HLA-DR4 Transgenic Mice Research Articles

A pharmacological protein kinase C/D inhibitor Gö6976 ameliorates collagen-induced arthritis

Abstract Toll-like receptor (TLR) signaling has been implicated with the initiation and progression of rheumatoid arthritis (RA). Disruption of TLR signaling at early stages of RA might thereby provide an opportunity to halt the RA progression and ameliorate outcomes. We previously found that protein kinase D1 (PKD1) is essential for proinflammatory responses mediated by MyD88-dependent TLRs, and TLR-dependent PKD1 activation is inhibited by Gö6976. However, it is currently unknown whether PKD1 plays a role in pathogenesis of RA and whether Gö6976 has ameliorating effects on arthritis. Here, we investigated whether PKD1 contributes to the TLR-mediated proinflammatory responses in human synovial cells, and to development and progression of type II collagen (CII)-induced arthritis (CIA; an experimental animal model for human RA). We found that PKD1 is constitutively hyperactivated in synoviocytes of RA patients, and is rapidly activated in response to TLR/IL-1R ligands. Silencing PKD1 expression in RA synoviocytes resulted in substantial inhibition of both spontaneous and TLR/IL-1R-mediated expression of cytokines/chemokines. Although Gö6976 had no effect on the TCR-induced cell proliferation and cytokine production, synergistic effects of TCR and TLR2, as well as TLR2 alone, on the spleen cell cytokine production and proliferation were significantly inhibited in the presence of Gö6976. In addition, daily treatment with Gö6976 significantly reduced serum levels of IFNg and anti-CII IgG, and the incidence and severity of arthritis in humanized HLA-DR1 transgenic mice immunized with CII. Our data suggest that PKD1 might play an important role in proinflammatory responses in RA and CIA, and could be a therapeutic target for arthritis.

A humanized HLA-DR4 mouse model for autoimmune myocarditis

Myocarditis, the principal cause of dilated cardiomyopathy and heart failure in young adults, is associated with autoimmunity to human cardiac α-myosin (hCAM) and the DR4 allele of human major histocompatibility II (MHCII). We developed an hCAM-induced myocarditis model in human HLA-DR4 transgenic mice that lack all mouse MHCII genes, demonstrating that immunization for 3weeks significantly increased splenic T-cell proliferative responses and titres of IgG1 and IgG2c antibodies, abolished weight gain, provoked cardiac inflammation and significantly impaired cardiac output and fractional shortening, by echocardiography, compared to adjuvant-injected mice. Neither cardiac dilatation nor fibrosis occurred at this time point but prolonging the experiment was associated with mortality. Treatment with mixtures of hCAM derived peptides predicted to have high affinity for DR4 significantly preserved ejection fraction and fractional shortening. Our new humanized mouse model of autoimmune cardiomyopathy should be useful to refine hCAM-derived peptide treatment.

Avian and Human Seasonal Influenza Hemagglutinin Proteins Elicit CD4 T Cell Responses That Are Comparable in Epitope Abundance and Diversity.

Avian influenza viruses remain a significant concern due to their pandemic potential. Vaccine trials have suggested that humans respond poorly to avian influenza vaccines relative to seasonal vaccines. It is important to understand, first, if there is a general deficiency in the ability of avian hemagglutinin (HA) proteins to generate immune responses and, if so, what underlies this defect. This question is of particular interest because it has been suggested that in humans, the poor immunogenicity of H7 vaccines may be due to a paucity of CD4 T cell epitopes. Because of the generally high levels of cross-reactive CD4 T cells in humans, it is not possible to compare the inherent immunogenicities of avian and seasonal HA proteins in an unbiased manner. Here, we empirically examine the epitope diversity and abundance of CD4 T cells elicited by seasonal and avian HA proteins. HLA-DR1 and HLA-DR4 transgenic mice were vaccinated with purified HA proteins, and CD4 T cells to specific epitopes were identified and quantified. These studies revealed that the diversity and abundance of CD4 T cells specific for HA do not segregate on the basis of whether the HA was derived from human seasonal or avian influenza viruses. Therefore, we conclude that failure in responses to avian vaccines in humans is likely due to a lack of cross-reactive CD4 T cell memory perhaps coupled with competition with or suppression of naive, HA-specific CD4 T cells by memory CD4 T cells specific for more highly conserved proteins.

