Human leukocyte immunoglobulin-like receptors in health and disease.

Human leukocyte immunoglobulin (Ig)-like receptors (LILR) are a family of 11 innate immunomodulatory receptors, primarily expressed on lymphoid and myeloid cells. LILRs are either activating (LILRA) or inhibitory (LILRB) depending on their associated signalling domains (D). With the exception of the soluble LILRA3, LILRAs mediate immune activation, while LILRB1-5 primarily inhibit immune responses and mediate tolerance. Abnormal expression and function of LILRs is associated with a range of pathologies, including immune insufficiency (infection and malignancy) and overt immune responses (autoimmunity and alloresponses), suggesting LILRs may be excellent candidates for targeted immunotherapies. This review will discuss the biology and clinical relevance of this extensive family of immune receptors and will summarise the recent developments in targeting LILRs in disease settings, such as cancer, with an update on the clinical trials investigating the therapeutic targeting of these receptors.

Towards a better understanding of human iNKT cell subpopulations for improved clinical outcomes.

Invariant natural killer T (iNKT) cells are a unique T lymphocyte population expressing semi-invariant T cell receptors (TCRs) that recognise lipid antigens presented by CD1d. iNKT cells exhibit potent anti-tumour activity through direct killing mechanisms and indirectly through triggering the activation of other anti-tumour immune cells. Because of their ability to induce potent anti-tumour responses, particularly when activated by the strong iNKT agonist αGalCer, they have been the subject of intense research to harness iNKT cell-targeted immunotherapies for cancer treatment. However, despite potent anti-tumour efficacy in pre-clinical models, the translation of iNKT cell immunotherapy into human cancer patients has been less successful. This review provides an overview of iNKT cell biology and why they are of interest within the context of cancer immunology. We focus on the iNKT anti-tumour response, the seminal studies that first reported iNKT cytotoxicity, their anti-tumour mechanisms, and the various described subsets within the iNKT cell repertoire. Finally, we discuss several barriers to the successful utilisation of iNKT cells in human cancer immunotherapy, what is required for a better understanding of human iNKT cells, and the future perspectives facilitating their exploitation for improved clinical outcomes.

FcγRIIB controls antibody-mediated target cell depletion by ITIM-independent mechanisms.

Many therapeutic antibodies deplete target cells and elicit immunotherapy by engaging activating Fc gamma receptors (FcγRs) on host effector cells. These antibodies are negatively regulated by the inhibitory FcγRIIB (CD32B). Dogma suggests inhibition is mediated through the FcγRIIB immunoreceptor tyrosine-based inhibition motif (ITIM), negatively regulating immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling from activating FcγR. To assess this, we generated experimental models expressing human (h)FcγRIIB on targets or effectors, lacking or retaining ITIM signaling capacity. We demonstrate that signaling through the hFcγRIIB ITIM is dispensable for impairing monoclonal antibody (mAb)-mediated depletion of normal and malignant murine target cells through three therapeutically relevant surface receptors (CD20, CD25, and OX40) affecting immunotherapy. We demonstrate that hFcγRIIB competition with activating FcγRs for antibody Fc, rather than ITIM signaling, is sufficient to impair activating FcγR engagement, inhibiting effector function and immunotherapy.

HIF activation enhances FcγRIIb expression on mononuclear phagocytes impeding tumor targeting antibody immunotherapy.

BACKGROUND: Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb. METHODS: We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. RESULTS: We report that TAMs are FcγRIIbbright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes. CONCLUSION: Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.

Human inhibitory leukocyte Ig-like receptors: from immunotolerance to immunotherapy.

In recent decades, immunotherapeutic strategies have been used to treat a wide range of pathologies, many of which were previously incurable, such as cancer and autoimmune disorders. Despite this unprecedented success, a considerable number of patients fail to respond to currently approved immunotherapies or develop resistance over time. Therefore, there is an urgent need to develop the next generation of immune-targeted therapies. Various members of the Ig superfamily play essential roles in regulating leukocyte functions. One such group, the leukocyte Ig-like receptors (LILRs), have been implicated in both innate and adaptive immune regulation. Human inhibitory LILRs (LILRBs) are primarily expressed on leukocytes and mediate their signaling through multiple cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. Engagement of LILRBs by endogenous and pathogenic ligands can markedly suppress immune responses, leading to tolerance or immunoevasion, whereas blocking these inhibitory receptors can potentiate immune responses. In this Review, we discuss the immunoregulatory functions of human LILRBs and the potential of targeting them to manipulate immune responses in various pathologies.

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.

Cyclophosphamide Enhances Cancer Antibody Immunotherapy in the Resistant Bone Marrow Niche by Modulating Macrophage FcγR Expression.

Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated cancer. Many hematologic and solid tumors are resistant to therapeutic antibodies in the bone marrow (BM), but not in the periphery (e.g., spleen). We previously showed that cyclophosphamide (CTX) sensitizes the BM niche to antibody therapeutics. Here, we show that (i) BM resistance was induced not only by the tumor but also by the intrinsic BM microenvironment; (ii) CTX treatment overcame both intrinsic and extrinsic resistance mechanisms by augmenting macrophage activation and phagocytosis, including significant upregulation of activating Fcγ receptors (FcγRIII and FcγRIV) and downregulation of the inhibitory receptor, FcγRIIB; and (iii) CTX synergized with cetuximab (anti-EGFR) and trastuzumab (anti-Her2) in eliminating metastatic breast cancer in the BM of humanized mice. These findings provide insights into the mechanisms by which CTX synergizes with antibody therapeutics in resistant niche-specific organs and its applicability in treating BM-resident tumors.

New revelations from an old receptor: Immunoregulatory functions of the inhibitory Fc gamma receptor, FcγRIIB (CD32B).

The Fc gamma receptor IIB (FcγRIIB/CD32B) was generated million years ago during evolution. It is the sole inhibitory receptor for IgG, and has long been associated with the regulation of humoral immunity and innate immune homeostasis. However, new and surprising functions of FcγRIIB are emerging. In particular, FcγRIIB has been shown to perform unexpected activatory roles in both immune-signaling and monoclonal antibody (mAb) immunotherapy. Furthermore, although ITIM signaling is an integral part of FcγRIIB regulatory activity, it is now clear that inhibition/activation of immune responses can occur independently of the ITIM. In light of these new findings, we present an overview of the established and noncanonical functions of FcγRIIB and discuss how this knowledge might be exploited therapeutically.

Corrigendum: In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.

This corrects the article DOI: 10.1038/nbt.3836.

In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;KrasG12D/+;Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.

Resistance is futile: Targeting the inhibitory FcγRIIB (CD32B) to maximize immunotherapy.

Monoclonal antibodies (mAb) are central to the treatment of several types of malignancy. However, these reagents are subject to particular types of resistance. Several resistance mechanisms are regulated by the inhibitory FcγRIIB. We recently developed mAbs to block FcγRIIB and provided in vivo proof-of-concept for their ability to overcome FcγRIIB-mediated resistance.

Leukocyte Ig-Like receptor B1 restrains dendritic cell function through increased expression of the NF-κB regulator ABIN1/TNIP1.

Inhibitory receptors of the human leukocyte immunoglobulin-like receptor family are constitutively expressed on all myeloid cell types and regulate their functional activity. We demonstrate that ligation of the human leukocyte antigen class I-specific receptor LILRB1, during the differentiation of monocytes to dendritic cells in vitro, results in increased expression of the nuclear factor κB inhibitor protein ABIN1 (also known as TNIP1). Similarly increased expression of ABIN1/TNIP1 was observed in the "immunosuppressive" monocyte populations of patients with non-Hodgkin lymphoma ex vivo. Reducing expression of ABIN1/TNIP1 using small interfering ribonucleic acid allows dendritic cells and immunosuppressive monocytes to respond to stimulation by allowing nuclear factor κB translocation to the nucleus (P < 0.001), increasing cell surface expression of antigen presentation and costimulatory molecules (P < 0.01), increasing phagocytic capacity (P < 0.001), secreting proinflammatory cytokines (P < 0.01), and an increasing ability to stimulate T cell responses (P < 0.05). Our study, therefore, identifies an important functional role for ABIN1/TNIP1 in mediating the effects of LILRB1 ligation-induced inhibitory effects on immune responses. Our findings suggest that inhibiting the LILRB1-ABIN1/TNIP1 pathway in antigen-presenting cells could be a therapeutic approach to stimulate antitumor immune responses. Conversely, stimulation of the pathway may also ameliorate autoimmune diseases in which TNIP1 is a susceptibility gene.

B Cells Promote Pancreatic Tumorigenesis.

Three recent studies, approaching the question from different angles and using different and/or overlapping models, provide compelling evidence for the involvement of tumor-infiltrating B cells in the initiation and progression of pancreatic ductal adenocarcinoma. These studies highlight the need for a better understanding of pancreatic tumor-immune system interactions and the immunologic mechanisms that promote or inhibit tumorigenesis, paving the way for better treatment strategies.

FcγR requirements leading to successful immunotherapy.

Monoclonal antibody (mAb) immunotherapy is currently experiencing an unprecedented amount of success, delivering blockbuster sales for the pharmaceutical industry. Having experienced several false dawns and overcoming technical issues which limited progress, we are now entering a golden period where mAbs are becoming a mainstay of treatment regimes for diseases ranging from cancer to autoimmunity. In this review, we discuss how these mAbs are most likely working and focus in particular on the key receptors that they interact with to precipitate their therapeutic effects. Although their targets may vary, their engagement with Fcγ receptors (FcγRs) on numerous immune effector cells is almost universal, and here we review their roles in delivering successful immunotherapy.

Development and Characterization of Monoclonal Antibodies Specific for Mouse and Human Fcγ Receptors.

