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. 2022 Dec 8;6(2):e202201533. doi: 10.26508/lsa.202201533

Figure 5. Anti-CD45/SLAMF6–bispecific antibody inhibits SLAMF6 enrichment in the synapse but still augments T cell activation.

Figure 5.

(A) A schematic representation of anti-CD45/SLAMF6 (CD45/SLAMF6) binding to inhibit SLAMF6 clustering with CD3 in the IS. (B) αCD45/SLAMF6 antibody binding was quantified using an ELISA assay: αCD45 and αSLAMF6 binding was assessed against immobilized, recombinant ectodomains of CD45 and SLAMF6, respectively. (C) Jurkat T cells were transfected with GFP-tagged SLAMF6, and Raji B cells were stained with LifeAct Far Red and preloaded with SEE (2 ng/ml). Jurkat T cells were then co-cultured with Raji B cells for 30 min. Synapse formation with enrichment of SLAMF6 in the IS was visualized (top row). To visualize the distribution of CD45, we next transfected the Jurkat T cells with OFPSpark-tagged CD45 and GFP-tagged SLAMF6. In the Jurkat T–Raji B co-cultures, exclusion of CD45 was coupled with enrichment of SLAMF6 in the IS (middle row). Finally, we pretreated Jurkat T cells with anti-CD45/SLAMF6 10 µg/ml for 15 min (bottom image). Exclusion of CD45 was now associated with a lack of enrichment of SLAMF6 in the IS (bottom row). Images are representative of at least 40 cell conjugates per each experimental condition from two independent experiments. Scale bar is 5 μm. Percent of cell conjugates with SLAMF6 enrichment in the IS was quantified; results are summarized in the bar graph. (D) Jurkat T cells were treated with αCD3 and αCD45/SLAMF6 at three different concentrations: 0.1, 1, and 10 µg/ml of the bispecific antibody. After 24 h, IL-2 levels were analyzed by ELISA. (E) Raji B cells were preloaded with different concentrations of SEE and co-cultured with Jurkat T cells in the absence (blue) or presence (magenta) of 1 µg/ml of αCD45/SLAMF6 antibody. IL-2 levels were analyzed for at least three independent experiments (n = 3). (F) Raji B cells were preloaded with SEE and co-cultured with T cells at increasing concentrations of αCD45/SLAMF6 antibody. IL-2 levels were analyzed for at least three independent experiments (n = 3). (G) Raji B cells and Jurkat T cells were co-cultured either in the presence of, or after T cell pretreatment with, αCD45/SLAMF6. Specifically, in the first experimental condition, αCD45/SLAMF6 was added as Jurkat T–Raji B conjugates formed (supporting in trans antibody ligation), whereas in the second experimental condition, Jurkat T cells were pretreated with αCD4/SLAMF6 for 30 min, washed, and subsequently co-cultured with the Raji B cells, supporting in cis antibody ligation on T cells before addition of the B cells. IL-2 levels were analyzed for at least three independent experiments (n = 3). *P < 0.05 for an unpaired t test.