Passive therapy with humanized anti-staphylococcal enterotoxin B antibodies attenuates systemic inflammatory response and protects from lethal pneumonia caused by staphylococcal enterotoxin B-producing Staphylococcus aureus

ABSTRACTDrugs such as linezolid that inhibit bacterial protein synthesis may be beneficial in treating infections caused by toxigenic Staphylococcus aureus. As protein synthesis inhibitors have no effect on preformed toxins, neutralization of pathogenic exotoxins with anti-toxin antibodies may be beneficial in conjunction with antibacterial therapy. Herein, we evaluated the efficacy of human-mouse chimeric high-affinity neutralizing anti-staphylococcal enterotoxin B (SEB) antibodies in the treatment of experimental pneumonia caused by SEB-producing S. aureus. Since HLA class II transgenic mice mount a stronger systemic immune response following challenge with SEB and are more susceptible to SEB-induced lethal toxic shock than conventional mice strains, HLA-DR3 transgenic mice were used. Lethal pneumonia caused by SEB-producing S. aureus in HLA-DR3 transgenic mice was characterized by robust T cell activation and elevated systemic levels of several pro-inflammatory cytokines and chemokines. Prophylactic administration of a single dose of linezolid 30 min prior to the onset of infection attenuated the systemic inflammatory response and protected from mortality whereas linezolid administered 60 min after the onset of infection failed to confer significant protection. Human-mouse chimeric high-affinity neutralizing anti-SEB antibodies alone, but not polyclonal human IgG, mitigated this response and protected from death when administered immediately after initiation of infection. Further, anti-SEB antibodies as well as intact polyclonal human IgG, but not its Fab or Fc fragments, protected from lethal pneumonia when followed with linezolid therapy 60 min later. In conclusion, neutralization of superantigens with high-affinity antibodies may have beneficial effects in pneumonia.

SCIB1, a huIgG1 antibody DNA vaccination, combined with PD-1 blockade induced efficient therapy of poorly immunogenic tumors.

PurposeWe have previously shown that supraoptimal signaling of high avidity T cells leads to high expression of PD-1 and inhibition of proliferation. This study was designed to see if this effect could be mitigated by combining a vaccine that stimulates high avidity T cells with PD-1 blockade.Experimental DesignWe investigated the anti-tumor effect of a huIgG1 antibody DNA vaccine (SCIB1) and PD-1 blockade.ResultsVaccination of HLA-DR4 transgenic mice with SCIB1 induced high frequency and avidity T cell responses that resulted in survival (40%) of mice with established B16F1-DR4 tumors. SCIB1 vaccination was associated with increased infiltration of CD4 and CD8 T cells within the tumor but was also associated with upregulation of PD-L1 within the tumor environment. PD-1 blockade also resulted in increased CD8 T cell infiltration and an anti-tumor response with 50% of mice showing long term survival. In line with our hypothesis that PD-1/PD-L1 signaling results in inhibition of proliferation of high avidity T cells at the tumor site, the combination of PD-1 blockade with vaccination, enhanced the number and proliferation of the CD8 tumor infiltrate. This resulted in a potent anti-tumor response with 80% survival of the mice.ConclusionsThere is a benefit in combining PD-1 blockade with vaccines that induce high avidity T cell responses and in particular with SCIB1.

Abstract A035: Citrullinated α-enolase as a novel target for cancer immunotherapy

