Functional requirements for inhibitory signal transmission by the immunomodulatory receptor CD300a

Abstract

Background: Activation signals can be negatively regulated by cell surface receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). CD300a, an ITIM bearing type I transmembrane protein, is expressed on many hematopoietic cells, including subsets of lymphocytes.

Results: We have taken two approaches to further define the mechanism by which CD300a acts as an inhibitor of immune cell receptor signaling. First, we have expressed in Jurkat T cells a chimeric receptor consisting of the extracellular domains of killer-cell immunoglobulin-like receptor (KIR)2DL2 fused to the transmembrane and cytoplasmic segments of CD300a (KIR-CD300a) to explore surrogate ligand-stimulated inhibition of superantigen stimulated T cell receptor (TCR) mediated cell signaling. We found that intact CD300a ITIMs were essential for inhibition and that the tyrosine phosphorylation of these ITIMs required the src tyrosine kinase Lck. Tyrosine phosphorylation of the CD300a ITIMs created docking sites for both src homology 2 domain containing protein tyrosine phosphatase (SHP)-1 and SHP-2. Suppression of SHP-1 and SHP-2 expression in KIR-CD300a Jurkat T cells with siRNA and the use of DT40 chicken B cell lines expressing CD300a and deficient in several phosphatases revealed that SHP-1, but not SHP-2 or the src homology 2 domain containing inositol 5' phosphatase SHIP, was utilized by CD300a for its inhibitory activity.

Conclusion: These studies provide new insights into the function of CD300a in tuning T and B cell responses.

Figure 1

The ITIMs of CD300a are essential for the inhibition of BCR stimulated activation. (A) DT40 chicken B cells expressing CD300a WT or CD300a 4F were loaded with Fluo-4 and Fura-Red. Cells were stimulated with anti-chicken BCR plus isotype control antibody (black line) or anti-chicken BCR plus anti-CD300a mAb (grey line) for 30 seconds and then co-crosslinked with a secondary antibody (GAM). Fluorescence emission was measured in a flow cytometer. Ca²⁺ mobilization is expressed as the ratio of Fluo-4/Fura-Red as a function of time. These results are representative of three independent experiments. (B) DT40 chicken B cells expressing CD300a WT or CD300a 4F were transiently transfected with a NFAT luciferase reporter plasmid and stimulated with GAM plus anti-chicken BCR plus isotype control or anti-chicken BCR plus anti-CD300a mAb. The measured luciferase activity was normalized to the activity obtained with cells treated with PMA plus ionomycin. Data are presented as percentage of inhibition of CD300a vs. isotype control and they are the average ± SEM for three separate experiments.

Figure 2

Schematic representation of the chimeric KIR-CD300a receptors. Structure of the chimeric molecules bearing the extracellular domain of KIR2DL2 and the transmembrane and cytoplasmic domains of CD300a is represented. The construct is fused to the hemagglutinin (HA) tag at the C-terminus. The shady dotted regions in the cytoplasmic tail correspond to the ITIMs in the CD300a receptor. The tyrosine in each of the 4 ITIMs was mutated to phenylalanine to generate the mutant chimeric construct KIR-CD300a 4F.

Figure 3

KIR-CD300a mediates inhibition of SED stimulated Jurkat T cell activation. (A) Gating strategy for assessing CD69 expression on superantigen activated cells. Jurkat T cells were electronically gated by size and forward scatter and by the absence of expression of CD19, a marker of 721.221 cells, and then the expression of CD69 was assessed. The gating strategy for assessing CD25 expression was the same. (B) KIR CD300a WT and KIR-CD300a 4F Jurkat T cells were co-cultured with 721.221 or 721.221-Cw3 cells, loaded or not with SED. Cultures were harvested and Jurkat T cells were assessed for CD69 expression by flow cytometry. Results are representative of three independent experiments. (C) Cells were cultured as in B, loaded (white bar) or not (black bars) with SED, and the expression of CD25 was assessed. The bar graph represents average ± SEM of the percentage of CD25+ Jurkat T cells. Results are from three independent experiments. (D) Untransfected E6.1, KIR-CD300a WT and KIR-CD300a 4F Jurkat T cells were transiently transfected with a NFAT luciferase reporter plasmid. Following coculture with 721.221, 721.221-Cw3 and 721.221-Cw6 loaded (withe bars) or not (black bars) with SED, cells were lysed and supernatants were assayed for luciferase activity. Data were normalized by the activity obtained with cells treated with PMA plus ionomycin. Results are representative of three independent experiments.

