Modulation of lymphocyte function with inhibitory CD2: loss of NK and NKT cells

Analysis of the NK cell developmental pathway suggests that CD2 expression may be important in regulating NK maturation. To test this hypothesis, we developed mice containing only an inhibitory CD2 molecule by linking the extracellular domain of CD2 to an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) motif. Mice containing the CD2 Tg(ITIM) transgene, introduced into a CD2 KO background, have no morphologically detectable lymph nodes, although development of the thymus appears normal. In addition, these mice had major loss of both NK and NKT subsets in peripheral organs, while T and B cell frequencies were intact. Expression of CD2 was low on T cells and lacking on B cells and functional defects were observed in these populations. NKT cells expressing CD4 were absent, while the CD8⁺ and double negative NKT cells were retained. Small subsets of NK cells were detected but expression of CD2 on these cells was very low or absent, and their maturation was impaired. Based on the phenotype described here, we believe that these mice represent a unique model to study lymphoid organ and lymphocyte development.     

Immune checkpoint blockade and its combination therapy with small-molecule inhibitors for cancer treatment

Initially understood for its physiological maintenance of self-tolerance, the immune checkpoint molecule has recently been recognized as a promising anti-cancer target. There has been considerable interest in the biology and the action mechanism of the immune checkpoint therapy, and their incorporation with other therapeutic regimens. Recently the small-molecule inhibitor (SMI) has been identified as an attractive combination partner for immune checkpoint inhibitors (ICIs) and is becoming a novel direction for the field of combination drug design. In this review, we provide a systematic discussion of the biology and function of major immune checkpoint molecules, and their interactions with corresponding targeting agents. With both preclinical studies and clinical trials, we especially highlight the ICI + SMI combination, with its recent advances as well as its application challenges.     

The crystal structure of the extracellular domain of the inhibitor receptor expressed on myeloid cells IREM-1

The immune receptors expressed on myeloid cells (IREM) are type I transmembrane proteins encoded on human chromosome 17 (17q25.1), whose function is believed to be important in controlling inflammation. To date, three IREM receptors have been identified. IREM-1 functions as an inhibitory receptor, whereas IREM-2 and IREM-3 serve an activating function. Here, we report the crystal structure of IREM-1 extracellular domain at 2.6 A resolution. The overall fold of IREM-1 resembles that of a V-type immunoglobulin domain, and reveals overall close homology with immunoglobulin domains from other immunoreceptors such as CLM-1, TREM-1, TLT-1 and NKp44. Comparing the surface electrostatic potential and hydrophobicity of IREM-1 with its murine homologous CLM-1, we observed unique structural properties for the complementary determining region of IREM-1, which suggests that they may be involved in recognition of the IREM-1 ligand. Particularly interesting is the structural conformation and physical properties of the antibody's equivalent CDR3 loop, which we show to be a structurally variable region of the molecule and therefore could be the main structural determinant for ligand discrimination and binding. In addition, the analysis of the IREM-1 structure revealed the presence of four structurally different cavities. Three of these cavities form a continuous hydrophobic groove on the IREM-1 surface, which point to a region of the molecule capable of accommodating potential ligands.     

Immune-related, lectin-like receptors are differentially expressed in the myeloid and lymphoid lineages of zebrafish

The identification of C-type lectin (Group V) natural killer (NK) cell receptors in bony fish has remained elusive. Analyses of the Fugu rubripes genome database failed to identify Group V C-type lectin domains (Zelensky and Gready, BMC Genomics 5:51, 2004) suggesting that bony fish, in general, may lack such receptors. Numerous Group II C-type lectin receptors, which are structurally similar to Group V (NK) receptors, have been characterized in bony fish. By searching the zebrafish genome database we have identified a multi-gene family of Group II immune-related, lectin-like receptors (illrs) whose members possess inhibiting and/or activating signaling motifs typical of Group V NK receptors. Illr genes are differentially expressed in the myeloid and lymphoid lineages, suggesting that they may play important roles in the immune functions of multiple hematopoietic cell lineages.     

TIGIT-Fc alleviates acute graft-versus-host disease by suppressing CTL activation via promoting the generation of immunoregulatory dendritic cells

Graft-versus-host disease (GVHD) is the most common complication and major limitation of allogeneic hematopoietic stem cell transplantation. The CD226/TIGIT-CD155 signal is critical for the cross-talk between T cells and dendritic cells (DCs). Studies have shown that blockade of the CD226-CD155 interaction, using an anti-CD226 antibody, can significantly ameliorate GVHD. It has also been reported that a TIGIT-Fc fusion protein exerts immunosuppressive effects by binding to CD155 on DCs. Here, we used a mouse allogeneic acute GVHD model to explore the therapeutic potential and mechanism of action of TIGIT-Fc. C57/BL6 and Balb/c mice were used as hematopoietic cell graft donors and recipients, respectively. In the TIGIT-Fc-treated mice, GVHD symptom occurrence and mortality were delayed compared to that in isotype control group mice. Histopathological analyses revealed that following TIGIT-Fc treatment, liver and small intestine tissue damage was reduced with minimal lymphocytic infiltration. The percentage of CD8⁺IFN-γ⁺ and CD8⁺ granzyme B⁺ cells significantly decreased in the TIGIT-Fc group. Moreover, treatment with TIGIT-Fc, even after the onset of GVHD, ameliorated symptoms and prolonged survival. TIGIT-Fc also inhibited CD8⁺ T cell activation in vitro; this was dependent on the presence of CD155 on bone marrow-derived dendritic cells (BMDCs) and on IL-10 production. In addition, TIGIT–CD155 ligation triggered both Erk phosphorylation and STAT3 nuclear translocation. These data indicate that TIGIT plays an important role in the development of GVHD and is an ideal molecular target to treat acute GVHD.     

