Relationship between CD8-dependent antigen recognition, T cell functional avidity, and tumor cell recognition

Abstract

Effective immunotherapy using T cell receptor (TCR) gene-modified T cells requires an understanding of the relationship between TCR affinity and functional avidity of T cells. In this study, we evaluate the relative affinity of two TCRs isolated from HLA-A2-restricted, gp100-reactive T cell clones with extremely high functional avidity. Furthermore, one of these T cell clones, was CD4- CD8- indicating that antigen recognition by this clone was CD8 independent. However, when these TCRs were expressed in CD8- Jurkat cells, the resulting Jurkat cells recognized gp100:209-217 peptide loaded T2 cells and had high functional avidity, but could not recognize HLA-A2+ melanoma cells expressing gp100. Tumor cell recognition by Jurkat cells expressing these TCRs could not be induced by exogenously loading the tumor cells with the native gp100:209-217 peptide. These results indicate that functional avidity of a T cell does not necessarily correlate with TCR affinity and CD8-independent antigen recognition by a T cell does not always mean its TCR will transfer CD8-independence to other effector cells. The implications of these findings are that T cells can modulate their functional avidity independent of the affinity of their TCRs.

Fig. 1

Immune Reactivity of T Cell Clones R6C12 and T4H2. T cell clones R6C12 and T4H2 were selected for this study based on their ability to recognize tumor cells and their functional avidity. a Tumor recognition by T cell clones R6C12 and T4H2 was assessed using interferon-γ release assays. HLA-A2⁺ gp100⁺ (624 MEL, 1300 MEL, 1383 MEL), HLA-A2⁻ gp100⁺ (624-28 MEL, 586 MEL), and HLA-A2⁻ gp100⁻ (RCC 131) cells were cocultured overnight with T cell clone R6C12 and T4H2 and the amount of interferon-γ released was measured by ELISA. As controls, the amount of interferon-γ released by TIL 1235 (anti-MART-1) and TIL 1383I (anti-tyrosinase) when cocultured with the same targets was measured. The amount of interferon-γ released was measured by ELISA. b The functional avidity of T cell clones R6C12 and T4H2 was measured in interferon-γ release assays. T cells were stimulated with T2 cells loaded with decreasing concentrations of gp100:209–217 peptide. The amount of interferon-γ released was measured by ELISA. Relative avidity was defined as the minimum amount of peptide loaded on T2 cells that could elicit significant cytokine release. Significant cytokine release was considered to be at least twice background and greater than 100 pg/ml. Each point represents the average of triplicate wells and is a representative of three replicate experiments. It should be noted that the data in a and b were from the same experiment. As a result, the specificity controls for b can be found in panel a

Fig. 2

Cell surface phenotype of T cell clones R6C12 and T4H2. The expression of T cell markers on T cell clones R6C12 and T4H2 was measured by immunofluorescence. Cells were stained with FITC conjugated anti-CD3, CD4, and anti-CD8 monoclonal antibodies or PE conjugated anti-TCR αβ, Vβ8, and Vβ17 monoclonal antibodies. The log fluorescence of 10⁴ live cells was measured using a FACScan flow cytometer

Fig. 3

Functional Avidity of TCR transduced Jurkat cells. The relative sensitivity of R6C12 and T4H2 TCR transduced Jurkat cells was measured using IL-2 release assays using T2 cells incubated with different concentrations of gp100 209–217 peptide. The amount of IL-2 released was measured by ELISA. Relative avidity was defined as the minimum amount of peptide loaded on T2 cells that could elicit significant cytokine release. Significant cytokine release was considered to be at least twice background and greater than 100 pg/ml. Each point represents the average of triplicate wells and is a representative of three replicate experiments. It should be noted that the data in this figure and in Table 1 were from the same experiment with T2 cells loaded with 10 μg/ml gp100 209–217 peptide data point being shown in both. As a result, the specificity controls for this figure can be found in Table 1