The CD277 molecule is expressed in both T and NK cells 1, 13 (Supporting Information Fig. 1 and 2). CD277 has three

isoforms BTN3A1, BTN3A2 and BTN3A3, with (BTN3A1 and BTN3A3) or without (BTN3A2) the B30.2 domain in their cytoplasmic part 5 (Fig. 5A). The used mAb (clone 20.1) does not discriminate between the Ig domains of the three BTN3A isoforms, which share a very high level of identity (>95%). Moreover, the CD277 mAb recognizes in a similar manner all the different isoforms expressed in an ectopic cellular PF-6463922 order model (Fig. 5B). Quantitative PCRs were performed to determine the different relative levels of mRNA expression for each isoform in T and NK cells isolated from human PBMCs (Fig. 5C). Both BTN3A1 and BTN3A2 represented the main forms expressed by CD4+ and CD8+ T-cell subsets whereas the decoy form, BTN3A2 was the unique form strongly expressed by NK cells (Fig. 5C and D). BTB3A3 is poorly expressed in these immune cells. These results are further confirmed using available data from GEO omnibus (data not shown). MAPK Inhibitor Library mw To identify a role for these two major CD277 isoforms (Fig. 5D), the KGHYG-1 NK cell line was nucleofected with constructs encoding for FLAG epitope tagged BTN3A1 or BTN3A2. This cell line expresses the natural cytotoxicity receptor, NKp30 and stimulation of this receptor by specific antibodies is able to induce IFN-γ production in this NK cell line (data not

shown). The overexpression of the BTN3 isoforms is monitored by anti-FLAG mAb cell surface staining (Fig. 6A). The transiently transfected NK cells were stimulated by anti-NKp30 and/or anti-FLAG mAbs, and the IFN-γ production assessed by FACS analysis (Fig. 6B). The NKp30 stimulation, but not BTN3A1 or BTN3A2 triggering alone, induces IFN-γ production.

However, co-engagement of NKp30 with a CD277 isoform, modulates the NKp30-induced IFN-γ production. BTN3A1 stimulation seems to increase this cytokine production, whereas BTN3A2 stimulation decreases the NKp30-induced IFN-γ production. These results suggest a differential functional role of these two CD277 isoforms in NK cells. In this study, we describe differential effects of the CD277 molecule as a co-regulator of the immune signal in T cells Methamphetamine but not in NK cells (Fig. 1). There is no effect noted on NK cells consistent with the selective expression of the BTN3A2 isoform that lacks much of the cytosolic domain (Fig. 5). However, in the context where only the BTN3A2 isoform is co-engaged, this molecule could induce some negative signals in NK cells (Fig. 6). CD277 cross-linking elicits a robust co-stimulation of T-cell proliferation, cytokine production and CD25 expression. We showed that the stimulation of BTN3/CD277 proteins with a home-made mAb (clone 20.1, 1) increases, in a dose-dependent manner, the rates of early and late T-cell activation events induced by a combination of CD3+/−CD28 mAbs (Figs. 3 and 4).