Immunoglobulin-like Transcript Research Articles

Increased ILT2 expression contributes to dysfunction of CD56dimCD16+NK cells in chronic hepatitis B virus infection

Natural killer (NK) cells play a crucial role in the control of human viral infections but their activity is significantly impaired in patients infected with chronic hepatitis B (CHB). The mechanism that contributes to NK cell dysfunction in CHB needs further elucidation. In this study, we analyzed the expression and function of the novel inhibitory receptor immunoglobulin-like transcript-2 (ILT2) on NK cells from 131 CHB patients and 36 healthy controls. We observed that ILT2 expression on circulating CD56dimCD16+NK cells was increased in immune-tolerant, immune-active and HBeAg-negative hepatitis patients compared with inactive carriers and controls. The frequency of ILT2+CD56dimNK cells was positively correlated with serum viral load in immune-tolerant patients. The percentage of ILT2+CD56dimNK cells decreased along with HBV load in CHB patients who received antiviral therapy. Functional analysis showed that ILT2+CD56dimNK cells in CHB patients had significantly reduced degranulation and IFN-γ production. Upregulation of ILT2 was associated with high levels of apoptosis in CD56dimCD16+NK cells from CHB patients. ILT2 blockade was shown to increase the cytotoxicity and IFN-γ production of CD56dimNK cells in some CHB patients. Finally, ILT2 was found to be moderately upregulated by TGF-β1, which was increased in immune-tolerant, immune-active and HBeAg-negative hepatitis patients. Our results show that chronic HBV infection increases the levels of the inhibitory receptor ILT2 on CD56dimNK cells and inhibits their functions, providing a new mechanism of NK-cell disability in CHB patients.

A highly diverse set of novel immunoglobulin-like transcript (NILT) genes in zebrafish indicates a wide range of functions with complex relationships to mammalian receptors.

Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout, and carp. NILTs typically encode activating or inhibitory transmembrane receptors with extracellular immunoglobulin (Ig) domains. Although predicted to provide immune recognition in ray-finned fish, we currently lack a definitive framework of NILT diversity, thereby limiting our predictions for their evolutionary origin and function. In order to better understand the diversity of NILTs and their possible roles in immune function, we identified five NILT loci in the Atlantic salmon (Salmo salar) genome, defined 86 NILT Ig domains within a 3-Mbp region of zebrafish (Danio rerio) chromosome 1, and described 41 NILT Ig domains as part of an alternative haplotype for this same genomic region. We then identified transcripts encoded by 43 different NILT genes which reflect an unprecedented diversity of Ig domain sequences and combinations for a family of non-recombining receptors within a single species. Zebrafish NILTs include a sole putative activating receptor but extensive inhibitory and secreted forms as well as membrane-bound forms with no known signaling motifs. These results reveal a higher level of genetic complexity, interindividual variation, and sequence diversity for NILTs than previously described, suggesting that this gene family likely plays multiple roles in host immunity.

Abstract LB216: Pre-clinical characterization of NGM831, an ILT3 antagonist antibody for the treatment of solid tumors

Abstract Suppressive myeloid cells inhibit antitumor immunity by preventing T cell responses. Immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. Our team recently identified fibronectin as a functional ligand for ILT3. The fibronectin-ILT3 interaction serves as a “stromal checkpoint” through which the extracellular matrix actively promotes myeloid cell suppression in the tumor microenvironment. We now report the pre-clinical characterization of NGM831, a humanized IgG1 (N297G) antibody that binds to ILT3 of human and cynomolgus monkey origin with high specificity and sub-nM affinity and blocks the interaction of ILT3 with both its reported ligands, fibronectin and ApoE. In a gene expression profiling experiment, treatment of tolerogenic dendritic cells with NGM831 in the presence of fibronectin decreased their expression of inhibitory and scavenger receptors (CD163, MRC1, MARCO, STAB1, LILRB2) and markers of dendritic cell immaturity (CD14, CD209, FOLR2) and increased their expression of genes involved in antigen presentation (TNFSF14, DCSTAMP, CD82, OLR1) and TH1 polarization (SPP1, TBX21), suggesting that these cells had been remodeled to a more stimulatory phenotype. Consistent with these observations, treatment of tolerogenic dendritic cells with NGM831 increased their production of pro-inflammatory cytokines in response to FcɣR ligation, restored their responsiveness to maturation stimuli such as lipopolysaccharide (LPS) and CD40 ligand (CD40L), and increased their production of chemokines that could induce the migration of other immune cells to sites of inflammation. In addition, NGM831 acted synergistically with pembrolizumab to increase the ability of dendritic cells to stimulate allogeneic T cells in mixed lymphocyte reactions. Taken together, these results demonstrate that NGM831 can block fibronectin-mediated immunosuppression and promote myeloid cell reprogramming. These data support the clinical development of the ILT3 antagonist antibody NGM831 alone and in combination with immune checkpoint blockade. NGM831 is expected to enter clinical trials for the treatment of advanced solid tumors in early 2022. Citation Format: Peirong Chen, Suzanne C. Crawley, Vicky Y. Lin, Varun Kapoor, Kevin J. Paavola, Hung-I Chen, Joshua S. Lichtman, Betty Li, Kalyani Mondal, Alan K. Kutach, Yan Wang, Daniel D. Kaplan, Julie M. Roda. Pre-clinical characterization of NGM831, an ILT3 antagonist antibody for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB216.

