B7 Costimulatory Molecules Research Articles

A transgenic mouse model for HLA-B*57:01-linked abacavir drug tolerance and reactivity.

Adverse drug reactions (ADRs) are a major obstacle to drug development, and some of these, including hypersensitivity reactions to the HIV reverse transcriptase inhibitor abacavir (ABC), are associated with HLA alleles, particularly HLA-B*57:01. However, not all HLA-B*57:01+ patients develop ADRs, suggesting that in addition to the HLA genetic risk, other factors may influence the outcome of the response to the drug. To study HLA-linked ADRs in vivo, we generated HLA-B*57:01-Tg mice and show that, although ABC activated Tg mouse CD8+ T cells in vitro in a HLA-B*57:01-dependent manner, the drug was tolerated in vivo. In immunocompetent Tg animals, ABC induced CD8+ T cells with an anergy-like phenotype that did not lead to ADRs. In contrast, in vivo depletion of CD4+ T cells prior to ABC administration enhanced DC maturation to induce systemic ABC-reactive CD8+ T cells with an effector-like and skin-homing phenotype along with CD8+ infiltration and inflammation in drug-sensitized skin. B7 costimulatory molecule blockade prevented CD8+ T cell activation. These Tg mice provide a model for ABC tolerance and for the generation of HLA-B*57:01-restricted, ABC-reactive CD8+ T cells dependent on both HLA genetic risk and immunoregulatory host factors.

Association of minimally invasive colorectal resection with elevated levels of plasma B7 homolog 1 (B7-H1) during the first month after surgery and effect on immune suppression and growth of residual metastases.

602 Background: The co-stimulatory molecule B7 homolog 1 (B7-H1) is a ligand for PD-1 which is an immunoinhibitory receptor of activated lymphocytes. PD-1 expression is upregulated on tumor infiltrating lymphocytes (TILs), and B7-H1 expressed on cancer cells may inhibit T-cell activation and proliferation. B7-H1 is also expressed on endothelial (EC) and intestinal epithelial cells as well as activated macrophages. B7H1 in EC is rapidly induced by IFN-gamma and TNF. B7-H1 silencing with siRNA inhibits tumor cell proliferation/invasion. B7H1 expression in keratinocytes (KC) directly downregulates CD8(+) T-cell effector function via PD-1 binding at sites of inflammation. Shed B7-H1 can be found in the blood. Surgery’s impact on plasma B7H1 levels is unknown. This study’s purpose was to evaluate plasma B7H1 levels during the first month after MICR for colorectal (CRC). Methods: MICR CRC patients in an IRB approved data/plasma bank with adequate plasma available were eligible. Clinical and pathological data were reviewed. Blood samples were collected preoperatively (PreOp) and at 6 post-operative (Postop) time points (POD 1, 3, 7-13, 14-20, 21-27, 28-41). B7-H1 levels were analyzed in duplicate using ELISA. The Wilcoxon test was used for analysis. Results: 88 CRC patients who had a MICR met inclusion criteria (28% rectal and 62% colon lesions). The mean PreOp B7-H1 level was 51.9±20.9 pg/ml. Significantly elevated mean plasma levels were noted on POD1 (64.9±24.2 pg/ml, n = 86, p = < 0.0001), POD3 (67.3±24.6 pg/ml, n= 72, p = < 0.0001), POD7-13 (69.2±22.6 pg/ml, n = 65, p = < 0.0001), POD14-20 (72.5±28.9 pg/ml, n=23,p=0.001), POD 21-27 (79.4±66.6 pg/ml, n = 13, p = 0.001), and on POD 28-41 (56.3 ±22.7 pg/ml, n = 20, p =0.02), when compared to PreOp levels. Conclusions: Plasma B7-H1 levels are elevated for over a month after MICR for CRC. The etiology of the early increase may be the acute inflammatory response whereas late elevations may be related to wound healing-related tissue remodeling. Elevated plasma B7-H1 may suppress TIL’s which may result in increased tumor growth. Further studies are warranted.

