B and T Lymphocyte Attenuator-Mediated Signal Transduction Provides a Potent Inhibitory Signal to Primary Human CD4 T Cells That Can Be Initiated by Multiple Phosphotyrosine Motifs

Abstract

The B and T lymphocyte attenuator (BTLA) is a recently identified member of the CD28 family of cell receptors. Initial reports demonstrated that mice deficient in BTLA expression were more susceptible to experimental autoimmune encephalomyelitis, indicating that BTLA was likely to function as a negative regulator of T cell activation. However, cross-linking of BTLA only resulted in a 2-fold reduction of IL-2 production, questioning the potency with which BTLA engagement blocks T cell activation. We established a model in which BTLA signaling could be studied in primary human CD4 T cells. We observed that cross-linking of a chimeric receptor consisting of the murine CD28 extracellular domain and human BTLA cytoplasmic tail potently inhibits IL-2 production and completely suppresses T cell expansion. Mutation of any BTLA tyrosine motifs had no effect on the ability of BTLA to block T cell activation. Only mutation of all four tyrosines rendered the BTLA cytoplasmic tail nonfunctional. We performed structure-function studies to determine which factors recruited to the BTLA cytoplasmic tail correlated with BTLA function. Using pervanadate as a means to phosphorylate the BTLA cytoplasmic tail, we observed both Src homology protein (SHP)-1 and SHP-2 recruitment. However, upon receptor engagement, we observed only SHP-1 recruitment, and mutations that abrogated SHP-1 recruitment did not impair BTLA function. These studies question whether SHP-1 or SHP-2 have any role in BTLA function and caution against the use of pervanadate as means to initiate signal transduction cascades in primary cells.

B and T Lymphocyte Attenuator Regulates B Cell Receptor Signaling by Targeting Syk and BLNK

“…In T cells, activation of BTLA leads to tyrosine phosphorylation of ITIM domains located within its cytoplasmic tail, which leads to recruitment of the phosphatase SHP1 and inhibition of undefined positive signaling molecules (6,8,11). Although BTLA recruits SHP1, the precise mechanisms involved in BTLA-mediated lymphocyte attenuation are unclear (6,8). Furthermore, little work has been done to elucidate the role of BTLA in other lymphocytes, including B cells.…”

“…In T cells it is unclear whether both SHP1 and SHP2 are recruited following activation of BTLA (5,6). Pervanadate treatment of murine T cells leads to recruitment of both SHP1 and SHP2 to BTLA (5,11), while in human T cells stimulated with artificial APCs only SHP1 is recruited (6).…”

“…The ligand for BTLA is the TNF receptor HVEM, which is unique in that it is the first example of an Ig superfamily receptor interacting with a TNF family member (4,5). In T cells, activation of BTLA leads to tyrosine phosphorylation of ITIM domains located within its cytoplasmic tail, which leads to recruitment of the phosphatase SHP1 and inhibition of undefined positive signaling molecules (6,8,11). Although BTLA recruits SHP1, the precise mechanisms involved in BTLA-mediated lymphocyte attenuation are unclear (6,8).…”

“…BTLA recruits the protein tyrosine phosphatase SHP1 in T and B cells, but beyond this, it is unclear how BTLA activity leads to suppressed lymphocyte activation (6,9,11). To determine ablated downstream signaling mechanisms following BTLA activation, BTLA was immunoprecipitated from human primary B cells after various stimulating conditions and was probed for protein interactions by Western blot analysis.…”

“…However, unlike PD-1 and CTLA-4, which bind members of the B7 Ig superfamily receptors, the established ligand for BTLA is the TNFR family member herpesvirus entry mediator (HVEM) (TNFRSF14) (3)(4)(5). HVEM binding leads to tyrosine phosphorylation of the cytoplasmic tail of BTLA, which contains a proposed Grb2/Grb2-related adaptor protein (Grap) docking site and two ITIMs (5)(6)(7)(8)(9)(10)(11). Biochemical analysis has revealed that phosphorylation at multiple tyrosines within the cytoplasmic tail of BTLA is necessary and required for efficient recruitment of protein tyrosine phosphatases and BTLA-mediated attenuation of T cell effector response and cell proliferation (5,8,10,11).…”