Killer Cell Lectin-like Receptor G1 Research Articles

Abstract 7532: NB005.1, a fully humanized anti-KLRG1 blocking mAb enhances effector function of human CD8 T-cells

Abstract Introduction: Immune checkpoint blockers (ICBs) have revolutionized treatment, enhancing survival and benefiting many patients. However, many patients fail to respond to the treatment, necessitating research for new targets and improved approaches. The Killer cell Lectin-like Receptor G1 (KLRG1), expressed on a subset of NK and T-cells harbors an immune receptor tyrosine-based inhibitory motif (ITIM) within its cytoplasmic domain, indicating its inhibitory function. On CD8+ T-cells, KLRG1 is expressed on highly differentiated antigen-specific effector memory T-cells (TEMs) and CD45RA+ effector memory T cells (TEMRA). These cells are known to exhibit strong cytotoxicity potential and display developmental plasticity following KLRG1 blockade. And 16-48% of CD8+ T-cells that infiltrate the tumor microenvironment (TME) are KLRG1 positive. We hypothesize that the blockade of KLRG1 should enhance the effector function of KLRG1+ CD8 T-cells in the TME and provide therapeutic benefit as a standalone or in combination with existing ICBs. Methods: Monoclonal antibodies (mAbs) targeting the human KLRG1 extracellular domain (ECD) were generated through mouse immunization and hybridoma technology. Purified mAbs from positive clones were screened using CHO-K1 and Jurkat cells overexpressing human or cynomolgus KLRG1 and human primary CD8 T-cells (n=3) to determine EC50 values. Epitope binning was performed, and high-affinity clones from each bin with cyno cross-reactivity were humanized to IgG1-Fc LALA variant. The Abs were evaluated for ligand blocking and enhancement of CD8 T-cell effector function through multiple functional assays. The assay measured enhancement of IFNγ or TNFα by KLRG1+ human CD8+ T-cells co-cultured with CHO-K1 expressing OKT3 and E-cadherin as artificial antigen-presenting cells (aAPCs) or PBMCs stimulated in with anti-CD3 and anti-CD28 Abs in presence of soluble E-cadherin. A human IgG1-LALA antibody (Ab) served as a negative control. PD-1 and KLRG1 synergy was evaluated in hPBMC assays. Results: NB005.1 demonstrated single digit nanomolar (nM) binding potency on human primary CD8 T-cells and Jurkat cells expressing human KLRG1 as identified by its EC50 value. Blocking of KLRG1 with NB005.1 on purified human CD8 T-cells in co-culture assay with aAPC expressing E-cad and OKT3 led to CD8 T-cell activation and increase in IFNγ production confirming inhibitory function of KLRG1. Similar result was obtained when the Ab was tested on hPBMCs stimulated with anti-CD3 and anti-CD28 Abs in presence of soluble E-cadherin. Conclusion: NB005.1, a fully humanized mAb, binds to human KLRG1 in the single digit nM range and effectively blocks the interaction with its ligand. This blockade enhances T-cell effector function as measured by IFNγ production, supporting the advancement of NB005.1 for preclinical testing, either as a standalone therapeutic or in combination with existing ICBs. Citation Format: Bishnu P. Nayak, Meiguang Xiong, Yajuan Xue, Binbin Wang, Shahrzad Lighvani, Shilpi Sharma, Dong Wang, Haojai Wang, Baiyang Wang, Yu Zhang, Xin Dong. NB005.1, a fully humanized anti-KLRG1 blocking mAb enhances effector function of human CD8 T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7532.

Prenatal Inflammation Promotes Enhanced Allergic Lung Inflammation By Programming Hyperactive ILC2s from Fetal Hematopoietic Progenitors

Our lab has recently demonstrated that prenatal inflammation, induced by maternal immune activation via viral-mimetic poly(I:C), specifically activates and expands lymphoid-biased developmentally-restricted hematopoietic stem cells (drHSCs) in the fetal liver, resulting in the generation of hyperactivated innate-like B1-B cells in the postnatal period that produce greater levels of IgM, IgG3, and IL-10 on a per-cell basis. We hypothesize that dysregulation and hyperactivation of long-lived fetal-derived innate immune compartments that persist across the lifespan underlies disease susceptibility into adulthood. Here we elucidate a mechanism by which prenatal inflammation imparts persistent changes to lung immunity and function by programming innate lymphoid cell output from fetal hematopoietic progenitors, resulting in adverse responses to allergen challenge into adulthood. To investigate how prenatal inflammation impacted fetal- and neonatal-derived group-2 innate lymphoid cells (ILC2s), we profiled changes to ILC2 establishment across early postnatal life. In response to prenatal inflammation, ILC2s were significantly expanded at postnatal day (P)9 and remained robustly expanded through adulthood, while also exhibiting a greater proliferation capacity through their peak expansion period postnatally (P14). At P14, prenatal inflammation also robustly increased IL-5 and IL-13 production from ILC2s after ex-vivo stimulation, concomitant with higher expression of killer cell lectin-like receptor G1 (KLRG1), indicative of an enhanced activation state. To determine hematopoietic origins of expanded and hyperactive ILC2s, we profiled changes to an immediate upstream precursor of the ILC lineage within the neonatal bone marrow (BM) and fetal liver after prenatal inflammation. At P14, prenatal inflammation did not alter the establishment of BM common helper-like innate lymphoid progenitors (CHILPs), whereas fetal liver CHILPs were robustly expanded by both cellularity and frequency at embryonic day (E)15.5 and E17.5 compared to saline treated controls. To test if the changes observed in mature ILC2s and their immediate precursors could be programmed at the stem cell level, we transplanted fetal liver drHSCs one day post-poly(I:C) or saline into irradiated adult recipients. One month post-transplantation, donor-derived ILC2s from the prenatal-inflammation condition produced more IL-13 cytokine within the lung microenvironment compared to saline controls, demonstrating that prenatal inflammation programs fetal hematopoietic precursors during development to generate hyperactive ILC2s. Single-cell RNA sequencing of over 26,000 lung ILC2s at P14 revealed extensive heterogeneity in response to prenatal inflammation. Unsupervised clustering analysis identified clusters unique to prenatal inflammation conditions, with transcriptional profiles significantly overlapping with ILC2 signatures identified in models of allergic airway inflammation (AAI). As prenatal inflammation induced functional and transcriptional shifts in ILC2s that regulate lung immunity, we profiled changes to the developing lung immune landscape in response ILC2 hyperactivation. ILC2 expansion and hyperactivation was concomitant with remodeling of multiple lung immune compartments, including expanded B-cells, T-cells, and eosinophils at P14. We acutely challenged mice intranasally with papain allergen to determine if prenatal inflammation modulated lung innate immune responses. ILC2s and eosinophils expanded in response to papain challenge and exhibited even greater expansion under prenatal inflammation conditions. As eosinophilia is a hallmark feature of AAI, we assessed respiratory mechanics using the FlexiVent system in adult mice to measure the underlying impact of prenatal inflammation on lung function in the context of acute allergen challenge. Exposure to prenatal inflammation alone caused a decrease in lung compliance and pressure-volume loops, indicative of pulmonary morbidity, that was further exacerbated by papain challenge compared to saline treated controls. Together, our data reveal a mechanism by which prenatal inflammation can program hyperactivity of ILC2s at the fetal HSC-level during embryonic development, resulting in lasting changes to postnatal lung immunity and lung disease susceptibility in offspring.

