Specific MHC Class Research Articles

G462(P) Capturing t–cell receptors. a potential new modality for targeting hepatic tumours and post–transplantation lymphoproliferative disease (ptld)

Background The immune system plays an important part in recognising infection and diseases including cancer. Malignant cells express specific proteins on their cell surface. It is widely believed that it is these proteins that the immune system uses to recognise tumours and eventually eradicate them. When this process goes wrong, a tumour forms. The immune response to cancers in general is widely accepted to play an important role in control of tumorigenic processes, by using tumour infiltrating lymphocytes. How this process works is still being established, however there remains a potential target for cancer specific immunomodulatory treatment regimens. Aim (1) To identify tumour specific MHC class I phosphopeptide antigens on lymphoblastoid cell lines LCL’s (an in vitro model for PTLD) as well as hepatic tumour tissues. (2) T-cells are immune cells which are notoriously difficult to maintain in long–term culture and as a result it is difficult to establish an ‘off the shelf’ T-cell product, however the aim of this project was to explore potential modalities for capturing the T-cell receptor (TCR), important in recognising tumour specific antigens and the resultant product could be used to establish a non patient–specific, but tumour specific product. Patients and methods Paediatric and adult patients were identified with hepatic malignancy and consented as per current policy. Cells were isolated and tumour specific phosphopeptide antigens were identified. These provide the targets for T-cells, and more specifically TCR’s. Having identified these antigens, modalities have been explored for expanding these cells. Hybridoma technology is long established in immortalising B-cells, and this study aims to explore its potential with immortalisation of T-cells. Results A number of novel phosphopeptide antigens have been identified both in vitro as well as on patient tissues. This information has been used to identify potential T-cell targets and by formation of hybridomas we have established a method for expanding specific T-cell’s in vitro. Although these hybridomas are currently unstable due to their tetraploid status, we aim to modify this protocol further to allow for stable expansion of hybridoma cells which possess the relevant TCR motif. Conclusions Identifying a modality for expanding cells with a specific TCR repertoire clearly allows us to target tumour specific phosphopeptide antigens and has the potential to be developed as an immunomodulatory therapy in patients with hepatic tumours or PTLD.

Accurate quantitation of MHC-bound peptides by application of isotopically labeled peptide MHC complexes.

Knowledge of the accurate copy number of HLA class I presented ligands is important in fundamental and clinical immunology. Currently, the best copy number determinations are based on mass spectrometry, employing single reaction monitoring (SRM) in combination with a known amount of isotopically labeled peptide. The major drawback of this approach is that the losses during sample pretreatment, i.e. immunopurification and filtration steps, are not well defined and must, therefore, be estimated. In addition, such losses can vary for individual peptides. Therefore, we developed a new approach in which isotopically labeled peptide-MHC monomers (hpMHC) are prepared and added directly after cell lysis, i.e. before the usual sample processing. Using this approach, all losses during sample processing can be accounted for and allows accurate determination of specific MHC class I-presented ligands. Our study pinpoints the immunopurification step as the origin of the rather extreme losses during sample pretreatment and offers a solution to account for these losses. Obviously, this has important implications for accurate HLA-ligand quantitation. The strategy presented here can be used to obtain a reliable view of epitope copy number and thus allows improvement of vaccine design and strategies for immunotherapy.

Strong vaccine-induced CD8 T-cell responses have cytolytic function in a chimpanzee clearing HCV infection.

A single correlate of effective vaccine protection against chronic HCV infection has yet to be defined. In this study, we analyzed T-cell responses in four chimpanzees, immunized with core-E1-E2-NS3 and subsequently infected with HCV1b. Viral clearance was observed in one animal, while the other three became chronically infected. In the animal that cleared infection, NS3-specific CD8 T-cell responses were observed to be more potent in terms of frequency and polyfunctionality of cytokine producing cells. Unique to this animal was the presence of killing-competent CD8 T-cells, specific for NS31258–1272, being presented by the chimpanzee MHC class I molecule Patr-A*03∶01, and a high affinity recognition of this epitope. In the animals that became chronically infected, T-cells were able to produce cytokines against the same peptide but no cytolysis could be detected. In conclusion, in the animal that was able to clear HCV infection not only cytokine production was observed but also cytolytic potential against specific MHC class I/peptide-combinations.

γδ T cells and their potential for immunotherapy.