Activation of Bombesin Receptor Subtype-3 Promotes Antigen-Presenting Action in Human Bronchial Epithelial Cells

Background: Bombesin receptor subtype-3 (BRS-3) is a member of the bombesin-like peptide receptor family. Our previous studies demonstrated that activation of the human BRS-3 plays a protective role in oxidation-injured human bronchial epithelial cells (HBEC). The present study was designed to determine the role of BRS-3 activation in the antigen-presenting action of HBEC and the corresponding proliferation and differentiation of T cells. Materials and Methods: In vivo, an asthma animal model was established and the expression and distribution of BRS-3 were analyzed by immunocytochemistry. In vitro, 2 kinds of B7 costimulatory molecules, i.e., B7H1 and B7DC, on HBEC were analyzed by flow cytometry. The antigen uptake by HBEC was examined by confocal microscopy and flow cytometry. The antigen-presenting-action-induced proliferation of T cells was determined by MTT assays. IFN-γ and IL-4 levels were measured by ELISA. All studies were performed in the absence or presence of the synthetic peptide P3513. Results: BRS-3 expression was induced in asthma animal models and mainly distributed in bronchial epithelial cells. HBEC express the costimulatory molecules B7H1 and B7DC. BRS-3 activation increased B7H1 expression but decreased B7DC expression on HBEC. BRS-3 activation also increased the antigen uptake by HBEC and the subsequent T cell proliferation. In addition, BRS-3 activation promoted the releases of IFN-γ, but not IL-4, in the supernatant of cocultured HBEC and T cells. Conclusion: These data suggest that HBEC can present antigen to T cells and BRS-3 activation promotes the process of antigen presentation and subsequent T cell proliferation and Th1 differentiation.

Cancer cell-expressed B7-H3 regulates the differentiation of tumor-associated macrophages in human colorectal carcinoma.

Co-stimulatory molecule B7 homolog 3 protein (B7-H3) has been described as an important tumor antigen in various human tumors. The exact role of B7-H3 in tumor progression and its receptor are still ambiguous. The phenotype and the function of tumor-associated macrophages (TAMs) in human solid tumors are complicated and could contribute to the shaping of the tumor microenvironment. In the present study, B7-H3 expression and lymphocyte infiltration were investigated by immunohistochemistry in 117 colorectal carcinoma (CRC) patients. B7-H3 expression was positively associated with the infiltrating density of macrophage in CRC tissues, and B7-H3 expression and the infiltrating density of macrophages were negatively associated with the overall survival rate of patients. The putative B7-H3 receptor was found on activated monocytes and macrophages, indicating the direct function of B7-H3 signal on macrophages. Additional results revealed that during the differentiation of TAMs, B7-H3 promoted the polarization of type 2 macrophages (M2s) and switch of the M1 phenotype to the M2 phenotype. Thus, B7-H3 signaling promotes M2 differentiation via the putative receptor on monocytes and macrophages. Targeting the manipulation of TAMs through the B7-H3 pathway may be valuable for the development of novel immunotherapeutic strategies against human CRC.

Enhanced immunotherapy by combining a vaccine with a novel murine GITR ligand fusion protein

Immunotherapy was significantly enhanced in a murine tumor model by combining a vaccine with a fusion protein designed to target the glucocorticoid-induced tumor necrosis factor (TNF) receptor related gene (GITR) on the surface of T cells. The recombinant poxvirus-based vaccine platform included Modified Vaccinia virus Ankara (rMVA) and fowlpox (rF) vectors as the driver immunogens both engineered to express the human carcinoembryonic antigen (CEA) and three murine costimulatory molecules B7.1, ICAM-1, LFA-3 (designated TRICOM). In previous studies, mice expressing human CEA as a transgene (CEA.Tg mice) vaccinated with rMVA/rF-CEA-TRICOM overcame CEA immune tolerance by inducing anti-CEA‒specific immunity and regression of CEA-expressing tumors. The murine GITR ligand fusion protein (mGITRL-FP) consisted of a mouse IgG2a Fc region, a yeast-derived coiled GCN4 pII and the extracellular GITR-binding domain of murine GITR ligand. The design maximized valency and the potential to agonize the GITR receptor. Combined treatment of the vaccine and mGITRL-FP mediated a more robust tumor regression, leading to sustained improvement in overall survival. The enhanced immunotherapeutic effect was linked to the generation of a strong CD8+ T cell antitumor immune response. A treatment schedule with mGITRL-FP administered prior to the priming rMVA-CEA-TRICOM vaccination was of paramount importance. The mechanism of action for the enhanced antitumor effects resided in the depletion of immune cells, particularly FoxP3+ regulatory T cells, that express high GITR levels following activation. The results provide evidence that targeting GITR with mGITRL-FP in concert with a cancer vaccine represents a potential novel approach to more effective immunotherapy.

Förster Resonance Energy Transfer to Study TCR-pMHC Interactions in the Immunological Synapse.

