Cytotoxic T Lymphocyte Antigen 4 (Ctla-4) Engagement Delivers an Inhibitory Signal through the Membrane-Proximal Region in the Absence of the Tyrosine Motif in the Cytoplasmic Tail

Abstract

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a T cell costimulation receptor that delivers inhibitory signals upon activation. Although the tyrosine-based motif (165YVKM) within its cytoplasmic tail has been shown to associate in vitro with Src homology 2 domain–containing tyrosine phosphatase (SHP-2) and phosphatidylinositol 3 kinase upon phosphorylation, the mechanism of negative signaling remains unclear. Here, we report a new mechanism of negative signaling based on the analysis of murine T cell clones transfected with various mutants of CTLA-4. Upon T cell activation by cross-linking with anti-CD3 and anti-CD28 antibodies, CTLA-4 engagement inhibited both proliferation and interleukin 2 production in tyrosine mutants as well as in wild-type CTLA-4 transfectants. Furthermore, the mutant CTLA-4 lacking most of the cytoplasmic region strongly suppressed interleukin 2 production as well. These data suggest that negative signals by CTLA-4 could be mediated through the membrane-proximal region of CTLA-4 but not through the YVKM motif and that the association of CTLA-4 with SHP-2 is not required for CTLA-4–mediated suppression of T cell activation.

In vivo overexpression of CTLA‐4 suppresses lymphoproliferative diseases and thymic negative selection

“…1B). These results are consistent with our previous observation on CTLA-4-transfected Th1 clones [24] and demonstrate that the cytoplasmic tail of CTLA-4 controls the cell surface expression and intracellular localization of CTLA-4 in vivo. Upon stimulation with anti-CD3 and -CD28 Ab, CTLA-4 expression is up-regulated on all subpopulations [double-negative (DN), DP, CD4 singlepositive (SP), CD8SP] in normal and Tg thymocytes (Fig.…”

“…We have shown previously that the cytoplasmic region, including the SHP-2/PI3K binding sites, is not required for CTLA-4-mediated inhibitory function in Th1 clones [24]. An explanation for this difference might be found in the difference of the activation status of T cells: T cell clones are maintained by occasional Ag stimulation, are cultured in IL-2 and have somewhat of a semi-activated status, whereas in vivo thymocytes are primarily naive cells.…”

“…To elucidate the mechanism of CTLA-4-mediated inhibition in vivo, we established two of each of the Tg mouse lines expressing either a full-length CTLA-4 (FL-Tg) or a mutant CTLA-4 with a truncated cytoplasmic tail lacking the tyrosine motif (TR-Tg) [24], under the control of the human CD2 promoter [29]. One of each of the lines of FL-Tg and TR-Tg mice was selected for further analysis of the in vivo function of CTLA-4.…”

“…Full-length and truncated CTLA-4 cDNA, previously described (only six amino acids remain in the truncated form) [24], were subcloned into an expression vector containing the human CD2 promoter [29]. The insert was injected into oocytes from C57BL/6 mice.…”

“…Although it has been suggested that several molecules, such as Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2) and phosphatidyl inositol 3-kinase (PI3K), may mediate the negative signal through binding to the phosphorylated tyrosine motif (YVKM) present in the cytoplasmic region of CTLA-4 [18][19][20], the precise inhibitory mechanism remains controversial [21][22][23]. We and others have previously shown that the PI3K/ SHP-2 binding sites are not essential for CTLA-4-mediated inhibitory signals in T cell clones/cell lines [24][25][26]. We have also shown that the same tyrosine motif controls the cell surface expression and intracellular localization of CTLA-4 through association with clathrin-mediated endocytosis [27,28].…”