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A transplantable tumor model allowing investigation of NY-BR-1-specific T cell responses in HLA-DRB1*0401 transgenic mice.

ABSTRACT: BACKGROUND:NY-BR-1 has been described as a breast cancer associated differentiation antigen with intrinsic immunogenicity giving rise to endogenous T and B cell responses. The current study presents the first murine tumor model allowing functional investigation of NY-BR-1-specific immune responses in vivo. METHODS:A NY-BR-1 expressing tumor model was established in DR4tg mice based on heterotopic transplantation of stable transfectant clones derived from the murine H2 compatible breast cancer cell line EO771. Composition and phenotype of tumor infiltrating immune cells were analyzed by qPCR and FACS. MHC I binding affinity of candidate CTL epitopes predicted in silico was determined by FACS using the mutant cell line RMA-S. Frequencies of NY-BR-1 specific CTLs among splenocytes of immunized mice were quantified by FACS with an epitope loaded Db-dextramer. Functional CTL activity was determined by IFN? catch or IFN? ELISpot assays and statistical analysis was done applying the Mann Whitney test. Tumor protection experiments were performed by immunization of DR4tg mice with replication deficient recombinant adenovirus followed by s.c. challenge with NY-BR-1 expressing breast cancer cells. RESULTS:Our results show spontaneous accumulation of CD8+ T cells and F4/80+ myeloid cells preferentially in NY-BR-1 expressing tumors. Upon NY-BR-1-specific immunization experiments combined with in silico prediction and in vitro binding assays, the first NY-BR-1-specific H2-Db-restricted T cell epitope could be identified. Consequently, flow cytometric analysis with fluorochrome conjugated multimers showed enhanced frequencies of CD8+ T cells specific for the newly identified epitope in spleens of immunized mice. Moreover, immunization with Ad.NY-BR-1 resulted in partial protection against outgrowth of NY-BR-1 expressing tumors and promoted intratumoral accumulation of macrophages. CONCLUSION:This study introduces the first H2-Db-resctricted CD8+ T cell epitope-specific for the human breast cancer associated tumor antigen NY-BR-1. Our novel, partially humanized tumor model enables investigation of the interplay between HLA-DR4-restricted T cell responses and CTLs within their joint attack of NY-BR-1 expressing tumors.

SUBMITTER: Das K

PROVIDER: S-EPMC6743128 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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A transplantable tumor model allowing investigation of NY-BR-1-specific T cell responses in HLA-DRB1*0401 transgenic mice.

Das Krishna K Eisel David D Vormehr Mathias M Müller-Decker Karin K Hommertgen Adriane A Jäger Dirk D Zörnig Inka I Feuerer Markus M Kopp-Schneider Annette A Osen Wolfram W Eichmüller Stefan B SB

BMC cancer 20190913 1

<h4>Background</h4>NY-BR-1 has been described as a breast cancer associated differentiation antigen with intrinsic immunogenicity giving rise to endogenous T and B cell responses. The current study presents the first murine tumor model allowing functional investigation of NY-BR-1-specific immune responses in vivo.<h4>Methods</h4>A NY-BR-1 expressing tumor model was established in DR4tg mice based on heterotopic transplantation of stable transfectant clones derived from the murine H2 compatible b ...[more]

PMID: 31519152

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Project description:A more effective vaccine against Mycobacterium tuberculosis is needed, and a number of M. tuberculosis vaccine candidates are currently in preclinical or clinical phase I and II studies. One of the strategies to select M. tuberculosis (protein) targets to elicit a CD8(+) or CD4(+) T-cell response is to gauge the binding of candidate peptides to major histocompatibility complex (MHC) class I or class II molecules, a prerequisite for successful peptide presentation and to expand antigen-specific T cells. We scanned 61 proteins from the M. tuberculosis proteome for potential MHC class II-presented epitopes that could serve as targets for CD4(+) T-cell responses. We constructed a peptide microarray consisting of 7,466 unique peptides derived from 61 M. tuberculosis proteins. The peptides were 15-mers overlapping by 12 amino acids. Soluble recombinant DRB1*0101 (DR1), DRB1*1501 (DR2), and DRB1*0401 (DR4) monomers were used to gauge binding to individual peptide species. Out of 7,466 peptides, 1,282, 674, and 1,854 peptides formed stable complexes with HLA-DR1, -DR2, and -DR4, respectively. Five hundred forty-four peptides bound to all three MHC class II molecules, 609 bound to only two, and 756 bound to only a single MHC class II molecule. This allowed us to rank M. tuberculosis proteins by epitope density. M. tuberculosis proteins contained "hot spots," i.e., regions with enriched MHC class II binding epitopes. Two hundred twenty-two peptides that formed MHC class II-peptide complexes had previously been described as exclusively recognized by IgG in sera from patients with active pulmonary tuberculosis, but not in sera from healthy individuals, suggesting that these peptides serve as B-cell and CD4(+) T-cell epitopes. This work helps to identify not only M. tuberculosis peptides with immunogenic potential, but also the most immunogenic proteins. This information is useful for vaccine design and the development of future tools to explore immune responses to M. tuberculosis.

Project description:ObjectiveAntibodies toward the citrullinated form of the synovial antigen vimentin are specific for rheumatoid arthritis (RA) and are associated with HLA-DRB1*0401. This suggests that T cells specific for peptides derived from citrullinated vimentin presented in the context of HLA-DRB1*0401 may contribute to the etiopathogenesis of RA. The aim of this study was to identify immunodominant epitopes from citrullinated vimentin presented by HLA-DRB1*0401 and to characterize the resulting T cell responses.MethodsWe first predicted an HLA-binding T cell epitope from citrullinated vimentin based on the binding motif of HLA-DRB1*0401 and then confirmed its affinity. A class II major histocompatibility complex (MHC) tetramer loaded with the citrullinated form of vimentin aa 59-78 (cit-vimentin aa 59-78) was constructed and used to screen for specific T cells in HLA-DRB1*0401-transgenic mice, patients with RA, and healthy control subjects. Additionally, the cytokine output following cit-vimentin aa 59-78 challenge was analyzed in patients and healthy control subjects by multicolor flow cytometry and Luminex-based analysis.ResultsThe citrullinated form of vimentin aa 59-78 bound to HLA-DRB1*0401, but the native form could not. Subsequently, cit-vimentin aa 59-78-specific T cells were detected in immunized mice and in the periphery of both HLA-DR*0401-positive healthy control subjects and HLA-DR*0401-positive patients with RA, using class II MHC tetramers, CD154 up-regulation, and intracellular cytokine measurements. As demonstrated in cell culture supernatants, the production of cytokines (predominantly interferon-γ) in response to cit-vimentin aa 59-78 was significantly higher in patients compared with controls.ConclusionHere, we describe a posttranslational modification of an RA candidate autoantigen toward which HLA-DRB1*0401-restricted T cells can be detected in both patients with RA and healthy controls but for which a proinflammatory response is observed uniquely in patients with RA.

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A transplantable tumor model allowing investigation of NY-BR-1-specific T cell responses in HLA-DRB1*0401 transgenic mice.

Publications

A transplantable tumor model allowing investigation of NY-BR-1-specific T cell responses in HLA-DRB1*0401 transgenic mice.

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