T-cell antigen recognition is remarkably efficient: when scanning the surface of antigen-presenting cells (APCs), T-cells can detect the presence of just a few single antigenic peptide/MHCs (pMHCs), which are often vastly outnumbered by structurally similar non-stimulatory endogenous pMHCs (Irvine et al., Nature 419(6909):845-849, 2002; Purbhoo et al., Nat Immunol 5(5):524-530, 2004; Huang et al., Immunity 39(5):846-857, 2013). How T-cells achieve this is still enigmatic, in particular in view of the rather moderate affinity that TCRs typically exert for antigenic pMHCs, at least when measured in vitro (Davis et al., Ann Rev Immunol 16:523-544, 1998). To shed light on this in a comprehensive manner, we have developed a microscopy-based assay, which allows us to quantitate TCR-pMHC interactions in situ, i.e., within the special confines of the nascent immunological synapse of a T-cell contacting a planar-supported lipid bilayer functionalized with the costimulatory molecule B7-1, the adhesion molecule ICAM-1, and pMHCs (Huppa et al., Nature 463(7283):963-967, 2010) (Fig. 1). Binding measurements are based on Förster resonance energy transfer (FRET) between site-specifically labeled pMHCs and TCRs, which are decorated with recombinant site-specifically labeled single-chain antibody fragments (scFV) derived from the TCRβ-reactive H57-597 antibody (Huppa et al., Nature 463(7283):963-967, 2010). FRET, a quantum-mechanical phenomenon, involves the non-radiative coupling of dipole moments of two adjacent fluorophores, a donor molecule and an acceptor molecule. FRET efficiency is inversely proportional to the sixth power of the inter-dye distance. Hence, it can be employed as a molecular ruler (Stryer and Haugland, Proc Natl Acad Sci, USA 58(2):719-726, 1967) or, as is the case here, to score for interactions of appropriately labeled molecules. To facilitate both quantitative and single-molecule readout, it is important to conjugate donor and acceptor dyes in a site-specific manner.While SLBs mimic some but certainly not all properties of a plasma membrane of a living cell, their use features a number of operational advantages: SLBs can be prepared in a fluid state, thereby facilitating the spatial rearrangements that accompany the formation of an immunological synapse (Grakoui et al., Science 285(5425):221-227, 1999). The imaging of a three-dimensional binding process is reduced to two dimensions, which saves time and fluorophore-emitted photons and allows for fast measurements. Furthermore, images can be acquired in noise-attenuated total internal reflection (TIR) mode, so far a necessity for single-molecule detection within the immunological synapse. Importantly, the stimulatory potency of pMHCs is very well preserved compared to cell surface-embedded pMHCs. Hence, while in principle artificial, SLBs are still a good approximation of the physiologic scenario a T-cell encounters when approaching an APC. Vice versa, the reconstitutive approach offers unique opportunities to interrogate the influence of accessory molecules on T-cell antigen recognition in a highly quantitative manner.In this chapter we will provide recommendations for the production of proteins used for SLB decoration as well as hands-on protocols for the production of SLBs. We will describe in detail how to perform and analyze FRET-based experiments to determine synaptic binding constants. In the "Notes" section, we will provide some information regarding the microscope setup as well as the mathematical and biophysical foundation underlying data analysis.

Anti-Tumor Activities of XBP1 Antigen-Specific Cytotoxic T Lymphocytes Are Enhanced By HDAC6 Inhibitor ACY241

The effects of histone deacetylase (HDAC) inhibition on immune effector cells may have significant clinical implications; however, this has not yet been elucidated. The goal of this study was to investigate the immunomodulatory potential of the selective HDAC6 inhibitor ACY241 in combination with a cancer vaccine to enhance the efficacy of antigen-specific cytotoxic T lymphocytes (CTL) and the specific activities against tumor cells. Here, we report the effects of ACY241 treatment on antigen expression, immune activation, proliferation, and functional activities of XBP1 antigen-specific cytotoxic T lymphocytes (XBP1-CTL). The antigen-specific CTL were generated in vitro by repeated stimulation with novel immunogenic heteroclitic HLA-A2 XBP1 peptides (YISPWILAV, YLFPQLISV), as described previously by our group (Bae et al. Leukemia 2011; Bae et al. Oncoimmunology 2014l; Bae et al. Leukemia 2016). We found that treatment with ACY241 up-regulated key co-stimulatory (CD28, CD40L) and activation (CD38, CD69, CD137) molecules on XBP1-CTL, without inducing expression of co-inhibitory checkpoints (PD1, LAG3, CTLA4, VISTA). In addition, ACY241 increased the frequency of memory CTL subsets and enhanced their anti-tumor activities (cytotoxic activity, Th1-type cytokine production, CTL proliferation) against HLA-A2+ and XBP1+ multiple myeloma, breast cancer, and colon cancer cells. The XBP1-CTL responses were dramatically increased in combination with ACY241, including higher levels of tumor-specific CD107a up-regulation, perforin release, IFN-g/IL-2/TNF-a cytokine production and proliferation of the CD3+CD8+ T cells expressing CD28/CD38 in response to the specific XBP1 peptides. ACY241 also enhanced the expression of various tumor-associated antigens (XBP1, CD138, CS1, BCMA, CD44), MHC class I/II molecules, along with co-stimulatory B7 molecules (CD80, CD86) on HLA-A2+ myeloma (U266), breast cancer (MDA-MB231) and colon cancer (SW480) cell lines. Furthermore, in vitro ACY241 treatment consistently decreased the frequency of immune suppressor cells including myeloid-derived suppressor cells (CD14- CD15+/CD11b+ CD33+/HLA-DRlow) and regulatory T cells (CD25+ FOXP3+/CD3+ CD4+) in peripheral blood or bone marrow mononuclear cells from multiple myeloma patients in a dose-dependent manner. In conclusion, our data demonstrates the immunomodulatory effects of selective HDAC6 inhibition by ACY241 and supports its potential role for improving tumor-specific CTL function and tumor cell recognition when used in combination with antigen-specific cancer vaccine. DisclosuresBae:OncoPep Inc.: Consultancy, Equity Ownership. Chauhan:Stemline Therapeutics: Consultancy. Hideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Munshi:OncoPep Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Anderson:OncoPep Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

