The size of CD148 ectodomain modulates its inhibitory effect on T-cell activation. (A) Schematic depiction of the CD148 constructs used in this study. The ectodomains comprised either the full-length form of CD148 or the truncated form of CD148 or the ectodomain from CD43 (CD43-CD148). Catalytically inactive (PTPneg) forms of full-length and truncated CD148 had a cysteine to serine substitution (C1140S) in the phosphatase domain. All constructs contained an N-terminal FLAG tag to stain and match surface expression levels by FACS and a C-terminal GFP tag for imaging. (B) Control (untransduced or vector-transduced) 2B4 T cells or 2B4 T cells expressing comparable levels of the indicated CD148 construct (supplemental Figure 1) were stimulated with either CHO cells expressing I-E^k, presenting the cognate MCC peptide (left) or plate-immobilized anti-mouse CD3ε (right) and IL-2 secretion analyzed after 14 to 18 hours. (C) Control or transduced B3Z T cells expressing comparable levels of the indicated CD148 construct (supplemental Figure 4A) were stimulated with CHO cells expressing cognate pMHC as a single-chain trimer and IL-2 secretion analyzed after 14 to 18 hours. (D) 2B4 T-cells transduced with the indicated CD148 constructs were stimulated with plate-immobilized anti-mouse CD3ε and IL-2 secretion examined after 14 to 18 hours. Three different CD43-CD148 transduced clones were tested. Error bars represent the SD of the mean from at least 3 replicates. For (B) and (C), IL-2 secretion data were normalized with 0% and 100%, representing unstimulated and stimulated vector-transduced controls, respectively. In (D), IL-2 secretion was normalized to full-length CD148 transduced cells.