KLRG1 is reduced on NK cells in SLE patients, inversely correlates with disease activity and is modulated by hydroxychloroquine in vitro

Killer cell lectin-like receptor G 1 (KLRG1), a transmembrane receptor with inhibitory capacity expressed in human immune cells, emerged as a novel susceptibility gene for systemic lupus erythematosus (SLE). The aim of this study was to investigate the expression of KLRG1 in SLE patients compared to healthy controls (HC) on both NK and T cells and to evaluate its possible involvement in SLE pathogenesis. Eighteen SLE patients and twelve healthy controls were enrolled. Peripheral blood mononuclear cells (PBMCs) from these patients were phenotypically characterized by immunofluorescence and flow cytometry. The effect of the hydroxychloroquine (HCQ) in vitro on KLRG1 expression and its signaling mediated functions in NK cells were analyzed. KLRG1 expression was significantly reduced on the analyzed immune cell populations in SLE patients compared to HC, especially on total NK cells. Moreover, expression of KLRG1 on total NK cells inversely correlated with the SLEDAI-2K. A direct association between KLRG1 expression on NK cells and patients' treatment with HCQ was observed. In vitro treatment with HCQ increased KLRG1 expression on NK cells. In HC, KLRG1+ NK cells showed reduced degranulation and IFNγ production, while in SLE patient, this inhibition occurred only for the IFNγ production. With this study we revealed a reduced expression and an impaired function of KLRG1 on NK cells in SLE patients. These results suggest a possible role of KLRG1 in the pathogenesis of SLE and as a novel biomarker of this disease.

KLRG1 Depletion Is a Novel Therapeutic Strategy for Patients with Mature T and NK/T-Cell Lymphomas

Outcomes for patients with aggressive T-cell lymphomas (TCLs) remain dismal. Killer cell lectin-like receptor G1 (KLRG1) is a co-inhibitory immune checkpoint receptor expressed on late differentiated CD8+ T effector memory (TEM) and TEMRA cells. Monotherapy with KLRG1 neutralizing antibodiesextended survival in solid tumor models. Here, we report the therapeutic potential of a novel and distinct strategy characterized by depletion of KLRG1+ malignant cells in hematologic malignancies and specifically in TCLs. We defined the molecular epidemiology of KLRG1 by determining tumor cell-specific RNA and protein expression on T cell subsets from 8 healthy donors, 26 TCL lines, 15 patient-derived xenografts (PDXs), and 165 tumor and blood specimens representing various subtypes of T and NK/TCLs. Microarray andRNA-seqdata demonstrated highertranscript levels of KLRG1 in patients with T-LGLL and PDXs of T-PLL and HSTCL relative to other subtypes, which were corroborated by protein expression. We quantified tumor cell-specific protein expression of KLRG1 by IHC by generating H scores (% of KLRG1+ tumor cells × measure of staining intensity) ranging from 0-300 across 141 primary TCL biopsies. PTCL-NOS and AITL had heterogeneous KLRG1 expression, whereas ALCL, CTCL, and EATL demonstrated none. The majority of patients with T-PLL (median 180), δ/γ TCLs (median 140), and NK/TCLs (median 100) exhibited higher H scores relative to other subtypes. Up to 99% of the CD3+/CD8+/CD57+ and up to 97% of the CD3-/CD56+/CD57+/CD16+/CD94+ leukemic cells in the blood of patients with T-LGLL (n=12) and CLPD-NK (n=12) respectively expressed KLRG1 protein (Fig.1). None of the TCL cell lines expressed surface KLRG1. Therefore, SMZ1 cells (TP63 rearranged PTCL-NOS line) were stably transduced to express KLRG1. We utilized two human KLRG1 binding antibodies, 13F12F2 (a commercial mIgG2a Ab) and mAb208 (a humanized afucosylated mIgG2a Ab) for in vitro and in vivo studies. Antibody binding capacity assays confirmed a higher number of KLRG1 receptors per cell on SMZ1 KLRG1+ (175,800), HSTCL (23,586), and CD8+ T cells (16,326) compared with B-cell lymphoma cells (242). Competition binding assays demonstrated that concentrations of 0.1 ug/ml of mAb208 saturated receptors on SMZ1 KLRG1+ cells. mAb208 induced a 2-5-fold dose-dependent increase in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), but not apoptosis of SMZ1 KLRG1+ cells relative to SMZ1 KLRG1- WT and isotype control (CTL). This was exhibited across a variety of human and murine effectors such as human PBMCs, human and murine macrophages, and human and murine complements. Reporter-based murine FcγRIV ADCC bioassay demonstrated a dose-dependent 2-6 fold induction of bioluminescence by mAb208 in SMZ1 KLRG1+ cells relative to WT (p=<0.0001). In 4/4 tested T-LGLL patient samples, mAb208 induced ADCC of CD8+/CD57+ LGLs with autologous PBMCs (p=0.03) relative to CTL. Finally, mAb208 induced selective ADCC of KLRG1+ CD8+ TEM and TEMRA cells while sparing KLRG1- naive and central memory T cells in 3 healthy donors (p=0.06). Duvelisib (Duv), a dual PI3K-δ/γ inhibitor (PI3Ki), has demonstrated a high ORR of 50% in phase II trials in TCLs and its ability to reprogram macrophages to an M1 phenotype in TCL PDXs. Thus, we hypothesized that the combination of PI3Ki with anti-KLRG1 mAbs would enhance the clearance of apoptotic and opsonized KLRG1+ TCL cells by proinflammatory macrophages in several PTCL subtypes. We tested this combination in vivo using SMZ1 KLRG1+ subcutaneous xenografts in NSG mice. After 17 days of treatment, combination (mAb208 + Duv)- treated mice had a marked reduction in their mean tumor volumes (159 mm3) relative to mAb208 (1003 mm3 ; p=0.03), Duv (465.6 mm3 ; p=0.03) and CTL (2104 mm3 ; p=0.03) mice resulting in prolonged survival (Fig. 2). Studies in PDX models of T-PLL and HSTCL with varying levels of KLRG1 expression (30% and 80% respectively) have been initiated and will be reported at the meeting. Selective depletion of tumor cells with an anti-KLRG1 depleting mAb in specific subtypes of TCLs, T-LGLL, and CLPD-NK is effective in vitro and in vivo, and its combination with Duv has marked activity in an aggressive PTCL-NOS xenograft model. A multicenter phase I/II trial of the KLRG1 depleting antibody ABC008 (Abcuro) in patients with R/R T-LGLL has been initiated. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Sex differences exist in adult heart group 2 innate lymphoid cells