Vγ9Vδ2 (also termed Vγ2Vδ2) T cells, a major human peripheral blood γδ T cell subset, recognize microbial (E)-4-hydroxy-3-methylbut-2-enyl diphosphate and endogenous isopentenyl diphosphate in a TCR-dependent manner. The recognition does not require specific accessory cells, antigen uptake, antigen processing, or MHC class I, class II, or class Ib expression. This subset of T cells plays important roles in mediating innate immunity against a wide variety of infections and displays potent and broad cytotoxic activity against human tumor cells. Because γδT cells express both natural killer receptors such as NKG2D and γδ T cell receptors, they are considered to represent a link between innate and adaptive immunity. In addition, activated γδ T cells express a high level of antigen-presenting cell-related molecules and can present peptide antigens derived from destructed cells to αβ T cells. Utilizing these antimicrobial and anti-tumor properties of γδ T cells, preclinical and clinical trials have been conducted to develop novel immunotherapies for infections and malignancies. Here, we review the immunological properties of γδ T cells including the underlying recognition mechanism of nonpeptitde antigens and summarize the results of γδ T cell-based therapies so far performed. Based on the results of the reported trials, γδ T cells appear to be a promising tool for novel immunotherapies against certain types of diseases.

Phase I/II Trial Of Immucin a Pan HLA, Anti-MUC1 Signal Peptide Vaccine, In Multiple Myeloma Patients With Residual Disease Or Progression Following Autologous Stem Cell Transplantation

BackgroundAnti-multiple myeloma (MM) vaccinations have recently emerged as a strategy to eradicate MM cells in patients with residual tumor mass. However, most vaccination studies reported transient immune responses, involving CD8+ T-cells or antibodies responses in patients with predefined MHC repertoire.Signal peptide (SP) domains, a 13-50 amino acid long peptides, have been demonstrated to present an exceptionally high number of antigen specific MHC class I, class II and B-cell epitopes per sequence length. Therefore, they are suitable to induce a robust immune response combining CD8+, CD4+ T-cells and antibodies across HLA barrier. Following this rationale we developed ImMucin, A 21-mer therapeutic vaccine encoding the entire SP domain of the MUC1 tumor-associated antigen (TAA), which is over expressed by most carcinomas and hematological tumors including MM. MethodsA phase I/II study to assess the safety (primary endpoint) and efficacy (secondary endpoint) of ImMucin was conducted in MM subjects with stable or progressive residual asymptomatic MUC1+ disease who have already had upfront auto-SCT. Patients recruited into the study initially received 6 bi-weekly vaccination of intra-dermal 100ug ImMucin plus 250ug hGM-CSF. Based on immune and clinical response eligible patient could receive additional 6 bi-weekly vaccination of 100ug or 250ug ImMucin plus 250ug hGM-CSF.Immunomonitoring analysis included; ImMucin-specific IFN-g production in CD4+ and CD8+ T-cells, HLA-A2.1 tetramer binding in CD8+ T-cells, proliferation and antibodies production. Positive response in these assays vs. pre vaccination levels were; x2 increase in IFN-g and >0.5%+ T-cells, any increase in tetramer binding levels, x2 increase and stimulation index (SI)>2 and any increase in anti-ImMucin titer respectively. Results15 patients, median age 58 years participated in the study. The median number of prior therapies was 2. Median time from diagnosis and from auto-SCT to vaccination were 25 months (12-143) and 15 months (3-134) respectively. 9 patients had post auto-SCT residual MM and 6 had an evidence for biochemical progression. 11 patients completed the vaccination program while 4 discontinued the program due to PD. ImMucin was shown to be safe with no vaccine related grade ≥3 adverse events (AEs). Patients had temporary, grade 1,2 AEs, including injection related rash (n=8), fatigue (n=6), weakness, self-resolving fever and muscle pain (n=5).IFN-g production response to ImMucin was highly positive in all patients with 4-80 folds increase for CD4+ and 18-80 folds increase for CD8+. Mean baseline and peak post vaccination levels for ImMucin-specific CD4+ T-cells were 0.21% and 4.07%, respectively (P<0.001, t-test) and of CD8+ T-cells 0.21% and 11.76%, respectively (P<0.0001, t-test).All 4 HLA-A2.1 positive patients demonstrated < x2 increase in tetramer-positive cells after vaccination, with mean levels of 0.33% and 2.11% at baseline and peak post vaccination, respectively. Proliferative response to ImMucin increased signifcantly in all patients with mean baseline and peak of 3.24 and 15.92, respectively (P<0.024, t-test). An increased ImMucin humoral response of up to 40 folds from baseline levels was detected in 10 of 15 patients (66.6%). The vaccination efficacy was further confirmed by CD8+ T-cells and antibody mediated cytotoxicity against autologous BM cells. In these assays MUC1 control epitope didn't show positive activation.Since ImMucin target MUC1's SP domain which is not part of soluble MUC1 (sMUC1), a reduction in sMUC1 following vaccination can reflect a specific tumor destruction. A reduction of up to 17 folds, 5129.44 pg/ml as a mean levels at baseline vs. 792.33 pg/ml at maximal response (P<0.002, t-test) was detected in 9 of 10 patients, initially presenting abnormally elevated sMUC1 levels.Response assessment revealed disease stabilization in the majority of the patients, lasting for up to 29 months post study completion (ongoing follow up), including in patients entering study with PD. ConclusionsThe ImMucin therapeutic vaccine is directed to the less studied SP domain of MUC1 TAA. The current phase I/II results confirmed its high safety profile. ImMucin induced a robust CD8+ and CD4+ specific T-cell response in all patients and a marked anti-tumor humoral response. Further studies exploring ImMucin's efficacy in patients with residual myeloma are under development. Disclosures:Carmon:Vaxil BioTherapeutics Ltd. : Employment, Equity Ownership, Patents & Royalties. Avivi:Vaxil BioTherapeutics Ltd. : Consultancy. Riva:Vaxil BioTherapeutics Ltd. : Employment. Shapira:Vaxil BioTherapeutics Ltd. : Consultancy.