The role of B7 family costimulatory molecules and indoleamine 2,3-dioxygenase in primary Sjögren's syndrome and systemic sclerosis.

B7 costimulatory molecules are present on antigen-presenting cells (APCs) and influence intracellular expression of indoleamine 2,3-dioxygenase (IDO), a molecule with important immunoregulatory functions. We determined the frequency of activated (CD11b+) monocytes expressing B7-1, B7-2, B7-H1, and B7-H2 molecules, and that of CD3+ and CD4+ T cells expressing the corresponding CD28, CTLA-4, PD-1, and ICOS receptors in peripheral blood samples of 20 healthy adults and 9 SSc and 15 pSS patients using flow cytometry. We also examined the intracellular expression of IDO. The expression of CD28 was lower in both SSc and pSS patients. The frequency of CTLA-4 was increased in pSS. The expression of ICOS, a stimulator of T cell activation, was elevated in pSS, but not in SSc, while that of its corresponding costimulatory molecule, B7-H2, was strongly decreased in SSc compared to controls. The frequency of PD-1 expressing T lymphocytes was decreased in both pSS and SSc. The frequency of IDO-expressing APCs, as well as intracellular IDO content in T cells was higher in pSS than in controls. Our investigation identified a number of differences in B7 costimulation between SSc and pSS patients which may play a role in the distinct pathogenesis and clinical features of these autoimmune disorders.

Systemic activation of antigen-presenting cells via RNA-loaded nanoparticles

ABSTRACTWhile RNA-pulsed dendritic cell (DC) vaccines have shown promise, the advancement of cellular therapeutics is fraught with developmental challenges. To circumvent the challenges of cellular immunotherapeutics, we developed clinically translatable nanoliposomes that can be combined with tumor-derived RNA to generate personalized tumor RNA-nanoparticles (NPs) with considerable scale-up capacity. RNA-NPs bypass MHC restriction, are amenable to central distribution, and can provide near immediate immune induction.We screened commercially available nanoliposomal preparations and identified the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as an efficient mRNA courier to antigen-presenting cells (APCs). When administered intravenously, RNA-NPs mediate systemic activation of APCs in reticuloendothelial organs such as the spleen, liver, and bone marrow. RNA-NPs increase percent expression of MHC class I/II, B7 co-stimulatory molecules, and maturation markers on APCs (all vital for T-cell activation). RNA-NPs also increase activation markers on tumor APCs and elicit potent expansion of antigen-specific T-cells superior to peptide vaccines formulated in complete Freund's adjuvant. We demonstrate that both model antigen-encoding and physiologically-relevant tumor-derived RNA-NPs expand potent antitumor T-cell immunity. RNA-NPs were shown to induce antitumor efficacy in a vaccine model and functioned as a suitable alternative to DCs in a stringent cellular immunotherapy model for a radiation/temozolomide resistant invasive murine high-grade glioma.Although cancer vaccines have suffered from weak immunogenicity, we have advanced a RNA-NP formulation that systemically activates host APCs precipitating activated T-cell frequencies necessary to engender antitumor efficacy. RNA-NPs can thus be harnessed as a more feasible and effective immunotherapy to re-program host-immunity.

The Salmonella Effector SteD Mediates MARCH8-Dependent Ubiquitination of MHC II Molecules and Inhibits T Cell Activation.

