Reciprocal changes in tumor antigenicity and antigen-specific T cell function during tumor progression

Abstract

Two seemingly incompatible models exist to explain the progression of cancers in immunocompetent hosts. The cancer immunosurveillance hypothesis posits that recognition of transformed cells by the immune system results in the generation of an effector response that may impede tumor growth. Clinically detectable cancer results from the emergence of tumor variants that escape this selective pressure. Alternatively, induction of immune tolerance to tumor antigens may enable cancer progression. We established a model where changes in the function of tumor-specific T cells and in tumor antigen expression could be followed during cancer progression. Early recognition of antigen led to activation, expansion, and effector function in tumor-specific CD4+ T cells resulting in the outgrowth of tumors expressing substantially reduced levels of antigen. Antigen loss was not complete, however, and levels remained above the threshold required for tumor-specific T cell recognition in vivo. In the face of persisting antigen, T cell tolerance ensued, leading to an impaired ability to mediate further antigen loss. Together, these studies establish that the processes of immunosurveillance and tumor editing coexist with a process in which the functional tumor-specific T cell repertoire is also "edited," reconciling two hypotheses historically central to our attempts to understand host antitumor immunity.

Figure 1.

Recognition of antigen by tumor-specific CD4⁺ T cells. CFSE-labeled HA-specific CD4⁺ T cells were transferred into mice inoculated with RencaHA 10 d earlier. On days 7 and 21 after T cell transfer, expansion of the transgenic CD4⁺ T cells was revealed by staining with anti-TCR clonotypic antibody and anti-Thy1.1. (A) FACS profiles of the transgenic CD4⁺ T cells in hilar draining LNs. CFSE histograms of the gated cells are also shown. Percentage of the gated population is displayed in each dot plot. For CFSE profiles, percentage of the divided cells in the gated population is indicated in each histogram. For comparison, CFSE-labeled HA-specific CD4⁺ T cells were transferred into tumor-free mice. Some were given vacHA the next day and analyzed 6 d later. (B) Percentage of the transgenic CD4⁺ T cells in spleen. Spleen cells from the same mice as in A were stained and analyzed by flow cytometry. The results are representative of three experiments with three mice per group.

Figure 2.

Early recognition of HA results in effector function of tumor-specific T cells. (A) T cell transfer was conducted as described in Fig. 1. IFN-γ ELISPOT was performed with cells from hilar LNs in each well ± HA peptide. The number of IFN-γ–positive spots was obtained by subtracting the number of spots in the no peptide group from the number of spots in the peptide group. The results are mean ± SD of pooled data from two separate experiments. (B) Activated CD4⁺ T cells provide help for CTL priming. Purified CD8⁺ T cells from CL4 transgenic mice on Thy1.1 background were transferred either alone or together with HA-specific CD4⁺ T cells (Thy1.2⁺) to tumor-free or tumor-bearing mice (10 d tumor). 7 d after T cell transfer, cells from draining LNs were stained for the transferred CD8⁺ T cells with antibody to Thy1.1 and CD8. For CD4⁺ T cells, cells were stained with anti-TCR clonotypic antibody and anti-CD4. Percentage of the gated population is given in each dot plot. The results are representative of two experiments with three mice per group.

Figure 3.

Immunoediting of RencaHA by the endogenous repertoire and by transferred, HA-specific CD4⁺ T cells. Mice were injected with 10⁶ tumor cells i.v. Mice in group D received HA-specific CD4⁺ T cells 10 d posttumor. All mice were killed at 25 d posttumor inoculation. Lung tissue was harvested, and sections were stained with anti-HA antibody and developed with NovaRed (red staining). Sections were counterstained with hematoxylin. (A, C, and D) BALB/c mice; (B) BALB/c Rag 2^−/− mice.

Figure 4.

HA mRNA frequencies on tumor explants as revealed by qRT-PCR. Tumor explants from mice with and without HA-specific T cell transfer were generated as described. Each explant or RencaHA cell line was assayed in triplicate using probe/primer set specific for HA. Samples were simultaneously assayed for HPRT which was used as an internal reference. The cDNA concentration of each sample was adjusted so that HPRT amplification was equivalent in all samples. Data shown are representative of two separate reactions with similar results.

Figure 5.

Edited tumor cells are recognized by naïve HA-specific CD4⁺ T cells. Mice were challenged with RencaHA. 10 d later, half of the mice received HA-specific CD4⁺ T cells. 21 d after T cell transfer, the tumors were harvested from the lungs and briefly expanded in vitro in the absence of drug selection. A second cohort of mice was then injected with either Renca WT cells, RencaHA cells that had never been passaged in vivo (RencaHA^hi), RencaHA cells explanted from BALB/c mice that did not receive T cell transfer (RencaHA^endog), or RencaHA cells explanted from mice that had received HA-specific CD4⁺ T cells (RencaHA^TCR-Tg). 10 d after tumor challenge, all mice were injected with naïve CFSE-labeled HA-specific CD4⁺ T cells. 7 d later cells were isolated from hilar LNs. Lymphocytes were stained with anti-Thy1.1 antibody and anti-TCR clonotypic antibody and analyzed by flow cytometry. Results are gated on Thy1.1⁺ cells.

Figure 6.

The kinetics of tumor-specific CD4⁺ T cell proliferation during tumor progression. Mice that received HA-specific CD4⁺ (Thy1.1^+/−) T cells were provided with BrdU-containing water at the specified interval. On the day of analysis, cells from the hilar LNs were isolated and stained for surface markers CD4 and Thy1.1, followed by intercellular BrdU staining. The data are representative of two separate experiments with similar results.

Figure 7.

Antigen-experienced, tumor-specific T cells have a decreased ability to edit tumor. HA-specific CD4⁺ T cells were transferred into tumor-free or tumor-bearing mice. 21 d later, spleens and LNs were harvested from the mice. CD4⁺ T cells were enriched and retransferred into mice with established RencaHA tumor. 3 wk after T cell transfer, tumor explants were harvested and processed. Shown are real-time RT-PCR results for HA mRNA frequencies on tumor explants. Each sample was assayed in triplicate, with HPRT included as internal reference. Each group consisted of two to three mice.