The CD85/LIR-1/ILT2 Inhibitory Receptor Is Expressed by All Human T Lymphocytes and Down-Regulates Their Functions 1 Daniele Saverino,* Marina Fabbi, ² Fabio Ghiotto,* Andrea Merlo,* Silvia Bruno,* Daniela Zarcone,* Claudya Tenca,* Micaela Tiso, ² Giuseppe Santoro, ‡ Giuseppe Anastasi, ‡ David Cosman, § Carlo E. Grossi,* and Ermanno Ciccone 2 * The inhibitory molecule CD85/LIR-1/ILT2 has been detected previously on the surface of a small proportion of T lymphocytes. In this study, evidence is provided that, although only a fraction of CD3  cells are stained by mAb specific for CD85/LIR-1/ILT2 on their surface, this inhibitory receptor is present in the cytoplasm of all T lymphocytes, and that it is detectable on the surface of all T cell clones by the M402 mAb. Biochemical analyses further demonstrate that CD85/LIR-1/ILT2 is present in all T clones analyzed, and that the protein is tyrosine-phosphorylated. Expression of mRNA coding for CD85/LIR-1/ILT2 has been assessed by RT-PCR. Notably, in the NKL cell line and in one T cell clone, amplification of the messenger required 30 cycles only, whereas, in other T cell clones, an amplification product was detected by increasing the number of cycles. CD85/LIR-1/ILT2 inhibits CD3/TCR-mediated activation in both CD4  and CD8  clones, and it down-regulates Ag recognition by CD8  cells in a clonally distributed fashion. Addition of anti-ILT2 HP-F1 mAb in the cytolytic assay enhances target cell lysis mediated by Ag-specific CTL. This could be due to interference of the mAb with receptor/ligand interactions. In contrast, HP-F1 mAb cross-linking triggers inhibitory signals that reduce cytotoxicity. CD85/LIR-1/ILT2 also controls responses to recall Ags and, in low responders, its engagement sharply increases T cell proliferation. The inhibitory function of the molecule is also confirmed by its ability to reduce CD3/TCR-induced intracellular Ca 2 mobilization. The Journal of Immunology, 2000, 165: 3742–3755. T he functional outcome of T lymphocytes depends on a balance between two opposite effects, i.e., activation when appropriate peptides are presented to the TCR in the presence of an efficient costimulation, and inhibition as a conse- quence of the interaction of inhibitory molecules with their ligands (1). When activation prevails, an efficient immune response is mounted, whereas T cell anergy occurs when inhibitory signals are overwhelming. In addition to CTLA-4 (CD152), present in all ac- tivated T cells, other molecules that counterbalance T cell activa- tion have been described. These inhibitory molecules belong to distinct families, such as killer cell inhibitory receptor (KIR) 3 (2) and leukocyte Ig-like receptor (LIR)/Ig-like transcript (ILT) (3) that map on human chromosome 19. KIR molecules are specific for HLA-class I loci and are expressed on the surface of a small proportion (2– 4%) of T lymphocytes (4). The LIR/ILT molecular family has been recently characterized in several studies (reviewed in Ref. 5) and consists of at least 10 genes coding for proteins of the Ig superfamily (3). The products of some of these genes, such as LIR-1/ILT2, are surface membrane inhibitory receptors. LIR- 1/ILT2 is found on the surface of a proportion of NK cells (23– 77%), on a small percentage of T lymphocytes (4 –20%), and on all B cells, monocytes, and dendritic cells (6 – 8). LIR-1/ILT2 consists of four C2 Ig domains and exhibits a molecular mass of 110 kDa under both reducing and nonreducing conditions (6, 7). The inhib- itory function of this molecule is mediated by tyrosine phosphor- ylation of four immunoreceptor tyrosine-based inhibitory motif- like sequences in its cytoplasmic tail. Tyrosine phosphorylation leads to the recruitment of Src homology protein-1, Src homology protein-2, and Src homology 2 domain-containing inositol phos- phatase. Recruitment and activation of these phosphatases down- regulates the signaling mediated by activatory receptors (3, 8, 9). Inhibition has been assessed by down-regulation of NK and T lymphocyte cytolytic functions. Ligands for LIR-1/ILT2 are non- classical class I HLA-G protein (10), some alleles of HLA-A, and -B loci and the human cytomegalovirus UL18 gene product, a viral homologue of HLA-class I (6). The first domain of LIR-1/ILT2 interacts with the relatively nonpolymorphic 3 domain of HLA- class I and the analogous region of UL18 (11). mAbs specific for LIR-1/ILT2 have been produced, namely HP-F1, M402, and M405 (8, 12). In addition, mAb VMP55 and GHI/75, defined at the Fifth Workshop on Leukocyte Ags as specific for CD85 (13, 14), have been recently shown to recognize the LIR-1/ILT2 inhibitory re- ceptor (15), because anti-CD85 mAb react with ILT2 transfectants. In addition, peptide maps of the CD85 Ag are identical with those of ILT2. This study demonstrates that CD85/LIR-1/ILT2 is present in the cytoplasm and on the surface of all T lymphocytes. The receptor is functional and regulates human T cells by inhibiting CD3/TCR- mediated activation of both CD4 + and CD8 + clones, and Ag rec- ognition by CD8 + cells. In addition, CD85/LIR-1/ILT2 controls responses to recall Ags and, in low responders, its blocking by the HP-F1 mAb sharply increases T cell proliferation. *Department of Experimental Medicine, Human Anatomy Section, University of Genova, ² Monoclonal Antibody Unit, National Institute for Cancer Research, Genova, Italy; ‡ Department of Biomorphology, Human Anatomy Section, University of Messina, Messina, Italy; and § Immunex, Seattle, WA Received for publication April 17, 2000. Accepted for publication July 7, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by grants from the Consiglio Nazionale delle Ricerche, from Ministero della Universita ` e Ricerca Scientifica e Tecnologica, and from Asso- ciazione Italiana Ricerca sul Cancro (to C.E.G. and E.C.). 2 Address correspondence and reprint requests to Dr. Ermanno Ciccone, Department of Experimental Medicine, Institute of Human Anatomy, University of Genova Via De Toni 14, 16132 Genova, Italy. E-mail address: cicc@anatomiau.unige.it 3 Abbreviation used in this paper: KIR, killer cell inhibitory receptor; LIR, leukocyte Ig-like receptor; ILT, Ig-like transcript; GAM, goat anti-mouse. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00