Abstract Stressful conditions in the tumor microenvironment induce autophagy in cancer cells to promote their survival, however, autophagy also causes post-translational modification of proteins which are recognized by the immune system. In particular, modified self-antigens can trigger CD4+ T cell responses that can be exploited to boost antitumor immune defenses. We have previously investigated the ability of CD4 cells to target tumor-specific self-antigens modified by citrullination, which converts arginine residues in proteins to citrulline. These studies showed that vimentin, which is frequently expressed in cells during epithelial-to-mesenchymal transition of metastasizing epithelial tumors, is citrullinated and is a good target for anti-tumor immunity (Ref. 1). Immunization with citrullinated vimentin peptides induced IFNγ- and granzyme B-secreting CD4 T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 days after tumor implant, resulted in long term survival in 60-90% of animals with no associated toxicity. This antitumor response was dependent on CD4 cells and not CD8+ T cells. Due to its ubiquitous expression and abundance in most cells, the glycolytic enzyme α-enolase is a protein that is often citrullinated and degraded during autophagy and may represent a further novel antitumor target. In this study we demonstrate that immunization of C57Bl, HLA-DR4 and HLA-DP4 transgenic mice with citrullinated enolase peptides induces strong Th1/cytotoxic CD4 responses that efficiently target tumor cells. The Th1 cell repertoire to citrullinated enolase is also detectable in healthy donors and cancer patients. Immunization of mice with citrullinated enolase peptides led to tumor therapy in HLA-DP4 mice with established B16-DP4 tumors (70% survival p = 0.0058) and in HLA-DR4 transgenic mice with established B16F1-DR4 melanoma (50% survival; p = 0.0048) or Pan02-DR4 pancreatic tumors (survival 50%; p = 0.0076). The response was partially mediated by CD4 cytotoxic T cells as tumor therapy was observed against the HLA-DR4-expressing lung tumor LLC2 (40% survival; p = 0.0142) but no survival advantage was witnessed against LLC2 tumors which do not express class II MHC. As MHC-II is not expressed by the majority of tumors unless induced by IFNγ we designed an HLA-DR4 construct under expression of an IFNγ inducible promoter. Immunization of HLA-DR4 mice with citrullinated enolase peptides led to tumor therapy against the established B16F1-IFNγ inducible DR4 melanoma (90% survival p>0.0001). These results suggest that, similar to citrullinated vimentin, citrullinated α-enolase is a promising novel target for human cancer immunotherapy.

Preclinical identification of vaccine induced protective correlates in human leukocyte antigen expressing transgenic mice infected with Coccidioides posadasii

There is an emerging interest to develop human vaccines against medically-important fungal pathogens and a need for a preclinical animal model to assess vaccine efficacies and protective correlates. HLA-DR4 (DRB1∗0401 allele) transgenic mice express a human major histocompatibility complex class II (MHC II) receptor in such a way that CD4+ T-cell response is solely restricted by this human molecule. In this study HLA-DR4 transgenic mice were immunized with a live-attenuated vaccine (ΔT) and challenged by the intranasal route with 50–70 Coccidioides posadasii spores, a potentially lethal dose. The same vaccination regimen offers 100% survival for C57BL/6 mice. Conversely, ΔT-vaccinated HLA-DR4 mice displayed 3 distinct manifestations of Coccidioides infection including 40% fatal acute (FAD), 30% disseminated (DD) and 30% pulmonary disease (PD). The latter 2 groups of mice had reduced loss of body weight and survived to at least 50days postchallenge (dpc). These results suggest that ΔT vaccinated HLA-DR4 mice activated heterogeneous immunity against pulmonary Coccidioides infection. Vaccinated HLA-DR4 mice displayed early expansion of Th1 and Th17 cells and recruitment of inflammatory innate cells into Coccidioides-infected lungs during the first 9dpc. While contraction rates of Th cells and the inflammatory response during 14–35dpc significantly differed among the 3 groups of vaccinated HLA-DR4 mice. The FAD group displayed a sharply reduced Th1 and Th17 response, while overwhelmingly recruiting neutrophils into lungs during 9–14days. The FAD group approached moribund by 14dpc. In contrast, vaccinated HLA-DR4 survivors gradually contracted Th cells and inflammatory response with the greatest rate in the PD group. While vaccinated HLA-DR4 mice are susceptible to Coccidioides infection, they are useful for evaluation of vaccine efficacy and identification of immunological correlates against this mycosis.

Influence of HLA-DR polymorphism and allergic sensitization on humoral immune responses to intact pneumococcus in a transgenic mouse model.

Asthma is independently associated with HLA-DR3 and increased risks of pneumococcal diseases. We aimed to determine whether HLA-DR polymorphism (HLA-DRB1*03), sensitization to house dust mite (HDM), or their interaction affects humoral immune responses to pneumococcal polysaccharide and protein antigens of intact pneumococci. Induction of serum titers of anti-pneumococcal polysaccharide and anti-surface protein IgM and IgG in response to immunization with intact pneumococci (Pn) serotype 14 was determined using humanized HLA-DR3 and DR2 transgenic mice. Transgenic mice were sensitized by injecting HDM and challenged with intranasal HDM. Mice were subsequently immunized with heat-killed Pn14 at day 24. Serum titers of anti-phosphorylcholine (PC) IgM and IgG, anti-pneumococcal polysaccharide, capsular type 14 (PPS14) IgM and IgG, and anti-pneumococcal surface protein A (PspA) IgG were measured. We included a total of 44 mice (22 DR3 and 22 DR2 mice) and half of mice in each group were sensitized with HDM (i.e. 22 HDM-sensitized and 22 control mice). HDM-sensitized mice, irrespective of HLA-DR polymorphism, had significantly lower humoral immune responses. HLA-DR3 mice, irrespective of HDM sensitization, elicited a significantly lower anti-PC IgG response. In contrast, the anti-PspA IgG response was higher in DR3 relative to DR2 mice. The effect of HDM sensitization on lowering humoral immune responses to Pn14 was observed in DR3 mice regardless of the nature of the antigen, whereas such decreases were observed only for the anti-PPS14 IgG and anti-PC IgM responses in DR2 mice. HDM sensitization lowered humoral immune responses to intact pneumococcus and this effect was significantly modified by the HLA-DR polymorphism.