Phase 1 first-in-human study of anti–ILT3 mAb MK-0482 as monotherapy and in combination with pembrolizumab in advanced solid tumors: Dose escalation results.

2505 Background: Immunoglobulin-like transcript 3 (ILT3) is an inhibitory receptor associated with immune tolerance and T-cell suppression within the tumor microenvironment. MK-0482, a novel humanized IgG4 mAb targeting ILT3, is undergoing phase 1 evaluation ± pembrolizumab (pembro) in advanced solid tumors (NCT03918278; MK-0482-001). Dose escalation data are presented. Methods: Eligible patients with advanced solid tumors were enrolled into sequentially escalating dose cohorts of MK-0482 monotherapy (0.2-2250 mg) or MK-0482 (7.5-2250 mg) + pembro 200 mg; both were administered IV Q3W for up to 35 cycles or until progressive disease (PD), unacceptable toxicity, death, or withdrawal. Patients receiving MK-0482 monotherapy could cross over to MK-0482 + pembro after PD. Primary objectives were safety, tolerability, and determination of the recommended phase 2 dose (RP2D). Secondary and exploratory objectives included assessing pharmacokinetics (PK), blood receptor occupancy (RO), anti–drug antibodies (ADA), and objective response rate (ORR) per RECIST v1.1 by investigator assessment. Results: Seventy-five patients were enrolled (n = 29, MK-0482 monotherapy; n = 46, MK-0482 + pembro); 8 patients crossed over to receive the combination. Median age was 63 years (range, 34-86); 73% had ECOG PS 1, 72% received ≥2 lines of prior anticancer therapy, and 32% received prior PD-1/PD-L1 inhibitors. Treatment-related AEs (TRAEs; any-grade/grade 3 or 4) were reported in 34%/7% of patients with MK-0482 monotherapy, 67%/4% with MK-0482 + pembro, and 50%/13% in those who crossed over. The most common TRAEs (≥10% of all patients) were pyrexia (10%) with MK-0482 monotherapy; fatigue (24%) and arthralgia, diarrhea, hyperthyroidism, hypothyroidism, and pruritus (11% each) with MK-0482 + pembro; and arthralgia (25%), hyperthyroidism and hypothyroidism (13% each) for patients who crossed over. Dose-limiting toxicities occurred in 2 patients who received MK-0482 + pembro: 1 grade 5 myositis (MK-0482 750 mg; the only TRAE leading to death) and 1 grade 2 myositis (MK-0482 2250 mg). Preliminary PK and blood RO data suggested that target-mediated drug disposition of MK-0482 was likely saturated in blood mononuclear cells at doses ≥75 mg. ADA to MK-0482 was observed in ̃20% of patients, but no clear impact on MK-0482 PK was observed. A confirmed ORR of 15% (8 PR) was observed in patients who received MK-0482 + pembro, including patients who crossed over; no confirmed responses were observed in patients who received MK-0482 monotherapy. MK-0482 750 mg + pembro was selected as the RP2D based on the totality of data. Conclusions: MK-0482 ± pembro was generally well tolerated, and combination therapy provided modest antitumor activity in patients with heavily pretreated advanced solid tumors. The RP2D of MK-0482 + pembro is under further evaluation in tumor-specific cohorts. Clinical trial information: NCT03918278.