SummaryThe SPI-2 type III secretion system (T3SS) of intracellular Salmonella enterica translocates effector proteins into mammalian cells. Infection of antigen-presenting cells results in SPI-2 T3SS-dependent ubiquitination and reduction of surface-localized mature MHC class II (mMHCII). We identify the effector SteD as required and sufficient for this process. In Mel Juso cells, SteD localized to the Golgi network and vesicles containing the E3 ubiquitin ligase MARCH8 and mMHCII. SteD caused MARCH8-dependent ubiquitination and depletion of surface mMHCII. One of two transmembrane domains and the C-terminal cytoplasmic region of SteD mediated binding to MARCH8 and mMHCII, respectively. Infection of dendritic cells resulted in SteD-dependent depletion of surface MHCII, the co-stimulatory molecule B7.2, and suppression of T cell activation. SteD also accounted for suppression of T cell activation during Salmonella infection of mice. We propose that SteD is an adaptor, forcing inappropriate ubiquitination of mMHCII by MARCH8 and thereby suppressing T cell activation.

A phase I study of recombinant (r) vaccinia-CEA(6D)-TRICOM and rFowlpox-CEA(6D)-TRICOM vaccines with GM-CSF and IFN-α-2b in patients with CEA-expressing carcinomas

Prime-boost vaccination with recombinant (r) vaccinia(V)-CEA(6D)-TRICOM (triad of co-stimulatory molecules B7.1, ICAM-1 and LFA-3) and rFowlpox(F)-CEA(6D)-TRICOM infect antigen-presenting cells and direct expression of co-stimulatory molecules. We hypothesized that co-administration of vaccine with GM-CSF and interferon alpha (IFN-α) would have efficacy in CEA-expressing cancers. Patients with CEA-expressing cancers received the rV-CEA(6D)-TRICOM vaccine subcutaneously (s.c.) on day 1 followed by GM-CSF s.c. to the injection site on days 1-4. In Cycle 1, patients received thrice weekly s.c. injections of IFN-α-2b the week after rV-CEA(6D)-TRICOM. In Cycles 2-4, patients received thrice weekly s.c. injections of IFN-α-2b the same week that rF-CEA(6D)-TRICOM was given. The first cohort received no IFN followed by dose escalation of IFN-α in subsequent cohorts. Thirty-three patients were accrued (mean 59.8years). Grade 3 toxicities included fatigue and hyperglycemia. Grade 4-5 adverse events (unrelated to treatment) were confusion (1), elevated aspartate transaminase (AST)/alanine transaminase (ALT) (1), and sudden death (1). No patients had a partial response, and eight patients exhibited stable disease of ≥3months. Median progression-free survival and overall survival (OS) were 1.8 and 6.3months, respectively. Significantly higher serum CD27 levels were observed after vaccine therapy (p=0.006 post 1-2 cycles, p=0.003 post 3 cycles, p=0.03 post 4-7 cycles) and 42% of patients assayed developed CEA-specific T cell responses. Pre-treatment levels of myeloid-derived suppressor cells correlated with overall survival (p=0.04). Administration of IFN-α led to significantly increased OS (p=0.02) compared to vaccine alone. While the vaccine regimen produced no clinical responses, IFN-α administration was associated with improved survival.

Dose-enhanced combined priming regimens for refractory acute myeloid leukemia and middle-and-high-risk myelodysplastic syndrome: a single-center, retrospective cohort study.

ObjectiveTo assess chemotherapeutic regimens for refractory acute myeloid leukemia (AML) and middle-and-high-risk myelodysplastic syndrome (MDS).MethodsBetween 2004 and 2014, 44 patients with refractory AML and 36 patients with MDS were treated with new priming regimens (CHAG, CHTG, CHMG, or CTMG), and 77 patients with refractory AML and 52 patients with MDS were treated with conventional priming regimens (CHG or CAG). This was a single-center retrospective analysis of remission, adverse event, mortality, and survival. The capacity of clinical features (including the expression of co-stimulatory molecule B7.1 on tumor cells) to influence survival was assessed by multivariate Cox regression.ResultsComplete and partial remission rates (RRs) were significantly higher in AML patients treated with new regimens compared to conventional ones (68.2% vs 13.6%, P<0.05). Complete and partial remission were also significantly higher in patients with MDS treated with new regimens (55.6% vs 19.4%, P<0.05). However, although survival advantages were observed in the first year, the new regimens did not significantly improve 3-year overall survival (P>0.05). Patients administered the new regimens experienced more severe and sustained myelosuppression (P<0.05), but no severe adverse events or treatment-related deaths were observed. The rate of non-hematological side effects did not differ significantly between treatment regimens (P>0.05). Both RR and B7.1 expression were significantly higher in patients with AML-M2 and M5 (P<0.05).ConclusionThe new priming regimens improved the RR, lowered the recurrence rate, and improved survival in AML and middle-and-high-risk MDS, without significantly increasing adverse events.

