Figure 1.

The E2 TCR consisting of the TRAV8-2/8-4 TCRα chain and the TRBV5-4 TCRβ chain recognizes HIP11 in the context of DQ2. (A) Peptide-specific proliferation of the E2 T cell clone was assessed using cells from an autologous EBV transformed B cell line as antigen presenting cells and pulsing with 10 µg/mL of the cognate HIP11 peptide in the presence or absence of anti-DR (black bar) or anti-DQ (grey bar) blocking antibody. (B) HLA restriction was further defined using cells from partially HLA-matched third-party donors as antigen presenting cells. Irradiated PBMC from each third party donor were pulsed with the cognate HIP11 peptide at concentrations of 0, 1, and 10 µg/mL. Substantial proliferation was observed only in response to the DQA1*05:01-DQB1*02:01 (HLA-DQ2) positive antigen presenting cells, indicating a DQA1*05:01-DQB1*02:01-restricted response. All data are represented as stimulation index (SI) values, calculated by normalizing the proliferation of the T cell clone based on [3H] thymidine incorporation of un-stimulated wells. (C) To confirm DQ2 restriction, the E2 T cell clone was cultured in the presence of either an autologous B cell line or K562 lines expressing DR3 or DQ2. After 18h, cells were harvested and stained for CD4 and CD25, and CD25 expression was monitored by flow cytometry. Data is representative of 3 independent experiments. (D) Two TCR alpha sequences and one beta sequence were identified from the E2 clone. Each TCR alpha/beta combination, E2a and E2b, was expressed in 5KC T-hybridoma cells with an NFAT-reporter. ZsGreen-1 is expressed upon T cell activation. The E2a and E2b TCR transductants were cultured with the HIP-11 peptide in the presence of K562 cells expressing DQ2 followed by evaluation of ZsGreen-1 expression by flow cytometry.