Alexandra Klevenz

Cross-presentation of oral antigens by liver sinusoidal endothelial cells leads to CD8 T cell tolerance.

After ingestion, oral antigens distribute systemically and provoke T cell stimulation outside the gastrointestinal tract. Within the liver, scavenger liver sinusoidal endothelial cells (LSEC) eliminate blood‐borne antigens and induce T cell tolerance. Here we investigated whether LSEC contribute to oral tolerance. Oral antigens were efficiently cross‐presented on H‐2Kb by LSEC to naive CD8 T cells. Cross‐presentation efficiency in LSEC but not dendritic cells was increased by antigen‐exposure to heat or low pH. Mechanistically, cross‐presentation in LSEC requires endosomal maturation, involves hsc73 and proteasomal degradation. H‐2Kb‐restricted cross‐presentation of oral antigens by LSEC in vivo induced CD8 T cell priming and led to development of CD8 T cell tolerance in two independent experimental systems. Adoptive transfer of LSEC from mice fed with antigen (ovalbumin) into RAG2–/– knockout mice, previously reconstituted with naive ovalbumin‐specific CD8 T cells, prevented development of specific cytotoxicity and expression of IFN‐γ in CD8 T cells. Using a new transgenic mouse line expressing H‐2Kb only on endothelial cells, we have demonstrated that oral antigen administration leads to tolerance in H‐2Kb‐restricted CD8 T cells. Collectively, our data demonstrate a participation of the liver, in particular scavenger LSEC, in development of CD8 T cell tolerance towards oral antigens.

Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire

The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability.

Dickkopf-3 Acts as a Modulator of B Cell Fate and Function

The mechanisms responsible for the generation of a mature B1 and B2 cell compartment are still poorly understood. In this study, we demonstrated that absence of Dickkopf-3 (DKK3) led to changes in the composition of the B cell compartment, which were due to an altered development and maintenance program of B cells. Development of B2 cells was impaired at the pre- and immature B cell stage, resulting in decreased numbers of follicular B cells in adult DKK3-deficient mice. Furthermore, DKK3 limited B1 cell self-maintenance in the periphery, by decreasing the survival and proliferation behavior of B1 cells. DKK3 may act via the BCR signaling pathway, as Ca2+ influx upon BCR stimulation was increased and SiglecG, a molecule shown to inhibit Calcium signaling, was downregulated in the absence of DKK3. DKK3-deficient mice exhibited altered Ab responses and an increased secretion of the cytokine IL-10. Additionally, DKK3 limited autoimmunity in a model of systemic lupus erythematosus. In summary, we identified DKK3 as a novel modulator interfering with B cell fate as well as the maintenance program of B cells, leading to changes in B cell immune responses.

ERAP1 overexpression in HPV-induced malignancies: A possible novel immune evasion mechanism

ABSTRACT Immune evasion of tumors poses a major challenge for immunotherapy. For human papillomavirus (HPV)-induced malignancies, multiple immune evasion mechanisms have been described, including altered expression of antigen processing machinery (APM) components. These changes can directly influence epitope presentation and thus T-cell responses against tumor cells. To date, the APM had not been studied systematically in a large array of HPV+ tumor samples. Therefore in this study, systematic expression analysis of the APM was performed on the mRNA and protein level in a comprehensive collection of HPV16+ cell lines. Subsequently, HPV+ cervical tissue samples were examined by immunohistochemistry. ERAP1 (endoplasmic reticulum aminopeptidase 1) was the only APM component consistently altered – namely overexpressed – in HPV16+ tumor cell lines. ERAP1 was also found to be overexpressed in cervical intraepithelial neoplasia and cervical cancer samples; expression levels were increasing with disease stage. On the functional level, the influence of ERAP1 expression levels on HPV16 E7-derived epitope presentation was investigated by mass spectrometry and in cytotoxicity assays with HPV16-specific T-cell lines. ERAP1 overexpression did not cause a complete destruction of any of the HPV epitopes analyzed, however, an influence of ERAP1 overexpression on the presentation levels of certain HPV epitopes could be demonstrated by HPV16-specific CD8+ T-cells. These showed enhanced killing toward HPV16+ CaSki cells whose ERAP1 expression had been attenuated to normal levels. ERAP1 overexpression may thus represent a novel immune evasion mechanism in HPV-induced malignancies, in cases when presentation of clinically relevant epitopes is reduced by overactivity of this peptidase.

