A novel inhibitory receptor (ILT3) expressed on monocytes, macrophages, and dendritic cells involved in antigen processing - PubMed (original) (raw)

Comparative Study

A novel inhibitory receptor (ILT3) expressed on monocytes, macrophages, and dendritic cells involved in antigen processing

M Cella et al. J Exp Med. 1997.

Abstract

Immunoglobulin-like transcript (ILT) 3 is a novel cell surface molecule of the immunoglobulin superfamily, which is selectively expressed by myeloid antigen presenting cells (APCs) such as monocytes, macrophages, and dendritic cells. The cytoplasmic region of ILT3 contains putative immunoreceptor tyrosine-based inhibitory motifs that suggest an inhibitory function of ILT3. Indeed, co-ligation of ILT3 to stimulatory receptors expressed by APCs results in a dramatic blunting of the increased [Ca2+]i and tyrosine phosphorylation triggered by these receptors. Signal extinction involves SH2-containing protein tyrosine phosphatase 1, which is recruited by ILT3 upon cross-linking. ILT3 can also function in antigen capture and presentation. It is efficiently internalized upon cross-linking, and delivers its ligand to an intracellular compartment where it is processed and presented to T cells. Thus, ILT3 is a novel inhibitory receptor that can negatively regulate activation of APCs and can be used by APCs for antigen uptake.

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Figures

Figure 1

Figure 1

Amino acid sequence of human ILT3, aligned with ILT1 and ILT2. Since ILT1 and ILT2 consist of four Ig-SF domains, only the two domains with higher homology to ILT3 were included in the alignment. The alignment was generated by the Clustal method. Gaps (dashes) were introduced to maximize homologies. Amino acids identical to the consensus are indicated by dots. Conserved cysteines involved in disulfide bonds in the extracellular domains are identified by asterisks. Cytoplasmic tyrosine-based motifs potentially involved in signal transduction and/or endocytosis are boxed. ILT3 has no N-linked glycosylation sites (N-X-S/T). Amino acid residues are numbered on the right side, beginning with the first residue of each of the predicted domains. ss, signal sequence; Ig1–4, Ig-SF extracellular domains; cp, connecting peptide; tm, transmembrane domain; cy, cytoplasmic domain. ILT3 cDNA sequence is available from EMBL/GenBank/DDBJ under accession number U82979.

Figure 2

Figure 2

Expression of ILT3 in monocytes, primary DCs, monocyte-derived DCs, and macrophages. (A) mAb ZM3.8 stains CD14+ monocytes in PBMC and CD83+ DCs in a monocyte-enriched population (right). On the contrary, CD3+ T cells, CD16+ NK cells, or CD20+ B cells are not stained (left). PBMCs and monocyte-enriched populations were subjected to two-color staining. Lymphocytes (left) and monocytes (right) were gated using forward and side scatter (FSC and SSC) parameters. (B) ILT3 is expressed on CD14+ monocytes and on a subset of CD14-/HLA-DRhigh cells, which corresponds to circulating primary DCs (46). Monocytes were enriched from peripheral blood and subjected to three-color staining using anti-CD14 (FITC), ZM3.8 (PE), and anti-HLA-DR (APC). (C) Monocyte-derived DCs express both CD1a and ILT3. (D) Macrophages express low levels of both CD16 and ILT3. DCs and macrophages were derived from monocytes under appropriate culture conditions and subjected to two-color staining. Negative controls were located in the lower left quadrants.

Figure 2

Figure 2

Expression of ILT3 in monocytes, primary DCs, monocyte-derived DCs, and macrophages. (A) mAb ZM3.8 stains CD14+ monocytes in PBMC and CD83+ DCs in a monocyte-enriched population (right). On the contrary, CD3+ T cells, CD16+ NK cells, or CD20+ B cells are not stained (left). PBMCs and monocyte-enriched populations were subjected to two-color staining. Lymphocytes (left) and monocytes (right) were gated using forward and side scatter (FSC and SSC) parameters. (B) ILT3 is expressed on CD14+ monocytes and on a subset of CD14-/HLA-DRhigh cells, which corresponds to circulating primary DCs (46). Monocytes were enriched from peripheral blood and subjected to three-color staining using anti-CD14 (FITC), ZM3.8 (PE), and anti-HLA-DR (APC). (C) Monocyte-derived DCs express both CD1a and ILT3. (D) Macrophages express low levels of both CD16 and ILT3. DCs and macrophages were derived from monocytes under appropriate culture conditions and subjected to two-color staining. Negative controls were located in the lower left quadrants.