Monitoring the immune response to vaccination with an inactivated vaccine associated to bovine neonatal pancytopenia by deep sequencing transcriptome analysis in cattle

Bovine neonatal pancytopenia (BNP) is a new fatal, alloimmune/alloantibody mediated disease of new-born calves induced by ingestion of colostrum from cows, which had been vaccinated with a specific vaccine against the Bovine Virus Diarrhoea Virus (BVDV). The hypothesis of pathogenic MHC class I molecules in the vaccine had been put up, but no formal proof of specific causal MHC class I alleles has been provided yet. However, the unique features of the vaccine obviously result in extremely high specific antibody titres in the vaccinated animals, but apparently also in further molecules inducing BNP. Thus, a comprehensive picture of the immune response to the vaccine is essential. Applying the novel approach of next generation RNA sequencing (RNAseq), our study provides a new holistic, comprehensive analysis of the blood transcriptome regulation after vaccination with the specific BVDV vaccine. Our RNAseq approach identified a novel cytokine-like gene in the bovine genome that is highly upregulated after vaccination. This gene has never been described before in any other species and might be specific to ruminant immune response. Furthermore, our data revealed a very coordinated immune response to double-stranded (ds) RNA or a dsRNA analogue after vaccination with the inactivated single-stranded (ss) RNA vaccine. This would suggest either a substantial contamination of the vaccine with dsRNA from host cells after virus culture or a dsRNA analogue applied to the vaccine. The first option would highlight the potential risks associated with virus culture on homologous cells during vaccine production; the latter option would emphasise the potential risks associated with immune stimulating adjuvants used in vaccine production.

Death Receptor–Independent Activation-Induced Cell Death in Human Melanoma Antigen–Specific MHC Class I–Restricted TCR-Engineered CD4 T Cells

Engaging CD4 T cells in antitumor immunity has been quite challenging, especially in an Ag-specific manner, because most human solid tumors usually do not express MHC class II molecules. We have recently shown that human CD4 T cells engineered to express a human melanoma-associated antigenic epitope, MART-127-35, specific MHC class I-restricted transgenic TCR function as polyfunctional effectors that can exhibit a helper as well as cytolytic effector function, in an epitope-specific and MHC class I-restricted manner (Chhabra et al. 2008. J. Immunol. 181: 1063-1070; Ray et al. 2010. Clin. Immunol. 136: 338-347). TCR-engineered (TCReng) CD4 T cells therefore have translational potential, and clinical trials with MHC class I TCReng CD4 T cells are under way. In this study, we show that although TCReng CD4 T cells could be useful in cancer immunotherapy, they are also susceptible to epitope-specific activation-induced cell death (AICD). We also show that the AICD in TCReng CD4 T cells is a death receptor-independent process and that JNK and p53 play critical roles in this process as pharmacological inhibitors targeting JNK activation and p-53-mediated transcription-independent mitochondria-centric death cascade rescued a significant fraction of TCReng CD4 T cells from undergoing AICD without affecting their effector function. Our data offer novel insights toward AICD in TCReng CD4 T cells and identify several potential targets to interfere with this process.

Mesoporous Silicon Microparticles Enhance MHC Class I Cross-Antigen Presentation by Human Dendritic Cells

The mesoporous silicon microparticles (MSMPs) are excellent vehicles for releasing molecules inside the cell. The aim of this work was to use MSMPs to deliver viral specific MHC class I restricted epitopes into human antigen presenting cells (monocyte derived dendritic cells, MDDCs) to facilitate their capture, processing, and presentation to CD8+ (cytotoxic) T lymphocytes. We show for the first time that MSMPs vehiculation of antigenic peptides enhances their MHC class I presentation by human MDDCs to CD8 T lymphocytes.