BackgroundGroup 2 innate lymphoid cells (ILC2s) are the most dominant ILCs in heart tissue, and sex-related differences exist in mouse lung ILC2 phenotypes and functions; however, it is still unclear whether there are sex differences in heart ILC2s.ResultsCompared with age-matched wild-type (WT) male mice, 8-week-old but not 3-week-old WT female mice harbored an obviously greater percentage and number of heart ILC2s in homeostasis. However, the percentage of killer-cell lectin-like receptor G1 (Klrg1)− ILC2s was higher, but the Klrg1+ ILC2s were lower in female mice than in male mice in both heart tissues of 3- and 8-week-old mice. Eight-week-old Rag2−/− mice also showed sex differences similar to those of age-matched WT mice. Regarding surface marker expression, compared to age-matched male mice, WT female mice showed higher expression of CD90.2 and Ki67 and lower expression of Klrg1 and Sca-1 in heart total ILC2s. There was no sex difference in IL-4 and IL-5 secretion by male and female mouse heart ILC2s. Increased IL-33 mRNA levels within the heart tissues were also found in female mice compared with male mice. By reanalyzing published single-cell RNA sequencing data, we found 2 differentially expressed genes between female and male mouse heart ILC2s. Gene set variation analysis revealed that the glycine, serine and threonine metabolism pathway was upregulated in female heart ILC2s. Subcluster analysis revealed that one cluster of heart ILC2s with relatively lower expression of Semaphorin 4a and thioredoxin interacting protein but higher expression of hypoxia-inducible lipid droplet-associated.ConclusionsThese results revealed greater numbers of ILC2s, higher expression of CD90.2, reduced Klrg1 and Sca-1 expression in the hearts of female mice than in male mice and no sex difference in IL-4 and IL-5 production in male and female mouse heart ILC2s. These sex differences in heart ILC2s might be due to the heterogeneity of IL-33 within the heart tissue.

Med1 Controls Effector CD8+ T Cell Differentiation and Survival through C/EBPβ-Mediated Transcriptional Control of T-bet.

Effector CD8+ T cells are crucial players in adaptive immunity for effective protection against invading pathogens. The regulatory mechanisms underlying CD8+ T cell effector differentiation are incompletely understood. In this study, we defined a critical role of mediator complex subunit 1 (Med1) in controlling effector CD8+ T cell differentiation and survival during acute bacterial infection. Mice with Med1-deficient CD8+ T cells exhibited significantly impaired expansion with evidently reduced killer cell lectin-like receptor G1+ terminally differentiated and Ly6c+ effector cell populations. Moreover, Med1 deficiency led to enhanced cell apoptosis and expression of multiple inhibitory receptors (programmed cell death 1, T cell Ig and mucin domain-containing-3, and T cell immunoreceptor with Ig and ITIM domains). RNA-sequencing analysis revealed that T-bet- and Zeb2-mediated transcriptional programs were impaired in Med1-deficient CD8+ T cells. Overexpression of T-bet could rescue the differentiation and survival of Med1-deficient CD8+ effector T cells. Mechanistically, the transcription factor C/EBPβ promoted T-bet expression through interacting with Med1 in effector T cells. Collectively, our findings revealed a novel role of Med1 in regulating effector CD8+ T cell differentiation and survival in response to bacterial infection.

