Restricted T-cell Responses Research Articles

Longitudinal Characterization of the Mumps-Specific HLA-A2 Restricted T-Cell Response after Mumps Virus Infection.

Waning of the mumps virus (MuV)-specific humoral response after vaccination has been suggested as a cause for recent mumps outbreaks in vaccinated young adults, although it cannot explain all cases. Moreover, CD8+ T cells may play an important role in the response against MuV; however, little is known about the characteristics and dynamics of the MuV-specific CD8+ T-cell response after MuV infection. Here, we had the opportunity to follow the CD8+ T-cell response to three recently identified HLA-A2*02:01-restricted MuV-specific epitopes from 1.5 to 36 months post-MuV infection in five previously vaccinated and three unvaccinated individuals. The infection-induced CD8+ T-cell response was dominated by T cells specific for the ALDQTDIRV and LLDSSTTRV epitopes, while the response to the GLMEGQIVSV epitope was subdominant. MuV-specific CD8+ T-cell frequencies in the blood declined between 1.5 and 9 months after infection. This decline was not explained by changes in the expression of inhibitory receptors or homing markers. Despite the ongoing changes in the frequencies and phenotype of MuV-specific CD8+ T cells, TCRβ analyses revealed a stable MuV-specific T-cell repertoire over time. These insights in the maintenance of the cellular response against mumps may provide hallmarks for optimizing vaccination strategies towards a long-term cellular memory response.

The similarity of class II HLA genotypes defines patterns of autoreactivity in idiopathic bone marrow failure disorders

AbstractIdiopathic aplastic anemia (IAA) is a rare autoimmune bone marrow failure (BMF) disorder initiated by a human leukocyte antigen (HLA)-restricted T-cell response to unknown antigens. As in other autoimmune disorders, the predilection for certain HLA profiles seems to represent an etiologic factor; however, the structure-function patterns involved in the self-presentation in this disease remain unclear. Herein, we analyzed the molecular landscape of HLA complexes of a cohort of 300 IAA patients and almost 3000 healthy and disease controls by deeply dissecting their genotypic configurations, functional divergence, self-antigen binding capabilities, and T-cell receptor (TCR) repertoire specificities. Specifically, analysis of the evolutionary divergence of HLA genotypes (HED) showed that IAA patients carried class II HLA molecules whose antigen-binding sites were characterized by a high level of structural homology, only partially explained by specific risk allele profiles. This pattern implies reduced HLA binding capabilities, confirmed by binding analysis of hematopoietic stem cell (HSC)-derived self-peptides. IAA phenotype was associated with the enrichment in a few amino acids at specific positions within the peptide-binding groove of DRB1 molecules, affecting the interface HLA-antigen-TCR β and potentially constituting the basis of T-cell dysfunction and autoreactivity. When analyzing associations with clinical outcomes, low HED was associated with risk of malignant progression and worse survival, underlying reduced tumor surveillance in clearing potential neoantigens derived from mechanisms of clonal hematopoiesis. Our data shed light on the immunogenetic risk associated with IAA etiology and clonal evolution and on general pathophysiological mechanisms potentially involved in other autoimmune disorders.

Myc Expression and Tumor-Infiltrating T Cells Are Associated with Response in Patients (Pts) with Relapsed/Refractory Diffuse Large B-Cell Lymphoma (r/r DLBCL) Treated with Tisagenlecleucel in the Juliet Trial

Myc Expression and Tumor-Infiltrating T Cells Are Associated with Response in Patients (Pts) with Relapsed/Refractory Diffuse Large B-Cell Lymphoma (r/r DLBCL) Treated with Tisagenlecleucel in the Juliet Trial

Abstract B092: Tumor infiltrating T-cells from renal cell carcinoma patients recognize neoantigens derived from point and frameshift mutations

