Immunomodulation of T- and NK-cell Responses by a Bispecific Antibody Targeting CD28 Homolog and PD-L1.

Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific, mAb targeting CD28 homolog (CD28H), a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells, with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation, induced NK-cell cytotoxicity of PD-L1-expressing tumor cells, and activated tissue-resident memory CD8+ T cells. Mechanistically, the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1-induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells, including CD8+ T cells, tissue-resident memory T cells, and NK cells, in a tumor-specific manner that may lead to induction of durable, therapeutic antitumor responses.

Design and Efficacy of a Monovalent Bispecific PD-1/CTLA4 Antibody That Enhances CTLA4 Blockade on PD-1+ Activated T Cells.

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1- T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. SIGNIFICANCE: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy.See related commentary by Burton and Tawbi, p. 1008.This article is highlighted in the In This Issue feature, p. 995.

LILRB3 (ILT5) is a myeloid cell checkpoint that elicits profound immunomodulation.

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress toward identifying the function of this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAbs) was generated. LILRB3-specific mAbs bound to discrete epitopes in Ig-like domain 2 or 4. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.

Interaction of the LILRB1 inhibitory receptor with HLA class Ia dimers.

Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) has been reported to interact with a wide spectrum of HLA class I (HLA-I) molecules, albeit with different affinities determined by allelic polymorphisms and conformational features. HLA-G dimerization and the presence of intracellular Cys residues in HLA-B7 have been shown to be critical for their recognition by LILRB1. We hypothesized that dimerization of classical HLA class Ia molecules, previously detected in exosomes, might enhance their interaction with LILRB1. A soluble LILRB1-Fc fusion protein and a sensitive cellular reporter system expressing a LILRB1-ζ chimera were employed to assess receptor interaction with different HLA class Ia molecules transfected in the human lymphoblastoid 721.221 cell line. Under these conditions, intracellular Cys residues and HLA-I dimerization appeared associated with increased LILRB1 recognition. On the other hand, a marginal interaction of LILRB1 with primary monocytic cells, irrespective of their high HLA-I expression, was enhanced by type I interferon (IFN). This effect appeared disproportionate to the cytokine-induced increase of surface HLA-I expression and was accompanied by detection of HLA class Ia dimers. Altogether, the results support that a regulated assembly of these noncanonical HLA-I conformers during the immune response may enhance the avidity of their interaction with LILRB1.

Allele-specific recognition by LILRB3 and LILRA6 of a cytokeratin 8-associated ligand on necrotic glandular epithelial cells.

The LILRs are a family of receptors that regulate the activities of myelomonocytic cells. We found that specific allelic variants of two related members of the LILR family, LILRB3 and LILRA6, interact with a ligand exposed on necrotic glandular epithelial cells. The extracellular domains of LILRB3 and LILRA6 are very similar and their genes are highly polymorphic. A commonly occurring allele, LILRB3*12, displayed particularly strong binding of these necrotic cells and further screening of the products of LILRB3 alleles identified motifs that correlated with binding. Immunoprecipitation of the ligand from epithelial cell lysates using recombinant LILRB3*12, identified cytokeratins 8, 18 and 19. Purified proteins obtained from epithelial cell lysates, using anti-cytokeratin 8 antibodies, were able to activate LILRB3*12 reporter cells. Knock-down of cytokeratin 8 in epithelial cells abrogated expression of the LILRB3 ligand, while staining with recombinant LILRB3*12 showed co-localisation with cytokeratin 8 and 18 in permeabilised breast cancer cells. Necrosis is a common feature of tumours. The finding of a necrosis-associated ligand for these two receptors raises the possibility of a novel interaction that alters immune responses within the tumour microenvironment. Since LILRB3 and LILRA6 genes are highly polymorphic the interaction may influence an individual's immune response to tumours.

TARM1 Is a Novel Leukocyte Receptor Complex-Encoded ITAM Receptor That Costimulates Proinflammatory Cytokine Secretion by Macrophages and Neutrophils.

We identified a novel, evolutionarily conserved receptor encoded within the human leukocyte receptor complex and syntenic region of mouse chromosome 7, named T cell-interacting, activating receptor on myeloid cells-1 (TARM1). The transmembrane region of TARM1 contained a conserved arginine residue, consistent with association with a signaling adaptor. TARM1 associated with the ITAM adaptor FcRγ but not with DAP10 or DAP12. In healthy mice, TARM1 is constitutively expressed on the cell surface of mature and immature CD11b(+)Gr-1(+) neutrophils within the bone marrow. Following i.p. LPS treatment or systemic bacterial challenge, TARM1 expression was upregulated by neutrophils and inflammatory monocytes and TARM1(+) cells were rapidly recruited to sites of inflammation. TARM1 expression was also upregulated by bone marrow-derived macrophages and dendritic cells following stimulation with TLR agonists in vitro. Ligation of TARM1 receptor in the presence of TLR ligands, such as LPS, enhanced the secretion of proinflammatory cytokines by macrophages and primary mouse neutrophils, whereas TARM1 stimulation alone had no effect. Finally, an immobilized TARM1-Fc fusion protein suppressed CD4(+) T cell activation and proliferation in vitro. These results suggest that a putative T cell ligand can interact with TARM1 receptor, resulting in bidirectional signaling and raising the T cell activation threshold while costimulating the release of proinflammatory cytokines by macrophages and neutrophils.

