Gamma delta T cell receptors confer autonomous responsiveness to the insulin-peptide B:9-23

Abstract

The range and physical qualities of molecules that act as ligands for the gammadelta T cell receptors (TCRs) remain uncertain. Processed insulin is recognized by alphabeta T cells, which mediate diabetes in non-obese diabetic (NOD) mice. Here, we present evidence that gammadelta T cells in these mice recognize processed insulin as well. Hybridomas generated from NOD spleen and pancreatic lymph nodes included clones expressing gammadelta TCRs that responded specifically to purified islets of Langerhans and to an insulin peptide, but not to intact insulin. The gammadelta TCRs associated with this type of response are diverse, but a cloned gammadelta TCR was sufficient to transfer the response. The response to the insulin peptide was autonomous as demonstrated by stimulating single responder cells in isolation. This study reveals a novel specificity for gammadelta TCRs, and raises the possibility that gammadelta T cells become involved in islet-specific autoimmunity.

Figure 1. The SP9D11 hybridoma: absence of TCR-β expression

A: SP9D11 cells and an ins2 B:9–23 reactive TCRαβ⁺ hybridoma (BDC12-4.1) were stained with mAbs specific for TCR-β and CD3ε, and analyzed by flow cytometry. B: SP9D11 cells were stained with mAbs specific for TCR-δ or TCR-Vγ4 (TRGV4) and CD3ε, and analyzed by flow cytometry. Data shown are representative of multiple (>3) independent staining experiments.

Figure 2. SP9D11 cells specifically respond to purified islets of Langerhans and to the insulin B:9–23 peptide, but not to intact insulin

Responses were measured using the LacZ enzyme activity assay and are shown as optical density (O.D.) values; the fold increase over the signal produced by the responders alone (SI) is also shown over each column. Data shown represent mean values +/− standard error of the mean of 3–4 separate determinations in a single experiment, representative of at least 3 similar stimulation experiments. A: 2×10⁴ SP9D11 cells were incubated overnight alone, with NOD islets (100 size-balanced islets/well), with an equivalent amount of adrenal tissue (based on DNA content), or with the ins2 B:9–23 peptide (100 µg/ml). The background signal of NOD islets alone is also shown. B: 2×10⁴ SP9D11 cells were incubated overnight alone, or with islets derived from ins1/ins2 double gene knockout mice expressing an insulin transgene with the wild-type nucleotide sequence (100 size-balanced islets/well). C: 2×10⁴ SP9D11 cells were incubated overnight alone (PBS), with intact bovine insulin (100 µg/ml) or with ins2 B:9–23 peptide (100 µg/ml). D: 2×10⁴ SP9D11 cells were incubated overnight alone (PBS), with intact human insulin (100 µg/ml) or with ins2 B:9–23 peptide (100 µg/ml).

Figure 3. The SP9D11 γδ TCR confers responsiveness to insulin peptide B:9–23

A: SP9D11 TCR-γ and δ genes were expressed by viral transduction in a TCRαβ-loss variant of hybridoma 5KC-73.8.20 to generate transfectoma 5KC-TCR:SP9D11. The transfectoma cells were selected for GFP-expression (not shown), then stained with mAbs specific for TCR-δ and CD3ε or TCR-Vγ4 (TRGV4), and analyzed cytofluorimetrically. Data shown are representative of 3 independent staining experiments. B: The transfectoma 5KC-TCR:SP9D11 (black columns) and the recipient cell line 5KC-73.8.20α⁻β⁻ (open columns) were incubated at 4×10⁴ cells/well (no APCs) overnight with the insulin peptide B:9–23 (200 µg/ml), TT (200 µg/ml) or anti CD3ε mAbs in solution (10 µg/ml), and responses were measured by IL-2 ELISA. Data shown represent mean values +/− standard error of the mean of 3–4 separate determinations in a single experiment, representative of at least 3 similar stimulation experiments.

Figure 4. The SP9D11 hybridoma: response to the insulin peptide B:9–23

A: 2×10⁴ SP9D11 cells (subclone 5) and equal numbers of three αβ TCR⁺ hybridomas with known specificity for peptide B:9–23 (BDC12-4.1; BDC12-4.4; BDC8-1.1alpha1) were cultured with peptide either alone (no APCs) or in the presence of 1×10⁵ NOD spleen cells (plus APCs). Responses were measured with the lacZ activity assay and are shown as optical density values (O.D.). B: 2×10⁴ SP9D11 cells were cultured with peptide for 24 hours (no accessory cells). Responses were measured with the lacZ activity assay and are shown as optical density values (O.D.). The insert shows the stimulation index (S.I.) calculated as fold increase over background responses. C: 1×10⁵ SP9D11 cells were cultured with peptide for 24 hours (no accessory cells), then the supernatant was harvested for an IL-2 ELISA assay. The cells secreted IL-2 in response to the B:9–23 peptide. Data shown represent mean values +/− standard error of the mean of 3 separate determinations in a single experiment, representative of >3 similar stimulation experiments. D: Individual SP9D11 cells were cultured alone (left panel) or with peptide (right panel), then fixed, overlaid with X-gal, and examined microscopically. Magnification: ×40

Figure 4. The SP9D11 hybridoma: response to the insulin peptide B:9–23

A: 2×10⁴ SP9D11 cells (subclone 5) and equal numbers of three αβ TCR⁺ hybridomas with known specificity for peptide B:9–23 (BDC12-4.1; BDC12-4.4; BDC8-1.1alpha1) were cultured with peptide either alone (no APCs) or in the presence of 1×10⁵ NOD spleen cells (plus APCs). Responses were measured with the lacZ activity assay and are shown as optical density values (O.D.). B: 2×10⁴ SP9D11 cells were cultured with peptide for 24 hours (no accessory cells). Responses were measured with the lacZ activity assay and are shown as optical density values (O.D.). The insert shows the stimulation index (S.I.) calculated as fold increase over background responses. C: 1×10⁵ SP9D11 cells were cultured with peptide for 24 hours (no accessory cells), then the supernatant was harvested for an IL-2 ELISA assay. The cells secreted IL-2 in response to the B:9–23 peptide. Data shown represent mean values +/− standard error of the mean of 3 separate determinations in a single experiment, representative of >3 similar stimulation experiments. D: Individual SP9D11 cells were cultured alone (left panel) or with peptide (right panel), then fixed, overlaid with X-gal, and examined microscopically. Magnification: ×40

Figure 5. Responses of the hybridoma SP9D11 and the transfectoma expressing the SP9D11 γδ TCR to the insulin peptide B:9–23 and derivatives

A: 2×10⁴ SP9D11 cells were incubated overnight alone (PBS), with the indicated peptide antigens at 100 µg peptide/ml or with 1 µg/ml anti CD3ε mAb in solution. Responses were measured using the LacZ enzyme activity assay, and are expressed as fold increase over the signal produced by the responders alone (SI). The OD value of SP9D11 cells without antigen was 0.029±0.004 at 4 hours. B: 3×10⁴ 5KC-TCR:SP9D11 cells were incubated overnight either alone (open columns) or together with NOD spleen cells (1×10⁵) (black columns) and the indicated peptide antigens. (1) = 100 µg/ml; (2) = 200 µg/ml. Responses were measured by IL-2 ELISA. Data shown represent mean values +/− standard error of the mean of 3 separate determinations in a single experiment, representative of multiple (>3) similar stimulation experiments.