Specific engagement of the CD94/NKG2-A killer inhibitory receptor by the HLA-E class Ib molecule induces SHP-1 phosphatase recruitment to tyrosine-phosphorylated NKG2-A: evidence for receptor function in heterologous transfectants

Abstract

It has been recently demonstrated that the CD94/NKG2-A killer inhibitory receptor (KIR) specifically recognizes the HLA-E class Ib molecule. Moreover, the apparent CD94-mediated specific recognition of different HLA class Ia allotypes, transfected into the HLA-defective cell line 721.221, indeed depends on their selective ability to concomitantly stabilize the surface expression of endogenous HLA-E molecules, which confer protection against CD94/ NKG2-A + effector cells. In the present study, we show that a selective engagement of the CD94/NKG2-A inhibitory receptor with a specific monoclonal antibody (mAb) (Z199) was sufficient to induce tyrosine phosphorylation of the NKG2-A subunit and SHP-1 recruitment. These early biochemical events, commonly related to negative signaling pathways, were also detected upon the specific interaction of NK cells with an HLA-E + 721.221 transfectant (.221-AEH), and were prevented by pre-incubation of .221-AEH with an anti-HLA class I mAb. Furthermore, mAb cross-linking of the CD94/NKG2-A receptor, segregated from other NK-associated molecules by transfection into a rat basophilic leukemia cell line (RBL-2H3), promoted tyrosine phosphorylation of NKG2-A and co-precipitation of SHP-1, together with an inhibition of secretory events triggered via Fc 4 RI. Remarkably, interaction of CD94/NKG2-A + RBL cells with the HLA-E + .221-AEH transfectant specifically induced a detectable association of SHP-1 with NKG2-A, constituting a more formal evidence for the receptor-HLA class I interaction.

Complexes of HLA-G Protein on the Cell Surface Are Important for Leukocyte Ig-Like Receptor-1 Function

“…The cell lines used in this work are the EBV-transformed class I MHCnegative human B cell line 721.221 (23), 721.221 transfectants, RBL (rat basophilic leukemia) cells, and RBL cells transfected with LIR-1 (24).…”

“…5, E and F). This is probably because virtually all decidual NK cells express one or more inhibitory receptors capable of interacting with the HLA-G molecules, including LIR-1 and KIR2DL4 (16), or with the HLA-E molecule, including CD94/NKG2A (24,37). Among these three receptors, KIR2DL4 was demonstrated to play a predominant role in preventing the NK-mediated lysis of HLA-G by decidual NK cells (16).…”

“…To directly test the interaction between LIR-1 and the various HLA-G transfectants, we used the RBL cells transfected with LIR-1 (18,22,24). RBL/LIR-1 transfectants were labeled with [ 3 H]serotonin, incubated with anti-TNP mAb of the IgE isotype, and cocultured with the various .221/HLA-G transfectants, previously haptenized with TNP.…”

Transcriptional Control of MurineCD94Gene: Differential Usage of Dual Promoters by Lymphoid Cell Types

“…Because of their initial characterization, experimental evidence has demonstrated a role for the heterodimeric CD94/NKG2 receptors in the regulation of immune responses in NK cells (11)(12)(13)(14) as well as subsets of T cells (15,16). Interestingly, while some of the NKG2 gene products, such as NKG2A, form inhibitory heterodimers with CD94, others such as NKG2C and E form activating complexes in association with CD94 and a signaling adaptor molecule DAP12 (14,17).…”

The NKG2 natural killer cell receptor family: comparative analysis of promoter sequences

“…12,13 In contrast, inhibitory NKG2 proteins (NKG2-A and -B) carry immunoreceptor tyrosine-based inhibition motifs (ITIMs). 12,14 Despite their distinct signaling capabilities, NKG2-A, -B and -C receptors have been found to recognize the same ligand, the non-classical HLA class I molecule HLA-E. 15,16 We have previously characterized gene structure and genomic organization of human NKG2 members. 10,17,18 We found that NKG2-A, -C, -E and -F are closely linked (as listed) and of the same transcriptional orientation.…”