Endogenous T Cell Receptors Drive Non-Specific Suppression by Foxp3 MHC Class I-Restricted T Cell Receptor-Transduced Polyclonal CD4+ T Cells in Models of Graft-Versus-Host Disease.

AbstractAbstract 3152Alloreactive immune responses directed against malignant cells in recipients of allogeneic hematopoietic stem cell transplants are able to cure patients with hematological cancers. However, such immune responses may cause severe morbidity when directed against healthy recipient tissue, resulting in graft-versus-host disease (GvHD).Naturally occurring regulatory T cells (Tregs) are CD4+ T cells characterized by their expression of the transcription factor Foxp3. Whilst adoptively transferred polyclonal Tregs suppress GvHD in several murine models, their lack of specificity may compromise beneficial immunity against malignancy or infection. The generation of MHC class I-restricted, alloantigen-specific Tregs would allow them to recognize antigen presented directly on GvHD target tissues, concentrating their sites of activation at these tissues and possibly reducing the potential for non-specific immune suppression.We have generated ‘converted' Tregs through retroviral transfer of genes encoding Foxp3 and specific MHC class I-restricted T cell receptors (TCRs) into polyclonal conventional CD4+ T cells. We used the 2C-TCR, which recognizes the MHC class I molecule H-2Ld, expressed in Balb/c and other H-2d mice, in complex with the ubiquitously expressed peptide p2Ca; and the MH-TCR, which recognizes the MHC class I molecule H-2Db, expressed in B6 and other H-2b mice, in complex with the male peptide WMHHNMDLI.In vitro, Foxp3 2C-TCR-transduced B6 polyclonal CD4+ T cells were hyporesponsive to stimulation and suppressed the alloreactive proliferative response of B6 CD4+ and CD8+ T cells to Balb/c splenocytes, consistent with the acquisition of regulatory function. When adoptively transferred to lethally irradiated Balb/c recipients of MHC-mismatched B6 bone marrow and conventional T cells, Foxp3 2C-TCR-transduced B6 polyclonal CD4+ T cells significantly reduced early proliferation of donor T cells, weight loss and GvHD score in the recipients. Similarly, polyclonal CD4+ T cells transduced with Foxp3 and the MH-TCR caused marked suppression of allogeneic responses both in vitro and in vivo. However, while both the 2C-TCR and the MH-TCR conferred specificity to their cognate antigens in vitro, the potent suppression in these in vivo models was independent of the cognate antigen for the transduced TCRs. This non-specific suppression was markedly reduced when class I-restricted TCRs were transduced into OT-II Rag1-/- CD4+ T cells that are transgenic for a single endogenous TCR.These findings demonstrate an important role for the endogenous TCRs in driving non-specific suppression by polyclonal CD4+ T cells transduced with Foxp3 and class I-restricted TCRs, and suggest that strategies to downregulate endogenous TCRs will be required to achieve antigen-specific suppression in TCR gene-modified regulatory T cells. Disclosures:No relevant conflicts of interest to declare.

Degenerate Recognition of MHC Class I Molecules with Bw4 and Bw6 Motifs by a Killer Cell Ig-like Receptor 3DL Expressed by Macaque NK Cells

The killer cell Ig-like receptors (KIRs) expressed on the surface of NK cells recognize specific MHC class I (MHC-I) molecules and regulate NK cell activities against pathogen-infected cells and neoplasia. In HIV infection, survival is linked to host KIR and MHC-I genotypes. In the SIV macaque model, however, the role of NK cells is unclear due to the lack of information on KIR-MHC interactions. In this study, we describe, to our knowledge, the first in-depth characterization of KIR-MHC interactions in pigtailed macaques (Macaca nemestrina). Initially, we identified three distinct subsets of macaque NK cells that stained ex vivo with macaque MHC-I tetramers loaded with SIV peptides. We then cloned cDNAs corresponding to 15 distinct KIR3D alleles. One of these, KIR049-4, was an inhibitory KIR3DL that bound MHC-I tetramers and prevented activation, degranulation, and cytokine production by macaque NK cells after engagement with specific MHC-I molecules on the surface of target cells. Furthermore, KIR049-4 recognized a broad range of MHC-I molecules carrying not only the Bw4 motif, but also Bw6 and non-Bw4/Bw6 motifs. This degenerate, yet peptide-dependent, MHC reactivity differs markedly from the fine specificity of human KIRs.

