B6 or allogeneic BALB/c donors. The allogeneic [Siglec-G−/−àB6Ly5.2] animals demonstrated significantly worse survival than the [B6àB6Ly5.2] animals (p<0.0001). We determined the converse, i.e. analyzed the role of Siglec-G on radio-resistant host APCs by generating [B6Ly5.2àB6] and [B6Ly5.2àSiglec-G−/−] BM chimeras and utilized them as recipients. [B6Ly5.2àSiglec-G−/−] chimeras demonstrated similar survival as [B6Ly5.2àB6] chimeras. These data collectively demonstrate that Siglec-G expression only on the host radiosensitive APCs is critical for protection from GVHD. To confirm the role of increased DAMPs in causing greater mortality we tested whether the intensity of conditioning affects the serum level of DAMPs (HMGB1, proinflammatory cytokines) and found that significantly greater levels of DAMPs were observed in the mice that received 13Gy than 8 Gy. Furthermore, consistent with the increased levels of DAMPs, Siglec-G−/− animals showed higher GVHD only after 13Gy radiation but not after 8Gy conditioning. Because responses to non-infectious DAMPs are regulated by Siglec-G through its interaction with CD24 we next hypothesized that enhanced CD24-Siglec-G interaction would mitigate GVHD following myeloablative conditioning. We first characterized stimulation of allogeneic T cell responses by Siglec-G−/− APCs. We utilized CD24−/− and WT BALB/c T cells as responders in an MLR with B6 and Siglec-G−/−stimulators. We found that Siglec-G−/− APCs expanded the CD24−/− T cells more than WT-B6 APCs. We next tested in vivo whether enhanced CD24-Siglec-G interaction would mitigate GVHD. We utilized a novel CD24 fusion protein (day-1, 100mg/mouse) and found that it decreased GVHD mortality only in the WT but not in the Siglec-G−/− animals. Together our data demonstrate a critical role for CD24-Siglec-G interactions in regulating GVHD and suggest that administration of the novel CD24 fusion protein may be an innovative strategy to mitigate GVHD. Disclosures:No relevant conflicts of interest to declare.

Identification of Previously Uncharacterized Delta 4-Positive Inflammatory Plasmacytoid Dendritic Cells That Play Important Roles in Eliciting Graft-Versus-Host Disease in Mice

Abstract 337Graft-versus-host disease (GVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Host antigen-presenting cells (APCs) are known to be essential for presenting alloantigens to activate donor T cells to become effector cells mediating GVHD after allo-HSCT. However, APCs are heterogeneous populations. The identity of APC subset(s) that directs effector differentiation of alloantigen-activated T cells and by which mechanism this effect may be achieved remain largely unknown. The Notch signaling pathway controls cell proliferation, differentiation and survival. Upon interaction with Notch ligands of the δ-like family (Dll1, Dll3 and Dll4) and Jagged family (J1, J2), Notch receptors (Notch 1, 2, 3, and 4) are cleaved by γ-secretase and translocate into the nucleus to modify gene transcription. We have recently demonstrated that activation of Notch receptors in donor T cells is critical to the production of alloreactive effector T cells producing multiple inflammatory cytokines (e.g., IFN-γ, TNF-α and IL-17) during GVH reaction (Blood 2011). Building on these findings, we hypothesized that: 1) Notch ligand(s) derived from APCs may be important for directing effector differentiation of alloantigen-activated T cells, and 2) the expression of Notch ligand(s) may differentiate the capability of APCs to prime GVH responses. Using mouse models of GVHD, here we report the identification of previously uncharacterized Dll4-positive (Dll4+) inflammatory plasmacytoid dendritic cells (i-pDCs) and their roles in eliciting allogeneic T-cell responses. Host-derived Dll4+ i-pDCs occurred in the spleen of allo-HSCT recipients one day after transplantation, peaked by three days and declined by seven days. In contrast, host-derived inflammatory conventional DCs (i-cDCs) were Dll4-negative (Dll4−) and rapidly diminished by three days after transplantation. Notably, donor-derived DCs which occurred seven days after HSCT did not express Dll4. In vitro mixed lymphocyte-reaction (MLR) assay showed that these host-derived Dll4+ i-pDCs induced approximately 2.5-fold and 7-fold more IFN-γ- and IL-17-producing effector T cells than Dll4− i-cDCs, respectively. Addition of neutralizing antibody specific to Dll4 to the MLR cultures markedly reduced the production of IFN-γ and IL-17 in donor T cells stimulated by host Dll4+ i-pDCs, but had minimal impact on donor T cells cultured in the presence of Dll4− i-cDCs. These results suggest that Dll4+ i-pDCs may play important roles in directing effector differentiation of alloantigen-activated T cells. Further characterization of biological properties of Dll4+ i-pDCs revealed that as compared to unstimulated host pDCs at steady state conditions, Dll4+ i-pDCs expressed higher levels of antigen-presenting and costimulatory molecules, upregulated other Notch ligands (e.g.,J1 and J2) on their surface and produced more Ifnb and Il23. Notably, Dll4+ i-pDCs were mainly located in the spleen and intestine of mice receiving allogeneic HSCT. In vivo administration of Dll4 antibody reduced donor alloreactive effector T cell producing IFN-γ, IL-17 and TNF-α in GVHD target organs (in particular of the intestine), leading to reduction of GVHD and significantly improved survival of mice after allogeneic HSCT. Furthermore, adoptive transfer of in vitro generated Dll4+ i-pDCs caused severe GVHD in MHC-II-deficient mice (in which host DCs are incapable to elicit GVHD). Our findings identify that Dll4+ i-pDCs may represent a previously uncharacterized inflammatory APC population developed during GVH reaction. These Dll4+ i-pDCs and their-derived Dll4 are critical for directing differentiation of alloreactive effector T cells and may be beneficial therapeutic targets for modulating GVHD. Disclosures:No relevant conflicts of interest to declare.