Abstract Mutation-derived neoantigens are important targets of T-cell mediated reactivity towards tumors. Their unique tumor-restriction poses an advantage compared to shared tumor antigens in that they are in principle both foreign and tumor specific, hence presumably less impacted by T-cell tolerance and for therapeutic applications less prone to mediate immune-related destruction of noncancerous tissue. Moreover, the mutational burden and predicted number of neoantigens correlate to favorable clinical outcome and benefit from immune checkpoint therapy. Neoantigen-reactive T-cells have been detected across a number of solid cancers, ranging from immunogenic tumors such as melanoma and non-small cell lung cancer to less immunogenic tumors such as breast cancer. Renal cell carcinomas (RCCs) are among medium-range mutational burden tumors and present with the highest pan-cancer number and proportion of frameshift mutations, a mutation type considered to be highly immunogenic. However, to our knowledge, yet no reports have described neoantigen-specific T-cells in this malignancy. In this study, the mutational landscape and HLA (human leukocyte antigen) profile of tumors from six renal cell carcinoma patients were analyzed by whole-exome sequencing (WXS) of DNA from tumor fragments (TFs), autologous tumor cell lines (TCLs) and tumor-infiltrating lymphocytes (TILs, germline reference). Hereafter the online MuPeXi tool was used to predict binding of mutated peptide sequences of 9-11mer length to the HLAs of each patient, using a rank score < 2 for selection of peptide binding, hereby creating patient-specific libraries of putative neo-peptides. TILs extracted from the patients tumors were screened for T-cell recognition of the peptide libraries by use of a novel high-throughput platform based on DNA barcode labeled peptide-MHC multimers, and responses were verified by conventional fluorochrome labeled MHC multimers. In four of six patients WXS was performed on both TF and TCL, in two of six patients only on TF. The average mutational burden of the six patients was 271 for TF (range 146–381, n=6) and 289 for TCL (range 182-404, n=4). Prediction of HLA-restricted peptides within the mutated sequences resulted in patient specific libraries of average 269 peptides for TF and TCL combined (range 126-443, n=6). Half of the peptides were predicted from both sources (52%, range 28-74%, n=4) compared to 20% (range 8-31%, n=4) predicted solely from TF and 29% (range 18-41%, n=4) predicted solely from TCL. The proportion of predicted peptides derived from frameshift mutations out of total mutations was 16% (range 7-24%, n=6). A total of 67 neoantigen-specific T-cell responses were detected across all patients by use of a novel high-throughput DNA barcode screening platform, with the number of detected responses ranging from 4-30 and spanning 3-5 HLA restrictions per patient. Of note, we detected a number of T-cell responses towards HLA-C restricted peptides, which have previously been poorly described. For several patients, the number of HLA-C restricted T-cell responses observed was substantially higher than for both HLA-A and -B, highlighting the importance of including this HLA type for neoepitope analyses. In the four patients in whom peptides were predicted from both TF and TCL, the distribution of responses was 37% on TF (range 14-60%, n=4), 36% on TCL (range 29-43%, n=4) and 27% (range 0-43%) on TF+TCL combined. The proportion of responses towards frameshift mutations was 17% (range 0-24%, n=6) of total responses. The identification of neoantigen-specific T-cells within tumors from RCC patients is an important step towards the use of neoantigens as therapeutic targets and predictors of response to immunotherapy in this cancer subtype. Moreover, our study points toward the importance of broad peptide prediction platforms covering multiple sources for WXS and mutational analyses covering both point and frameshift mutations. Citation Format: Sofie Ramskov, Ulla Kring Hansen, Anne-Mette Bjerregaard, Amalie Kai Bentzen, Marco Donia, Rikke Andersen, Zoltan Szallasi, Inge Marie Stentoft Svane, Aron Charles Eklund, Sine Reker Hadrup. Tumor infiltrating T-cells from renal cell carcinoma patients recognize neoantigens derived from point and frameshift mutations [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B092.