Impaired natural killer cell phenotype and function in idiopathic and heritable pulmonary arterial hypertension.

BACKGROUND: Beyond their role as innate immune effectors, natural killer (NK) cells are emerging as important regulators of angiogenesis and vascular remodeling. Pulmonary arterial hypertension (PAH) is characterized by severe pulmonary vascular remodeling and has long been associated with immune dysfunction. Despite this association, a role for NK cells in disease pathology has not yet been described. METHODS AND RESULTS: Analysis of whole blood lymphocytes and isolated NK cells from PAH patients revealed an expansion of the functionally defective CD56(-)/CD16(+) NK subset that was not observed in patients with chronic thromboembolic pulmonary hypertension. NK cells from PAH patients also displayed decreased levels of the activating receptor NKp46 and the killer immunoglobulin-like receptors 2DL1/S1 and 3DL1, reduced secretion of the cytokine macrophage inflammatory protein-1ß, and a significant impairment in cytolytic function associated with decreased killer immunoglobulin-like receptor 3DL1 expression. Genotyping patients (n=222) and controls (n=191) for killer immunoglobulin-like receptor gene polymorphisms did not explain these observations. Rather, we show that NK cells from PAH patients exhibit increased responsiveness to transforming growth factor-ß, which specifically downregulates disease-associated killer immunoglobulin-like receptors. NK cell number and cytotoxicity were similarly decreased in the monocrotaline rat and chronic hypoxia mouse models of PAH, accompanied by reduced production of interferon-γ in NK cells from hypoxic mice. NK cells from PAH patients also produced elevated quantities of matrix metalloproteinase 9, consistent with a capacity to influence vascular remodeling. CONCLUSIONS: Our work is the first to identify an impairment of NK cells in PAH and suggests a novel and substantive role for innate immunity in the pathobiology of this disease.

Filament-associated TSGA10 protein is expressed in professional antigen presenting cells and interacts with vimentin.

Testis-specific gene antigen 10 (TSGA10) encodes an 82-kDa protein expressed during development, and in testis and brain tissues. We report its expression in human monocyte-derived dendritic cells (DC) and macrophages in vitro and in murine spleen CD11c(+) cells ex vivo. An interaction between DC/macrophage-derived TSGA10 and vimentin, as well as a few other major cytoskeletal proteins (e.g., actin-γ1), was identified by pull-down and mass spectroscopy assays. The interaction between TSGA10 and vimentin was further confirmed by immunoprecipitation and immunolocalisation in transfected RAW267 and HEK293 cell lines. TSGA10 formed filamentous structures in transfected COS-1 cells and was observed in cellular projections. We propose that TSGA10 could influence the function of antigen presenting cells (APC) via its interaction with cytoskeletal proteins such as vimentin.

The inhibitory receptor LILRB4 (ILT3) modulates antigen presenting cell phenotype and, along with LILRB2 (ILT4), is upregulated in response to Salmonella infection.

BACKGROUND: Leukocyte Ig-like receptors (LILR) are a family of innate immune receptors with immunomodulatory functions. High-level expression of the receptors LILRB2 (ILT4) and LILRB4 (ILT3) is a feature of tolerogenic antigen presenting cells and has been observed in cancer and transplant situations. There are relatively few studies regarding these receptors in the context of infection and it is not yet clear how LILRB4 exerts its inhibitory effects. RESULTS: We studied the effects of LILRB4 ligation on antigen presenting cell phenotype, and the expression of LILRB2 and LILRB4 on Salmonella-infected antigen presenting cells. Ligation of LILRB4 throughout in vitro culture of dendritic cells led to an upregulation of the co-stimulatory protein CD86. Alterations in the production of IL-8 and IL-10 by LILRB4-ligated macrophages were also observed. Infection with Salmonella typhimurium or TLR stimulation with Salmonella components led to an upregulation of LILRB2 and LILRB4. CONCLUSION: Our results indicate that the inhibitory effects of LILRB4 do not result from a failure to upregulate co-stimulatory proteins. In addition to the high level expression that can render antigen presenting cells tolerogenic, there may be a role for lower level expression and activity of LILRB2 and LILRB4 in response to TLR signalling during an immune response to bacterial infection.

Alternative mRNA splicing creates transcripts encoding soluble proteins from most LILR genes.

Leucocyte Ig-like receptors (LILR) are a family of innate immune receptors expressed on myeloid and lymphoid cells that influence adaptive immune responses. We identified a common mechanism of alternative mRNA splicing, which generates transcripts that encode soluble protein isoforms of the majority of human LILR. These alternative splice variants lack transmembrane and cytoplasmic encoding regions, due to the transcription of a cryptic stop codon present in an intron 5' of the transmembrane encoding exon. The alternative LILR transcripts were detected in cell types that express their membrane-associated isoforms. Expression of the alternative LILRB1 transcript in transfected cells resulted in the release of a soluble approximately 65 Kd LILRB1 protein into culture supernatants. Soluble LILRB1 protein was also detected in the culture supernatants of monocyte-derived DC. In vitro assays suggested that soluble LILRB1 could block the interaction between membrane-associated LILRB1 and HLA-class I. Soluble LILRB1 may act as a dominant negative regulator of HLA-class I-mediated LILRB1 inhibition. Soluble isoforms of the other LILR may function in a comparable way.