Prevention of Acute Graft-Versus-Host Disease in Human/Mouse Xenogeneic SCID Mouse Model by Humanized Anti-CD74 Monoclonal Antibody, Milatuzumab

AbstractAbstract 4105 Introduction:Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation. Prevention and treatment of GVHD remains a major challenge, because current T-cell depletion and mainstay immunosuppressive therapies compromise preexisting T-cell immunity, leading to severe infections and disease relapse. Thus, novel anti-GVHD agents that can spare protective T-cell memory are critically needed. Milatuzumab (hLL1) is a humanized anti-CD74 antagonist IgG1κ monoclonal antibody (mAb) currently under clinical investigation as a therapeutic mAb for relapsed or refractory B-cell malignancies. Since CD74 is widely expressed in both hematopoietic and non-hematopoietic antigen-presenting cells (APCs), and because host APCs, especially non-hematopoietic APCs, play an important role in initiating GVHD, whereas donor APCs contribute and are required to maximize GVHD, we reasoned milatuzumab could affect recipient or donor APCs, thereby modulating GVHD. We report herein the in vitro effect of milatuzumab on the survival and function of human blood APCs and T cells, including CMV-specific T cells, and the in vivo therapeutic efficacy on preventing acute GVHD in a humanized SCID mouse model. Methods:The effects of milatuzumab on APCs and T cells in human peripheral blood mononuclear cells (PBMCs) were assessed by multi-color flow cytometry. The effect of milatuzumab on the proliferation of T cells and T cell subsets in allogeneic mixed lymphocyte reactions (allo-MLRs) was measured by CFSE labeling of allogeneic PBMCs that were mixed to each other and cultured for 11 days before flow cytometric analysis of the proliferating cells that lost fluorescence. The impact of milatuzumab on preexisting anti-viral T-cell immunity was evaluated by intracellular staining of CD3+CD8+IFN-γ+T cells in allo-MLRs upon stimulation with HLA-A2-restricted cytomegalovirus (CMV) pp65 peptide (NLVPMVATV). The therapeutic effect of milatuzumab on acute GVHD was evaluated in a human PBMC-transplanted SCID mouse model, in which GVHD is developed and mediated by engrafted human T cells and DCs. Results:Milatuzumab moderately reduces the number of myeloid DC type 1 (mDC1), myeloid DC type 2 (mDC2), and B cells in PBMCs, but has little effect on plasmacytoid DC (pDC), monocytes, or T cells, which correlates with the level of CD74 expression on these cells. As a consequence, milatuzumab inhibits the proliferation of total T cells, CD4 and CD8 T cell subsets in allo-MLRs. In a human/mouse xenogeneic SCID mouse model, milatuzumab effectively prevents the onset and manifestations of acute GVHD, suppresses the serum levels of human interferon-g and IL-5, eliminates the infiltration of human lymphocytes in GVHD target organs (lung, liver and spleen), and significantly promotes the survival of animals (90% versus 20% for controls, P=0.0012). Furthermore, exposure to milatuzumab does not affect the number of CMV-specific, IFN-γ-producing, human CD8+ T cells in allo-MLRs. Conclusion:CD74 is a potential target for antibody-mediated mitigation of GVHD, as demonstrated by the encouraging results obtained with milatuzumab. Further exploration of milatuzumab as a new therapeutic agent for GVHD is warranted. Disclosures:Cardillo:Immunomedics, Inc: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.