Figure 3

Figure 3

(A) SDS-PAGE analysis of ILT3 immunoprecipitated from 125I-labeled, ILT3-transfected Jurkat T cells and monocytes. ILT3 appears as a ∼55-kD band in ILT3-transfected cells and as a ∼58–60-kD band in monocytes. (B) When ILT3 is precipitated from 32P-labeled cells, it is detectable as a constitutively phosphorylated molecule.

Figure 3

Figure 3

(A) SDS-PAGE analysis of ILT3 immunoprecipitated from 125I-labeled, ILT3-transfected Jurkat T cells and monocytes. ILT3 appears as a ∼55-kD band in ILT3-transfected cells and as a ∼58–60-kD band in monocytes. (B) When ILT3 is precipitated from 32P-labeled cells, it is detectable as a constitutively phosphorylated molecule.

Figure 4

Figure 4

Intracellular Ca2+ mobilization induced in monocytes and macrophages via CD11b (A), HLA-DR (B), and FcγRIII (C) are inhibited upon cross-linking with ILT3 (D–F). G shows that in the absence of a cross-linking antibody, ILT3 does not inhibit Ca2+ flux triggered by the 3.8B1 anti-HLA-DR mAb.

Figure 5

Figure 5

(A) Protein tyrosine phosphorylation stimulated by treatment of monocytes with the anti-HLA-DR mAb 3.8B1 is inhibited by co-ligation of HLADR with ILT3. Monocytes were incubated with medium alone (lane 1), with ZM3.8 mAb (antiILT3; lane 2), with 3.8B1 mAb (anti-HLA-DR; lane 3) or with both mAbs in the absence (lane 4) or in the presence (lane 5) of a secondary cross-linking antibody. Cell lysates were separated on SDS-PAGE, transferred to nitrocellulose, and probed with HRP-coupled antiphosphotyrosine mAb 4G10. (B) Association of SHP-1 with ILT3 is increased upon ILT3 cross-linking. Monocytes were stimulated with ZM3.8 (lane 2) or with a control IgG (5.133 mAb) (lane 1) coated on plastic plates. Cells were kept at 37°C for 2 min, harvested, and lysed. ILT3 was immunoprecipitated with ZM3.8. Proteins were separated on SDSPAGE, transferred to nitrocellulose, and probed with anti-SHP-1, followed by HRP-conjugated goat anti–rabbit Ig. The migration of SHP-1 is marked by the arrow.

Figure 5

Figure 5

(A) Protein tyrosine phosphorylation stimulated by treatment of monocytes with the anti-HLA-DR mAb 3.8B1 is inhibited by co-ligation of HLADR with ILT3. Monocytes were incubated with medium alone (lane 1), with ZM3.8 mAb (antiILT3; lane 2), with 3.8B1 mAb (anti-HLA-DR; lane 3) or with both mAbs in the absence (lane 4) or in the presence (lane 5) of a secondary cross-linking antibody. Cell lysates were separated on SDS-PAGE, transferred to nitrocellulose, and probed with HRP-coupled antiphosphotyrosine mAb 4G10. (B) Association of SHP-1 with ILT3 is increased upon ILT3 cross-linking. Monocytes were stimulated with ZM3.8 (lane 2) or with a control IgG (5.133 mAb) (lane 1) coated on plastic plates. Cells were kept at 37°C for 2 min, harvested, and lysed. ILT3 was immunoprecipitated with ZM3.8. Proteins were separated on SDSPAGE, transferred to nitrocellulose, and probed with anti-SHP-1, followed by HRP-conjugated goat anti–rabbit Ig. The migration of SHP-1 is marked by the arrow.

Figure 6

Figure 6

ZM3.8 mAb is internalized upon cross-linking. ZM3.8 bound to monocytes on ice did not disappear from the cell surface when the cells were shifted at 37°C, in the absence of a cross-linking antibody (▴). After cross-linking with a F(ab′)2 biotin-labeled secondary antibody, ZM3.8 was rapidly internalized, becoming undetectable by FACS® analysis in 30–60 min (□). In fixed cells, only a minimal decrease of the median fluorescence intensity was detected over time (▪). Cell surface– bound ZM3.8 was revealed by PE-conjugated goat anti–mouse IgG1 (▴) or by PE-conjugated streptavidin (□, ▪). The MFI was determined by FACS® analysis. The percentage decrease of MFI as compared to a control sample kept at 4°C was used as a measure of internalization.

Figure 7

Figure 7

Presentation of ZM3.8 mAb to a T cell clone specific for mouse IgG1 by irradiated monocytes. ZM3.8 mAb (▪) is presented 50– 100-fold more efficiently than anti-FcγRI (□) and 400–500-fold more efficiently than anti-TNP (•) and anti-NKAT4 KIR (○) mAbs, which do not stain monocytes.

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