Induction of antigen-specific T cell anergy: An early event in the course of tumor progression

Abstract

The priming of tumor-antigen-specific T cells is critical for the initiation of successful anti-tumor immune responses, yet the fate of such cells during tumor progression is unknown. Naive CD4(+) T cells specific for an antigen expressed by tumor cells were transferred into tumor-bearing mice. Transient clonal expansion occurred early after transfer, accompanied by phenotypic changes associated with antigen recognition. Nevertheless, these cells had a diminished response to peptide antigen in vitro and were unable to be primed in vivo. The development of antigen-specific T cell anergy is an early event in the tumor-bearing host, and it suggests that tolerance to tumor antigens may impose a significant barrier to therapeutic vaccination.

Figure 1

Characteristics of A20 wild type and A20HA in vitro, and kinetics of tumor growth in vivo. (A) Flow cytometric analysis of CD80 (B7–1) and CD86 (B7–2) expression on A20 cells. (B) Recognition of A20HA by anti-HA/I-E^d TCR transgenic T cells in vitro. A20HA was explanted from tumor-bearing mice, and a single-cell suspension was made by mechanical dissociation and passage through nylon mesh. Explanted A20HA cells (•), A20HA cells passaged in vitro (□), or A20 wild-type cells passaged in vitro (▴) were irradiated with 10,000 centigrays and plated at the indicated cell number in 96-well microtiter plates together with 2 × 10⁵ freshly isolated anti-HA/I-E^d TCR transgenic splenocytes per well. Incorporation of [³H]thymidine was determined after three days in culture. (C) BALB/c mice were injected intravenously (i.v.) with 1 × 10⁶ A20WT (□) or A20HA (•) tumor cells on day zero and were inspected twice weekly for the development of tumors. Ten mice were included in each group. (D) Nine days after i.v. injection with 1 × 10⁶ A20WT or A20HA tumor cells, BALB/c mice received 2.5 × 10⁶ CD4⁺ TCR transgenic T cells specific for HA/I-E^d. Mice were inspected as above and euthanized when tumors were evident.

Figure 2

Changes in clonotype-positive T cells after transfer into tumor-bearing mice. BALB/c mice were injected (i.v.) with 1 × 10⁶ A20WT or A20HA tumor cells. Nine days later all mice, including a group not challenged with tumor, received 2.5 × 10⁶ anti-HA/I-E^d TCR⁺ transgenic T cells i.v. Four mice per group were sacrificed on days +6, +13, and +20 after the adoptive transfer of T cells. Flow cytometric analysis was performed on purified splenic T cells stained with FITC-conjugated goat anti-mouse CD4 and biotinylated rat anti-clonotypic TCR antibody (mAb 6.5) followed by PE-conjugated streptavidin. For each sample 100,000 gated events were collected. (A) Representative two-color FACS analysis of splenocytes obtained 6 days after T cell transfer. (B) Change in the percentage of CD4⁺ anti-HA TCR⁺ T cells over time after T cell transfer. Values represent the mean ± SE of the percentage of cells expressing the clonotypic TCR for four mice. Background staining was less than 0.05%. (C) In vitro proliferative response of clonotype⁺ CD4⁺ T cells to stimulation with HA 110–120 peptide. Purified T cells (4 × 10⁴ per well) from the mice in A were mixed with fresh splenocytes (8 × 10⁴ per well) from naive BALB/c mice to which HA peptide (12.5 μg/ml) was added. ³H incorporation was assayed after 3 days of incubation and is shown as cpm from which values for medium alone were subtracted. Values represent the mean (±SE) cpm/absolute number of clonotypic T cells per well from four mice in each group.

Figure 3

Phenotypic changes associated with antigen recognition on CD4⁺ TCR clonotype⁺ T cells after adoptive transfer into tumor-bearing mice. BALB/c mice were injected (i.v.) with 1 × 10⁶ A20WT or A20HA tumor cells. Nine days later all mice received 2.5 × 10⁶ anti-HA/I-E^d TCR⁺ transgenic T cells i.v. Fifteen days after transfer, T cells from non-tumor-bearing mice (patterned bars), mice bearing A20WT (hatched bars), or A20HA (solid bars) were isolated as in Fig. 2 and stained with Cy-Chrome-labeled anti-mouse CD4, biotinylated anti-TCR clonotype mAb 6.5 followed by PE-labeled streptavidin and FITC-conjugated anti-mouse CD44, anti-mouse CD45RB, or anti-mouse CD62L. Live gating on CD4⁺ T cells was set, and 100,000 events were collected per sample. Mean fluorescence intensity + SE is shown for each activation marker expressed by CD4⁺TCR clonotype⁺ T cells (four mice per group).

Figure 4

HA-specific T cells fail to respond to in vivo priming in mice bearing A20HA but not A20WT tumors. BALB/c mice were given 1 × 10⁶ A20WT or A20HA tumor cells i.v. Nine days later, all mice, including a group not challenged with tumor, received 2.5 × 10⁶ anti-HA/I-E^d TCR⁺ transgenic T cells. Half the mice in each group were immunized with 1 × 10⁷ pfu of vacc-HA subcutaneously (s.c.) on day 2, 9, or 16 after T cell transfer and were sacrificed for analysis six days after immunization (days 8, 15, or 22). Unimmunized controls received 0.1 ml of HBSS s.c. (A) Purified T cells from unimmunized (solid bars) and vacc-HA-immunized animals (patterned bars) were analyzed by two-color flow cytometry staining for CD4 versus anti-HA TCR clonotype as in Fig. 2. Values represent mean + SE of percentage of T cells expressing the clonotypic TCR. T cells were cultured with media alone or HA peptide (12.5 μg/ml) plus fresh BALB/c splenocytes. Forty-eight hours later, supernatants were collected and assayed for γ-interferon (B) and IL-2 (C) by ELISA (R & D Systems). Values are the mean + SE of triplicate cultures from four animals in each group. Data are expressed as the amount of cytokine produced per 100 clonotype-positive cells. Values for T cells cultured in media alone were less than 10% of the values for stimulated T cells. (D) HA-specific proliferative response of clonotype-positive T cells from immunized versus unimmunized animals. Values represent the mean proliferation of T cells from which values for medium alone were subtracted. Four mice per group were analyzed.