Myelin oligodendrocyte glycoprotein-associated disease is associated with BANK1, RNASET2 and TNIP1 polymorphisms

AimHere we aimed to compare association of common immune-related genetic variants with three autoimmune central nervous system (CNS) demyelinating diseases, namely myelin oligodendrocyte glycoprotein-associated disease (MOGAD), multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). MethodsIn this retrospective cross-sectional study, 26 common immune-related single nucleotide polymorphisms were genotyped in 102 patients with MOGAD, 100 patients with MS, 198 patients with NMOSD and 541 healthy control subjects recruited from Guangzhou, China. ResultsAmong all tested genetic variations, one polymorphism, B cell scaffold protein with ankyrin repeats 1 (BANK1) rs4522865 was associated with multiple disorders, namely MOGAD (OR = 1.94, 95% CI:1.19–3.17, P = 0.0059) and NMOSD (OR = 1.69, 95% CI:1.17–2.45). Besides BANK1 rs4522865, two other non-HLA loci, ribonuclease T2 (RNASET2) rs9355610 (OR = 0.47, 95% CI: 0.26–0.85) and TNFAIP3 interacting protein 1 (TNIP1) rs10036748 (OR = 1.76, 95% CI: 1.16–2.71), were associated with MOGAD. In addition, NMOSD was associated with signal transducer and activator of transcription 4 (STAT4) rs7574865 (OR = 1.58, 95% CI: 1.12–2.24) and general transcription factor Iii (GTF2I) rs73366469 (OR = 1.60, 95% CI:1.12–2.29), while MS was associated with a killer cell lectin like receptor G1 (KLRG1) rs1805673 (OR = 0.61, 95% CI: 0.40–0.94) and T-box transcription factor 21 (TBX21) rs17244587 (OR = 2.25, 95% CI: 1.25–4.06). ConclusionThe current study suggests for the first time three non-HLA susceptibility loci for MOGAD. In addition, comparison of association of 26 immune-related polymorphisms with three autoimmune CNS demyelinating diseases demonstrates substantial difference in genetic basis of those disorders.

Infiltration of Mature KLRG1 Expressing Cytotoxic T Cells in Oral Lichen Planus.

Oral lichen planus (OLP) is a chronic inflammatory disease affecting oral mucosa. Its pathogenesis includes T cell infiltration. T cells may be naïve or in response to antigen stimulation, progress through differentiation stages. The differentiated states of T cells in OLP mucosa have not previously been reported. Available OLP microarray gene expression data from Gene Expression Omnibus were analyzed for markers of T cell cytotoxicity. Immunohistochemical studies of T cell subset markers CD4 and CD8 and the T cell differentiation marker killer cell lectin-like receptor G1 (KLRG1) were performed on paraffin embedded formalin fixed oral mucosa biopsy samples from 10 patients with OLP. Gene expression analysis of OLP oral mucosa samples disclosed increased transcript expression of KLRG1, CD8A, and granzyme K (GZMK). By immunohistochemistry, prominent CD4 + and CD8 + T cell infiltration was seen in all patient samples. KLRG1 + T cells were abundant, constituting a mean of 51% (range 40-65%) of the number of CD8 + T cells. KLRG1 + T cells localized at the epithelium and lamina propria junction, infiltrating both basal and intraepithelial regions and adjacent to both basal and intraepithelial keratinocytes. OLP oral mucosa T cell infiltration includes KLRG1 + highly differentiated cytotoxic T cells, suggesting continued antigen exposure driving T cells to a highly differentiated phenotype. The known phenotype of these cells, together with microarray detected increases in cytotoxic molecules, suggests that highly differentiated cytotoxic T cells contribute to oral mucosa injury in OLP.

Expression of KLRG1 on subpopulations of lymphocytes in the peripheral blood of patients with locally advanced nasopharyngeal carcinoma and prognostic analysis

AbstractObjectiveWe aimed to investigate the relationship between the expression of killer cell lectin like receptor G1 (KLRG1) in peripheral blood lymphocytes, and the clinical characteristics and prognosis in patients with locally advanced nasopharyngeal carcinoma (NPC), and to dynamically evaluate the changes of KLRG1 expression after treatment.MethodsA total of 82 patients with locally advanced NPC and 44 healthy individuals were recruited in this study. The expression of KLRG1 on T, natural killer, and natural killer T cells in peripheral blood mononuclear cells (PBMCs) was detected by flow cytometry. Its relationship with clinical characteristics and prognosis of patients, as well as changes in KLRG1 expression in PBMCs after treatment, were investigated.ResultsThe expression of KLRG1 in CD8+ T cells in PBMCs of NPC patients was 46.50 (29.10, 69.80)%, significantly higher than 32.50 (17.38, 61.00) % in healthy controls (Z = −2.290, p = 0.022). The expression of KLRG1 in CD3+ CD56+ natural killer T cells in PBMCs of patients was 29.50 (6.21, 78.00) %, significantly higher than 1.47 (0.62, 8.88)% in healthy controls (Z = −5.371, p &lt; 0.001). The expression levels of KLRG1(+)CD8+ T cells in PBMCs of patients were higher in the age ≥50 years (Z = −3.413, p &lt; 0.001), pathology of keratinizing squamous cell carcinoma (H = 10.541, p = 0.005, and EGFR(+) groups (Z = −2.006, p = 0.043). Patients with higher KLRG1(+) expression in CD8+ T cells had a lower progression‐free survival (p = 0.029) and a lower distant metastasis‐free survival (p = 0.023). KLRG1 expression on T cells increased after radiotherapy.ConclusionKLRG1 can be harnessed as a potential target in designing novel therapy to encompass a broader range of NPC patients.