Brain atrophy in picornavirus‐infected FVB mice is dependent on the H‐2Dbclass I molecule

Brain atrophy is a common feature of numerous neurologic diseases in which the role of neuroinflammation remains ill-defined. In this study, we evaluated the contribution of major histocompatibility complex class I molecules to brain atrophy in Theiler's murine encephalomyelitis virus (TMEV)-infected transgenic FVB mice that express the Db class I molecule. FVB/Db and wild-type FVB mice were evaluated for changes in neuroinflammation, virus clearance, neuropathology, and development of brain atrophy via T2-weighted MRI and subsequent 3-dimensional volumetric analysis. Significant brain atrophy and hippocampal neuronal loss were observed in TMEV-infected FVB/Db mice, but not in wild-type FVB mice. Brain atrophy was observed at 1 mo postinfection and persisted through the 4-mo observation period. Of importance, virus-infected FVB/Db mice elicited a strong CD8 T-cell response toward the immunodominant Db-restricted TMEV-derived peptide, VP2121-130, and cleared TMEV from the CNS. In addition, immunofluorescence revealed CD8 T cells near virus-infected neurons; therefore, we hypothesize that class I restricted CD8 T-cell responses promote development of brain atrophy. This model provides an opportunity to analyze the contribution of immune cells to brain atrophy in a system where persistent virus infection and demyelination are not factors in long-term neuropathology.-Huseby Kelcher, A. M., Atanga, P. A., Gamez, J. D., Cumba Garcia, L. M., Teclaw, S. J., Pavelko, K. D., Macura, S. I., Johnson. A. J. Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2Db class I molecule.

Characteristics of HLA-E Restricted T-Cell Responses and Their Role in Infectious Diseases.

Human HLA-E can, in addition to self-antigens, also present pathogen-derived sequences, which elicit specific T-cell responses. T-cells recognize their antigen presented by HLA-E highly specifically and have unique functional and phenotypical properties. Pathogen specific HLA-E restricted CD8+ T-cells are an interesting new player in the field of immunology. Future work should address their exact roles and relative contributions in the immune response against infectious diseases.

Reduced mucosal associated invariant T-cells are associated with increased disease severity and Pseudomonas aeruginosa infection in cystic fibrosis.

BackgroundPrimary defects in host immune responses have been hypothesised to contribute towards an inability of subjects with cystic fibrosis (CF) to effectively clear pulmonary infections. Innate T-lymphocytes provide rapid pathogen-specific responses prior to the development of classical MHC class I and II restricted T-cell responses and are essential to the initial control of pulmonary infection. We aimed to examine the relationship between peripheral blood lymphocyte phenotype and clinical outcomes in adults with CF.MethodsWe studied 41 subjects with CF and 22, age matched, non-smoking healthy control subjects. Lymphocytes were extracted from peripheral blood samples and phenotyped by flow-cytometry. Lymphocyte phenotype was correlated with sputum microbiology and clinical parameters.ResultsIn comparison to healthy control subjects, mucosal associated invariant T (MAIT)-lymphocytes were significantly reduced in the peripheral blood of subjects with CF (1.1% versus 2.0% of T-lymphocytes, P = 0.002). MAIT cell concentration was lowest in CF subjects infected with P. aeruginosa and in subjects receiving treatment for a pulmonary exacerbation. Furthermore a reduced MAIT cell concentration correlated with severity of lung disease.ConclusionReduced numbers of MAIT cells in subjects with CF were associated with P. aeruginosa pulmonary infection, pulmonary exacerbations and more severe lung disease. These findings provide the impetus for future studies examining the utility of MAIT cells in immunotherapies and vaccine development. Longitudinal studies of MAIT cells as biomarkers of CF pulmonary infection are awaited.

Intradermal photosensitisation facilitates stimulation of MHC class-I restricted CD8 T-cell responses of co-administered antigen