Nck adaptor proteins modulate differentiation and effector function of T cells

Understanding the molecular mechanisms regulating T cell reactivity is required for successful reprogramming of immune responses in medical conditions, characterized by dysfunctions of the immune system. Nck proteins are cytoplasmic adaptors mediating diverse cellular functions, including TCR signaling. By enhancing TCR signal strength, Nck proteins influence thymic selection and regulate the size and sensitivity of the peripheral T cell repertoire. Here, we investigated the contribution of Nck proteins to CD4+ T cell differentiation and effector function using Nck.T−/− mice. Impaired GC formation and reduced Tfh were observed in Nck.T−/− mice after immunization with T cell‐dependent antigens. Th2/Tfh‐related cytokines, such as IL‐4, IL‐10, and IL‐21, were decreased in Nck.T−/− mice T cells. Moreover, an increased susceptibility to cell death of Tfh cells in Nck.T−/− mice was associated with decreased levels of Akt phosphorylation. As a result of this dysregulation in Tfh cells of Nck.T−/− mice, we found impaired production and affinity maturation of antibodies against T cell‐dependent antigens. Thus, Nck proteins not only participate in thymic selection and generation of the peripheral T cell repertoire but also are involved in the differentiation and effector functions of CD4+ T cells.

Dickkopf-3 Contributes to the Regulation of Anti-Tumor Immune Responses by Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are known to limit immune responses in vivo by multiple soluble factors. Dickkopf-3 (DKK3), a secreted glycoprotein, has recently been identified as a novel immune modulator. Since DKK3 has been reported to be produced by MSCs, we investigated whether DKK3 contributes to the immune suppression of anti-tumor responses by MSCs. Whereas wild-type MSCs inhibited immune responses against two different transplantation tumors, DKK3-deficient MSCs did not affect the rejection process. Increased CD8+ T cell and reduced M2-type macrophages infiltration was observed in tumors inoculated together with DKK3-deficient MSCs. Thus, DKK3 could alter the composition of the tumor stroma, thereby supporting the MSCs-mediated suppression of immune responses against these tumor transplants.

Abstract B31: Identification of target T cell epitopes for a therapeutic HPV16 vaccine

