Skewed T-cell Research Articles

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes.

The domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing. Peripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, alpha/beta T cell receptor transcripts and gamma/delta T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5' reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs. Unsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and gamma/delta T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis demonstrated a skewed T-cell receptor alpha gene usage and a restricted T-cell receptor gamma junctional length in CD8+ cytotoxic T cells compared to other alpha/beta T cell subsets. Among myeloid cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster. Our study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

A phase 3 randomized controlled trial of a COVID-19 recombinant vaccine S-268019-b versus ChAdOx1 nCoV-19 in Japanese adults.

We assessed S-268019-b, a recombinant spike protein vaccine with a squalene-based adjuvant, for superiority in its immunogenicity overChAdOx1 nCoV-19 vaccine among adults in Japan. In this multicenter, randomized, observer-blinded, phase 3 study, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-naïve participants (aged ≥ 18years, without prior infection or vaccination against SARS-CoV-2) were randomized (1:1) to receive either S-268019-b or ChAdOx1 nCoV-19 as two intramuscular injections given 28days apart. Participants who provided consent for a booster administration received S-268019-b at Day 211. The primary endpoint was SARS-CoV-2 neutralizing antibody (NAb) titer on Day 57; the key secondary endpoint was the seroconversion rate for SARS-CoV-2 NAb titer on Day 57. Other endpoints included anti-SARS-CoV-2 S-protein immunoglobulin (Ig)G antibody titer and safety. The demographic and baseline characteristics were generally comparable between S-268019-b (n = 611) and ChAdOx1 nCoV-19 (n = 610) groups. S-268019-b showed superior immunogenicity over ChAdOx1 nCoV-19, based on their geometric mean titers (GMTs) and GMT ratios of SARS-CoV-2 NAb on Day 57 by cytopathic effect assay (GMT [95% confidence interval {CI}] 19.92 [18.68, 21.23] versus 3.63 [3.41, 3.87]; GMT ratio [95% CI] 5.48 [5.01, 6.00], respectively; two-sided p-values < 0.0001). Additionally, NAb measured using a cell viability assay also showed similar results (GMT [95% CI] 183.25 [168.04, 199.84] versus 24.79 [22.77, 27.00]; GMT ratio [95% CI] 7.39 [6.55, 8.35] for S-268019-b versus ChAdOx1 nCoV-19, respectively; p < 0.0001). The GMT of anti-SARS-CoV-2 S-protein IgG antibody was 370.05 for S-268019-b versus 77.92 for ChAdOx1 nCoV-19 on Day 57 (GMT ratio [95% CI] 4.75 [4.34, 5.20]). Notably, immune responses were durable through the end of the study. S-268019-b elicited T-helper 1 skewed T-cell response, comparable to that of ChAdOx1 nCoV-19. After the first dose, the incidence of solicited systemic treatment-related adverse events (TRAEs) was higher in the ChAdOx1 nCoV-19 group, but after the second dose, the incidence was higher in the S-268019-b group. Headache, fatigue, and myalgia were the most commonly reported solicited systemic TRAEs, while pain at the injection site was the most frequently reported solicited local TRAE following both doses in both groups. No serious treatment-related adverse serious TRAEs events were reported in the two groups. S-268019-b was more immunogenic than ChAdOx1 nCoV-19 vaccine and was well tolerated (jRCT2051210151).

P27 Successful treatment of severe eczema and plane warts with Dupilumab in a child with Dedicator of Cytokinesis 8 (DOCK8) deficiency (Autosomal recessive form of hyper-IgE syndrome)

Abstract A six-year-old girl first presented with severe eczema at the age of 6 months, which was difficult to control with topical treatments. She was found to have severely elevated total IgE and multiple food allergies. Due to recurrent ear and chest infections, and persistent varicella zoster, she was screened for a primary immunodeficiency and found to have a homozygous exon 29-46 deletion in the DOCK8 gene. Her eczema remained challenging, with coalescing warts on her limbs. She started Methotrexate with an initial good response; however, the frequency of infections and hospitalizations increased. After the diagnosis of DOCK8 deficiency was confirmed, she started Dupilumab, resulting in the complete resolution of her eczema and warts. She is awaiting haematopoietic stem cell transplant (HSCT) . DOCK8 deficiency is an autosomal recessive hyper-IgE syndrome that presents early in life with severe eczema, skin abscesses, multiple food allergies, recurrent respiratory tract infections, and severe viral (including skin) infections. Older patients invariably develop bronchiectasis and are at high risk of malignancy. Patients have a combined immunodeficiency, with T-cell lymphopenia, reduced thymic output and T-cell skewing to a Th2 response. Children with this condition often present to dermatologists because of severe eczema, which is usually difficult to treat with standard topical treatments. The decision to treat with systemic immunosuppression is often challenging due to recurrent infections. Although HSCT is the curative treatment for DOCK8 deficiency, Dupilumab can be used to reduce inflammation and infections, and improve quality of life in patients awaiting HSCT.

Mustard seed major allergen Sin a1 activates intestinal epithelial cells and also dendritic cells that drive type 2 immune responses.