Changes in the expression of T-cell factor-1 in follicular helper T cells reflect the condition of systemic lupus erythematosus patients

ObjectiveThis study aimed to identify changes in T-cell factor-1 (TCF1) in circulating T-cell subsets of systemic lupus erythematosus (SLE) patients and to explore their significance in SLE. MethodsPeripheral blood was collected from 41 SLE patients and 45 healthy controls (HCs). TCF1 in follicular helper T (TFH), follicular regulatory T (TFR) and regulatory T (Treg) cells was analyzed by flow cytometry. Interleukin-21 (IL-21), programmed death receptor 1 (PD-1) and killer cell lectin-like receptor G1 (KLRG1) were detected and compared among TFH subsets sorted according to TCF1 and CD62L expression. Correlation analyses were conducted between TCF1-related TFH cell subsets and autoantibody levels, systemic lupus erythematosus disease activity index (SLEDAI), and plasmablast levels of SLE patients. ResultsAbsolute numbers of TCF1- TFH and TCF1+ Treg cells were increased in SLE patients. According to TCF1 and CD62L expression, circulating TFH cells and Tregs were sorted into four subsets. CD62L+TCF1+ TFH cell percentages were decreased, and CD62L-TCF1- TFH cells were increased. CD62L+TCF1+ Treg cell percentages were increased, and CD62L-TCF1- Treg cell percentages were decreased. KLRG1+ percentages in CD62L-TCF1-TFH were higher in SLE patients than in HCs. Functionally, CD62L+TCF1+ TFH cells had stronger IL-21 secretion, while CD62L-TCF1-TFH cells had weaker IL-21 secretion and lower PD-1 expression. TCF1- and CD62L-TCF1- TFH numbers were positively correlated with anti-ribosomal P, anti-dsDNA and SLEDAI. ConclusionThe increased TFH cells in SLE patients were mostly TCF1-negative subsets with weakened function. Changes in TCF1-related subsets can reflect the condition and abnormal humoral immunity of SLE patients.

POS1342 DEPLETION OF KLRG1+ T CELLS IN A FIRST-IN-HUMAN CLINICAL TRIAL OF ABC008 IN INCLUSION BODY MYOSITIS (IBM)

BackgroundInclusion body myositis (IBM), a relentlessly progressive autoimmune skeletal muscle disease, has no effective available pharmacological therapy. A prominent feature of IBM on microscopy is highly differentiated effector CD8+ cytotoxic T (Tc) cells invading non-necrotic myofibers [1]. These Tc cells, known to be relatively resistant to apoptosis, express markers including killer cell lectin-like receptor G1 (KLRG1) [2]. ABC008, a first-in-class humanized, afucosylated monoclonal antibody (mAb) specific for KLRG1, selectively depletes these highly differentiated Tc cells while sparing other blood cell populations, e.g., naïve, central memory, and regulatory T cells and B cells. ABC008 has been designed to treat diseases mediated by these Tc cells, including IBM and T-cell large granular lymphocytic leukemia (T-LGLL). IBM and rheumatoid arthritis overlap clinically with T-LGLL and share similar expansions of large granular lymphocytes (LGLs), which also express KLRG1. We report here our preliminary data from our ongoing trial of ABC008 in IBM (NCT04659031).ObjectivesEvaluate the safety, pharmacodynamics (PD), and pharmacokinetics (PK) of ABC008 administered subcutaneously (SC) in adults with IBM.MethodsIn this first-in-human, open-label, single ascending dose trial with 3 + 3 design evaluating ABC008 SC, eligible participants must have clinicopathologically defined, clinically defined, or probable IBM according to the European Neuromuscular Centre 2011 research diagnostic criteria [3] and an IBM Functional Rating Scale (IBMFRS) score ≤38. Four dose cohorts are planned: ABC008 0.1, 0.5, 2.0, and 5.0 mg/kg. PD, PK, safety, and disease severity assessments are performed pre-dose (Day 0) and during the 6-month follow-up period.ResultsFive of the 6 (83.3%) participants were male with baseline mean age = 65.7 years, mean IBM disease duration = 6.8 years, and mean IBMFRS score = 27.5 (Table 1). Each received a single dose of ABC008 SC: Cohorts 1 (C1) and 2 (C2) received 0.1 and 0.5 mg/kg and have completed 168 and 56 days of follow-up, respectively.Table 1.Baseline DemographicsBaseline CharacteristicsABC008 SC Treatment GroupsCohort 10.1 mg/kg SC (n=3)Cohort 20.5 mg/kg SC (n=3)ABC008 Overall (N=6)Age (years), mean ± SD64.0 ± 11.3667.3 ± 6.6665.7 ± 8.52Male sex, n (%)3 (100)2 (66.7)5 (83.3)Caucasian3 (100)3 (100)6 (100)Body Mass Index (kg/m2)28.5 ± 3.5828.3 ± 4.2528.4 ± 3.52Disease Duration (years), mean ± SD9.7 ± 5.973.9 ± 4.486.8 ± 5.70IBMFRS score, mean ± SD30.0 ± 4.0825.0 ± 6.1627.5 ± 5.80Abbreviations: IBMFRS, Inclusion Body Myositis Functional Rating Scale; n or N, number; SC, subcutaneous; SD, standard deviation.Maximum depletion of CD8+KLRG1+ cells for C1 and C2 ranged from 46-96% and 98-99%, respectively (Figure 1A), with depletion evident by the next assessment on Day 1. Recovery in C1 began at Day 84 with Day 168 depletion at 29-71%. Other hematologic parameters generally were stable (e.g., T regulatory and B cells). CD8+CD57+ LGLs, mostly KLRG1+, were also depleted (Figure 1B). Preliminary PK showed that ABC008 SC displays a long absorption phase and slow clearance properties typical of mAb therapies. No severe adverse events (AEs) or discontinuations due to AEs have been reported. One unrelated serious AE of fall with muscle tear in a C1 participant with a prior history of falls occurred.Figure 1.Key pharmacodynamic parameters in Study ABC008-IBM-101. Baseline and change (A) of KLRG1+CD8+ T cells and (B) CD8+CD57+ large granular lymphocytes (LGLs). C1 and C2 are Cohorts 1 and 2; P1, P2, and P3 are Participants 1, 2, and 3.ConclusionIn study participants with IBM, single SC doses of 0.1 and 0.5 mg/kg of ABC008 resulted in the depletion of CD8+KLRG1+ cells and CD8+CD57+ LGLs with clear evidence of a dose response for KLRG1+ T cell depletion and no apparent safety signals. Based on these results, a study evaluating ABC008 for the treatment of T-LGLL is planned.