Dendritic Cell-Secreted Cytotoxic T-Lymphocyte-Associated Protein-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7.

The remarkable functional plasticity of professional antigen-presenting cells (APCs) allows the adaptive immune system to respond specifically to an incredibly diverse array of potential pathogenic insults; nonetheless, the specific molecular effectors and mechanisms that underpin this plasticity remain poorly characterized. Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), the target of the blockbuster cancer immunotherapeutic ipilimumab, is one of the most well-known and well-studied members of the B7 superfamily and negatively regulates T cell responses by a variety of known mechanisms. Although CTLA-4 is thought to be expressed almost exclusively among lymphoid lineage hematopoietic cells, a few reports have indicated that nonlymphoid APCs can also express the CTLA-4 mRNA transcript and that transcript levels can be regulated by external stimuli. In this study, we substantially build upon these critical observations, definitively demonstrating that mature myeloid lineage dendritic cells (DC) express significant levels of intracellular CTLA-4 that they constitutively secrete in microvesicular structures. CTLA-4(+) microvesicles can competitively bind B7 costimulatory molecules on bystander DC, resulting in downregulation of B7 surface expression with significant functional consequences for downstream CD8(+) T-cell responses. Hence, the data indicate a previously unknown role for DC-derived CTLA-4 in immune cell functional plasticity and have significant implication for the design and implementation of immunomodulatory strategies intended to treat cancer and infectious disease.

Blocking B7-1/CD28 Pathway Diminished Long-Range Brain Damage by Regulating the Immune and Inflammatory Responses in a Mouse Model of Intracerebral Hemorrhage.

Acute brain injuries can activate bidirectional crosstalk between the injured brain and the immune system. The immune system, particularly T lymphocytes and cytokines, has been implicated in the progression of brain injury after intracerebral hemorrhage (ICH). Co-stimulatory molecules B7-1 (CD80)/B7-2 (CD86) binding cognate receptor provides a secondary signaling to T cell activation. The aim of our study was to explore the effects of anti-B7-1 antibody on the development and prognosis of cerebral hemorrhage and to investigate the possible underlying mechanism. Mice were inner canthus veniplex administered with anti-B7-1 antibody at 10min and 24h after ICH and sacrificed on the third day after ICH. Immune function was assessed via splenocyte proliferation assay and organism index, respectively. IFN-γ and IL-4 were detected by enzyme-linked immuno sorbent assay. The cerebral edema was evaluated via brain water content. The levels of autophagy and apoptosis related proteins were measured by western blotting analysis. In addition, functional outcome was studied with pole-climbing test and morris water maze. The mice were weighed on 0, 1, 3, 14 and 21days after ICH. The treatment with anti-B7-1 antibody significantly lowered immune function, and reduced the latency of water maze on 18 and 20days, the ratio of IFN-γ/IL-4 as well as body weight on day 3 after cerebral hemorrhage. Our study suggests that in the cerebral hemorrhage mice brain anti-B7-1 antibody may reduce long-range brain damage by reversing immune imbalance.

IMPS-36RNA NANOPARTICLE VACCINES RE-PROGRAM HOST-IMMUNITY AGAINST GLIOBLASTOMA INDUCING THERAPEUTIC ANTI-TUMOR ACTIVITY

INTRODUCTION: Glioblastoma (GBM) remains invariably associated with poor patient outcomes thus necessitating development of more targeted therapeutics. To circumvent the challenges associated with cellular therapeutics, we developed a novel treatment platform, which leverages the use of commercially available and clinically translatable nanoparticles (NPs) that can be combined with tumor derived RNA to peripherally activate T cells against GBM antigens. Although cancer vaccines have suffered from weak immunogenicity, we have advanced a NP vaccine formulation that can dramatically reshape a host's regulatory immune profile through combinatorial delivery of tumor RNA encoding for GBM antigens and RNAs encoding for immunomodulatory molecules to mediate long-lived T cell persistence. OBJECTIVE: We sought to assess if vaccination with amplified tumor derived RNA encapsulated in lipophilic NPs could be assembled to target antigen presenting cells (APCs) in vivo and induce therapeutic anti-tumor immunity in pre-clinical murine GBM models. RESULTS: We identified a clinically translatable NP formulation for the delivery of RNA to APCs that induces in vivo gene expression and preserves RNA stability over time. We showed that RNA-NPs induce global changes in B7 co-stimulatory molecules and MHC class II expression on CD11c expressing APCs. RNA-NPs vaccines increase systemic levels of monocyte chemotactic protein-1 (MCP-1) and increase the percentage of CD11c+ cells at target sites. White blood cells from target organs (spleen and livers) of vaccinated mice induce de novo antigen specific immune responses in naive mice. T cell immunity elicited by RNA-NP vaccination was superior to peptide vaccines in CFA, enhanced through co-encapsulation of RNA encoding GM-CSF or viral PAMPs (pathogen associated molecular patterns) from hepatitis C, and engendered therapeutic anti-tumor efficacy against a radiation and temozolomide resistant GBM murine model. CONCLUSION: Combinatorial RNA-NPs represent a novel therapeutic platform for GBM patients that can be commercialized as an ‘off the shelf’ vaccine to re-program host-immunity.

