Chemotherapy enhances cross-presentation of nuclear tumor antigens

Abstract

Cross-presentation of tumor antigen is essential for efficient priming of naïve CD8⁺ T lymphocytes and induction of effective anti-tumor immunity. We hypothesized that the subcellular location of a tumor antigen could affect the efficiency of cross-presentation, and hence the outcome of anti-tumor responses to that antigen. We compared cross-presentation of a nominal antigen expressed in the nuclear, secretory, or cytoplasmic compartments of B16 melanoma tumors. All tumors expressed similar levels of the antigen. The antigen was cross-presented from all compartments but when the concentration was low, nuclear antigen was less efficiently cross-presented than antigen from other cellular locations. The efficiency of cross-presentation of the nuclear antigen was improved following chemotherapy-induced tumor cell apoptosis and this correlated with an increase in the proportion of effector CTL. These data demonstrate that chemotherapy improves nuclear tumor antigen cross-presentation and could be important for anti-cancer immunotherapies that target nuclear antigens.

Figure 1. Schematic illustration and characterization of B16 tumor cells expressing differentially localized antigen.

(A) Schematic depiction of gene constructs used to generate B16 parental (B16.par) tumors expressing EGFP-CL4 recombinant protein in secretory (B16.Sec), nuclear (B16.Nuc), or cytoplasmic (B16.Cyto) cellular compartments. (B) Confocal fluorescence microscopy demonstrated targeted expression of EGFP-CL4 in distinct subcellular compartments within respective tumors (magnification, ×60). (C) Quantitative imaging flow cytometry confirmed confocal microscopy observations. (D) EGFP ELISA was used to compare the amount of EGFP present in whole cell lysates (W.C.L) and supernatants (Sup.) of respective tumors. A standard curve of recombinant EGFP protein was used to calculate the amount of EGFP. A representative experiment is shown out of two independent ELISAs in triplicates ± SEM. (E) Relative intensity and stability of EGFP expression was determined by live-cell flow cytometry. Mean fluorescent intensities (MFI) are shown in figure. (F) In vivo growth pattern of respective tumors after s.c. inoculation in F1 mice. Data are means of 10 mice from two independent experiments. ***p<0.001. One-way ANOVA, followed by a Bonferroni post-test.

Figure 2. Nuclear localized tumor antigen was not cross-presented as efficiently as cytoplasmic and secretory counterparts.

(A) The EGFP-CL4 antigen expression profile of B16 parental cells (B16 par.) or, B16 par. mixed with 1 (19.4), 5 (97), 10 (194), or 20% (388 nmol) of B16.Nuc (N), B16.Cyto (C), or B16.Sec (S) CL4 antigen-containing cells, was confirmed on the day of tumor inoculation by flow cytometry. For simplicity, Figure 2A only shows the profile of B16.Nuc mixtures, as they were not different to B16.Cyto or B16.Sec (B) CFSE-labelled CL4-specific CD8+ T cells were intravenously injected into F1 mice bearing 8-day-old subcutaneous tumors, and the proliferation of these T cells in the tumour draining lymph node examined on day 11 post-tumor inoculation by flow cytometry. Data are representative of two independent experiments, each involving five mice per group. Bars begin at the Mean ± SEM baseline proliferation for B16 parental cells.

Figure 3. The apoptosis-inducing agent gemcitabine has a direct effect on B16 tumors in vitro and in vivo.

(A) The relative in vitro sensitivity of B16 parental, B16.Nuc, B16.Cyto, and B16.Sec tumor cells to gemcitabine was assessed by MTT assay, after a 48 hr incubation with drug. With relevent concentrations of gemcitabine that kill 50% of respective tumors shown. (B) In vivo growth of B16 parental only, and B16 parental mixed with 10% (194 nmols) of B16.Sec-, Cyto-, or Nuc-CL4 antigen-containing tumors, after subcutaneous inoculation of F1 mice on day 0, and intraperitoneal administration of a single dose of gemcitabine (240 µg/g) or saline on day 6. Bar graph shows tumor sizes on day 11. For simplicity, Figure 3B only shows data from the 10% antigen concentration experiment as this was similar to that of 1 and 5%. Mean ± SEM. Six mice per group from two independent experiments. One-way ANOVA followed by a Bonferroni post test against all groups.

Figure 4. Gemcitabine improves the cross-presentation efficiency of nuclear localized antigen.

F1 mice were inoculated on day 0 with B16 parental only (B16 only), or B16 parental mixed with 1 (19.4), 5 (97), or 10% (194 nmol), B16.Nuc (N), B16.Cyto (C), or B16.Sec (S) CL4 antigen-containing tumors, and a single dose of gemcitabine or saline administered on day 6. CFSE-labelled CL4 T cells were transferred on day 8, and the (A) proliferation and (B) Interferon gamma expression of CL4-specific CD8⁺ T cells in tumour draining lymph nodes of mice assessed on day 11. Mean ± SEM. Six mice per group from two independent experiments. One-way ANOVA followed by a Bonferroni post test against all groups. ns  =  not significant, *p<0.05, ***p<0.001.