Chemotherapy and radiation therapy elicits tumor specific T cell responses in a breast cancer patient

Abstract

Background: Experimental evidence and clinical studies in breast cancer suggest that some anti-tumor therapy regimens generate stimulation of the immune system that accounts for tumor clinical responses, however, demonstration of the immunostimulatory power of these therapies on cancer patients continues to be a formidable challenge. Here we present experimental evidence from a breast cancer patient with complete clinical response after 7 years, associated with responsiveness of tumor specific T cells.

Methods: T cells were obtained before and after anti-tumor therapy from peripheral blood of a 63-years old woman diagnosed with ductal breast cancer (HER2/neu+++, ER-, PR-, HLA-A*02:01) treated with surgery, followed by paclitaxel, trastuzumab (suspended due to cardiac toxicity), and radiotherapy. We obtained a leukapheresis before surgery and after 8 months of treatment. Using in vitro cell cultures stimulated with autologous monocyte-derived dendritic cells (DCs) that produce high levels of IL-12, we characterize by flow cytometry the phenotype of tumor associated antigens (TAAs) HER2/neu and NY-ESO 1 specific T cells. The ex vivo analysis of the TCR-Vβ repertoire of TAA specific T cells in blood and Tumor Infiltrating Lymphocytes (TILs) were performed in order to correlate both repertoires prior and after therapy.

Results: We evidence a functional recovery of T cell responsiveness to polyclonal stimuli and expansion of TAAs specific CD8+ T cells using peptide pulsed DCs, with an increase of CTLA-4 and memory effector phenotype after anti-tumor therapy. The ex vivo analysis of the TCR-Vβ repertoire of TAA specific T cells in blood and TILs showed that whereas the TCR-Vβ04-02 clonotype is highly expressed in TILs the HER2/neu specific T cells are expressed mainly in blood after therapy, suggesting that this particular TCR was selectively enriched in blood after anti-tumor therapy.

Conclusions: Our results show the benefits of anti-tumor therapy in a breast cancer patient with clinical complete response in two ways, by restoring the responsiveness of T cells by increasing the frequency and activation in peripheral blood of tumor specific T cells present in the tumor before therapy.

Keywords: Breast cancer; CTLA-4; Chemotherapy; HER2/neu; T cells; TCR repertoire; Type I alpha dendritic cells.

Figures

Fig. 1

Chemotherapy restores deficient immune response in breast cancer patient. a. Representative contour plots of SSC-A vs CD3 in Lymphocyte cells (by SSC-A vs FSC-A) after 72 h of polyclonal stimulation with anti-CD3/CD28/CD2 micro beads, 2d-aDCs or a combination of micro beads in addition to 2d-aDCs in healthy donors (n = 12), and breast cancer patients (n = 8) (before and after anti-TTx), numbers inside correspond to MFI of CD3 in lymphocyte gate (SSC-A vs FSC-A). b. Contour plots of CD69 percentage expression in T cells (CD3+) as in panel A, numbers correspond to the percentage of CD69 expressing cells in CD3+ T cells. c. Delta (from left to right) of IFN-γ, IL-8, IL-1β, and IL-6 cytokines relative to unstimulated control in healthy donors (Dashed bars) (n = 6) and MCC-002 breast cancer patient (before and after anti-TTx white and black bars respectively), bars show SEM. X: not detected. Results of experiments presented in panel c are representative of three performed

Fig. 2

2d-aDCs induce expansion and activation of CD8+ NY-ESO1 and HER2-specific T cells in a breast cancer patient after anti-TTx. a. Percentage of TEMRA HER2/neu-specific CD8+ T cells subsequent to induction of in situ 2d-aDCs or 2d-stDCs in total PBMCs (MCC-002) induced for 6 days comparing samples obtained before (left column) and after anti-TTx (right column) pulsed with HER2/neu369–377 peptide. b. Percentage of TEMRA NY-ESO 1-specific CD8+ T cells from patient MCC-002 derived from naïve CD8+ T cells in co-culture with 2d-aDCs or 2d-stDCs-pulsed/unpulsed with NY-ESO 1157–165 and stimulated with DCs for 14 days and boosted with corresponding DCs for 6 additional days, comparing samples obtained before (left column) and after anti-TTx (right column). X: not done. Results of experiments presented in panels a and b are representative of two performed