The protection or treatment of several immunological disorders is dependent on the antigen-specific and cytotoxic CD8 T cells. However, vaccines aimed at stimulating CD8 T-cell responses are typically ineffective because vaccine antigens are primarily processed by the MHC class-II and not the MHC class-I pathway of antigen presentation: the latter requires cytosolic delivery of antigen. In order to facilitate targeting of antigen to cytosol, the antigen was combined with the photosensitiser TPCS2a (disulfonated tetraphenyl chlorin) and administered intradermally to mice. The photosensitiser was activated by illumination of the injection site. This photochemical internalization (PCI) strongly increased the stimulation of CD8 T-cell responses as measured by antigen-specific proliferation and secretion of pro-inflammatory cytokines. Fluorescence microscopy showed that delivery to cytosol was TPCS2a dependent and occurred by light-induced disruption of TPCS2a- and antigen-containing endosomes. PCI-based vaccination prevented growth of malignant B16 cells as compared with vaccination without PCI. In conclusion, PCI represents a potent tool for delivery of antigens to cytosol for stimulation of cytotoxic CD8 T-cell responses. This study demonstrated a first proof-of-principle for PCI-mediated immunisation with potential application in cancer immunotherapy.

Antigen-specific immunomodulation for type 1 diabetes by novel recombinant antibodies directed against diabetes-associates auto-reactive T cell epitope

The trimolecular complex composed of autoreactive T-cell receptor, MHC class II, and an autoantigenic peptide plays a central role in the activation of pathogenic Islet-specific CD4+ T cells in type 1 diabetes (T1D). We isolated and characterized novel antibodies against autoreactive T-cell epitopes associated with T1D. Our antibodies mimic the specificity of the T-cell receptor (TCR), while binding MHC class II/peptide complexes in an autoantigen peptide specific, MHC-restricted manner. The isolated TCR-like antibodies were directed against the minimal T-cell epitope GAD-555–567 in the context of the HLA-DR4-diabetic-associated molecule. A representative high-affinity TCR-like antibody clone (G3H8) enabled the detection of intra- and extra-cellular DR4/GAD-555–567 complexes in antigen presenting cells. I561M single mutation at the central position (P5) of the GAD-555–567 peptide abolished the binding of G3H8 to the DR4/GAD complex, demonstrating its high fine TCR-like specificity. The G3H8 TCR-like antibody significantly inhibited GAD-555–567 specific, DR4 restricted T-cell response in vitro and in vivo in HLA-DR4 transgenic mice. Our findings constitute a proof-of-concept for the utility of TCR-like antibodies as antigen-specific immunomodulation agents for regulating pathogenic T-cells and suggest that TCR-like antibodies targeting autoreactive MHC class II epitopes are valuable research tools that enable studies related to antigen presentation as well as novel therapeutic agents that may be used to modulate autoimmune disorders such as T1D.

Exploring the HLA-A Associations in EBV-Associated Classical Hodgkin Lymphoma : HLA-A Genotype Modifies the HLA-A*02:01 Restricted Cytotoxic T-Cell and Cytokine Response to EBV