Prognostic significance of the immune checkpoint HLA-G/ILT-4 in the survival of patients with gastric cancer

ObjectiveHuman leukocyte antigen-G (HLA-G) and its receptors, including immunoglobulin-like transcripts (ILT)-2 and ILT-4, are closely associated with cancer development and clinical outcomes of patients. However, the clinical significance of HLA-G and ILT-2/-4 in gastric cancer (GC) is limited. MethodsIn this study, the percentage of HLA-G-, ILT-2 and ILT-4 positive tumor cells in 127 GC lesion suspensions of tumor cells gated for epithelialcelladhesionmolecule(EpCAM) was determined using multicolor flow cytometry and their clinical significance was evaluated. ResultsOur data showed that the median percentages of HLA-G-, ILT-2, and ILT-4 expressing GC cells were 18.0%, 67.80%, and 1.42%, respectively, and co-expression of HLA-G/ILT-2, HLA-G/ILT-4, and ILT-2/ILT-4 was 16.9%, 1.42%, and 1.70%, respectively. Kaplan-Meier survival results revealed that besides post-operation N status (p = 0.006), M status (p = 0.001), and AJCC clinical stage (p < 0.001), only high percentage of ILT-4+ GC cells was a significant factor for worse survival of patients with GC (overall survival [OS]: 42.9 months vs. 84.5 months; p = 0.031). However, among female patients with GC (n = 31), high percentage of either HLA-G+ (OS: 18.5 months vs. 89.3 months; p = 0.001) or ILT-4+ (OS: 17.9 months vs. 85.8 months; p = 0.002) GC cells was markedly associated with a poor prognosis. ConclusionOur findings revealed that among HLA-G, ILT-2, and ILT-4, only a high percentage of ILT-4+ GC cells was significantly related to poor prognosis in the entire cohort of patients with GC. However, high percentage of HLA-G+ and ILT-4+ GC cells is associated with poor clinical outcome among female patients with GC.

Tumor-derived immunoglobulin like transcript 5 induces suppressive immunocyte infiltration in colorectal cancer.

Infiltration of immunosuppressive cells in the tumor microenvironment (TME) induced colorectal cancer (CRC) progression and its resistance to immunotherapy. Identification of tumor‐specific factors to modulate inhibitory immunocyte infiltration would provide alternative and novel targets for CRC immunotherapy. Immunoglobulin‐like transcript (ILT) 5 is a negative regulator of myeloid cell activation. However, its expression and functional role in solid tumors is still unknown. Using human CRC tissues and cell lines, we found that ILT5 was highly expressed in CRC cells compared with normal colorectal epithelial cells. Enriched ILT5 in tumor cells was correlated with advanced tumor stages and poor patient survival. Our subsequent in vitro and in vivo studies revealed that tumor‐derived ILT5 inhibited the infiltration of T cells, especially that of CD8+ T cells in the TME, creating suppressive T‐cell contexture. Furthermore, ILT5 directed M2‐like polarization of tumor‐associated macrophages (TAMs). Inhibition of tumor‐derived ILT5 restored the immunosuppressive T‐cell and TAM contexture, and restricted CRC progression. Our findings identified ILT5 expression in solid tumor cells for the first time and raised ILT5 as a potential immunotarget and prognostic predictor in CRC.

HLA Class Ib-receptor interactions during embryo implantation and early pregnancy.