Core fucosylation of N-linked glycans: a novel player in memory CD8+ T cell differentiation following acute viral infection

Abstract Core fucosylation (CF) of N-linked glycans is indispensable for normal physiology, but whether CF regulates any aspect of T cell activation, differentiation, or function remains unknown. Fucosyltransferase 8 (Fut8) is the only enzyme in mammals that catalyzes CF and following activation, both expression of Fut8 and CF synthesis increases on CD8+ T cells. To test the hypothesis that CF of N-glycans regulates memory T cell differentiation following acute viral infection, we generated mice with a floxed Fut8 gene. WT (CD4-Cre−) and Fut8−/− (CD4-Cre+) mice were infected with LCMV Armstrong (LCMV-Arm) and the differentiation of LCMV-specific CD8+ T cells was analyzed by measuring the surface expression of killer-cell lectin like receptor G1 (KLRG1) and the α chain of interleukin 7 receptor (CD127) to identify short-lived effector and memory precursor T cells, respectively. LCMV-specific CD8+ T cells from both WT and Fut8−/− followed the same pattern of expansion and contraction without any significant difference in the overall frequency of LCMV-specific CD8+ T cells. However, Fut8−/− retained a higher percentage of CD127+KLRG1− and a lower percentage of KLRG1+CD127− CD8 T cells throughout the course of infection suggesting CF influences the progression to long-term memory. At day 42 post infection, mice were infected with virulent Listeria monocytogenes expressing the LCMV epitope GP33–41. Both WT and Fut8−/− GP33-specific CD8+ T cells expanded equally. However, Fut8−/− retained a higher frequency of GP33-specific CD8+ T cells and did not undergo contraction until &amp;gt;3 weeks post-2ry challenge. Taken together, our findings suggest that CF of N-linked glycans regulates memory CD8+ T cell differentiation following acute viral infection. Supported by grants from NIH (R21 AI159401)

E3 Ubiquitin Ligase Riplet Is Expressed in T Cells and Suppresses T Cell-Mediated Antitumor Immune Responses.

The E3 ubiquitin ligase Riplet mediates retinoic acid-inducible gene-I polyubiquitination and is essential for viral-induced expression of type I IFNs in dendritic cells and macrophages. The function of Riplet in innate immunity has been well demonstrated; however, its role in adaptive immunity during the antitumor immune response is unclear. In this study, we examined the role of Riplet in the T cell-mediated antitumor immune response. Riplet was expressed in T cells and upregulated in CD8+ T cells in response to TCR-mediated stimulation. Furthermore, PR domain containing 1, eomesodermin, and killer cell lectin-like receptor G1 expression was increased in effector CD8+ T cells by Riplet knockout in vitro, which suggests that Riplet is involved in the effector function of CD8+ T cells. Our results indicated that Riplet deficiency augmented the antitumor response of MO4 (OVA-expressing melanoma)-bearing mice treated with OVA peptide-pulsed dendritic cells. Moreover, both CD4+ and CD8+ T cells played important roles in Riplet-mediated augmentation of the antitumor immune response. In tumor-draining lymph nodes, the Th1 response was promoted, and the induction of OVA-specific CD8+ T cells and IFN-γ production were enhanced by Riplet deficiency. Furthermore, the IFN-γ response and OVA-specific cytotoxicity of CD8+ T cells in tumor tissue were augmented by Riplet deficiency. The expression of Cxcl9fluorescence-minus-one and Cxcl10 mRNA was also enhanced in the tumor microenvironment by Riplet knockout, consistent with the augmented recruitment of CTLs. Overall, we clarified a function of Riplet in T cells, which is to suppress the antitumor immune response through modulating Th1 and CTLs.

Neural-Cadherin Influences the Homing of Terminally Differentiated Memory CD8 T Cells to the Lymph Nodes and Bone Marrow.

Memory T (TM) cells play an important role in the long-term defense against pathogen reinvasion. However, it is still unclear how these cells receive the crucial signals necessary for their longevity and homeostatic turnover. To understand how TM cells receive these signals, we infected mice with lymphocytic choriomeningitis virus (LCMV) and examined the expression sites of neural cadherin (N-cadherin) by immunofluorescence microscopy. We found that N-cadherin was expressed in the surroundings of the white pulps of the spleen and medulla of lymph nodes (LNs). Moreover, TM cells expressing high levels of killer cell lectin-like receptor G1 (KLRG1), a ligand of N-cadherin, were co-localized with N-cadherin+ cells in the spleen but not in LNs. We then blocked N-cadherin in vivo to investigate whether it regulates the formation or function of TM cells. The numbers of CD127hiCD62Lhi TM cells in the spleen of memory P14 chimeric mice declined when N-cadherin was blocked during the contraction phase, without functional impairment of these cells. In addition, when CD127loKLRG1hi TM cells were adoptively transferred into anti–N-cadherin–treated mice compared with control mice, the number of these cells was reduced in the bone marrow and LNs, without functional loss. Taken together, our results suggest that N-cadherin participates in the development of CD127hiCD62Lhi TM cells and homing of CD127loKLRG1hi TM cells to lymphoid organs.

Immune Activation Induces Telomeric DNA Damage and Promotes Short-Lived Effector T Cell Differentiation in Chronic HCV Infection.