Urate Oxidase (Rasburicase) to Inhibit Graft Versus Host Disease (GVHD) After Myeloablative HLA-Matched Allogeneic Hematopoietic Cell Transplantation (HCT).

Abstract 3063 Background:Dying mammalian cells release danger signals that stimulate antigen presenting cells (APCs) to promote an immune response. It has been demonstrated that uric acid released from injured cells alerts the immune system to cell death, acts as a danger signal to stimulate cytotoxic T cell responses and that elimination of uric acid in mouse models reduces this immune response (Ref: Nature 2003; 425:516–521). Rasburicase is a recombinant urate-oxidase enzyme that catalyzes oxidation of uric acid into an inactive soluble metabolite and is currently used to prevent tumor lysis syndrome. We hypothesized that rasburicase administered during myeloablative conditioning prior to allogeneic HCT will reduce the serum levels of uric acid and thereby may decrease the incidence of acute GVHD via inhibition of danger signal-mediated activation of host APCs. Methods:In this pilot trial at the Massachusetts General Hospital, between 2007 and 2010, 23 patients (median age: 41 years, range: 19–59) with hematologic malignancies in complete remission (AML, n=13; ALL, n=8; MDS, n=1; MPD, n=1;) received myeloablative preparative regimens (Bu/Cy, n=14; Cy/TBI, n=7; Bu/Flu, n=2) followed by GCSF-mobilized HLA-matched (MRD, n=18; MUD, n=5) peripheral blood HCT. GVHD prophylaxis consisted of cyclosporine or tacrolimus and methotrexate for MRD transplants and tacrolimus/MTX and anti-thymocyte globulin (Thymoglobulin) for MUD transplants. Rasburicase was administered beginning on the first day of conditioning therapy, at a dose of 0.20 mg/kg intravenously daily for 5 consecutive days. Outcomes were compared to 44 controls at this institution from the same time period identified using retrospective chart review. Patients in the control group received allopurinol during the conditioning as a part of the institutional guidelines. Associations between categorical variables were evaluated using Fisher’s exact test. Overall and disease-free survival (OS and DFS) were estimated using the method of Kaplan and Meier. Results:The mean serum uric acid concentration was 0.2 mg/dl (range, 0–1.7) on 6 consecutive days, beginning the day after the first dose of rasburicase. Engraftment was achieved in all patients, and the median times to neutrophil (at least 0.5 × 109/ul) and platelet engraftment (at least 20 × 109/ul) were 18 days (range, 7–26 × 109/ul) and 16 days (range, 8–57 × 109/ul), respectively. The only serious toxicity caused by rasburicase was intravascular hemolysis in one patient who was found to have G6PD deficiency; this patient received only 2 doses of rasburicase. Greater than or equal to grade II-IV acute GVHD occurred in 5 out of 23 patients (22%), 4/18 in MRD and 1/5 in MUD recipients: grade II, n=1; grade III, n=2; and grade IV, n=2. When compared with 44 patients (AML 28, ALL 5, MDS 4, NHL 5, MPD 1, CML 1), comparable to rasburicase-treated patients with respect to age, gender, disease status, donor sources, conditioning and GVHD prophylaxis, who received myeloablative HCT (MRD, n=32; MUD, n=12) at the MGH during the same time period as the patients in the rasburicase-treated group, there was significantly less grade II or higher aGVHD in the rasburicase group (rasburicase: 22% vs. institutional control: 48% [total 21/44, 13/32 in MRD and 8/12 MUD recipients], Fisher’s exact test, p=0.033). There was no significant difference in the incidence and severity of chronic GVHD between the two groups (rasburicase, 61%, control 48%; Fisher’s exact test, p=0.721). At 3 years, there was no significant difference in disease-free survival (DFS) or overall survival (OS) between the rasburicase-treated and control groups (DFS: rasburicase vs. control 51.2% vs. 40.4%, p=0.97; OS: rasburicase vs. control 55.3% vs. 44.9%, p=0.88). Conclusion:Rasburicase can be safely administered during myeloablative conditioning, results in a profound lowering of serum uric acid levels and has the potential to reduce the incidence of acute GVHD. Based on these data a prospective randomized phase II trial is planned in order to verify whether rasburicase inhibits acute GVHD after myeloablative HCT and to study the possible mechanisms of protection from GVHD. Disclosures:Off Label Use: Rasburicase for GVHD prevention in allo-HSCT.