AbstractAbstract 3635 Background:Class I HLA genes are associated with risk of developing EBV-associated classical Hodgkin Lymphoma (EBV-cHL). HLA-A*01:01 is associated with increased risk and HLA-A*02:01 with decreased risk, resulting in an almost 10-fold difference in odds of developing EBV-cHL between HLA-A*02:01 and HLA-A*01:01 homozygotes with a gradation of risk in intermediate genotypes. Class I HLA genes are involved in EBV-specific cytotoxic T cell (CTL) responses, keeping infection under tight control in healthy hosts. HLA associations with EBV-cHL suggest that EBV-specific immune responses at time of initial infection, during chronic latent infection or at time of oncogenesis are critical in the pathogenesis of EBV-cHL. Many immunodominant EBV peptides are known to be presented by HLA-A*02:01. Since HLA-A*01:01 appears to increase risk of disease even in those individuals with HLA-A*02:01, we investigated whether the presence of an HLA-A*01:01 allele modifies the CTL response to HLA-A*02:01-restricted EBV epitopes. Methods:Individuals with relevant HLA-A genotypes were selected from a pool of healthy adults who agreed to take part in this study; A*01:01/A*02:01 heterozygotes (n=15), A*02:01 homozygotes (n=12) and A*02:01/× heterozygotes (n=15), where x is neither A*01:01 or A*02:01. Assessment of A*02:01 restricted EBV response used IFN-γ ELISPOT and all 31 reported A*02:01 restricted EBV peptides. PBMCs were exposed to single EBV peptides and controls in duplicate. The highest negative control plus two standard deviations was taken as the cut-off for a “positive” response. Measures of response were: the number of peptides recognised; maximal response to any single peptide; and summative response to EBV peptides. Analyses examined all peptides, lytic peptides, latent peptides and peptides expressed in type II latency. Assessment of cytotoxicity by degranulation was performed by flow cytometric analysis of CD107 expression after exposure to peptide pools. Supernatants from these stimulations were used to measure TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 and IL-17. Results were compared by Kruskall-Wallis analysis across groups and Mann-Whitney analysis between pairs of groups, using SPSS. Results:Satisfactory ELISPOTs were obtained from 31 donors. No difference in response to A*02:01 restricted EBV peptides was observed between A*01:01/A*02:01 heterozygotes and A*02:01 homozygotes. A*02:01/x heterozygotes recognised more EBV peptides than either A*01:01/A*02:01 heterozygotes (p=0.023) or A*02:01/A*02:01 homozygotes (p=0.049), an effect which was greatest for lytic peptides (p= 0.012). The single maximal response to any EBV peptide was higher in A*02:01/x heterozygotes, reaching significance for lytic peptide responses (p=0.042). The summative response was also higher in A*02:01/x heterozygotes, and for lytic peptides these differences were significant (p=0.022).A*02:01/x heterozygotes also demonstrated greater cytotoxicity assessed by CD107 than either A*01:01/A*02:01 heterozygotes or A*02:01 homozygotes. This effect was seen in response to latent and lytic peptides, with the differences greatest between A*02:01/x heterozygotes and A*02:01/A*01:01 heterozygotes.A*02:01/A*01:01 heterozygotes produced significantly higher levels of IL-10 (mean 1427 vs 155 pg/ml p= 0.03), IL-17 (84 vs 70 pg/ml p=0.02) and IL-5 (35 vs 29 pg/ml p= 0.04) in response to lytic peptides compared to both other HLA-A groups. IFN-g secretion was higher in A*02:01/x heterozygotes (2552 vs 552 pg/ml, p=0.007). Conclusions:The null hypothesis of this study, that all HLA-A*02:01 carriers should have the same response to HLA-A*02:01 restricted EBV peptides was disproved. A*02:01/x heterozygotes had CTL responses of greater breadth and magnitude than either A*01:01/A*02:01 heterozygotes or A*02:01 homozygotes, as measured by three different methods. A*01:01 did not inhibit CTL responses to A*02:01-restricted EBV peptides; however, in response to EBV lytic peptides, A*02:01/*01:01 heterozygotes demonstrated significant increases in cytokines associated with a Th2 response. These data suggest that overall HLA-A phenotype is important in determining CTL response through a single HLA allele. The cytokine profiles observed in the three groups following stimulation with EBV peptides may begin to explain the HLA-associated differences in risk of developing EBV-cHL. Disclosures:No relevant conflicts of interest to declare.

Identification and expansion of highly suppressive CD8+FoxP3+ regulatory T cells after experimental allogeneic bone marrow transplantation

AbstractFoxP3+ confers suppressive properties and is confined to regulatory T cells (Treg) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4+ Treg are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8+ population of FoxP3+ Treg that convert from CD8+ conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8+ Treg undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-β. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4+FoxP3+ population and is more potent in exerting class I–restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8+FoxP3+ Treg are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8+FoxP3+ Treg thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I–restricted T-cell responses after bone marrow transplantation.

Prevention of “Humanized” Diabetogenic CD8 T-Cell Responses in HLA-Transgenic NOD Mice by a Multipeptide Coupled-Cell Approach