Hepatitis C virus (HCV) leads to a high rate of chronic infection and T cell dysfunction. Although it is well known that chronic antigenic stimulation is a driving force for impaired T cell functions, the precise mechanisms underlying immune activation-induced T cell dysfunctions during HCV infection remain elusive. Here, we demonstrated that circulating CD4+ T cells from patients who are chronically HCV-infected exhibit an immune activation status, as evidenced by the overexpression of cell activation markers human leukocyte antigen-antigen D-related, glucose transporter 1, granzyme B, and the short-lived effector marker CD127- killer cell lectin-like receptor G1+ . In contrast, the expression of stem cell-like transcription factor T cell factor 1 and telomeric repeat-binding factor 2 (TRF2) are significantly reduced in CD4+ T cells from patients who are chronically HCV-infected compared with healthy participants (HP). Mechanistic studies revealed that CD4+ T cells from participants with HCV exhibit phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling hyperactivation on T cell receptor stimulation, promoting proinflammatory effector cell differentiation, telomeric DNA damage, and cellular apoptosis. Inhibition of Akt signaling during T cell activation preserved the precursor memory cell population and prevented inflammatory effector cell expansion, DNA damage, and apoptotic death. Moreover, knockdown of TRF2 reduced HP T cell stemness and triggered telomeric DNA damage and cellular apoptosis, whereas overexpression of TRF2 in CD4 T cells prevented telomeric DNA damage. These results suggest that modulation of immune activation through inhibiting Akt signaling and protecting telomeres through enhancing TRF2 expression may open therapeutic strategies to fine tune the adaptive immune responses in the setting of persistent immune activation and inflammation during chronic HCV infection.

Abstract 510: Impact of tisagenlecleucel product attributes on clinical outcomes in pediatric and young adult patients with relapsed or refractory acute lymphoblastic leukemia (r/r ALL)

Abstract Background Tisagenlecleucel CAR-T cell therapy showed durable clinical outcomes in pediatric/young adult patients with r/r ALL in ELIANA/ENSIGN trials. We examined the effect of tisagenlecleucel product attributes on clinical outcomes (Table). Methods Key product attributes of tisagenlecleucel were examined with univariate and multivariate (lasso, random forest) approaches (N=143) to evaluate the effects on efficacy (best overall response [BOR] ≤3 months, duration of remission, event-free survival [EFS], and safety (cytokine release syndrome [CRS] and neurological events). Results Univariate: High in vitro IFNγ release positively correlated with BOR and EFS. Relative and absolute number (no.) of early differentiated (ED) CD4+ and CD8+, activated (HLA-DR+CD38+) CD8+, and absolute no. of naïve CD4+ cells positively correlated with efficacy. Total CD4+ cells with central memory markers, LAG3+, CD25+ had weak positive correlation with grade 3/4 CRS. Relative no. of effector memory CD4+ and KLRG1+CD8+ and senescent CD4+ and CD8+ cells negatively correlated with efficacy. Median T-cell transduction efficiency by flow (23%; range, 1.5%-56%) and viability did not correlate with efficacy or safety. Also, the median CD4:CD8 cell ratio did not correlate with outcomes. Multivariate: ED CD4+ and CD8+, and activated CD8+ cells positively correlated with BOR. Naïve CD4+ cells positively correlated with survival, while KLRG1+CD8+ cells and effector memory CD4+ cells negatively correlated with survival. Conclusions Less mature T cells trended toward improved efficacy, while senescent and differentiated cells led to worse outcomes. No strong correlations to safety outcomes were observed. While phenotypes do not definitively distinguish outcomes, the analyses provide insight into the potential set of parameters to be used in predictive/causal modeling. Table. Univariate analysis of tisagenlecleucel product attributes and clinical outcomesProduct Attributes (CAR+ Cells)Correlation,a +/–BORDOREFSCRS G3/4NE G3/4IFNγ++++Transduction efficiency+Total CD4++++Total CD8+++++ED CD4+ cells+++++++ED CD8+ cells++++++TIM3+ CD4+ cellsTIM3+ CD8+ cells+++Activated CD4+ cells+++Activated CD8+ cells++++++HLA-DR+ CD4+ cells+HLA-DR+ CD8+ cells++++Naïve CD4+ cells++++++Naïve CD8+ cells+LAG3+ CD4+ cells++CD25+ CD4+ cells++Effector memory CD4+ cells– –– – –KLRG1+ CD4+ cells– –KLRG1+ CD8+ cells– – –– –– –Senescent CD4+ cells– – –Senescent CD8+ cells–Key: +++/– – –, P value &amp;lt;0.01; ++/– –, P value &amp;lt;0.05; +/–, P value &amp;lt;0.1 P values refer to Cox regression or Wilcoxon tests. Note: Central memory CD4+ and CD8+, LAG3+ CD8+, CD25+ CD8+ cells and CD4:CD8 ratio were analyzed but showed no significance. aComparison of ≤median to &amp;gt;median. BOR, best overall response; CAR, chimeric antigen receptor; CD, cluster of differentiation;CRS, cytokine release syndrome; DOR, duration of remission; ED, early differentiated;EFS, event-free survival; G, grade; HLA-DR, human leukocyte antigen-DR; IFN, interferon; KLRG1, Killer cell lectin-like receptor G1; LAG3, lymphocyte-activation gene 3; NE, neurological events; TIM3, T-cell immunoglobulin and mucin domain-3. Citation Format: Irina Gershgorin, Edward R. Waldron, Stephan A. Grupp, John E. Levine, Michael A. Pulsipher, Stella M. Davies, G. Doug Myers, Margit Jeschke, Boris Engels, Chris del Corral, Andre Baruchel. Impact of tisagenlecleucel product attributes on clinical outcomes in pediatric and young adult patients with relapsed or refractory acute lymphoblastic leukemia (r/r ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 510.