Functional Differences Exist between TNFα Promoters Encoding the Common −237G SNP and the Rarer HLA-B*5701-Linked A Variant

A large body of functional and epidemiological evidence have previously illustrated the impact of specific MHC class I subtypes on clinical outcome during HIV-1 infection, and these observations have recently been re-iterated in genome wide association studies (GWAS). Yet because of the complexities surrounding GWAS-based approaches and the lack of knowledge relating to the identity of rarer single nucleotide polymorphism (SNP) variants, it has proved difficult to discover independent causal variants associated with favourable immune control. This is especially true of the candidate variants within the HLA region where many of the recently proposed disease influencing SNPs appear to reflect linkage with ‘protective’ MHC class I alleles. Yet causal MHC-linked SNPs may exist but remain overlooked owing to the complexities associated with their identification. Here we focus on the ancestral TNFα promoter −237A variant (rs361525), shown historically to be in complete linkage disequilibrium with the ‘protective’ HLA-B*5701 allele. Many of the ancestral SNPs within the extended TNFα promoter have been associated with both autoimmune conditions and disease outcomes, however, the direct role of these variants on TNFα expression remains controversial. Yet, because of the important role played by TNFα in HIV-1 infection, and given the proximity of the −237 SNP to the core promoter, its location within a putative repressor region previously characterized in mice, and its disruption of a methylation-susceptible CpG dinucleotide motif, we chose to carefully evaluate its impact on TNFα production. Using a variety of approaches we now demonstrate that carriage of the A SNP is associated with lower TNFα production, via a mechanism not readily explained by promoter methylation nor the binding of transcription factors or repressors. We propose that the −237A variant could represent a minor causal SNP that additionally contributes to the HLA-B*5701-mediated ‘protective’ effect during HIV-1 infection.

Quantitative immunoproteomics analysis reveals novel MHC class I presented peptides in cisplatin-resistant ovarian cancer cells

Platinum-based chemotherapy is widely used to treat various cancers including ovarian cancer. However, the mortality rate for patients with ovarian cancer is extremely high, largely due to chemo-resistant progression in patients who respond initially to platinum based chemotherapy. Immunotherapy strategies, including antigen specific vaccines, are being tested to treat drug resistant ovarian cancer with variable results. The identification of drug resistant specific tumor antigens would potentially provide significant improvement in effectiveness when combined with current and emerging therapies. In this study, using an immunoproteomics method based on iTRAQ technology and an LC-MS platform, we identified 952 MHC class I presented peptides. Quantitative analysis of the iTRAQ labeled MHC peptides revealed that cisplatin-resistant ovarian cancer cells display increased levels of MHC peptides derived from proteins that are implicated in many important cancer pathways. In addition, selected differentially presented epitope specific CTL recognize cisplatin-resistant ovarian cancer cells significantly better than the sensitive cells. These over-presented, drug resistance specific MHC class I associated peptide antigens could be potential targets for the development of immunotherapeutic strategies for the treatment of ovarian cancer including the drug resistant phenotype.

Regulatory T Cells Enhance the Allogeneic Activity of Endothelial-Specific Cytotoxic T Lymphocytes – Protective Effect of Defibrotide

