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 D^b-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-D^b-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-D^b-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.

Keywords: CTL epitope; Differentiation antigen; HLA transgenic mice; NY-BR-1.

Fig. 1

Characterization of EO771/NY-BY-1 transfectant clones. EO771/NY-BR-1 transfectant clones generated by transfection of EO771 cells with a linearized plasmid pcDNA3.1-NY-BR-1 followed by limiting dilution were characterized for NY-BR-1 expression in vitro and the ability to form tumors in vivo. a NY-BR-1 protein expression (159 kDa) in the selected clones was analyzed by Western blot. β-actin (42 kDa) was used as a loading control. b HLA-DRB1*0401tg mice were injected s.c. on the right flank with 2 × 10⁵ EO771, EONY #9 or EONY #17 cells and tumor growth was monitored for 18 days post cell injection. Error bars represent SEM (n = 10). Tumor area was measured and statistical analysis was performed using a mixed linear model with random intercept for animal. Difference between cell lines was highly significant (p < 0.0001); pairwise comparisons: *** p < 0.0001; * p = 0.0157

Fig. 2

Characterization of tumor-infiltrating leukocytes in EO771 and EONY tumors. 2 × 10⁵ EO771 cells, EONY#9 cells or EONY#17 cells were injected s.c. into the right flank of HLA-DRB1*0401tg mice (n = 10). Tumor-infiltrating leukocytes were isolated 20 days post cell implantation and analyzed by flow cytometry. a Percentage of CD8⁺ T cells, CD4⁺ T cells and CD11b⁺F4/80⁺ macrophages among CD45⁺ leukocytes is depicted for the different tumors. b The frequency of TAMs expressing M1-associated markers HLA-DR4 and iNOS or M2-associated marker CD206. c The corresponding surface expression levels (MFI) on the positive cells is presented. Error bars depict SEM and statistical analysis performed using One-way-ANOVA with Tukey’s multiple comparisons (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001)

Fig. 3

Analysis of TAM polarization in EO771 and EONY#17 tumors. Tumor infiltrating leukocytes isolated from EO771 and EONY#17 tumors were enriched for CD11b⁺ cells using anti-CD11b microbeads. The expression of various genes associated with a M2-like or b M1-like macrophages was analyzed in the isolated CD11b⁺ cells by qRT-PCR. Log fold change of each gene normalized to the house-keeping gene is shown. Error bars represent SEM and statistical analysis was performed using Student’s t test and Bonferoni-Holm adjusted p values were calculated (* p < 0.05; ** p < 0.01; *** p < 0.001)

Fig. 4

Peptide binding affinities of putative H2^b-restricted NY-BR-1-specific CTL epitopes. Peptide binding affinity of putative CTL epitopes to H2 K^b and –D^b molecules was tested on RMA-S cells using a H2-D^b-specific monoclonal antibody B22.249 or b H2-K^b-specific monoclonal antibody E3–25. Surface expression of MHC I molecules stabilized by external addition of synthetic candidate epitopes was measured by flow cytometry to provide an estimate of peptide binding affinity. The H2-D^b-restricted E7-specific epitope E7_49–57 and the H2-K^b-restricted OVA-specific epitope OVA_257–264 served as positive controls

Fig. 5

NY-BR-1_1241-1249 is a natural H2-D^b-restricted CTL epitope. HLA-DRB1*0401tg mice were immunized i.p. either with 5 × 10⁸ pfu Ad.NY-BR-1 (n = 3) or with 5 × 10⁸ pfu Ad. Control (n = 3) and splenocytes were harvested 14 days post immunization. a Splenocytes were incubated overnight with 5 μg/mL of synthetic peptides representing predicted NY-BR-1 epitopes. IFNγ secreting CD8⁺ T cells were analyzed by IFNγ catch assay and the percentage of CD8⁺ T cells secreting IFNγ is depicted. Immunization with Ad.NY-BR-1 resulted in CD8⁺ T cells reactive against peptides NY-BR-1_1241-1249. b and c Splenocytes from immunized mice were stained with fluorescently labelled H2-D^b dextramers loaded with NY-BR-1_1241-1249 (NY-BR-1-specific dextramer) or HPV 16 E7_49–57 (control dextramer). b Proportion of dextramer⁺CD8⁺ T cells among viable CD14⁻CD3⁺ splenocytes from control mice (left panel) or from an Ad.NY-BR-1 immunized mice (right panel). c Representative dot plot depicting the proportion of dextramer⁺CD8⁺ T cells among viable CD14⁻CD3⁺ splenocytes from a control mouse (left panel) or from an Ad.NY-BR-1 immunized mouse (right panel)

Fig. 6

Immunization against NY-BR-1 delays tumor growth and diminishes accumulation of TAMs with reduced immunosuppressive phenotype. HLA-DRB1*0401tg mice were immunized i.p. with 5 × 10⁸ pfu Ad.NY-BR-1 (n = 10) or with 5 × 10⁸ pfu Ad. Control (n = 10) and 2 × 10⁵ EONY#17 cells were injected s.c. into the right flank 14 days post immunization. Tumor growth was monitored for 30 days followed by excision of tumors and isolation of tumor-infiltrating leukocytes. a Schematic representation of the experimental procedure. b Tumor area was measured and statistical analysis was performed using a mixed linear model with random intercept for animal. Difference in treatment was highly significant (p < 0.0001). Analysis was performed using SAS Version 9.4 (SAS Institute Inc., Cary, NC, USA.). c Tumor weight was also measured. d-h CD45⁺CD11b⁺F4/80⁺ macrophages were isolated by FACS and RNA was extracted for gene expression analysis. d Frequency of CD11b⁺F4/80⁺ macrophages among CD45⁺ cells, e frequency of HLA-DR4⁺ cells among macrophages and f level of HLA-DR4 surface expression on TAMs. c-f Statistical analysis was performed using Student’s t test and p values are indicated (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). Expression of g M2- and h M1-associated genes in TAMs was quantified by qRT-PCR. Log fold expression change of a gene normalized to the house-keeping gene is shown. Error bars represent SEM and Bonferoni-Holm adjusted p values were calculated (* p < 0.05; ** p < 0.01; *** p < 0.001)