Although the immune system intuitively must have an important role in embryo implantation and in the achievement of a pregnancy, the molecular details have for long been controversial. The role of the human leukocyte antigen (HLA) system has been debated. The unique HLA expression profile of the HLA Class Ia molecule HLA-C and the HLA Class Ib molecules HLA-E, HLA-F and HLA-G at the feto-maternal interface is now recognized. However, HLA Class Ib molecules may also have a role in embryo implantation and pregnancy success. The aim of this review was to evaluate the literature and recent discoveries on the role of the non-polymorphic HLA Class Ib molecules with a focus on HLA-F and HLA-G molecules at the time of implantation, including the interaction with uterine immune cells through the specific receptors immunoglobulin-like transcript 2 (ILT2), ILT4 and a number of killer cell immunoglobulin-like receptors (KIRs), and the importance of HLA-F and HLA-G genetic variation that influences fertility and time-to-pregnancy. Drawing on recent advances in basic and clinical studies, we performed a narrative review of the scientific literature to provide a timely update on the role of HLA Class Ib in embryo implantation, fertility and infertility. Pertinent studies were searched in PubMed/Medline using relevant key words. Both HLA-F and HLA-G interact with inhibitory or activating ILT2 or ILT4 receptors and KIRs on uterine immune cells, especially uterine natural killer (NK) cells that are highly abundant in the mid-secretory endometrium and in early pregnancy. The binding of HLA-G to ILT2 stimulates the secretion of growth-promoting factors from decidual NK cells. However, functional aspects of a HLA-F-receptor interaction remain to be clarified. Recent studies indicate that HLA-F and HLA-G are expressed in mid-secretory endometrium and HLA-G is expressed in the blastocyst. HLA-F fluctuates during the menstrual cycle with high levels during the implantation window. The level of HLA-F protein expression correlates with the number of CD56-positive NK cells in the mid-secretory endometrium. HLA-F and HLA-G gene polymorphisms, including a single nucleotide polymorphism (SNP) in a progesterone-responsive element, are associated with time-to-pregnancy. Depending on the SNP genotype, the effect of progesterone varies resulting in differences in HLA-F expression and thereby the interaction with receptors on the uterine NK cells. Studies suggest that the expression of HLA-G and HLA-F, both by the embryonic-derived trophoblast cells and by cells in the endometrium and decidua, and the interaction between HLA-G and HLA-F with specific receptors on uterine immune cells, stimulate and facilitate embryo implantation and placentation by secretion of growth factors, cytokines and angiogenic factors. A detailed understanding of the molecular mechanisms controlling the expression of HLA-F and HLA-G periconceptionally and in early pregnancy may improve the success of ART and holds promise for further insight into pathophysiological aspects of certain pregnancy complications.

Phase 1b/2 umbrella study of investigational immune and targeted combination therapies for patients with advanced clear cell renal cell carcinoma (ccRCC).

TPS404 Background: New treatments for first and subsequent lines of therapy for ccRCC are needed. An adaptive, open-label, rolling-arm, multicenter, phase 1b/2 umbrella platform trial in advanced ccRCC is being conducted to evaluate the safety and efficacy of multiple combinations of investigational agents targeting CTLA-4 (quavonlimab [MK-1308]), hypoxia-inducible factor 2α (belzutifan [MK-6482]), lymphocyte-activation gene 3 (favezelimab [MK-4280]), immunoglobulinlike transcript 4 (MK-4830), PD-1 (pembrolizumab), and vascular endothelial growth factor–tyrosine kinase inhibitor (VEGF-TKI; lenvatinib) for advanced ccRCC. Substudy 03A (NCT04626479) will evaluate first-line therapy combinations, and substudy 03B (NCT04626518) will evaluate therapies in patients experiencing progression on PD-1/PD-L1 inhibitors and VEGF-TKIs in combination or in sequence. Methods: Adults with histologically confirmed ccRCC and KPS score ≥70% are eligible. Patients in substudy 03A cannot have previously received systemic therapy for advanced ccRCC. Patients in substudy 03B must have progression (per RECIST v1.1) on or after treatment with a PD-1/PD-L1 inhibitor and a VEGF-TKI for advanced ccRCC. Both substudies will have a safety lead-in phase for experimental combinations with investigational agents to establish a recommended phase 2 dose, followed by an efficacy phase. Experimental arms will enroll approximately 80 (03A) and 50 (03B) patients. Patients will be randomly assigned 2:1 (03A) or 1:1 (03B) to an experimental arm or a reference arm (pembrolizumab 400 mg IV every 6 weeks [Q6W] + lenvatinib 20 mg orally once daily [QD]). Experimental arms in 03A are MK-1308A (coformulation of quavonlimab 25 mg and pembrolizumab 400 mg IV Q6W) + lenvatinib (20 mg orally QD), MK-4280A (coformulation of favezelimab 800 mg and pembrolizumab 200 mg IV every 3 weeks [Q3W]) + lenvatinib (20 mg orally QD), and belzutifan (120 mg orally QD) + lenvatinib (20 mg orally QD) + pembrolizumab (400 mg Q6W IV). Experimental arms in 03B are MK-1308A, MK-4280A, pembrolizumab (400 mg Q6W IV) + belzutifan (120 mg orally QD), lenvatinib (20 mg orally QD) + belzutifan (120 mg orally QD), and pembrolizumab (200 mg IV Q3W) + MK-4830 (800 mg IV Q3W). Treatment will continue until disease progression, unacceptable toxicity, or withdrawal of consent. Primary end points for the safety lead-in phase are safety and tolerability; coprimary end points for the efficacy phase are safety and objective response rate per RECIST v1.1 by blinded independent central review (BICR). Secondary end points during the efficacy phase are duration of response, progression-free survival (RECIST v1.1 by BICR), clinical benefit rate, and overall survival. Both substudies are recruiting patients in Australia, Canada, France, Israel, South Korea, Spain, New Zealand, the United Kingdom, and the United States. Clinical trial information: NCT04626479; NCT04626518.