The research progress of costimulatory molecule B7 family in hematological malignancy

The research progress of costimulatory molecule B7 family in hematological malignancy

O75. B7 Costimulation and intracellular indoleamine-2,3-dioxygenase expression in peripheral blood of healthy pregnant and preeclamptic women

Introduction B7 costimulatory molecules are expressed on antigen presenting cells (APCs) and are important regulators of T cell activation. We investigated their role in the development of the systemic maternal immune tolerance during healthy pregnancy (HP) and preeclampsia (PE). Objectives We also aimed to investigate the intracellular expression of indoleamine-2,3-dioxygenase (IDO) and plasma levels of tryptophane (TRP), kynurenine (KYN) and kynurenic acid (KYNA), important molecules with immunoregulatory properties, in order to describe their potential contribution to the pregnancy-specific maternal immune tolerance. Methods We determined the frequency of activated (CD11b+) monocytes expressing B7-1, B7-2, B7-H1, and B7-H2, and that of T cells and CD4+ T helper cells expressing CD28, CTLA-4, PD-1, and ICOS in peripheral blood samples of 20 PE, 20 HP and 14 non-pregnant (NP) women using flow cytometry. We also examined the intracellular expression of IDO applying flow cytometry and plasma levels of TRP, KYN and KYNA using high-performance liquid chromatography. Results A significant increase in the prevalence of CD28+ T cells was observed in HP compared to NP women. At the same time a decrease was shown in the expression of CTLA-4 on these cells. The frequency of B7-1 and B7-2 expressing monocytes and that of IDO expressing T lymphocytes was lower in PE than in HP. The prevalence of IDO-expressing T cells and monocytes was higher in HP compared to NP women. Plasma KYN, KYNA and TRP levels were lower, while at the same time, the KYN/TRP ratio was higher in HP than in NP women. We found a positive correlation between the expression of B7-2 and IDO within activated monocytes. Conclusions Costimulation via CD28 may not contribute to the immunosuppressive environment, at least in the third trimester of pregnancy. The development of the pregnancy-specific immune tolerance in the mechanism of B7 costimulation may be more related to the altered expression of B7 proteins on APCs rather than that of their receptors on T cells. The elevated intracellular IDO expression in monocytes and T cells, as well as higher plasma enzymatic IDO activity are likely to contribute to the systemic immunosuppressive environment in the third trimester characteristic for healthy gestation. Lower expression of B7-1 and B7-2 proteins on peripheral monocytes in PE might indicate a secondary regulatory mechanism in response to the ongoing systemic maternal inflammation. Immunosuppression by IDO may play an important role in the pregnancy-specific immune tolerance, and might be a contributing factor in the pathogenesis of PE.

Mature dendritic cells secrete functionally active CTLA-4 in microvesicles (IRC7P.430)

Abstract Cytotoxic T-Lymphocyte Associated protein-4 (CTLA-4), the target of the blockbuster cancer immunotherapeutic ipilimumab, is a regulatory molecule of the B7 superfamily known to inhibit T-cell responses by a variety of different mechanisms. While CTLA-4 expression and function have been well-characterized in T-cells, very little is known about the presence and/or role of CTLA-4 in other hematopoietic lineages. Previous reports have indicated that myeloid lineage dendritic cells (DC) might express the CTLA-4 mRNA transcript and that transcript levels could be regulated by external stimuli. In the present study, we substantially build upon these critical observations, definitively demonstrating that mature DC express significant levels of intracellular CTLA-4 which are constitutively secreted in Rab5+Rab11- microvesicular structures. We further demonstrate that CTLA-4-containing microvesicles are capable of competitively binding B7 costimulatory molecules with significant functional consequences for downstream T-cell responses both in vitro and in vivo. The data indicate a previously-unknown role for DC-derived CTLA-4 in the physiologic regulation of T-cell responses and have significant implication for the design and implementation of immunomodulatory strategies intended to treat cancer and infectious disease.