Pinpointing phosphotyrosine-dependent interactions downstream of the collagen receptor DDR1

Activation of the receptor tyrosine kinase DDR1 by collagen results in robust and sustained phosphorylation, however little is known about its downstream mediators. Using phosphopeptide mapping and site-directed mutagenesis, we here identified multiple tyrosine phosphorylation sites within DDR1. We found that Nck2 and Shp-2, two SH2 domain-containing proteins, bind to DDR1 in a collagen-dependent manner. The binding site of Shp-2 was mapped to tyrosine-740 of DDR1 within an ITIM-consensus sequence. Lastly, ablation of DDR1 in the mouse mammary gland resulted in delocalized expression of Nck2, suggesting that defects observed during alveologenesis are caused by the lack of the DDR1-Nck2 interaction.     

Association of Grb-2 and PI3K p85 with phosphotyrosile peptides derived from BTLA

B and T lymphocyte attenuator (BTLA) is a recently identified inhibitory receptor expressed by B and T cells. We previously identified two tyrosine-containing signaling motifs in the cytoplasmic domain of BTLA that interact with the SHP-1 and SHP-2 phosphatases. BTLA has a third conserved tyrosine-containing motif within the cytoplasmic domain, similar in sequence to a Grb-2 recruitment site. To identify specific interacting proteins that would be recruited to this motif, we carried out an unbiased screen by using synthetic peptides in active (e.g., phosphotyrosil-containing) or control (e.g., non-phosphorylated) forms as baits. Using mass spectrometry, we identified two specific interacting proteins, Grb-2 and the p85 subunit of PI3K. Further, we demonstrate that the interaction with Grb-2 is direct, whereas the recruitment of the p85 subunit by BTLA phosphotyrosile-containing peptides may be indirect via its association with Grb-2. These findings may provide biochemical basis for previously unexplained actions of BTLA.     

Comparative genomics indicates the mammalian CD33rSiglec locus evolved by an ancient large-scale inverse duplication and suggests all Siglecs share a common ancestral region

The CD33-related sialic acid binding Ig-like lectins (CD33rSiglecs) are predominantly inhibitory receptors expressed on leukocytes. They are distinguishable from conserved Siglecs, such as Sialoadhesin and MAG, by their rapid evolution. A comparison of the CD33rSiglec gene cluster in different mammalian species showed that it can be divided into subclusters, A and B. The two subclusters, inverted in relation to each other, each encode a set of CD33rSiglec genes arranged head-to-tail. Two regions of strong correspondence provided evidence for a large-scale inverse duplication, encompassing the framework CEACAM-18 (CE18) and ATPBD3 (ATB3) genes that seeded the mammalian CD33rSiglec cluster. Phylogenetic analysis was consistent with the predicted inversion. Rodents appear to have undergone wholesale loss of CD33rSiglec genes after the inverse duplication. In contrast, CD33rSiglecs expanded in primates and many are now pseudogenes with features consistent with activating receptors. In contrast to mammals, the fish CD33rSiglecs clusters show no evidence of an inverse duplication. They display greater variation in cluster size and structure than mammals. The close arrangement of other Siglecs and CD33rSiglecs in fish is consistent with a common ancestral region for Siglecs. Expansion of mammalian CD33rSiglecs appears to have followed a large inverse duplication of a smaller primordial cluster over 180 million years ago, prior to eutherian/marsupial divergence. Inverse duplications in general could potentially have a stabilizing effect in maintaining the size and structure of large gene clusters, facilitating the rapid evolution of immune gene families. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The conundrum of inhibitory signaling by ITAM-containing immunoreceptors: Potential molecular mechanisms

Immunoreceptor signals must be appropriately transduced and regulated to achieve effective immunity while controlling inflammation and autoimmunity. It is generally held that these processes are mediated by the interplay of distinct activating and inhibitory receptors via conserved activating (ITAM) and inhibitory (ITIM) signaling motifs. However, recent evidence indicates that under certain conditions incomplete phosphorylation of ITAM tyrosines leads to inhibitory signaling. This new regulatory function of ITAMs has been termed ITAMi (inhibitory ITAM). Here we discuss the potential molecular mechanisms of inhibitory signaling by ITAM-containing receptors.