HLA-G-ILT2 interaction contributes to suppression of bone marrow B cell proliferation in acquired aplastic anemia.

Acquired aplastic anemia (AA) is an autoimmune disease characterized by hematopoietic stem and progenitor cell destruction in bone marrow. The non-classic human leukocyte class I antigen (HLA-) G interacts with multiple cell subsets, such as T cells and B cells. HLA-G exerts powerful immune suppression by binding with its receptors, immunoglobulin-like transcripts (ILTs). Here, we compared 46 AA patients and 28 healthy controls. Soluble HLA-G levels in bone marrow supernatants from AA patients were higher than controls. The proportion of bone marrow B cells was decreased and the ILT2-expressing cells among CD19+ cells were increased in AA patients. In addition, the percentage of mature B cells among marrow B cells was increased in AA patient, while the percentage of pro-B plus pre-B cells was decreased. More immature B cells and pro-B plus pre-B cells expressed ILT2 in AA patients than in controls, while mature B cells expressing ILT2 did not differ significantly. Functional studies demonstrated that high-level soluble HLA-G inhibited bone marrow B cell proliferation by interacting with ILT2 in AA, and was blocked by anti-HLA-G and anti-ILT2 monoclonal antibodies. Together, these results suggest that the abnormal decrease of pro-B plus pre-B cells in AA patients was related to the enhanced suppression by the excess HLA-G and ILT2 proteins. Therapeutic blockade of the HLA-G-ILT2 interaction may help to normalize bone marrow B cell proliferation.

Immune Regulatory Functions of IgG in the Ontogeny of Human Natural Regulatory T Cells

The heavy constant region of IgG (Fc) is highly immunogenic for human natural regulatory T cells (nTreg). Mature IgG+ B cells prime Fc-specific Treg via recycling of surface immunoglobulin with an antigen-processing pathway that is very efficient in presenting immunodominant Fc peptides to Treg. Some of these peptides are pan-HLA binders, explaining the presence of Fc-specific Treg in circulation in healthy pediatric and adult subjects. Following IgG+ B cell priming, further Treg expansion occurs with the presentation of Fc peptides following IgG uptake and processing by CD14+ myeloid dendritic cells type 2 (cDC2) and CD4+ immunoglobulin-like transcript 4 (ILT-4+) tolerogenic DC that secretes IL-10 when stimulated by the Fc that enters cells prevalently via Fcg receptor II. Fc-specific Treg are important in regulating naive T cell differentiation and account for a key mechanism of success for intravenous immunoglobulin therapy (IVIG) in several inflammatory conditions, including Kawasaki disease (KD) a pediatric acute vasculitis of the coronary arteries.

Perspective of HLA-G Induced Immunosuppression in SARS-CoV-2 Infection.

COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has threatened public health worldwide. Host antiviral immune responses are essential for viral clearance and disease control, however, remarkably decreased immune cell numbers and exhaustion of host cellular immune responses are commonly observed in patients with COVID-19. This is of concern as it is closely associated with disease severity and poor outcomes. Human leukocyte antigen-G (HLA-G) is a ligand for multiple immune inhibitory receptors, whose expression can be upregulated by viral infections. HLA-G/receptor signalling, such as engagement with immunoglobulin-like transcript 2 (ILT-2) or ILT-4, not only inhibit T and natural killer (NK) cell immune responses, dendritic cell (DC) maturation, and B cell antibody production. It also induces regulatory cells such as myeloid-derived suppressive cells (MDSCs), or M2 type macrophages. Moreover, HLA-G interaction with CD8 and killer inhibitory receptor (KIR) 2DL4 can provoke T cell apoptosis and NK cell senescence. In this context, HLA-G can induce profound immune suppression, which favours the escape of SARS-CoV-2 from immune attack. Although detailed knowledge on the clinical relevance of HLA-G in SARS-CoV-2 infection is limited, we herein review the immunopathological aspects of HLA-G/receptor signalling in SARS-CoV-2 infection, which could provide a better understanding of COVID-19 disease progression and identify potential immunointerventions to counteract SARS-CoV-2 infection.

