CD8(+) T cell tolerance to a tumor-associated antigen is maintained at the level of expansion rather than effector function

Abstract

CD8+ T cell tolerance to self-proteins prevents autoimmunity but represents an obstacle to generating T cell responses to tumor-associated antigens. We have made a T cell receptor (TCR) transgenic mouse specific for a tumor antigen and crossed TCR-TG mice to transgenic mice expressing the tumor antigen in hepatocytes (gag-TG). TCRxgag mice showed no signs of autoimmunity despite persistence of high avidity transgenic CD8+ T cells in the periphery. Peripheral CD8+ T cells expressed phenotypic markers consistent with antigen encounter in vivo and had upregulated the antiapoptotic molecule Bcl-2. TCRxgag cells failed to proliferate in response to antigen but demonstrated cytolytic activity and the ability to produce interferon gamma. This split tolerance was accompanied by inhibition of Ca(2+) flux, ERK1/2, and Jun kinase phosphorylation, and a block in both interleukin 2 production and response to exogenous interleukin 2. The data suggest that proliferation and expression of specific effector functions characteristic of reactive cells are not necessarily linked in CD8+ T cell tolerance.

Figure 1.

FACS^® analysis of thymic and splenic T cells from B6, TCR-TG, and TCRxgag. (A) Thymocytes were stained with CD4, CD8, Vα3, and Vβ12 and analyzed for CD4/CD8 expression (top) or Vα3/Vβ12 expression (bottom). Vα3/Vβ12 plots are gated on CD4⁻/CD8⁺ thymocytes. (B) Splenocytes were stained with CD4 and CD8 (top) or Vα3; Vβ12 and CD8 (bottom). Vα3/Vβ12 plots are gated on CD8⁺ splenocytes. (C) For analysis of memory/activation phenotype of splenocytes, cells were stained with CD8 and either Ly-6C (top) or CD44 (bottom). All histograms are gated on CD8⁺ splenocytes.

Figure 2.

CTLs from TCR-TG and TCRxgag lyse gag-positive targets. 1.5 × 10⁷ TCR-TG (white symbols) and TCRxgag (black symbols) splenocytes were stimulated for 3 d with 5 × 10⁶ irradiated FBL cells in the presence of 5 U/ml IL-2. After 3 d, the cells were CD4 depleted and tested in a standard 4 h ⁵¹[Cr]-release assay against FBL, E10, or E10 together with FMuLVgag peptide at indicated concentrations. E:T ratio of 50:1 and 10:1 are shown, However, the final CD8⁺/TCR-αβ^hi ratio for TCRxgag compared with TCR is overestimated ∼5–15 times based on profiles in Fig. 1.

Figure 3.

TCRxgag mice do not proliferate or produce IL-2 but retain IFN-γ and TNF-α production after antigen stimulation. (A) To measure antigen-specific proliferation, 0.1 × 10⁶ CD4-depleted splenocytes from TCR-TG plus 0.9 × 10⁶ B6 splenocytes and 10⁶ splenocytes from TCRxgag mice were stimulated with 2 × 10⁴ irradiated FBL for 3 d, and then pulsed with ³[H]-thymidine for 8 h. For analysis of IL-2 production (B), 0.1 × 10⁶ CD4-depleted splenocytes from TCR-TG plus 0.9 × 10⁶ B6 and 10⁶ CD4-depleted TCRxgag were stimulated with 2 × 10⁴ irradiated FBL in a total volume of 200 μl in a 96-well round bottomed plate. After 72 h, 50 μl of culture supernatant was harvested and murine IL-2 measured by ELISA. (C) The antigen-specific response of TCRxgag in the presence of IL-2 was measured as in A, but with indicated amounts of IL-2 added to the wells. Intracellular staining for IFN-γ and TNF-α production (D) was performed after stimulation of TCR-TG or TCRxgag splenocytes with irradiated FBL. After 48 h, secretion of protein was prevented by addition of Golgi-Block reagent for 6 h and cells permeabilized by Cytofix/Cytoperm. The cells were stained with anti-CD8 and anti–IFN-γ or anti–TNF-α. Plots were gated on CD8⁺ cells.