Abstract 4693 BACKGROUND:Damage to the vascular endothelium is the primary event of transplant related complications and often precedes loss of organ function. Depending on the amount of co-stimulatory signals, endothelial cells can either act as stimulating or inhibiting antigen presenting cells (APC). On the other hand, numerous data indicate that CD4+CD25+FoxP3+ T cells (Treg cells) can attenuate alloresponses of conventional T lymphocytes against classical APC and thus qualify for clinical use in various transplant settings. However, it is unknown whether Treg cells also influence T cell – endothelial cell interactions. Defibrotide (DF) is a polydisperse mixture of single-stranded deoxyribonucleotides with anti-thrombotic and anti-inflammatory activity, known to modulate the antigenicity of vascular endothelial cells. METHODS:CD8+ T lymphocytes (CTL) were isolated by magnetic microbead separation of peripheral blood mononuclear cells (PBMC) from healthy human blood donors and stimulated with mito-inactivated cells of a human microvascular endothelial cell line (CDC/EU.HMEC-1, further referred to as EC) and other primary and transformed micro- and macrovascular ECs for 7 days in the presence of interleukin 2 (IL-2). Treg cells from the CTL donor were prepared by CD4 (untouched) and a double CD25 microbead separation as well as a CD127bright depletion, followed by anti-CD3/CD28 expansion in the presence of IL-2 and a phenotypic quality control. Treg cells were added to the CTL-EC co-culture (1:1:1) prior to 51Cr release or flow cytometric cytotoxicity assays. Additionally, Treg cells were also tested for their capacity to influence CTL lysis of Epstein-Barr-Virus-transformed B-LCL, which as classical APC were HLA-matched to the HMEC. Furthermore, EC targets were incubated in the presence or absence of DF (25μM) for 24 hrs to assess the drug's protective function on the allogenicity of EC. RESULTS:EC-stimulated CTL showed a specific MHC class I-restricted target lysis. Addition of Treg cells prior to the cytotoxicity assay and during the afferent immune phase surprisingly increased EC lysis by CTL. In contrast, Treg cells alone did not show any lytic activity against EC. As a control, conventional CD4+CD25- T cells did not influence CTL activity either. Treg cell-mediated enhancement was endothelial-specific, since B-LCL lysis was not influenced. Further subpopulation analysis revealed the existence of CD8+/CD28-/CD57+ CTL, requiring cell-to-cell contact with Treg cells for their increased activity towards EC. Importantly, DF could almost fully protect EC against lysis by allogeneic CD28- CTL and the Treg cell-mediated enhancement. Of note, DF exclusively protected EC and did influence T cell function nor viability, suggesting a strong tropism for the endothelial cell type. CONCLUSION:There is no doubt about the potential therapeutic efficacy of Treg cells to ameliorate outcome of allogeneic transplants, but the endothelium might require additional protective interventions to prevent specific alloreactivity, such as DF. Disclosures:Eissner:Gentium, Sp.A.: Consultancy. Iacobelli:Gentium SpA: Employment.

SIV-infected Chinese-origin rhesus macaques express specific MHC class I alleles in either elite controllers or normal progressors

SIV-infected Chinese-origin rhesus macaques express specific MHC class I alleles in either elite controllers or normal progressors

Cytotoxic CD8α + leucocytes have heterogeneous features in antigen recognition and class I MHC restriction in grouper

CD8 is a membrane glycoprotein found primarily on the surface of T lymphocytes such as cytotoxic T lymphocytes (CTL), natural killer cells (NK) and γδ T lymphocytes. It helps T lymphocytes to kill the infected cells that presents microbial antigen on the cell surface. However, analysis of fish cellular immunity has been limited because of the lack of CD8 antibodies in grouper. In this present study, we cloned full-length CD8α cDNAs from orange-spotted grouper ( Epinephelus coioides), an important fish species economically. The deduced protein of CD8α contained 227 amino acid residues in length and included one signal peptide, Ig superfamily V domain, hinge region, transmembrane domain, cytoplasmic tail and conserved binding motif associated with tyrosine kinase p56 lck. The molecular weight of the mature protein was estimated at 22.5 kDa and pI at 9.55. Phylogenetically, the predicted grouper CD8α protein was similar to CD8α from other marine fish species in which the identity was 50–60%. Real-time PCR revealed that CD8α transcript was constitutively expressed in thymus, head kidney, gill, spleen, gut and peripheral blood leucocyte (PBL); and the highest expression in thymus. CD8α transcript in the spleen of fish injected with nervous necrosis virus (NNV) was significantly up-regulated at 4 days post-injection compared to the untreated fish. Rabbit antiserum prepared against recombinant CD8α protein was able to recognize specifically the subset lymphocytes which have a diameter of 7 μm, a high nucleus/cytoplasm ratio and a ring-shaped cytoplasm. The cytotoxicity of CD8α + lymphocytes at one-week post-NNV infection was enhanced significantly against NNV-infected autologous fin cells in comparison with NNV-infected allogeneic or RSIV-infected autologous fin cells. Flow cytometry analysis revealed that both the number and mean fluorescence intensity (MFI) of CD8α + PBL were significantly increased at 7 days post-NNV infection. The specific cytotoxicity and MHC class I restriction of the lymphocytes sorted by rCD8α antibody are properties that can be attributed to CTL. In addition, low level of cytotoxicity was found in PBL against allogeneic targets as well as CD8α + effectors killed autologous targets nonspecifically, implicated presence of cytotoxic T subsets, possibly nonspecific cytotoxic cells (NCC) and γδ T lymphocytes, without MHC class I restriction. In conclusion, grouper cytotoxic CD8α + PBL have heterogeneous features in specific antigen recognition and class I MHC restriction.