First-in-Class Anti-immunoglobulin-like Transcript 4 Myeloid-Specific Antibody MK-4830 Abrogates a PD-1 Resistance Mechanism in Patients with Advanced Solid Tumors.

In this first-in-human study (NCT03564691) in advanced solid tumors, we investigated a novel first-in-class human IgG4 monoclonal antibody targeting the immunoglobulin-like transcript 4 (ILT4) receptor, MK-4830, as monotherapy and in combination with pembrolizumab. Patients with histologically/cytologically confirmed advanced solid tumors, measurable disease by RECIST v1.1, and evaluable baseline tumor sample received escalating doses of intravenous MK-4830 every 3 weeks as monotherapy (parts A and B) and in combination with pembrolizumab (part C). Safety and tolerability were the primary objectives. Pharmacokinetics, objective response rate per RECIST v1.1, and molecular biomarkers were also evaluated. Of 84 patients, 50 received monotherapy and 34 received combination therapy. No dose-limiting toxicities were observed; maximum tolerated dose was not reached. MK-4830 showed dose-related target engagement. Eleven of 34 patients in the dose-escalation phase who received combination therapy achieved objective responses; 5 previously had progressive disease on anti-PD-1/PD-L1 therapies. Exploratory evaluation of the association between response and pretreatment gene expression related to interferon-gamma signaling in tumors suggested higher sensitivity to T-cell inflammation with combination therapy than historically expected with pembrolizumab monotherapy, with greater response at more moderate levels of inflammation. This first-in-class MK-4830 antibody dosed as monotherapy and in combination with pembrolizumab was well tolerated with no unexpected toxicities, and demonstrated dose-related evidence of target engagement and antitumor activity. Inflammation intrinsic to the ILT4 mechanism may be facilitated by alleviating the myeloid-suppressive components of the tumor microenvironment, supporting the target of ILT4 as a potential novel immunotherapy in combination with an anti-PD-1/PD-L1 agent.

Dynamics of circulating dendritic cells and cytokines after kidney transplantation-No effect of remote ischaemic conditioning.

Inflammation resulting from ischaemia/reperfusion injury can cause kidney graft dysfunction, increase the risk of delayed graft function and possibly reduce long-term graft survival. Remote ischaemic conditioning may protect against ischaemia/reperfusion injury and mitigate the immunological response to the graft. We investigated the immunological effects of remote ischaemic conditioning on kidney transplantation from deceased donors in the randomized CONTEXT study. Three circulating dendritic cell (DC) subtypes identified in peripheral blood from kidney transplant recipients [myeloid DCs, plasmacytoid DCs and immunoglobulin-like transcript (ILT)3+ DCs] were measured at baseline, days 1, 3 and 5 and 1and 3months after transplantation. We also quantified 21cytokines at baseline, days 1 and 5 and 3months after transplantation. Neither DC counts nor cytokine levels differed between patients receiving remote ischaemic conditioning and controls; however, several parameters exhibited dynamic and parallel alterations in the two groups over time, reflecting the immunological response to the kidney transplantation and immunosuppression.

The Fibronectin-ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells.

Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3-fibronectin interaction represents a "stromal checkpoint" through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.

ILT4 in Colorectal Cancer Cells Induces Suppressive T Cell Contexture and Disease Progression.