IL-17A is rapidly triggered by staphylococcal superantigens and is pathogenic in toxic shock syndrome

Abstract Toxic shock syndrome (TSS) is a life-threatening illness characterized by high-grade fever, erythematous rash, hypotensive shock, and multi-organ dysfunction within hours of onset. It is caused by exposure to bacterial exotoxins known as superantigens (SAgs), which are produced by the common bacterium Staphylococcus aureus. During infection, SAgs activate up to 20% of T cells inducing a severe systemic inflammatory response that can lead to vascular leakage, multiple organ damage and death. However, the role of IL-17A, a potent inflammatory cytokine, in this potentially fatal reaction remains virtually unexplored. We have found that human peripheral blood mononuclear cells (PBMCs) stimulated with SAgs in vitro immediately up-regulate IL-17A mRNA (up to 12,000-fold) and produce substantial amounts of IL-17A protein within hours. Utilizing the cutting-edge RNA-Flow Cytometry technique, we identified a subset of memory T cells that was responsible for the rapid IL-17A response. We also investigated the effect of IL-17A signaling on downstream inflammatory cytokine and chemokine gene expression via IL-17A receptor blockade. Lastly, we used HLA-DR4 transgenic mice to demonstrate rapid IL-17A production in response to SAgs, similar to human PBMCs. Neutralizing IL-17A in this model greatly reduced tissue damage, morbidity and mortality. Taken together, our results reveal a novel role for memory T cells in TSS and define a previously unrecognized contribution by IL-17A to the initiation and pathogenesis of the syndrome.

HLA-DR3 transgenic mice expressing Nur77-GFP reveal early and robust activation of T cells by superantigens during Staphylococcus aureus pneumonia.

Abstract The role of superantigens (SAg) in the immunopathogenesis Staphylococcus aureus pneumonia has been shown by several indirect means. To directly demonstrate activation of T cells in vivo during staphylococcal pneumonia by SAg and to study the kinetics of T cell activation, HLA-DR3+.Nur77-eGFP+ mice were used. In these mice, rapid expression of green fluorescent protein (GFP) in T cells occurs only upon activation through the TCR, but not following other inflammatory stimuli. Experimental pneumonia was induced using isogenic strains of S. aureus either producing the SAg staphylococcal enterotoxin B (SEB), or not producing any SAg. Mice were sacrificed at different time points and expression of GFP was determined by flow cytometry. As SEB primarily activates CD4+ and CD8+ T cells bearing TCR Vβ8, the expression of GFP within the TCR Vβ8+ sub-populations was examined. Strong upregulation of GFP in the TCR Vβ8+ sub-population was appreciable in both CD4+ and CD8+ T cell subsets (20–25%) even as early as 4 hours only in mice infected with S. aureus strain producing SEB (SEB+SA), but not those infected with the isogenic strain that does not produce SEB (SEB-SA). Serum cytokine/chemokine levels mirrored this finding. By 24 hours, 80–90% of the CD4+ and CD8+ T cells bearing TCR Vβ8+ expressed GFP in mice infected with SEB+SA, compared to only 1–2% in mice infected with SEB-SA. The pattern of expression of CD69 expression, an early T cell activation marker, was distinct from that of GFP. Our novel study directly demonstrated T cell activation and established their activation kinetics in vivo during staphylococcal pneumonia. These results might be useful for timing of administration of novel drugs aimed at suppressing T cell activation during pneumonia.