Functional characterization of human T cell hyporesponsiveness induced by CTLA4-Ig.

During activation, T cells integrate multiple signals from APCs and cytokine milieu. The blockade of these signals can have clinical benefits as exemplified by CTLA4-Ig, which blocks interaction of B7 co-stimulatory molecules on APCs with CD28 on T cells. Variants of CTLA4-Ig, abatacept and belatacept are FDA approved as immunosuppressive agents in arthritis and transplantation, yet murine studies suggested that CTLA4-Ig could be beneficial in a number of other diseases. However, detailed analysis of human CD4 cell hyporesponsivness induced by CTLA4-Ig has not been performed. Herein, we established a model to study the effect of CTLA4-Ig on the activation of human naïve T cells in a human mixed lymphocytes system. Comparison of human CD4 cells activated in the presence or absence of CTLA4-Ig showed that co-stimulation blockade during TCR activation does not affect NFAT signaling but results in decreased activation of NF-κB and AP-1 transcription factors followed by a profound decrease in proliferation and cytokine production. The resulting T cells become hyporesponsive to secondary activation and, although capable of receiving TCR signals, fail to proliferate or produce cytokines, demonstrating properties of anergic cells. However, unlike some models of T cell anergy, these cells did not possess increased levels of the TCR signaling inhibitor CBLB. Rather, the CTLA4-Ig–induced hyporesponsiveness was associated with an elevated level of p27kip1 cyclin-dependent kinase inhibitor.

Cohort Study on GHA and New Combined Priming Chemotherapeutic Regimens in Treatment of Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome

To explore the clinical efficacy and adverse effects of GHA(G-CSF+homoharringtonin+cytarabine C) and new combined priming chemotherapeutic regimens(GHAA/GHTA) with high efficacy and low toxicity for treatment of relapsed and refractory acute myeloid leukemia(AML) and myelodysplastic syndrome(MDS), and to analyze the relation of above-mentioned regimens with the expression of co-stimuolating molecule B7.1. Standard GHA regimen consisting of G-CSF: 100 µg/(m2·d) subcutaneous injection, d 0-14; homoharringtonine: 1.0 mg/(m2·d) intravenous drip, d 1-14; Ara-C: 7.5-10 mg/(m2·d) subcutaneous injection, q12h, d 1-14. Other regimens as GHAA/GHTA were combined respectively with aclarubicin 20 mg d 1-7, or pirarubicin 20 mg d 1-7. 74 patients with refractory AML and 46 patients with MDS received these priming chemotherapy. The clinical efficacy and toxicity of above-mentioned priming chemotherapy were compared with 56 patients received routine chemotherapy (MA/TAE) respectively. And the expression of costimulatory molecule B7.1 on leukemia cells in patients of different subtypes was also detected by immunofluoressence and its relationship with clinical efficiency was explored. (1) for AML patients treated with priming chemotherapy, the total remission was 67.56% (CR 54.05%, PR 13.51%), which was much higher than that of patients received routine chemotherapy (P<0.05). The CR rate of AML-M2 and AML-M5 group (65.51%, 61.90% respectively) was much higher than that of AML other subtypes (P<0.05), and the longest remission period lasted for 4 years; (2) for MDS patients treated with priming chemotherapy, the total remission was 60.87% (CR 45.65%, PR 15.22%), which was also significantly higher than that of patients received routine chemotherapy (P<0.05); (3) in comparison with patients received standard GHA priming regimen, the remission rate of combined priming chemotherapy GHAA/GHTA was significantly higher both in patients with AML (85.18%) and MDS (81.25%); (4) side effects after chemotheropy, including granulocyte deficiency, thrombocytopenia and anemia etc, lasted for 7-14 days; the severe infection rate was 1%, there were no severe bleeding, digest effect and damage of function in heart, liver and kidney. The therapy-related mortality was zero. Compared with routine chemotherapy, priming chemotherapy proved significantly safe and effective (P<0.05); (5) the expression rate of costimulatory molecule B7.1 showed large variance between AML and MDS, it was higher in AML-M2/AML-M5 and lower in AML of other subtypes (P<0.05). In the same case, B7.1 expression was positive correlated with efficiency of priming chemotherapy. GHA priming chemotherapy, as well as other combination regimens GHAA/GHTA, are well-tolerated, effective regimens for refractory AML and advanced MDS, without severe side effects and therapy-related mortality. Especially the new regimens GHAA/GHTA has better efficacy, which are proved more efficient than conventional GHA. Efficiency of priming chemotherapy is positive correlated with B7.1 expression, its mechanism will be further explored.