1. A transplantable tumor model allowing investigation of NY-BR-1-specific T cell responses in HLA-DRB1*0401 transgenic mice
Krishna Das, et al. BMC Cancer. 2019 Sep 13;19(1):914. doi: 10.1186/s12885-019-6102-6.
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 IFNγ catch or IFNγ 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.
2. Identification of a naturally processed NY-ESO-1 peptide recognized by CD8+ T cells in the context of HLA-B51
Elke Jäger, et al. Cancer Immun. 2002 Sep 19;2:12.
NY-ESO-1 is one of the most immunogenic cancer antigens known to date, inducing humoral and cellular immune responses in a high proportion of patients with advanced NY-ESO-1-expressing cancers. The assessment of spontaneous and vaccine-induced CD8+ T cell responses has been limited to a small number of known NY-ESO-1 epitopes presented by MHC class I alleles. Recently, a new method to monitor NY-ESO-1-specific CD8+ T cell responses was introduced that does not depend on the individual MHC class I status and on predefined peptide epitopes. Antigen-presenting cells transduced with recombinant adenoviral vectors encoding NY-ESO-1 were used to stimulate CD8+ selected NY-ESO-1-specific T cells. Effector cells were tested for recognition of autologous B cell targets transfected with NY-ESO-1 using a recombinant vaccinia virus construct. Using a modified approach we identified the NY-ESO-1 p94-102 peptide as being recognized by CD8+ T cells in the context of HLA- B51. NY-ESO-1 p94-102 specific CD8+ T cells recognized naturally processed NY-ESO-1 presented by HLA-B51+ monocyte-derived dendritic and tumor cells. Transfection of target cells with NY-ESO-1 combined with different HLA class I alleles confirmed that the NY-ESO-1 peptide was naturally processed and recognized by HLA-B51-restricted CD8+ T cell lines and clones. Therefore, NY-ESO-1 p94-102 is a new candidate peptide antigen for cancer immunotherapy and for the monitoring of spontaneous and vaccine-induced NY-ESO-1-specific T cell responses in HLA- B51+ patients with NY-ESO-1 expressing malignancies.
3. Humoral and cellular immune responses against the breast cancer antigen NY-BR-1: definition of two HLA-A2 restricted peptide epitopes
Dirk Jäger, Julia Karbach, Claudia Pauligk, Inka Seil, Claudia Frei, Yao-Tseng Chen, Lloyd J Old, Alexander Knuth, Elke Jäger Cancer Immun. 2005 Dec 12;5:11.
Cancer immunotherapy depends on the identification of tumor-specific target antigens that are predominantly expressed in cancer cells and not in normal tissues. Here, we report the cloning and the expression analysis of the differentiation antigen NY-BR-1 that we have identified in a previous SEREX (serological analysis of recombinant cDNA expression libraries) screening. The cloning of the full length NY-BR-1 sequence led to the prediction of an open reading frame of 4.2 kb, encoding a protein of 158.9 kDa. NY-BR-1 mRNA expression analysis revealed tissue-specific expression in normal testis and breast tissues, as well as in 70% of breast tumors. We now show that NY-BR-1 is also sporadically expressed in normal prostate and in 32% of prostate tumors. Furthermore, we were able to identify two HLA-A2 restricted NY-BR-1 epitopes (p158-167 and p960-968) that are recognized by CD8+ T cell clones (NW1100-CTL-7 and NW1100-CTL-43, respectively), as determined by ELISPOT analysis and tetramer staining. Cotransfection assays of COS-7 cells also demonstrated that these two peptides are naturally processed and presented on HLA-A2 molecules. The identification of these two naturally processed NY-BR-1-specific CD8+ T cell epitopes opens the perspective for active immunotherapy of HLA-A2 positive patients with NY-BR-1 expressing tumors.