Fig. 3

Anti-TTx induces enrichment of CTLA-4 in tumor specific T cells. a. Expression of tetramer positive (pool of Her2/neu or viral tetramers) in CD8+ T cells after 6 days of stimulation with aDCs derived in situ and pulsed with a combination of HLA-A*02:01 restricted peptides (TAA peptide pool HER2/neu369–377, HER2/neu689–697 and HER2/neu435–443, or viral peptide pool CMVPP65, EBV280–288, and FLU58–66). b. Pie chart of naïve and memory sub-populations distribution in tetramer positive CD8+ T cells (TAA or viral specific) after stimulation with pulsed 2d-aDCs before and after anti-TTx (Blue; naïve, Grey; TCM, yellow; TEM; and green TEMRA). c. Fold increase of inhibitory receptors (PD1, BTLA, and CTLA-4) expression between tetramer positive over tetramer negative CD8+ T cells after stimulation with peptide pulsed 2d-aDCs, obtained before (white bars) and after (black bars) anti-TTx

Fig. 4

Anti-TTx induces the expansion, differentiation and CTLA-4 expression in Her2/neu369–377 tumor specific CD8+ T cells. a. Representative contour plots of tetramer specific CD8+ T cells staining for HER2/neu369–377 (KIFGSLAFL), HER2/neu689–697 (RLLQETELV), HER2/neu435–443 (ILHNGAYSL) and NY-ESO 1157–165 (SLLMWITQV), and two tetramers for viral antigens (NLVPMVATV – CMVPP65, and GILGFVFTL – influenza M1 FLU58–66), stained ex vivo in PBMCs from a breast cancer patient before and after anti-TTx in purified CD8+ T cells. The number inside the plots represents the percentage of tetramer-specific CD8+ T cells. b. Representative contour plots of tetramer specific CD8+ T cells after 7 days of in vitro stimulation of PBMCs with the corresponding peptide pulsed 2d-aDCs, the number inside the plots represents the percentage of tetramer-specific CD8+ T cells. Results of experiments presented in panels a, and B are representative of two performed. c. and d. Representative pie charts of ex vivo (c) or after in vitro PBMC stimulation (d), as described in a and b, with the percentage of the phenotype of naïve and memory sub-populations in CD8+ tetramer positive T cells for four TAAs and two viral HLA-A*02:01 peptides before and after anti-TTx. Blue; naïve, Orange; TSCM, Grey; TCM, yellow; TEM; and green TEMRA. Results of experiments presented in panels c and d are representative of two performed. e. Representative contour plots of CTLA-4 vs CD8 in tetramer positive CD8+ T cells for HER2/neu369–377 (KIFGSLAFL) and NY-ESO1157–165 (SLLMWITQV) tetramer + CD8+ T cells for two viral antigens (NLVPMVATV – CMVPP65, and GILGFVFTL – influenza M1 FLU58–66), in PBMCs stimulated with in situ 2d-aDCs from PBMCs obtained before and after anti-TTx as described in a and b. Results of experiments presented are representative of two performed

Fig. 5

Increased correlation of TCR Vβ families between tumor infiltrating lymphocytes and HER2/neu tetramer CD8+ T cells after chemotherapy. a. Dot plot example of an ex vivo flow cytometry analysis of TCR-Vβ families 13.2, 4 and 7.2 in CD8+ T cells HER2/neu369–377 tetramer negative vs. tetramer positive from before and after anti-TTx cells, each gate represents one TCR-Vβ family, PE positive correspond to Vβ13.2, the double positive cells corresponds to family Vβ4, and FITC positive corresponds to family Vβ7.2, numbers correspond to percentage of each family, left; Delta of the percentage of each of 24 families (after minus before therapy) in total CD8+ T cells (empty bars) or tetramer positive CD8+ T cells (black bars), right. b. Heat map of sequenced TCRs from total PBMCs (before and after anti-TTx) and Tumor Infiltrating Lymphocytes (TILs) from FFPE tumor tissue slice. Arrows points insert of highly expressed of TCR Vβ families and genes in TILs. c. Correlation plots of Vβ families of HER2/neu369–377 tetramer positive cells (red corresponds to before anti-TTx sample, and blue corresponds to after anti-TTx sample) analyzed by flow cytometry vs. the percentage of Vβ families obtained from TILs. Pre-chemotherapy r2: 0.164, p value: 0.017, post-chemotherapy r2: 0.477, p value: <0.0001 (linear regression analysis)