Figure 4. Slamf6^−/−Sh2d1a^−/− CD4⁺ T cells form stable conjugates with B cells, and ITSM phosphotyrosine motifs are required for inhibitory signals transmitted by Ly108 in vivo.

(a-b) Conjugation efficiency of WT, Sh2d1a^−/−, and Slamf6^−/− Sh2d1a^−/− SM CD4⁺ T cells with B cells pulsed with cognate peptide (LCMV gp66-77). (a) Representative flow cytometry plots, gated on CD4⁺ T cells. (b) Mean frequency of CD4⁺CD19⁺ conjugates in total CD4⁺ events. N = 2. (c-g) Roles of Ly108 tyrosines in vivo. (c-e) Slamf6^−/−Sh2d1a^−/− SM CD4⁺ T cells were transduced with GFP, Ly108-2 (“Ly108”), Ly108-Y3 mutant, or Ly108-AllF mutant RV and transferred into Sh2d1a^−/− recipient mice. An additional group received Sh2d1a^−/− SM cells transduced with RV-GFP. Mice were infected with LCMV and B cell responses in spleen were analyzed 8 days following infection. (c) Representative germinal center B cell FACS plots are shown, gated on total B cells (CD19⁺CD4⁻). (d) Quantitation of GC B cells as gated in (c). (e) Quantitation of the plasma cell response. (f-g) Slamf6^−/−Sh2d1a^−/− SM -Ly108-Y1 mutant or - Ly108-Y2 mutant CD4⁺ T cells were transduced with RV-GFP, -Ly108-2 (“Ly108”), and transferred into Sh2d1a^−/− recipient mice subsequently infected with LCMV. (f) Representative germinal center B cell FACS plots are shown, gated on total B cells (CD19⁺CD4⁻), analyzed at day 8 after infection. (g) Quantitation of GC B cells as gated in (f). Data are representative of 2 independent experiments. N = 4 or more per group. * P < 0.05, ** P < 0.005 Error bars are SEM.