The cytoplasmic domain of BTLA contains three tyrosine-containing motifs that are conserved in mice, rats, dogs, chimpanzees and humans. The most membrane-proximal tyrosine (Y245 in the BALB/c mouse strain) is a predicted recruitment site for growth-factor receptor-bound protein 2 (GRB2), whereas the second and third tyrosines (Y274 and Y299 in the BALB/c mouse strain) are both present in immunoreceptor tyrosine-based inhibitory motif (ITIM) sequences. Each tyrosine residue in these motifs can be phosphorylated after BTLA crosslinking, and both ITIMs are required to recruit the tyrosine phosphatases SHP1 and SHP2. Notably, HVEM / TNFRSF14 expression in the BJAB B-cell line could induce BTLA phosphorylation and SHP2 recruitment in the EL4 T-cell line through cell–cell interaction. At present, the targets of SHP1 and SHP2 recruited to BTLA are unknown, although it is possible that they also have a role in dephosphorylating signalling intermediates downstream of antigen receptors in lymphocytes or in specifically targeting the PI3K–PKB pathway, as proposed for PD1.
Despite the prediction that Y245 of BTLA might recruit GRB2, direct evidence of protein recruitment to this tyrosine-containing motif has been elusive. Nevertheless, this motif is similar to the YMN motif in CD28 and CTLA4 that recruits GRB2 and PI3K50. Recently published findings confirm that a phosphorylated peptide containing the BTLA N-terminal tyrosine motif can interact with GRB2 and the p85 subunit of PI3K in vitro62, although the functional effects of this interaction remain unexplored in vivo.
Most analyses of BTLA activity have focused on its actions in T cells. Crosslinking of BTLA on T cells using BTLA-specific antibodies inhibited T-cell proliferation in response to TCR ligation, although the inhibition was less effective on CD28 co-stimulation. In agreement with antibody crosslinking data, analysis in an antigen-specific priming system using CHO cells expressing HVEM / TNFRSF14 confirmed that HVEM & BTLA immune checkpoint pathway on T cells reduced T-cell proliferation. Furthermore, activation of CD28 by CD80 expressed by these CHO cells reduced the inhibition of T-cell proliferation mediated by HVEM & BTLA immune checkpoint pathway at high antigen doses, but did not affect the inhibition of T-cell proliferation at low antigen doses. This inhibition remained dependent on T-cell expression of BTLA. In other studies, an agonistic BTLA-specific antibody was identified, and treatment of T cells with this antibody inhibited the expression of the activation marker CD25, owing in part to a block in IL-2 secretion by the T cells, but did not reduce expression of another activation marker, CD69, or cause apoptosis. Together, these data show that HVEM & BTLA immune checkpoint pathway reduces T-cell activation, but, at present, its function on other cell types is less well documented.
Kenneth M. Murphy et al. Balancing co-stimulation and inhibition with BTLA and HVEM. Nature Reviews Immunology 6, 671-681