FcγRs are key regulators of the immune response, capable of binding to the Fc portion of IgG Abs and manipulating the behavior of numerous cell types. Through a variety of receptors, isoforms, and cellular expression patterns, they are able to fine-tune and direct appropriate responses. Furthermore, they are key determinants of mAb immunotherapy, with mAb isotype and FcγR interaction governing therapeutic efficacy. Critical to understanding the biology of this complex family of receptors are reagents that are robust and highly specific for each receptor. In this study, we describe the development and characterization of mAb panels specific for both mouse and human FcγR for use in flow cytometry, immunofluorescence, and immunocytochemistry. We highlight key differences in expression between the two species and also patterns of expression that will likely impact on immunotherapeutic efficacy and translation of therapeutic agents from mouse to clinic.

Antagonistic human FcγRIIB (CD32B) antibodies have anti-tumor activity and overcome resistance to antibody therapy in vivo.

Therapeutic antibodies have transformed cancer therapy, unlocking mechanisms of action by engaging the immune system. Unfortunately, cures rarely occur and patients display intrinsic or acquired resistance. Here, we demonstrate the therapeutic potential of targeting human (h) FcγRIIB (CD32B), a receptor implicated in immune cell desensitization and tumor cell resistance. FcγRIIB-blocking antibodies prevented internalization of the CD20-specific antibody rituximab, thereby maximizing cell surface accessibility and immune effector cell mediated antitumor activity. In hFcγRIIB-transgenic (Tg) mice, FcγRIIB-blocking antibodies effectively deleted target cells in combination with rituximab, and other therapeutic antibodies, from resistance-prone stromal compartments. Similar efficacy was seen in primary human tumor xenografts, including with cells from patients with relapsed/refractory disease. These data support the further development of hFcγRIIB antibodies for clinical assessment.

Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies.

Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo.

Upregulation of FcγRIIb on monocytes is necessary to promote the superagonist activity of TGN1412.

The anti-CD28 superagonist antibody TGN1412 caused life-threatening cytokine release syndrome (CRS) in healthy volunteers, which had not been predicted by preclinical testing. T cells in fresh peripheral blood mononuclear cells (PBMCs) do not respond to soluble TGN1412 but do respond following high-density (HD) preculture. We show for the first time that this response is dependent on crystallizable fragment gamma receptor IIb (FcγRIIb) expression on monocytes. This was unexpected because, unlike B cells, circulating monocytes express little or no FcγRIIb. However, FcγRIIb expression is logarithmically increased on monocytes during HD preculture, and this upregulation is necessary and sufficient to explain TGN1412 potency after HD preculture. B-cell FcγRIIb expression is unchanged by HD preculture, but B cells can support TGN1412-mediated T-cell proliferation when added at a frequency higher than that in PBMCs. Although low-density (LD) precultured PBMCs do not respond to TGN1412, T cells from LD preculture are fully responsive when cocultured with FcγRIIb-expressing monocytes from HD preculture, which shows that they are fully able to respond to TGN1412-mediated activation. Our novel findings demonstrate that cross-linking by FcγRIIb is critical for the superagonist activity of TGN1412 after HD preculture, and this may contribute to CRS in humans because of the close association of FcγRIIb-bearing cells with T cells in lymphoid tissues.

Conformation of the human immunoglobulin G2 hinge imparts superagonistic properties to immunostimulatory anticancer antibodies.

Monoclonal antibody (mAb) drugs that stimulate antitumor immunity are transforming cancer treatment but require optimization for maximum clinical impact. Here, we show that, unlike other immunoglobulin isotypes, human IgG2 (h2) imparts FcγR-independent agonistic activity to immune-stimulatory mAbs such as anti-CD40, -4-1BB, and -CD28. Activity is provided by a subfraction of h2, h2B, that is structurally constrained due its unique arrangement of hinge region disulfide bonds. Agonistic activity can be transferred from h2 to h1 by swapping their hinge and CH1 domains, and substitution of key hinge and CH1 cysteines generates homogenous h2 variants with distinct agonistic properties. This provides the exciting opportunity to engineer clinical reagents with defined therapeutic activity regardless of FcγR expression levels in the local microenvironment.

Inhibitory FcγRIIb (CD32b) becomes activated by therapeutic mAb in both cis and trans and drives internalization according to antibody specificity.

A major feature that distinguishes type I from type II anti-CD20 monoclonal antibodies (mAbs) and reduces their therapeutic efficacy is the tendency to internalize from the cell surface. We have shown previously that the extent of internalization correlates with the capacity of type I mAb to simultaneously engage both CD20 and the inhibitory Fcγ receptor, FcγRIIb, in a bipolar configuration. Here, we investigated whether mAbs directed at other B-cell surface receptors also engaged FcγRIIb and whether this interaction promoted internalization. Most mAbs engaged and activated FcγRIIb, with the strength of activation related to the level of mAb bound to the cell surface. However, engagement did not affect internalization of most mAb-ligated receptors, either in cell lines or primary chronic lymphocytic leukemia cells with the exception of CD19 and CD38. Furthermore, at high cell concentrations/density both cis and trans interactions between cell-surface bound mAb and FcγRIIb were evident, but trans interactions did not inhibit type I anti-CD20 mAb-mediated internalization. These data identify that FcγRIIb is engaged by many mAbs in both cis and trans configurations, triggering its activation, but that internalization via FcγRIIb occurs for only a select subset. These findings have implications when designing new antibody-based therapeutics.