PurposeImmune checkpoint blockade (ICB) therapy shows little or no clinical benefit in most colorectal cancer (CRC) patients, due to the immunosuppressive T cell contexture in the tumor microenvironment (TME). Immunoglobulin-like transcript (ILT) 4 is an immunosuppressive molecule in myeloid cells. ILT4 is enriched in solid tumor cells, facilitating their proliferation, invasion, and metastasis. However, the regulatory role of ILT4 in T cell immunity of CRC is still undetermined. Here, we aimed to explore how tumor cell-derived ILT4 orchestrates T cell infiltration, subset distribution, and function in CRC.MethodsA total of 145 paraffin-embedded cancer tissues and the corresponding clinicopathological information were collected from CRC patients. Immunohistochemical (IHC) staining and public database analyses determined the correlation of ILT4 expression with different T cell subset densities, IFN-γ levels, and patient outcomes. Paired Ig-like receptor B (PIR-B, ILT4 mouse ortholog)-overexpressing/-downregulated MC38 cells were subcutaneously injected into C57BL/6 mice as a CRC transplantation model. The frequencies, subsets, and IFN-γ levels of T cells in mouse blood and spleens were determined using flow cytometry and immunohistochemistry, respectively.ResultsHigh ILT4 expression in CRC cells was associated with decreased T cell infiltration, disease progression, and poor patient survival. T cell subset analyses indicated that ILT4-high patients showed reduced CD8+ T cell but elevated FOXP3+ regulatory T (Treg) cell frequencies in the TME. High ILT4 levels predicted lower IFN-γ production by tumor-infiltrating lymphocytes (TILs), especially by CD8+T cells in human CRC tissues. Moreover, PIR-B overexpression accelerated MC38 growth in mice, decreased CD3+/CD8+/IFN-γ+ T cell densities, and elevated Treg infiltration in the TME, blood, and spleens. PIR-B knockdown had the opposite effects.ConclusionILT4 in CRC cells induced immunosuppressive T cell subset infiltration and impaired IFN-γ production in TILs, suggesting that ILT4 might be a potential immunotherapeutic target and prognostic biomarker.

HLA-G/ILTs Targeted Solid Cancer Immunotherapy: Opportunities and Challenges.

Immune checkpoint inhibitors (ICIs) have become a promising immunotherapy for cancers. Human leukocyte antigen-G (HLA-G), a neoantigen, its biological functions and clinical relevance have been extensively investigated in malignancies, and early clinical trials with “anti-HLA-G strategy” are being launched for advance solid cancer immunotherapy. The mechanism of HLA-G as a new ICI is that HLA-G can bind immune cell bearing inhibitory receptors, the immunoglobulin-like transcript (ILT)-2 and ILT-4. HLA-G/ILT-2/-4 (HLA-G/ILTs) signaling can drive comprehensive immune suppression, promote tumor growth and disease progression. Though clinical benefits could be expected with application of HLA-G antibodies to blockade the HLA-G/ILTs signaling in solid cancer immunotherapy, major challenges with the diversity of HLA-G isoforms, HLA-G/ILTs binding specificity, intra- and inter-tumor heterogeneity of HLA-G, lack of isoform-specific antibodies and validated assay protocols, which could dramatically affect the clinical efficacy. Clinical benefits of HLA-G-targeted solid cancer immunotherapy may be fluctuated or even premature unless major challenges are addressed.

Phase 1, first-in-human trial of JTX-8064, an anti-LILRB2/ILT4 monoclonal antibody, as monotherapy and in combination with anti-PD-1 in adult patients with advanced solid tumors (INNATE).

TPS2672 Background: Leukocyte Immunoglobulin-like receptor B2 [LILRB2; immunoglobulin-like transcript 4 (ILT4)] is an immunoinhibitory protein expressed on the surface of myeloid cells and is a therapeutic target of interest in immuno-oncology. Published data showed that antagonism of LILRB2 resulted in the repolarization of human macrophages from an M2 (suppressive) to M1 (pro-inflammatory) phenotype, and enhancement of anti-tumor immunity in a mouse model (Chen 2018). JTX-8064 is a novel humanized IgG4 monoclonal antagonist antibody that selectively binds LILRB2 and prevents it from binding its ligands, classical and non-classical MHC I molecules. By blocking the ability of LILRB2 to bind HLA-A/B and/or HLA-G, a marker of immunotolerance on cancer cells, JTX-8064 has been shown to enhance pro-inflammatory cytokine production in macrophages (Cohen 2019). Additionally, blocking HLA-A/B-LILRB2 binding with JTX-8064 may augment antigen presentation and has been shown to lead to enhanced T cell activation and IFNg production (McGrath 2021). Using an ex vivo tumor explant model, we observed an IFNg-associated pharmacodynamic response in tumor tissue treated with JTX-8064 and a PD-1 inhibitor (PD-1i) that was not observed with PD-1i alone. Biomarkers were identified that predicted this JTX-8064 driven response (Hashambhoy-Ramsay 2020). It is hypothesized that JTX-8064 is a novel macrophage immune checkpoint inhibitor that may overcome mechanisms of resistance to PD-1i in tumors not responsive to JTX-8064 or PD-1i alone. Methods: The primary objectives of this open-label, phase 1, first-in-human, multicenter trial are to determine the safety and tolerability, and the recommended phase 2 dose (RP2D) of JTX-8064 as a monotherapy and in combination with a PD-1i, JTX-4014 (a Jounce investigational agent) or pembrolizumab, in patients with advanced solid tumors (NCT04669899). The INNATE study will consist of 4 stages: 1) JTX-8064 monotherapy dose escalation, 2) JTX-8064 dose escalation in combination with a PD-1i, 3) JTX-8064 monotherapy in indication-specific expansion cohorts and 4) JTX-8064 in combination with a PD-1i in indication-specific expansion cohorts. Stages 1 and 2 will employ an innovative interval i3 + 3 design with Bayesian decision framework to guide dose escalation. Safety, pharmacokinetic and receptor occupancy data will be considered during dose escalation. INNATE will assess pharmacodynamic and potential predictive biomarkers of response, and the expansion cohorts will explore multiple patient populations, including PD-(L)1i sensitive and PD-(L)1i-resistant (primary or acquired) patients to address current unmet medical needs. Enrolment in INNATE began in January 2021. Clinical trial information: NCT04669899.