To rationally design therapeutic human papillomavirus (HPV) vaccines, it is important to know which T cell epitopes are present on HPV-transformed cells. HPV affects the cellular antigen processing machinery, thus not every epitope derived from viral proteins is necessarily presented by Human Leukocyte Antigen (HLA)-molecules on the cancer cell surface. HPV16 has been identified as the causative agent in 50% of all cervical cancer cases and in approximately 95% of all extra-cervical mucosal HPV-induced tumors. The transforming potential of high-risk HPVs is mediated by two consistently expressed viral oncoproteins, E6 and E7. As the induction and maintenance of the malignant phenotype depend on these two proteins, they are ideal targets for immunotherapy. A therapeutic vaccine that is applicable to everyone without prior HLA typing needs to contain epitopes for all HLA-types. Definition of epitopes for the five major HLA supertypes allows >95% population coverage. To date, HPV T cell epitopes have mostly been determined for the most prevalent HLA type, HLA-A2. We now aim to identify HPV16 E6 and E7 T cell epitopes for the HLA-A3, HLA-A11 and HLA-A24 supertypes. Different in silico prediction algorithms were used to predict prospective epitopes from the HPV16 E6 and E7 proteins for the mentioned HLA supertypes. In total, 64 epitopes, comprised of 8- to 11-mer peptides, were predicted for HLA-A3, 90 epitopes for HLA-A11 and 115 epitopes for HLA-A24. In competition-based binding assays, 15 previously known binding peptides were confirmed and 93 novel binding peptides were identified. For the generation of peptide-specific short term T cell lines, peripheral blood mononuclear cells (PBMCs) from buffy coats with known HLA types were stimulated with previously identified HLA-matching peptides and cultured for twelve days. Several peptides induced interferon gamma (IFNγ)-responses. Immunogenicity of the four most promising candidate peptides for HLA-A24 is currently assessed by generation of peptide-specific long term T cell lines from healthy donors. Functional assays, such as IFNγ-ELISpot assays and cytotoxicity assays, are conducted to determine the best vaccine candidates. The presence of binding peptides on HPV16-transformed cells is additionally analyzed by mass spectrometry analysis of immunoprecipitated HLA-peptide complexes. In conclusion, we identified several new HPV16 E6 and E7 T cell epitopes. Verified epitopes are the basis of rational therapeutic vaccine design and also important for immunomonitoring purposes. Citation Format: Stephanie Hoppe, Jan Winter, Renata Blatnik, Julia Schessner, Lisa Dressler, Alina Steinbach, Hadeel Khallouf, Martin Wuehl, Alexandra Klevenz, Angelika B. Riemer. Identification of target T cell epitopes for a therapeutic HPV16 vaccine. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B31.

Abstract LB-235: Therapeutic human papillomavirus vaccine design based on epitopes identified on the tumor cell surface by mass spectrometry

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Detailed knowledge about T cell epitopes, which are bona fide presented on the surface of human papillomavirus (HPV)-transformed cells, is essential for rational design of therapeutic HPV vaccines. HPV affects the cellular antigen processing machinery, thus not every epitope derived from viral proteins is presented on human leukocyte antigen (HLA) molecules. Even the presented epitopes are displayed in low abundance. In this study, we developed a highly sensitive nano-UPLC-ESI-MS3 multiple-reaction monitoring mass spectrometry (MS) approach for direct detection of low-abundant epitopes on the cell surface. Several web-based prediction algorithms were used to predict prospective epitopes from the HPV16 E6 and E7 proteins. These candidate epitopes were tested for actual HLA binding in cellular binding assays. The presence of binding peptides on HPV16-transformed cells was analyzed by our MS technology. Immunogenicity of candidate peptides was assessed in vitro with short-term and long-term T cell line experiments, generated from peripheral blood mononuclear cells of healthy donors, and in vivo for a selected HLA-A2-restricted epitope in HLA-A2/DR-1 transgenic mice. To ensure >95% population coverage, prospective HPV epitopes were predicted for the HLA supertypes HLA-A1, A2, A3, A11, A24, B7, and B15. Close to 500 peptides were tested in competition-based binding assays and multiple novel binders were identified. MS epitope detection was first established for HLA-A2 epitopes, and is now extended to the other supertypes. In the immunogenicity assays, several peptides induced robust IFN-γ responses. In conclusion, several new HPV16 E6 and E7 epitopes were identified and validated in this study. They represent promising candidates for inclusion into a therapeutic HPV vaccine. Validated epitopes are the basis of rational therapeutic vaccine design and are also important for immunomonitoring purposes. Citation Format: Stephanie Hoppe, Renata Blatnik, Julia P. Schessner, Lisa Dressler, Alina Steinbach, Jan Winter, Martin Wuehl, Alexandra Klevenz, Hadeel Khallouf, Angelika B. Riemer. Therapeutic human papillomavirus vaccine design based on epitopes identified on the tumor cell surface by mass spectrometry. [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 LB-235. doi:10.1158/1538-7445.AM2015-LB-235