Figure 5. Ly108 recruits SHP-1 to the T:B immunological synapse.

(a) Activated WT and Sh2d1a^−/− AND TCR transgenic (PCC-specific) CD4⁺ T cells were lysed without stimulation or post-incubation with LPS-activated B cells that were untreated or pulsed with PCC peptide. Lysates were immunoprecipated for Ly108 and blotted for SHP-1 and Ly108. Total cell lysates (TCL) were examined for pERK activation and total ERK protein levels. NP = no peptide. (b-d) WT, Sh2d1a^−/−, and Slamf6^−/− Sh2d1a^−/− SM CD4⁺ T cell conjugates with LPS-activated B cells pulsed with cognate peptide (LCMV gp66-77) were stained with Hoechst (blue), and antibodies to CD4 (white) and SHP-1 (green). (b) Cells were examined from the side as a confocal projection in the x-y plane (top row), at 45° (middle row), and 90° (en face, bottom row) rotations in the y-z plane. (c) Representative immunofluorescence images of SHP-1 localization at the immune synapse. (d) Quantification of SHP-1 localization at the immune synapse. Data represent two independent experiments with over 40 conjugates scored / genotype for each experiment. (e) Activated Slamf6^−/− and Slamf6^−/−Sh2d1a^−/− SM CD4⁺ T cells expressing Ly108-GFP or Ly108-AllF-GFP (‘Ly108-AllF’) constructs were incubated with activated B cells pulsed with cognate peptide (LCMV gp66-77). Lysates were immunoprecipated for Ly108 and blotted for SHP-1 and Ly108. (f-h) Slamf6^−/− and Slamf6^−/−Sh2d1a^−/− SM CD4⁺ T cells were transfected with either Ly108-GFP (‘WTLy108’) or Ly108-AllF-GFP, and conjugated to WT B cell targets pulsed with cognate peptide (LCMV gp66-77). Cells were stained with Hoechst (blue) and antibodies against SHP-1 (red). Green is Ly108-GFP fluorescence. (f) Quantitation of SHP-1 localization at the synapse. (g) Representative immunofluorescence images of cells expressing Ly108-GFP. (h) Representative immunofluorescence images of cells expressing Ly108-AllF-GFP. Further examples are shown in Figure S4. Data for each experiment depicted is representative of two or more experiments.