CD300c is uniquely expressed on CD56 bright Natural Killer Cells and differs from CD300a upon ligand recognition

Abstract

Paired receptors on NK cells recognize similar ligands with varied strength of binding ability and perform different functions. The CD300 molecules are emerging as novel immune regulators in health and disease due to their interaction with their lipid-nature ligands. Particularly, the paired receptors CD300c and CD300a have been shown to elicit activating and inhibitory capabilities, respectively. In the current study, we seek to investigate the expression and function of CD300c on human NK cells. We demonstrate that IL-2 and IL-15 treatment significantly induce CD300c expression exclusively on CD56(bright) NK cells. CD300c up-regulation requires STAT5 and its expression is inhibited by IL-4. Consistently, IL-2 secreted from activated CD4(+) T cells specifically induces the expression of CD300c on CD56(bright) NK cells. Crosslinking CD300c with a specific antibody enhances the proficiency of CD56(bright) NK cells to degranulate and induce chemokine and cytokine secretion. We also show the differential binding of CD300a and CD300c to their ligands phosphatidylethanolamine (PE) and phosphatidylserine (PS) and their differential ability to affect CD56(bright) NK cell functions. Our results provide an insight into the novel set of paired receptors CD300a and CD300c that are distinctively expressed on CD56(bright) NK cells with varied effector functions.

Figure 1. CD300c is exclusively expressed on CD56^bright NK cells.

(A) The gating strategy of NK cells is shown to distinguish the CD56^bright and CD56^dim populations (upper panel). The dot plots in the bottom panel indicate the percentage of CD300c+ NK cells upon various cytokine stimuli for 40 hours. (B) The bar graph shows significant up-regulation of CD300c expression on CD56^bright subsets that are stimulated with IL-2 and IL-15. The data are from independent experiments from 4–6 donors. (C) The specific mRNA transcripts of CD300A and CD300C were determined by RT-PCR analysis on the sorted populations of different subsets of unstimulated, IL-2 and IL-15 stimulated NK cells. The y-axis denotes the normalized expression over 18sRNA and the corresponding ΔΔCt values are plotted. The data is from four healthy donors. The error bars indicate the average ± SEM.

Figure 2. Expression of CD300c is regulated by STAT5 and IL-4.

(A) Purified NK cells were stimulated with either IL-2 or IL-15 in the presence or absence of STAT5 inhibitor. Representative histograms show the expression of CD300c on different NK cell subsets that are either untreated (shaded), IL-2 or IL-15 plus vehicle DMSO as represented (thick line) and IL-2 or IL-15 plus STAT5 inhibitor (dotted line). (B) Bar graph represents the MFI of CD300c expression on CD56^bright NK cells with indicated treatments. (C) Representative histograms show the expression of CD300c on different NK cell subsets that are either untreated (shaded), IL-2 or IL-15 as represented (thick line) and IL-2 or IL-15 with IL-4 (dotted line). (D) Bar graph represents the MFI of CD300c expression on CD56^bright NK cells with indicated treatments. The data are from independent experiments from 4 donors. The error bars indicate the average ± SEM.

Figure 3. T cell-derived IL-2 induces the expression of CD300c on CD56^bright NK cells.

(A) NK cells form clusters when cultured either in the presence of recombinant IL-2 (rIL-2) or activated CD4⁺ T cells in a trans-well assay. (B) The histograms represent the percentage of CD300c⁺ CD56^bright NK cells cultured under several conditions in the transwell assay as described in Materials and Methods. (C) Bar graph represents the MFI of CD300c expression on CD56^bright NK cells cultured under the indicated conditions in the transwell assay. The data are from independent experiments from 4 donors. The error bars indicate the average ± SEM. rCD4 T-cells: resting CD4+ T cells; aCD4 T-cells: activated CD4+ T cells; α-IL-2: neutralizing anti-IL-2 mAb; rec IL-2: recombinant IL-2.

Figure 4. Crosslinking of CD300c induces CD56^bright NK cells to degranulate and cytokine secretion.

Purified NK cells were both left untreated or pre-treated with IL-2 and cross-linked with isotype or anti-CD300c (clone TX45) to assess NK cell functions. (A) The zebra plots represent the production of various cytokines and degranulation markers (CD107a/b) from CD56^bright NK cells, gated as in Fig. 1 (gated as CD56^bright CD16^neg). (B) Bar graphs represent the percentage of CD56^bright NK cells producing cytokines and expressing CD107a/b after different stimulation conditions. The data are from independent experiments from at least 4 donors. (C) Purified NK cells were both left untreated or pre-treated with IL-2 and crosslinked with isotype or anti-CD300c (clone TX45), and in the absence or presence of IL-12 and IL-18. The upper panel shows the percentage of IFN-γ⁺ CD56^bright NK cells. The lower panel represents the fold increase in the MFI IFN-γ⁺ CD56^bright NK cells. The Y axis on the left correspond to the white bars and the white symbols, while the Y axis on the right is for the black bars and the black symbols. The data are from independent experiments from 4 donors. The error bars indicate the average ± SEM.

Figure 5

(A) Binding of the purified fusion proteins CD300a-Ig (top graph) and CD300c-Ig (bottom graph) to dead Jurkat cells. Increasing concentrations of fluorochrome labeled CD300a-Ig and CD300c-Ig proteins were incubated with dead Jurkat cells and then acquired in a flow cytometer. LAIR1 R65K-Ig fusion protein served as negative control. Graphs represent the binding (MFI) of the fusion proteins to dead cells. The data are representative of two independent experiments. (B) ELISA assay shows binding of increasing concentrations of CD300a-Ig (top graph) and CD300c-Ig (bottom graph) fusion proteins to pure lipids that are coated on plates. LAIR1 R65K-Ig fusion protein served as negative control. The data is a representative of 2 independent experiments. (C) Binding of CD300-Ig fusion proteins to liposomes. Liposomes of specified compositions were prepared and coupled to a L1 biosensor. The binding of CD300a-Ig (top) and CD300c-Ig (bottom) was analyzed by allowing the proteins to pass through the L1 sensor. The curves represent the binding of fusion proteins to liposome coated L1 chips. (D) The binding (RU) for plateau values is shown in the bar graph and the error bars represent the average ± SEM. LAIR1 R65K-Ig fusion protein served as negative control. Results shown are from 3 independent experiments.

Figure 6. Purified NK cells were both left untreated or pre-treated with IL-2 and cross-linked with pure lipids coated on a plate as mentioned in Materials and Methods.

(A) The zebra plots represent the secretion of various cytokines and degranulation markers (CD107a/b) from CD56^bright NK cells, gated as in Fig. 1 (gated as CD56^bright CD16^neg) stimulated under the indicated conditions. (B) Bar graphs represent the percentage of TNF-α, MIP-1α and CD107a/b positive CD56^bright NK cells. The data are from independent experiments from 4 donors. The error bars indicate the average ± SEM.