The CD8+ T cell response to Brucella melitensis during acute vs. chronic infection (40.24)

Abstract In areas of endemic brucellosis, prevention of infection via vaccine would be far more cost-effective than the World Health Organization suggested regimen of antibiotics. As with other intracellular organisms, subunit or killed vaccines present very limited efficacy. The rationale for an improved Brucella melitensis vaccine requires a greater understanding of the host-pathogen relationship. Using bioinformatics coupled with practical experiments in the BALB/c model of brucellosis, we have identified two Brucella epitopes and show their capacity to elicit specific CD8+ T cells that kill effectively in vivo. Additionally, as seen by microarray, gene transcripts that contain these epitopes are significantly and differentially expressed following Brucella infection of macrophages. Specific CD8+ T cells are present and functional following infection; however, reactivation of disease still occurs in up to 30% of human patients after antibiotic treatment. Our current work using Brucella specific MHC Class I tetramers focuses on the presence and persistence of CD8+ T cell memory during chronic brucellosis. Investigation of the functional potential of this memory fraction of CD8+ T cells will fill a gap in our understanding of the immunological response to this often asymptomatic, persistent infection.

Activation Induced Cell Death (AICD) of Human Melanoma Antigen Specific MHC Class I TCR Engineered CD8 T Cells is an Intrinsic Process That Involves JNK and p53

Activation Induced Cell Death (AICD) of Human Melanoma Antigen Specific MHC Class I TCR Engineered CD8 T Cells is an Intrinsic Process That Involves JNK and p53

Vaccination of ponies with the IE gene of EHV-1 in a recombinant modified live vaccinia vector protects against clinical and virological disease

The control of EHV-1 infection by cytotoxic T-cell responses (CTL) via a reduction in cell associated viremia remains an important goal in horses. Unfortunately, current vaccines are inefficient at inducing these responses. We have identified the immediate early (IE) gene of EHV-1 as a potent stimulator of virus-specific CTL responses in ponies expressing a specific MHC class I serological haplotype (A3/B2). This study was designed to determine if vaccination of A3/B2 MHC I positive ponies with the IE gene could induce protection and immune responses associated with cell mediated immunity.Ponies expressing the MHC-I A3/B2 haplotype (A3/B2 vaccinates) and ponies with a different MHC I haplotype (either non-A3 vaccinates or A3-non-B2 vaccinates) were vaccinated with a recombinant modified vaccinia Ankara (rMVA) vector expressing the IE gene on 3 occasions and vaccinates and unvaccinated controls were challenge infected 8 weeks after the last vaccination. Interferon gamma (IFN-γ) mRNA and antibody titers were determined throughout the study and clinical signs, nasal virus shedding and viremia were determined following challenge infection.Vaccination of A3/B2 vaccinates conferred significant clinical protection and a significant reduction in EHV-1 viremia. IFN-γ mRNA increased significantly following vaccination in the A3/B2 vaccinates. Antibody titers remained low until after challenge infection, indicating that no accidental field acquired or recrudescent EHV-1 infection had occurred.In summary, this is an important study showing that vaccination of ponies with the EHV-1 IE protein provides not only reduction in clinical disease but also reduction of cell associated viremia, which is a prerequisite for the prevention of abortion and neurological disease.

Identification of Low Frequency Multiple Novel Aspergillus Fumigatus Specific MHC Class II Epitopes and Rapid Generation of An Aspergillus-Specific T-Cell Product Based On Activation Dependent Expression of CD154.