OBJECTIVEType 1 diabetes can be inhibited in standard NOD mice by autoantigen-specific immunotherapy targeting pathogenic CD8+ T-cells. NOD.β2mnull.HHD mice expressing human HLA-A2.1 but lacking murine major histocompatibility complex class I molecules develop diabetes characterized by CD8 T-cells recognizing certain autoantigenic peptides also targeted in human patients. These include peptides derived from the pancreatic β-cell proteins insulin (INS1/2 A2–10 and INS1 B5–14) and islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP265–273 and IGRP228–236). Hence, NOD.β2mnull.HHD mice represent a model system for developing potentially clinically translatable interventions for suppressing diabetogenic HLA-A2.1–restricted T-cell responses.RESEARCH DESIGN AND METHODSStarting at 4–6 weeks of age, NOD.β2mnull.HHD female mice were injected intravenously with syngeneic splenocytes to which various admixtures of the four above-mentioned peptides were bound by the cross-linking agent ethylene carbodiimide (ECDI).RESULTSTreatment with such cells bearing the complete cocktail of INS and IGRP epitopes (designated INS/IGRP-SPs) significantly inhibited diabetes development in NOD.β2mnull.HHD recipients compared with controls receiving splenocytes coupled with an irrelevant HLA-A2.1–restricted Flu16 peptide. Subsequent analyses found syngeneic splenocytes bearing the combination of the two ECDI-coupled IGRPs but not INS peptides (IGRP-SPs or INS-SPs) effectively inhibited diabetes development in NOD.β2mnull.HHD mice. This result was supported by enzyme-linked immunospot (ELISPOT) analyses indicating combined INS/IGRP-SPs diminished HLA-A2.1–restricted IGRP but not INS autoreactive CD8+ T-cell responses in NOD.β2mnull.HHD mice.CONCLUSIONSThese data support the potential of a cell therapy approach targeting HLA-A2.1–restricted IGRP autoreactive CD8 T-cells as a diabetes intervention approach in appropriate human patients.

Glycosidic Tn-based vaccines targeting dermal dendritic cells favor germinal center B-cell development and potent antibody response in the absence of adjuvant

AbstractIn vivo targeting of C-type lectin receptors is an effective strategy for increasing antigen uptake and presentation by dendritic cells (DCs). To induce efficient immune response, glycosylated tumor-associated Tn antigens were used to target DCs through binding to macrophage galactose-type lectin (MGL). The capacity of Tn-glycosylated antigens—and the multiple antigenic glycopeptide Tn3 therapeutic candidate vaccine—to target mouse and human MGL+ DCs are demonstrated, especially regarding dermal DCs. In mice, MGL+ CD103− dermal DCs efficiently captured and processed glycosylated Tn antigen in vivo, inducing a potent major histocompatibility complex (MHC) class II–restricted T-cell response. Intradermal immunization with Tn-glycopeptides induced high levels of Th2 cytokines—even in the presence of unmethylated cytosine-phosphate-guanosine—and was associated with increased expansion of the germinal center B-cell population. Therefore, MGL acts as an efficient endocytic antigen receptor on dermal DCs in vivo, able to prime Tn-specific T- and B-cell responses. Moreover, even in the absence of adjuvant, immunization with this glycosidic Tn-based vaccine induced high levels of anti-Tn antibody responses, recognizing human tumor cells. In vivo DC-targeting strategies, based on Tn-MGL interactions, constitute a promising strategy for enhancing antigen presentation and inducing potent antibody response.

Vaccination with a plasmid DNA encoding HER-2/neu together with low doses of GM-CSF and IL-2 in patients with metastatic breast carcinoma: a pilot clinical trial