Immune responses to Mycobacterial heat shock protein 70 accompany self-reactivity to human BiP in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease, and a member of human heat shock protein (HSP) 70 protein family, Binding Immunoglobulin Protein (BiP), has been identified as an important autoantigen for T and B cells. We herein focused on Mycobacterial (Myc) HSPs and immune responses to MycHSPs in RA patients. Serum titers of antibodies against MycHSP70 were significantly elevated in RA patients and correlated with serum anti-BiP antibody titers. A MycHSP70-derived HLA-DR4 major epitope was identified using the proliferative capacity of RA PBMCs as an indicator. The major epitope, MycHSP70287–306, was located at the corresponding position in the major epitope for human BiP336–355, and a strong correlation was found between the proliferation of PBMCs in response to MycHSP70287–306 and BiP336–355. The immunization of HLA-DR4 transgenic mice with MycHSP70 induced the proliferation of T cells and development of anti-BiP antibodies. In contrast, the oral administration of MycHSP70287–306 resulted in the amelioration of collagen-induced arthritis, serum antibody responses, and T cell proliferation. In conclusion, immune responses to MycHSP70 were associated with adaptive immunity against BiP in RA, and could be an important mechanism underlying the development of autoimmunity.

Autoimmune response to transthyretin in juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) is the most common pediatric rheumatological condition. Although it has been proposed that JIA has an autoimmune component, the autoantigens are still unknown. Using biochemical and proteomic approaches, we identified the molecular chaperone transthyretin (TTR) as an antigenic target for B and T cell immune responses. TTR was eluted from IgG complexes and affinity purified from 3 JIA patients, and a statistically significant increase in TTR autoantibodies was observed in a group of 43 JIA patients. Three cryptic, HLA-DR1-restricted TTR peptides, which induced CD4+ T cell expansion and IFN-γ and TNF-α production in 3 out of 17 analyzed patients, were also identified. Misfolding, aggregation and oxidation of TTR, as observed in the synovial fluid of all JIA patients, enhanced its immunogenicity in HLA-DR1 transgenic mice. Our data point to TTR as an autoantigen potentially involved in the pathogenesis of JIA and to oxidation and aggregation as a mechanism facilitating TTR autoimmunity.

Swift Intrahepatic Accumulation of Granulocytic Myeloid-Derived Suppressor Cells in a Humanized Mouse Model of Toxic Shock Syndrome.

Toxic shock syndrome (TSS) and other superantigen-mediated illnesses are associated with 'systemic' immunosuppression that jeopardizes the host's ability to fight pathogens. Here, we define a novel mechanism of 'local' immunosuppression that may benefit the host. Systemic exposure to staphylococcal enterotoxin B (SEB) rapidly and selectively recruited CD11b(+)Gr-1(high)Ly-6C(+) granulocytic myeloid-derived suppressor cells (MDSCs) to the liver of HLA-DR4 transgenic mice. Hepatic MDSCs inhibited SEB-triggered T cell proliferation in a reactive oxygen species-dependent manner, and ex vivo-generated human MDSCs also similarly attenuated the proliferative response of autologous T cells to SEB. We propose a role for MDSCs in mitigating excessive tissue injury during TSS.

Ocular myasthenia gravis induced by human acetylcholine receptor ϵ subunit immunization in HLA DR3 transgenic mice

Extraocular muscles (EOM) are preferentially involved in myasthenia gravis (MG) and acetylcholine receptor (AChR) antibody positive MG patients may occasionally present with isolated ocular symptoms. Although experimental autoimmune myasthenia gravis (EAMG) induced by whole AChR immunization closely mimics clinical and immunopathological aspects of MG, EOM are usually not affected. We have previously developed an EAMG model, which imitates EOM symptoms of MG by immunization of human leukocyte antigen (HLA) transgenic mice with α or γ-subunits of human AChR (H-AChR). To investigate the significance of the ϵ-subunit in ocular MG, we immunized HLA-DR3 and HLA-DQ8 transgenic mice with recombinant H-AChR ϵ-subunit expressed in Escherichia coli. HLA-DR3 transgenic mice showed significantly higher clinical ocular and generalized MG severity scores and lower grip strength values than HLA-DQ8 mice. H-AChR ϵ-subunit-immunized HLA-DR3 transgenic mice had higher serum anti-AChR antibody (IgG, IgG1, IgG2b, IgG2c and IgM) levels, neuromuscular junction IgG and complement deposit percentages than ϵ-subunit-immunized HLA-DQ8 transgenic mice. Control mice immunized with E. coli extract or complete Freund adjuvant (CFA) did not show clinical and immunopathological features of ocular and generalized EAMG. Lymph node cells of ϵ-subunit-immunized HLA-DR3 mice showed significantly higher proliferative responses than those of ϵ-subunit-immunized HLA-DQ8 mice, crude E. coli extract-immunized and CFA-immunized transgenic mice. Our results indicate that the human AChR ϵ-subunit is capable of inducing myasthenic muscle weakness. Diversity of the autoimmune responses displayed by mice expressing different HLA class II molecules suggests that the interplay between HLA class II alleles and AChR subunits might have a profound impact on the clinical course of MG.