A highly diverse set of novel immunoglobulin-like transcript (NILT) genes in zebrafish indicates a wide range of functions

Abstract Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout and carp. NILTs typically encode activating or inhibitory transmembrane receptors with extracellular immunoglobulin (Ig) domains. Although predicted to provide some level of immune recognition in ray-finned fish, we currently lack a definitive framework of NILT diversity, thereby challenging our ability to understand the evolutionary origin and specific function of these genes. In order to better understand the diversity of NILT genes and their possible roles in immune function, we identified 5 NILT loci in the Atlantic salmon (Salmo salar) genome, defined 86 NILT Ig domains within a 3 Mbp region of zebrafish (Danio rerio) chromosome 1, and described 41 NILT Ig domains as part of an alternate haplotype for this same genomic region. We then identified transcripts that define 43 different NILT genes and reflect an unprecedented range of sequence diversity and combinatorial diversity of Ig domains for a single family of non-recombining receptors. Zebrafish NILTs include a single putative activating receptor but extensive inhibitory and secreted forms as well as membrane-bound forms with no known signaling motifs. Whole genome sequences from different genetic backgrounds reveal at least three alternative haplotypes of the zebrafish NILT gene cluster that display gene content variation. This observation indicates that different individual fish can encode different combinations of NILTs which could impact their immune function. Furthermore, these results reveal a higher level of genetic complexity and sequence diversity for NILTs than previously described, suggesting that this gene family likely plays multiple roles in host immunity.

Prostaglandin I2 signaling licenses Treg suppressive function and prevents pathogenic reprogramming.

Tregs restrain both the innate and adaptive immune systems to maintain homeostasis. Allergic airway inflammation, characterized by a Th2 response that results from a breakdown of tolerance to innocuous environmental antigens, is negatively regulated by Tregs. We previously reported that prostaglandin I2 (PGI2) promoted immune tolerance in models of allergic inflammation; however, the effect of PGI2 on Treg function was not investigated. Tregs from mice deficient in the PGI2 receptor IP (IP KO) had impaired suppressive capabilities during allergic airway inflammatory responses compared with mice in which PGI2 signaling was intact. IP KO Tregs had significantly enhanced expression of immunoglobulin-like transcript 3 (ILT3) compared with WT Tregs, which may contribute to the impairment of the IP KO Treg's ability to suppress Th2 responses. Using fate-mapping mice, we reported that PGI2 signaling prevents Treg reprogramming toward a pathogenic phenotype. PGI2 analogs promoted the differentiation of naive T cells to Tregs in both mice and humans via repression of β-catenin signaling. Finally, a missense variant in IP in humans was strongly associated with chronic obstructive asthma. Together, these data support that PGI2 signaling licenses Treg suppressive function and that PGI2 is a therapeutic target for enhancing Treg function.

Tumor-derived ILT4 induces T cell senescence and suppresses tumor immunity

BackgroundCurrent immunotherapies including checkpoint blockade therapy have limited success rates in certain types of cancers. Identification of alternative checkpoint molecules for the development of effective strategies for tumor immunotherapy is...