Figure 5. The ‘EP’ motif in the CT of LAG3 disrupts co-receptor-Lck complex.

(a) Immunoblot analyses of CD4 and Lck in lysates of Lag3^−/− CD4⁺ T cell stimulated with CD3ε and CD28 Abs, following one hour incubation with 26 amino acid LAG3 ‘EP’ peptide motif or peptides with amino acid substitutions as depicted with 1–10-O pheanthroline (Phe) utilized as a positive control. (b) Analysis of phase separation and dissociation of Lck from membrane-tethered CD4^CT or CD8^CT in response to LAG3^CT or LAG3^CT-QP mutant, with quantification shown. Scale bar 1μm. (c) Representative super resolution STORM of molecular interactions between CD4 and Lck in CD4⁺ T cells isolated from the spleens and lymph nodes of Lag3^−/− mice, transduced with empty vector, LAG3 or LAG3 lacking the ‘EP’ motif (LAG3^ΔEP) and stimulated with TCRβ Abs. The co-localization index of CD4 and Lck was analyzed and is depicted. Scale bar 5 μm. (d) Immunoblot and quantification of co-immunoprecipitation western blot analyses of CD4 and Lck in Lag3^−/− CD4⁺ T cell lysates as above following addition of the LAG3 ‘EP’ peptide motif or peptide mutants in the presence or absence of competing Zn²⁺ or (e) alternative 2⁺ metals. Data in (a, b, d, e) are representative of 3–5 experiments, with statistical analysis performed using Student’s unpaired two-sided t test. For (c) data are representative of at least 2 experiments and 15 individual data sets with data represented as mean and statistics determined by Kruskal-Wallis test. P values are noted in figure.