Figure 4.

TCRxgag T cells exhibit deficiencies in Ras/MAPK and SAPK/JNK as well as in flux of Ca²⁺ after antigen stimulation. CD4-depleted splenocytes from TCR-TG and TCRxgag mice were stimulated with irradiated B6 splenocytes pulsed with 10 μg/ml gag-peptide. After stimulation, the cells were lysed in SDS-PAGE sample buffer at indicated time points. The samples were run on a SDS-PAGE gel and blotted. Blots were probed using primary Abs against phosphorylated forms of ERK1/2 (A) or JNK (B). Specific binding was detected by incubation with a secondary HRP-conjugated Ab and ECL Western blotting detection reagents. For analysis of Ca²⁺ flux (C), TCR-TG (black line) and TCRxgag (gray line) splenocytes expressing the Thy1.1 allomarker were depleted of CD4 cells and labeled with Indo-1. During the loading, cells were stained with Abs against Thy.1.1 and CD4. After washes, the cells were analyzed by flow cytometry in order to establish a base line. Control or peptide treated B6 Thy1.2 APC were added and after gating on Thy1.1⁺/CD4⁻ cells, the Ca²⁺ was followed over time. As a positive control, responder cells were treated with 1 μg/ml ionomycin.

Figure 5.

CTL from TCRxgag lyse gag-positive targets independently of Fas-expression. 1.5 × 10⁷ TCR-TG (black symbols) and TCRxgag (white symbols) splenocytes were stimulated for 3 d with 5 × 10⁶ irradiated FBL cells in the presence of 5 U/ml IL-2. After 3 d, the cells were CD4 depleted and tested in a standard ⁵¹[Cr]-release assay using ConA-induced blasts from B6 or Fas-negative lpr mice as targets. Target cells were labeled with ⁵¹[Cr] and used either untreated or after pulse with 5 μg/ml gag-peptide. E:T ratios were 50:1, 10:1, and 2:1.

Figure 6.

The tolerant cells in TCRxgag are a stable population and not deleted in thymectomized animals. 5-wk-old TCR-TG and TCRxgag mice were anaesthetized and their thymi removed. After 5 wk, splenocytes from control and thymectomized mice were (A) stained with CD8; Vα3 and Vβ12, and after gating on CD8⁺ cells, T cell receptor expression was analyzed. For analysis of lysis by thymectomized mice (B), 1.5 × 10⁷ TCR-TG and TCRxgag splenocytes from normal control (N) or adult-thymectomized (ATx) mice were stimulated with 5 × 10⁶ irradiated FBL cells in the presence of 5 U/ml IL-2. After 3 d, the cells were CD4 depleted and tested in a standard ⁵¹[Cr]-release assay against E10 or E10 pulsed with FMuLVgag. E:T ratio was 50:1.

Figure 7.

Tolerant TCRxgag cells CD8⁺ express high levels of Bcl-2. Fresh splenocytes from TCR and TCRxgag were stained for CD8, and then permeabilized and stained with antibodies for Bcl-2 or with isotype control antibodies diluted in permeabilization buffer. After three washes, cells were analyzed by flow cytometry for CD8 and Bcl-2 expression.

Figure 8.

TCRxgag T cells that have been expanded in vitro by repetitive in vitro stimulations have similar avidity as T cells from TCR-TG mice. 1.5 × 10⁷ splenocytes from TCR-TG or TCRxgag mice were stimulated with 5 × 10⁶ irradiated FBL in T25 flasks in a total volume of 10 ml supplemented with 20 U/ml IL-2. The cultures were restimulated every 7 d for 11 wk with 2 × 10⁶ irradiated FBL, 5 × 10⁶ irradiated B6 splenocytes as feeder cells and 20 U/ml IL-2. 8 d after last stimulation cultures were tested in proliferation assay. 5 × 10⁴ CD4-depleted cells from TCR-TG (white symbols) and TCRxgag (black symbols) cultures were stimulated with 5 × 10⁵ irradiated B6 splenocytes pulsed with indicated concentrations of peptide for 4 d in the presence of 5 U/ml IL-2, and then pulsed with ³[H]-thymidine for 8 h.