Figure 4

Tyrosine phosphorylation of CD300a requires the src kinase Lck. (A) KIR-CD300a WT and KIR-CD300a 4F Jurkat T cells were stimulated with medium or pervanadate for 3 minutes, or mixed with 721.221-Cw3 or 721.221-Cw6 and incubated at 37°C for 5 minutes. Cell lysates were immunopreciprecipitated with anti-KIR2DL2 (clone GL183) mAb and blotted separately for phosphotyrosine and HA. Results are representative of five independent experiments. (B) E6.1 Jurkat cells, Jurkat cells deficient in Lck or deficient in ZAP-70 were transiently transfected with a plasmid encoding KIR-CD300a and incubated at 37°C with 721.221-Cw3 and 721.221-Cw6 for 5 minutes. Cell lysates were immunoprecipitated with anti-KIR2DL2 (clone GL183) mAb and blotted separately for phosphotyrosine and HA. Results are representative of three independent experiments.

Figure 5

The phosphatases SHP-1 and SHP-2 associate with tyrosine phosphorylated CD300a ITIMs. KIR-CD300a Jurkat T cells were stimulated with medium or pervanadate for 3 minutes, or incubated for 5 minutes at 37°C with 721.221-Cw3 and 721.221-Cw6 cells. Then, cell lysates were immunopreciprecipitated with anti-KIR2DL2 (clone GL183) mAb and blotted separately for HA, SHP-1 and SHP-2. Results are representative of two independent experiments.

Figure 6

SHP-1, but not SHP-2 or SHIP, is required for CD300a mediated inhibition of BCR stimulated activation. (A) DT40 cells, DT40 cells lacking SHP-1, DT40 cells lacking SHP-2, or DT40 cells lacking SHIP, all expressing CD300a WT were loaded with Fluo-4 and Fura-Red. Then, cells were acquired in a flow cytometer and stimulated with anti-chicken BCR plus isotype control antibody (black line) or anti-chicken BCR plus anti-CD300a (grey line) mAb for 30 seconds and then co-crosslinked with a secondary antibody (GAM). Ca²⁺ mobilization is expressed as the ratio of Fluo-4/Fura-Red as a function of time. These results are representative of two independent experiments. (B) DT40 cell lines expressing CD300a WT were transiently transfected with a NFAT luciferase reporter plasmid and stimulated with anti-chicken BCR plus isotype control or anti-chicken BCR plus anti-CD300a mAb. Cells were lysed and supernatants assayed for luciferase activity. Results were normalized to the activity obtained when cells were treated with PMA plus ionomycin. Data are presented as percentage of inhibition of CD300a vs. isotype control and they are the average ± SEM for three separate experiments. (C) The indicated DT40 cells lines stably expressing CD300a WT and human SHP-1 WT, SHP-1 CS, SHP-2 WT or SHP-2 CS were loaded with Fluo-4 and Fura-Red. Ca²⁺ mobilization was assessed as in A. These results are representative of three independent experiments. (D) KIR-CD300a WT Jurkat T cells transfected with non target (NT), SHP-1 and SHP-2 siRNA were co-cultured with 721.221 or 721.221-Cw3 cells, loaded or not with SED. Cultures were harvested and Jurkat T cells were assessed for CD69 expression by flow cytometry. In the left panel, the percentage of inhibition of CD69 expression, calculated as shown in material and methods, is presented. The scatter plot represents the average ± SEM. In the center panel, the expression of SHP-1, SHP-2 and actin (loading control) was assessed by western blot analysis in lysates from siRNA transfected KIR-CD300a WT Jurkat T cells. In the right panel, the relative amount of SHP-1 mRNA and SHP-2 mRNA from siRNA transfected KIR-CD300a WT Jurkat T cells is shown. The bar graph represents the average ± SEM. Results are from three independent experiments.