A Comprehensive Preclinical Model Evaluating the Recombinant PRAME Antigen Combined With the AS15 Immunostimulant to Fight Against PRAME-expressing Tumors.

The PRAME tumor antigen is a potential target for immunotherapy. We assessed the immunogenicity, the antitumor activity, and the safety and the tolerability of a recombinant PRAME protein (recPRAME) combined with the AS15 immunostimulant (recPRAME+ AS15) in preclinical studies in mice and Cynomolgus monkeys. Four groups of 12 CB6F1 mice received 4 injections of phosphate-buffered saline (PBS), recPRAME, AS15, or recPRAME+AS15. Immunized mice were injected with tumor cells expressing PRAME (CT26-PRAME) 2 weeks or 2 months after the last injection. The mean tumor surface was measured twice a week. Two groups of 10 monkeys received 7 injections of saline or recPRAME+ AS15. T-cell responses were measured by flow cytometry using intracellular cytokine staining (ICS). In CB6F1 mice, repeated injections of recPRAME+ AS15 induced high PRAME-specific antibody titers and mostly CD4+ T cells producing cytokines. This immune response was long-lasting in these animals and was associated with protection against a challenge with PRAME-expressing tumor cells (CT26-PRAME) applied either 2 weeks or 2 months after the last injection; these data indicate the induction of an immune memory. In HLA-A02.01/HLA-DR1 transgenic mice, recPRAME+ AS15 induced both CD4+ and CD8+ T-cell responses, indicating that this antigen can be processed by the human leukocyte antigen and is potentially immunogenic in humans. In addition, a repeated-dose toxicity study in monkeys showed that 7 biweekly injections of recPRAME+ AS15 were well tolerated, and induced PRAME-specific antibodies and T cells. In conclusion, these preclinical data indicate that repeated injections of the PRAME cancer immunotherapeutic are immunogenic and have an acceptable safety profile.

Abstract 5012: Establishment of a transplantable, NY-BR-1 expressing breast cancer model in HLA-transgenic mice

Abstract Introduction: Breast cancer is one of the leading causes of cancer related deaths in women worldwide and current standard therapies show limited efficacy. However, immunotherapeutic approaches like adoptive T cell transfer might represent an attractive option for the treatment of breast carcinoma. The differentiation antigen NY-BR-1 appears as a suitable target for T cell immunotherapy against breast cancer as it is overexpressed in 60% of breast carcinomas compared to healthy breast. Objectives: The aim of this project is to establish a NY-BR-1-expressing, transplantable tumor model in HLA transgenic mice that would allow to investigate the functional role of NY-BR-1-specific HLA-restricted CD4+ T cells in vivo with respect to their capacity to sustain cytotoxic T lymphocytes (CTL)-mediated tumor attack. Furthermore, the capability of NY-BR-1-specific CD4+ T cells to interact with tumor-associated macrophages (TAMs) thereby potentially promoting differentiation of TAMs into immunostimulatory type-1 macrophages (M1) will be investigated. Materials and methods: Stable transfectant clones of the C57BL/6 derived lymphoma cell line EL4 and of the mammary adenocarcinoma cell line EO771 expressing NY-BR-1 were established. Furthermore, an H2Db-restricted, NY-BR-1-specific CTL epitope could be identified which upon peptide immunization of C57BL/6 mice resulted in the generation of NY-BR-1 specific, H2Db-restricted CTL lines, similarly to immunization with a rec. NY-BR-1 encoding adenovirus as observed in parallel experiments. Results and conclusions: Transplanted EL4 and EO771 derived transfectant clones stably expressing NY-BR-1 gave rise to subcutaneous tumors in H2-compatible, HLA-DR4 transgenic mice. In addition, a NY-BR-1-specific CTL line recognizing EL-4/NY-BR-1 expressing transfectants in vitro was successfully generated upon peptide immunization as described above. Studies to investigate the cooperative effect between the aforementioned CTL line and HLA-DR3- and HLA-DR4-restricted NY-BR-1-specifc CD4+ T cell lines, upon joint adoptive transfer into tumor bearing HLA-transgenic mice are currently ongoing. The results presented here demonstrate the first NY-BR-1 expressing mouse tumor model, allowing the investigation of NY-BR-1-specific immune responses in vivo. Citation Format: Krishna Das, Adriane Gardyan, Mathias Vormehr, Karin Mueller-Decker, Inka Zörnig, Dirk Jäger, Wolfram Osen, Stefan B. Eichmüller. Establishment of a transplantable, NY-BR-1 expressing breast cancer model in HLA-transgenic mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5012. doi:10.1158/1538-7445.AM2015-5012

Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis

Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by "dormant" M. tuberculosis and well recognized by T cells from latently M. tuberculosis-infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ(+)/TNF(+)) and IFN-γ(+) CD4(+) T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting.

Superantigens produced by catheter-associated Staphylococcus aureus elicit systemic inflammatory disease in the absence of bacteremia.

SAgs, produced by Staphylococcus aureus, play a major role in the pathogenesis of invasive staphylococcal diseases by inducing potent activation of the immune system. However, the role of SAgs, produced by S. aureus, associated with indwelling devices or tissues, are not known. Given the prevalence of device-associated infection with toxigenic S. aureus in clinical settings and the potency of SAgs, we hypothesized that continuous exposure to SAgs produced by catheter-associated S. aureus could have systemic consequences. To investigate these effects, we established a murine in vivo catheter colonization model. One centimeter long intravenous catheters were colonized with a clinical S. aureus isolate producing SAgs or isogenic S. aureus strains, capable or incapable of producing SAg. Catheters were subcutaneously implanted in age-matched HLA-DR3, B6, and AE(o) mice lacking MHC class II molecules and euthanized 7 d later. There was no evidence of systemic infection. However, in HLA-DR3 transgenic mice, which respond robustly to SSAgs, the SSAg-producing, but not the nonproducing strains, caused a transient increase in serum cytokine levels and a protracted expansion of splenic CD4(+) T cells expressing SSAg-reactive TCR Vβ8. Lungs, livers, and kidneys from these mice showed infiltration with CD4(+) and CD11b(+) cells. These findings were absent in B6 and AE(o) mice, which are known to respond poorly to SSAgs. Overall, our novel findings suggest that systemic immune activation elicited by SAgs, produced by S. aureus colonizing foreign bodies, could have clinical consequences in humans.

The hierarchy of two different immunodominant epitopes influences CD4+ T cell responses (APP5P.112)

Abstract A cell-free antigen processing system for MHC class II was recently reported that has proved to be effective in identifying physiologically relevant CD4+ T cell epitopes from proteins (Hartman/Kim, et al. Nat Med. 16, pp1333-40 (2010)). This system has also provided insights into the mechanism of epitope selection (Kim, et al. Nat Commun. 5:5369 (2014)). We applied this system to a mixture of two different antigens, H5N1-HA1 and LSA-NRC of malaria to examine if there would be a competition for epitope capture. While each dominant epitope from either protein was identified in the system individually, when both proteins were simultaneously present, HA1 dominant epitope was predominantly selected. The competition between different epitopes was verified in vivo by immunizing HLA-DR1 transgenic mice with a mixture of HA1 and LSA-NRC. A recall T cell proliferation showed that T cells responded only to the HA1 and its dominant epitope. These observations did not change even when DR1 mice were immunized with increasing molar ratio of LSA-NRC to HA1 (1:1, 2:1 or 4:1). However, T cell responded to both antigens if mice were immunized first with LSA-NRC dominant epitope followed by HA1 epitope. These findings show a hierarchy for the selection of dominant epitopes from different proteins during antigen processing. Immunodominance hierarchy among different antigens influences the epitope presentation to the same MHC II, which can be important factor for designing combination vaccines.

A Bell-Shaped Dose-Dependent Induction of Allergen-Specific Tetramer+ CD4 T Cells and Activated Lung ILC2s Following Epicutaneous Allergen Sensitization in HLA-DR4 Transgenic Mice

A Bell-Shaped Dose-Dependent Induction of Allergen-Specific Tetramer+ CD4 T Cells and Activated Lung ILC2s Following Epicutaneous Allergen Sensitization in HLA-DR4 Transgenic Mice