Abstract 1170Poster Board I-192Despite new antifungal drugs, invasive aspergillosis (IA) remains a major cause of morbidity and mortality in patients undergoing myeloablative chemotherapy and allogeneic stem cell transplantation. Invasion of Aspergillus sp. in immuncompetent individuals is primarily controlled by neutrophils, phagocytes and pathogen-specific TH1 CD4+ cells. Therefore, adoptive transfer of Aspergillus-specific CD4+ T-cells could protect patients at risk from IA. Favorable candidates for such an approach are GPI-anchored antigens, which have demonstrated induction of protective adaptive immunity in murine studies.To implement Aspergillus-specific CD4+ T-cells into the clinical setting, we aimed (i) to define which GPI-anchored antigen induces TH1 response, (ii) to map several epitopes and (iii) to generate large amounts of Aspergillus-specific TH1 cells within a short period.Several recombinant proteins were either synthesized or were received by J-P Latge. Amongst all tested proteins, only CRF-1 induced repeatedly TH1 response in healthy individuals.To identify Aspergillus-specific MHC class II epitopes, we mapped peripheral blood mononuclear cells (PBMC) of healthy individuals with a custom made peptide library of CRF-1 consisting of 95 overlapping 15mer peptides. The library was divided into a complete pool, subpools of 10 and 95 single peptides. No precursor frequencies were detected in interferon-gamma (IFN-g)-ELISPOT using the complete and sub-pools. After 7 days of stimulation with subpools, 9 peptides were identified producing high amount of IFN-g in at least 3 donors with different MHC class II alleles (n=6). CD4+ T-cells clones of one peptide were then established by limited dilution cloning. Restriction to DRB1*0401 was identified using LCLs and a tetramer was generated. The peptide-specific T-cell clones showed functional activity against ethanol-inactivated fungus or fungal extracts presented by dendritic cells.To generate Aspergillus-specific TH1 cell lines, we stimulated PBMC with the MHC class II DRB1*0401 restricted peptide. After 7 days of in vitro stimulation (IVS) with interleukin (IL)-2 5U/ml added every other day, tetramer staining was between 0.3 and 11% of CD4+ cells (mean 4.2%, n=5). After 14 days of IVS with 1 restimulation of autologous peptide-pulsed monocytes at a responder: stimulator ratio of 5:1 and IL-7 and IL-15 10ng/ml added after day 7, mean percentages of tetramer staining increased to 37% (range 20-57%, n=4) and absolute cell counts were duplicated. The expansion process was further optimized using IFN-g capture assay and CD154+ MicroBead Kit (Miltenyi). In both assays, PBMC were stimulated for 16 hours, separated by magnetic beads and co-cultured with irradiated autologous PBMC for 14 days using IL-2 5U/ml till day 7 and IL-7 and -15 10ng/ml thereafter. We were unable to expand specific CD4+ cells by IFN-g capture assay in 2 of 3 donors. In contrast, we found that Aspergillus-specific CD4+ cells selected by CD154+ showed comparable tetramer specificity and expansion but higher functional activity in intracellular cytokine assay and IFN-g ELISA than CD4+ cell lines generated from the same donor using the restimulation protocol. The CD4+ cell lines generated by CD154+ expression showed proliferative capacity in CFSE when restimulated with peptides and functional activity in IFN-g ELISA against fungal extracts (n=4).To generate Aspergillus-specific TH1 cell lines recognizing multiple MHC class II epitopes we stimulated PBMC with the previously identified 9 peptides of CRF-1 protein. After 14 days of expansion using CD154+ MicroBead Kit, we measured high IFN-g response towards 4 of 9 peptides (n=3). We are generating T-cell clones and will characterize their HLA-restriction.In summary, we have identified an immunodominant Aspergillus fumigatus protein including 9 MHC class II epitopes. One epitope was characterized specific for an abundant MHC class II allele. CD4+ TH1 cells specific for this epitope can be activated by dendritic cells after uptake of whole fungi or fungal extracts. Furthermore, we have established a GMP-applicable protocol for rapid generation (<14d) of CD4+ T-cell lines specific for Aspergillus fumigatus with low precursor frequency using CD154+ separation. These CD4+ T-cell lines demonstrate functional activity against peptides and fungal extracts that could be prophylactically administered to high risk patients. Disclosures:No relevant conflicts of interest to declare.

Phase 1 study of infusion of HER-2/neu specific T cells in patients with advanced stage HER-2/neu overexpressing cancers who have received a HER-2/neu vaccine (41.3)

Abstract Adoptive T cell therapy has great potential for the treatment of patients with advanced-stage cancers. We have previously reported that HER-2/neu (HER2) specific T cell expansion from peripheral blood mononuclear cells (PBMN) can be greatly facilitated by in vivo antigen specific MHC class II epitope-priming. To test whether vaccine primed T cells could be administered to patients, we assessed the safety and feasibility of infusion of ex vivo expanded HER2 specific T cells in patients who have advanced HER2 overexpressing cancers. Three escalating doses of T cells were given at 10 day intervals with cyclophosphamide used for host lymphodepletion prior to the first dose of T cells. To date, 5 of 10 subjects have been enrolled. The maximal T cell doses infused were 1x109- 41x109 cells (median 10x109) with and average 35% of CD4+ and 60% of CD8+ T cells. IFN-gamma ELISPOT was used to monitor antigen specific T cells in both the infused product and to track the cells in vivo. HER2 specific T cells could be generated at high levels in vivo after infusion. Moreover, the magnitude of HER2 specific CD4+ and CD8+ T cells post-infusion was maintained in responding patients, with precursor frequencies 1:253 and 1:286 in total PBMN after 130 and 450 days respectively. Epitope spreading, which was induced with immunization, was also maintained and in some cases augmented after adoptive T cell therapy. TCR Vβ analysis performed showed the outgrowth of several HER2 specific clones in the CD4+ population post infusion. Our preliminary data suggests that adoptive transfer of autologous HER2 specific polyclonal T cells is safe and results in persistent high levels of tumor specific T cell immunity in vivo.