BackgroundAdjuvant trastuzumab (Herceptin) treatment of breast cancer patients significantly improves their clinical outcome. Vaccination is an attractive alternative approach to provide HER-2/neu (Her2)-specific antibodies and may in addition concomitantly stimulate Her2-reactive T-cells. Here we report the first administration of a Her2-plasmid DNA (pDNA) vaccine in humans.Patients and MethodsThe vaccine, encoding a full-length signaling-deficient version of the oncogene Her2, was administered together with low doses of GM-CSF and IL-2 to patients with metastatic Her2-expressing breast carcinoma who were also treated with trastuzumab. Six of eight enrolled patients completed all three vaccine cycles. In the remaining two patients treatment was discontinued after one vaccine cycle due to rapid tumor progression or disease-related complications. The primary objective was the evaluation of safety and tolerability of the vaccine regimen. As a secondary objective, treatment-induced Her2-specific immunity was monitored by measuring antibody production as well as T-cell proliferation and cytokine production in response to Her2-derived antigens.ResultsNo clinical manifestations of acute toxicity, autoimmunity or cardiotoxicity were observed after administration of Her2-pDNA in combination with GM-CSF, IL-2 and trastuzumab. No specific T-cell proliferation following in vitro stimulation of freshly isolated PBMC with recombinant human Her2 protein was induced by the vaccination. Immediately after all three cycles of vaccination no or even decreased CD4+ T-cell responses towards Her2-derived peptide epitopes were observed, but a significant increase of MHC class II restricted T-cell responses to Her2 was detected at long term follow-up. Since concurrent trastuzumab therapy was permitted, λ-subclass specific ELISAs were performed to specifically measure endogenous antibody production without interference by trastuzumab. Her2-pDNA vaccination induced and boosted Her2-specific antibodies that could be detected for several years after the last vaccine administration in a subgroup of patients.ConclusionThis pilot clinical trial demonstrates that Her2-pDNA vaccination in conjunction with GM-CSF and IL-2 administration is safe, well tolerated and can induce long-lasting cellular and humoral immune responses against Her2 in patients with advanced breast cancer.Trial registrationThe trial registration number at the Swedish Medical Products Agency for this trial is Dnr151:785/2001.

Mouse Schwann cells activate MHC class I and II restricted T-cell responses, but require external peptide processing for MHC class II presentation

Schwann cells are the myelinating glia cells of the peripheral nervous system (PNS). In inflammatory neuropathies like the Guillain-Barré syndrome (GBS) Schwann cells become target of an autoimmune response, but may also modulate local inflammation. Here, we tested the functional relevance of Schwann cell derived MHC expression in an in vitro coculture system. Mouse Schwann cells activated proliferation of ovalbumin specific CD8+ T cells when ovalbumin protein or MHC class I restricted ovalbumin peptide (Ova 257–264 SIINFEKL) was added and after transfection with an ovalbumin coding vector. Schwann cells activated proliferation of ovalbumin specific CD4+ T cells in the presence of MHC class II restricted ovalbumin peptide (Ova 323–339 ISQAVHAAHAEINEAGR). CD4+ T-cell proliferation was not activated by ovalbumin protein or transfection with an ovalbumin coding vector. This indicates that Schwann cells express functionally active MHC class I and II molecules. In this study, however, Schwann cells lacked the ability to process exogenous antigen or cross-present endogenous antigen into the MHC class II presentation pathway. Thus, antigen presentation may be a pathological function of Schwann cells exacerbating nerve damage in inflammatory neuropathies.

Diabetes-Specific HLA-DR–Restricted Proinflammatory T-Cell Response to Wheat Polypeptides in Tissue Transglutaminase Antibody–Negative Patients With Type 1 Diabetes

OBJECTIVEThere is evidence of gut barrier and immune system dysfunction in some patients with type 1 diabetes, possibly linked with exposure to dietary wheat polypeptides (WP). However, questions arise regarding the frequency of abnormal immune responses to wheat and their nature, and it remains unclear whether such responses are diabetes specific.RESEARCH DESIGN AND METHODSIn type 1 diabetic patients and healthy control subjects, the immune response of peripheral CD3+ T-cells to WPs, ovalbumin, gliadin, α-gliadin 33-mer peptide, tetanus toxoid, and phytohemagglutinin was measured using a carboxyfluorescein diacetate succinimidyl ester (CFSE) proliferation assay. T–helper cell type 1 (Th1), Th2, and Th17 cytokines were analyzed in WP-stimulated peripheral blood mononuclear cell (PBMNC) supernatants, and HLA was analyzed by PCR.RESULTSOf 42 patients, 20 displayed increased CD3+ T-cell proliferation to WPs and were classified as responders; proliferative responses to other dietary antigens were less pronounced. WP-stimulated PBMNCs from patients showed a mixed proinflammatory cytokine response with large amounts of IFN-γ, IL-17A, and increased TNF. HLA-DQ2, the major celiac disease risk gene, was not significantly different. Nearly all responders carried the diabetes risk gene HLA-DR4. Anti-DR antibodies blocked the WP response and inhibited secretion of Th1 and Th17 cytokines. High amounts of WP-stimulated IL-6 were not blocked.CONCLUSIONST-cell reactivity to WPs was frequently present in type 1 diabetic patients and associated with HLA-DR4 but not HLA-DQ2. The presence of an HLA-DR–restricted Th1 and Th17 response to WPs in a subset of patients indicates a diabetes-related inflammatory state in the gut immune tissues associated with defective oral tolerance and possibly gut barrier dysfunction.