Neisseria gonorrhoeae Suppresses Dendritic Cell-Induced, Antigen-Dependent CD4 T Cell Proliferation

Neisseria gonorrhoeae is the second most common sexually transmitted bacterial pathogen worldwide. Diseases associated with N. gonorrhoeae cause localized inflammation of the urethra and cervix. Despite this inflammatory response, infected individuals do not develop protective adaptive immune responses to N. gonorrhoeae. N. gonorrhoeae is a highly adapted pathogen that has acquired multiple mechanisms to evade its host's immune system, including the ability to manipulate multiple immune signaling pathways. N. gonorrhoeae has previously been shown to engage immunosuppressive signaling pathways in B and T lymphocytes. We have now found that N. gonorrhoeae also suppresses adaptive immune responses through effects on antigen presenting cells. Using primary, murine bone marrow-derived dendritic cells and lymphocytes, we show that N. gonorrhoeae-exposed dendritic cells fail to elicit antigen-induced CD4+ T lymphocyte proliferation. N. gonorrhoeae exposure leads to upregulation of a number of secreted and dendritic cell surface proteins with immunosuppressive properties, particularly Interleukin 10 (IL-10) and Programmed Death Ligand 1 (PD-L1). We also show that N. gonorrhoeae is able to inhibit dendritic cell- induced proliferation of human T-cells and that human dendritic cells upregulate similar immunosuppressive molecules. Our data suggest that, in addition to being able to directly influence host lymphocytes, N. gonorrhoeae also suppresses development of adaptive immune responses through interactions with host antigen presenting cells. These findings suggest that gonococcal factors involved in host immune suppression may be useful targets in developing vaccines that induce protective adaptive immune responses to this pathogen.

Interleukin-21 Receptor-Mediated Signals Control Autoreactive T Cell Infiltration in Pancreatic Islets

It remains unclear how interleukin-21 receptor (IL-21R) contributes to type 1 diabetes. Here wehave shown that dendritic cells (DCs) in the pancreas required IL-21R not for antigen uptake, but to acquire the chemokine receptor CCR7 and migrate into thedraining lymph node. Consequently, less antigen, major histocompatibility complex (MHC) class II, and CD86 was provided to autoreactive effector cells inIl21r(-/-) mice, impairing CD4(+) Tcell activation, CD40:CD40L interactions, and pancreatic infiltration by autoreactive Tcells. CD40 crosslinking restored defective CD4(+) cell expansion and CD4 independently expanded autoreactive CD8(+) cells, but CD8(+) cells still required CD4(+) cells to reach the pancreas and induce diabetes. Diabetes induction by transferred Tcells required IL-21R-sufficient host antigen-presenting cells. Transferring IL-21R-sufficient DCs broke diabetes resistance in Il21r(-/-) mice. We conclude that IL-21R controls both antigen transport by DCs and the crucial beacon function of CD4(+) cells for autoreactive CD8(+) cells to reach the islets.

Polarization of host immune responses by helminth‐expressed glycans

Helminth parasites bias host CD4(+) T helper (Th) cells toward Th2 responses, drive alternative activation of macrophages, and expand T regulatory cells. Helminth-expressed carbohydrates play critical roles in driving much of this immune cell biasing. Studies on helminth glycans have focused on Lewis X, LDN, LDN-DF, other fucosylated structures, chitin, tyvelose, and trehalose, which interact with host antigen presenting cells (APCs) minimally via C-type lectins and/or Toll-like receptors (TLR). Here, we review recent findings on helminth glycan activation of APCs via C-type lectin/TLRs and introduce the concept that glycosylated helminth molecules require endocytosis to function as immune modulators. Second, we describe unpublished data showing that in vivo glycoconjugates comprising multiple copies of glycans on carriers are directly immune modulatory. Lastly, we discuss the observation that CD14 negatively regulates alternative activation of APCs during helminth infection. We close with a discussion on the use of immune modulatory glycans as vaccine adjuvants and as antiinflammatory therapeutics.

GVHD Prevention: An Ounce Is Better Than a Pound

The pathophysiology of acute graft-versus-host disease (aGVHD) is known to involve donor T cells responding to host histoincompatible allo-antigens presented by the host antigen presenting cells (APCs) and the subsequent induction of pro-inflammatory cytokines and cellular effectors that cause target organ damage. In a more general sense, GVHD can be considered as an immune response against foreign antigens that has gone awry. Similar to all immune responses, GVHD, can be understood as a process that consists of (A) triggers, (B) sensors, (C) mediators, and (D) effectors of GVHD.