Functions and clinical significance of KLRG1 in the development of lung adenocarcinoma and immunotherapy

BackgroundAs a marker of differentiation, Killer cell lectin like receptor G1 (KLRG1) plays an inhibitory role in human NK cells and T cells. However, its clinical role remains inexplicit. This work intended to investigate the predictive ability of KLRG1 on the efficacy of immune-checkpoint inhibitor in the treatment of lung adenocarcinoma (LUAD), as well as contribute to the possible molecular mechanisms of KLRG1 on LUAD development.MethodsUsing data from the Gene Expression Omnibus, the Cancer Genome Atlas and the Genotype-Tissue Expression, we compared the expression of KLRG1 and its related genes Bruton tyrosine kinase (BTK), C-C motif chemokine receptor 2 (CCR2), Scm polycomb group protein like 4 (SCML4) in LUAD and normal lung tissues. We also established stable LUAD cell lines with KLRG1 gene knockdown and investigated the effect of KLRG1 knockdown on tumor cell proliferation. We further studied the prognostic value of the four factors in terms of overall survival (OS) in LUAD. Using data from the Gene Expression Omnibus, we further investigated the expression of KLRG1 in the patients with different responses after immunotherapy.ResultsThe expression of KLRG1, BTK, CCR2 and SCML4 was significantly downregulated in LUAD tissues compared to normal controls. Knockdown of KLRG1 promoted the proliferation of A549 and H1299 tumor cells. And low expression of these four factors was associated with unfavorable overall survival in patients with LUAD. Furthermore, low expression of KLRG1 also correlated with poor responses to immunotherapy in LUAD patients.ConclusionBased on these findings, we inferred that KLRG1 had significant correlation with immunotherapy response. Meanwhile, KLRG1, BTK, CCR2 and SCML4 might serve as valuable prognostic biomarkers in LUAD.

FAM13A Regulates KLRG1 Expression and Interferon Gamma Production of Natural Killer Cells

Abstract The polymorphism of the gene FAM13A (family with sequence similarity 13, member A) is strongly linked to the risk of lung cancer and chronic obstructive pulmonary disease, which are among the leading causes of mortality and morbidity in lung-related diseases worldwide. However, the underlying molecular and cellular mechanisms through which FAM13A contributes to the pathogenesis of these diseases largely remain unclear. Here, using a Fam13a knock out (KO) mouse model, we showed that Fam13a depletion upregulated the expression of the terminal differentiation and inhibitory marker, KLRG1 (killer cell lectin-like receptor G1) in natural killer (NK) cells. NK cells from Fam13a-deficient mice showed impaired IFN-γ production either against target tumor cells or following various cytokine cocktail stimulations. Furthermore, the number of lung metastases induced by B16F10 melanoma cells was increased in Fam13a-KO mice. Collectively, our data suggest a key role of FAM13A in regulating NK cell functions, indicating that the key lung-disease risk gene FAM13A might contribute to the pathogenesis of several lung diseases via regulating NK cells.

Targeting androgen signaling in ILC2s protects from IL-33–driven lung inflammation, independently of KLRG1

Allergic asthma is more severe and frequent in women than in men. In male mice, androgens negatively control group 2 innate lymphoid cell (ILC2) development and function by yet unknown mechanisms. We sought to investigate the impact of androgen on ILC2 homeostasis and IL-33-mediated inflammation in female lungs. We evaluated the role of androgen receptor (AR) signaling and the contribution of the putative inhibitory receptor killer cell lectin-like receptor G1 (KLRG1). Subcutaneous pellets mimicking physiological levels of androgen were used to treat female mice together with mice expressing a reporter enzyme under the control of androgen response elements and mixed bone marrow chimeras to assess the cell-intrinsic role of AR activation within ILC2s. We generated KLRG1-deficient mice. We established that lung ILC2s express a functionally active AR that can be invivo targeted with exogenous androgens to negatively control ILC2 homeostasis, proliferation, and function. Androgen signaling upregulated KLRG1 on ILC2s, which inhibited their proliferation on E-cadherin interaction. Despite evidence that KLRG1 impaired the competitive fitness of lung ILC2s during inflammation, KLRG1 deficiency neither alters invivo ILC2 numbers and functions, nor did it lead to hyperactive ILC2s in either sexes. AR agonists can be used invivo to inhibit ILC2 homeostatic numbers and ILC2-dependent lung inflammation through cell-intrinsic AR activation. Although androgen signals in ILC2s to upregulate KLRG1, we demonstrate that KLRG1 is dispensable for androgen-mediated inhibition of pulmonary ILC2s.

Expression and Clinical Significance of KLRG1 and 2B4 on T Cells in the Peripheral Blood and Tumour of Patients with Cervical Cancer

ABSTRACT Background Killer cell lectin-like receptor G1 (KLRG1) and 2B4 play important roles in the immune regulation and immune tolerance to tumor cells by inhibiting T cell function. However, the clinical relevance of KLRG1 and 2B4 to cervical cancer remains to be understood. Methods We measured the frequency of KLRG1+ or 2B4+ cells in CD4+ or CD8 + T cells derived from peripheral blood or tumour biopsies in cervical cancer patients by flow cytometry. Results Compared with healthy controls, the level of KLRG1 and 2B4 on CD8 + T cells in the blood of the patients increased significantly (P = .0056 and .0441). KLRG1 level on CD8 + T cells and 2B4 level on CD4 + T cells in peripheral blood were significantly higher than in tumor tissues (P < .0001 and P = .0003). Higher KLRG1 level on blood-derived CD8 + T cells was observed in patients older than 54 years (P = .001) or tested to be HPV-negative (P = .026). Tumor-infiltrated CD8 + T cells demonstrated elevated KLRG1 level in patients having pelvic lymph node metastasis (P = .016). Increased 2B4 level on blood-derived CD8 + T cells was also observed in patients older than 54 years (P < .001). KLRG1 expression on both CD4 + T (P = .0158) and CD8 + T (P = .0187) cells in the peripheral blood increased after radiotherapy. Conclusion KLRG1 level on T cells was related to age and HPV in patients with cervical cancer, while 2B4 level on T cells was related to age, underlying their roles in the host immune response to cervical cancer. Radiotherapy can improve the immune function of patients.