Mamu-A⁎01/K b transgenic and MHC Class I knockout mice as a tool for HIV vaccine development

We have developed a murine model expressing the rhesus macaque (RM) Mamu-A⁎01 MHC allele to characterize immune responses and vaccines based on antigens of importance to human disease processes. Towards that goal, transgenic (Tg) mice expressing chimeric RM (α1 and α2 Mamu-A⁎01 domains) and murine (α3, transmembrane, and cytoplasmic H-2K b domains) MHC Class I molecules were derived by transgenesis of the H-2K bD b double MHC Class I knockout strain. After immunization of Mamu-A⁎01/K b Tg mice with rVV-SIVGag–Pol, the mice generated CD8 + T-cell IFN-γ responses to several known Mamu-A⁎01 restricted epitopes from the SIV Gag and Pol antigen sequence. Fusion peptides of highly recognized CTL epitopes from SIV Pol and Gag and a strong T-help epitope were shown to be immunogenic and capable of limiting an rVV-SIVGag–Pol challenge. Mamu-A⁎01/K b Tg mice provide a model system to study the Mamu-A⁎01 restricted T-cell response for various infectious diseases which are applicable to a study in RM.

25Characterization of a single peptide derived from Cytochrome P450 1B1 that elicits spontaneous HLA-A1 as well as HLA-B35 restricted CD8 T-cell responses in cancer patients

25Characterization of a single peptide derived from Cytochrome P450 1B1 that elicits spontaneous HLA-A1 as well as HLA-B35 restricted CD8 T-cell responses in cancer patients

25 Characterization of a single peptide derived from Cytochrome P450 1B1 that elicits spontaneous HLA-A1 as well as HLA-B35 restricted CD8 T-cell responses in cancer patients

25 Characterization of a single peptide derived from Cytochrome P450 1B1 that elicits spontaneous HLA-A1 as well as HLA-B35 restricted CD8 T-cell responses in cancer patients

Characterization of a single peptide derived from cytochrome P450 1B1 that elicits spontaneous human leukocyte antigen (HLA)-A1 as well as HLA-B35 restricted CD8 T-cell responses in cancer patients

Cytochrome P450 1B1 (CYP1B1) is widely expressed in human malignancies, but silent in most normal tissues. Importantly, the protein is believed to play an important role in the survival and growth of cancer cells in a stressed environment, e.g., as a result of hypoxia or chemotherapy. Thus, targeting of CYP1B1 represents a potentially successful strategy in the treatment of metastatic cancer, e.g., by therapeutic vaccination. Herein, we describe the characterization of a novel peptide from the CYP1B1 protein (CYP240), which is spontaneously recognized by CD8 T cells in cancer patients. Interestingly, the peptide binds to both human leukocyte antigen (HLA)-A1 and HLA-B35. Hence, peripheral blood lymphocytes from a total of 49 cancer patients (25 melanoma, 13 RCC, and 11 breast cancer; 41 HLA-A1 positive, 8 HLA-B35 positive) were analyzed for reactivity taking advantage of the EliSpot assay. Rare but strong responses were detected in HLA-A1-positive patients, and more frequent responses were detected in HLA-B35-positive patients. No reactivity against the peptide could be detected in healthy donors. Furthermore, we demonstrated that peptide-specific T cells were able to lyze target cells presenting the peptide on the surface. The characterized CYP240 peptide presented herein opens the avenue for more broader recruitment of patients in vaccination trials targeting CYB1B1.