Mustard seeds belong to the food category of mandatory labelling due to the severe reactions they can trigger in allergic patients. However, the mechanisms underlying allergic sensitization to mustard seeds are poorly understood. The aim of this work is to study type 2 immune activation induced by the mustard seed major allergen Sin a1 via the intestinal mucosa, employing an in vitro model mimicking allergen exposure via the intestinal epithelial cells (IECs). Sin a1 was isolated from the total protein extract and exposed to IEC, monocyte derived dendritic cells (DCs) or IEC/DC co-cultures. A system of consecutive co-cultures was employed to study the generic capacity of Sin a1 to induce type 2 activation leading to sensitization: IEC/DC, DC/T-cell, T/B-cell and stem cell derived mast cells (MCs) derived from healthy donors. Immune profiles were determined by ELISA and flow cytometry. Sin a1 activated IEC and induced type-2 cytokine secretion in IEC/DC co-culture or DC alone (IL-15, IL-25 and TSLP), and primed DC induced type 2 T-cell skewing. IgG secretion in the T-cell/B-cell phase was enhanced in the presence of Sin a1 in the first stages of the co-culture. Anti-IgE did not induce degranulation but promoted IL-13 and IL-4 release by MC primed with the supernatant from B-cells co-cultured with Sin a1-IEC/DC or -DC primed T-cells. Sin a1 enhanced the release of type-2 inflammatory mediators by epithelial and dendritic cells; the latter instructed generic type-2 responses in T-cells that resulted in B-cell activation, and finally MC activation upon anti-IgE exposure. This indicates that via activation of IEC and/or DC, mustard seed allergen Sin a1 is capable of driving type 2 immunity which may lead to allergic sensitization.

CD22 CAR T Cell-Related IEC-HS Is Associated with an IFN-γ Cytokine Signature

Introduction Chimeric antigen receptor T-cell therapy targeting CD22 (CAR22) shows promise for patients with relapsed / refractory B-cell acute lymphoblastic leukemia (ALL) and large B cell lymphoma (LBCL) (Fry et al. Nat Med 2018; Baird et al. Blood 2020). The overall response rate for CAR22 in adults who received prior CD19 CAR therapy is 100% and 68% in ALL and LBCL respectively (Tandem 2023) (NCT04088890). However, 33% of pediatric ALL patients who received CAR22 developed immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS) (Shah et al. JCO 2020). IEC-HS is characterized by hyperferritinemia, cytopenias, coagulopathy and end organ damage. Pediatric ALL data suggest many cytokines including IFN-γ, IL-1β, IL-18 and TNF-α are upregulated in IEC-HS (Ishii et al. JCI 2020, Lichtenstein et al. Blood 2021). Here, we report the clinical features and cytokines associated with IEC-HS after CAR22 infusion in predominantly adult subjects with ALL and LBCL. Methods Plasma was collected weekly from Day 0 to 28 for 54 patients (ALL: n = 16, LBCL: n = 38; adult: n = 51, pediatrics: n = 3) who were treated with CAR22 manufactured using an automated, closed-system Miltenyi CliniMACS Prodigy device. Three dose levels were tested (ALL: 3 x 10 5 cells/kg, LBCL dose level 1 (DL1): 1 x 10 6 cells/kg, LBCL dose level 2 (DL2): 3 x 10 6 cells/kg). Clinical characteristics associated with IEC-HS were retrospectively collected and graded according to consensus criteria (Hines et al. TCT 2023). Eighty cytokines were quantified in batch via multiplexed fluorescence immunoassay (Luminex) to generate mean fluorescent indices (MFIs). Unsupervised hierarchical clustering using Euclidean distance and ward.D2 linkage were employed to analyze cytokine dynamics. Statistical analyses were performed using R (v 4.2.2). Results 8 of 54 (15%) patients developed IEC-HS, all were ≥18 years old. 5 patients had LBCL, 3 had ALL. 5 cases were grade 2, 3 were grade 4. Median onset was D+12 (range 8 - 21). All patients had prior CRS with median onset at D+1 (range 0 - 9). The 3 ALL patients had ongoing CRS and spectral overlap with IEC-HS at time of diagnosis whereas LBCL patients' CRS resolved prior to IEC-HS onset. IEC-HS patients had significant increases in inflammatory markers (ferritin, CRP, LDH, triglycerides), coagulation markers (INR, PTT), cytopenias (hemoglobin, platelets) and transaminases (Table 1). Patients most commonly received high-dose glucocorticoids, tocilizumab and anakinra. Additional agents for grade 4 IEC-HS included anti-thymoglobulin, dasatanib and emapalumab. 5 of 8 patients developed serious infections requiring IV antimicrobials including bacteremia, invasive fungal infections and viral reactivation. 5 patients with IEC-HS died after achieving complete response: 3 due to IEC-HS and concomitant infection, 1 from t-AML and 1 lost to follow up. IEC-HS occurred more frequently at higher doses in LBCL where multiple doses were tested (DL2: 33% vs DL1: 7%, p = 0.08). Peak serum CAR22 concentration was markedly elevated in patients who developed IEC-HS compared to those who did not (median 648 vs 42 cells/µL, p&amp;lt;0.001). Unsupervised hierarchical clustering performed on serial cytokine measurements identified a signature of IFN-γ-related cytokines including CXCL9, CXCL10, TNFα, FasL, IL6 and IL18 as being closely related in IEC-HS (Figure 1). Most cytokines in the signature peaked at D+14, consistent with onset of clinical IEC-HS. Peak levels of nearly all cytokines in the interferon-γ cluster were significantly elevated in IEC-HS patients compared to patients who did not develop IEC-HS, and this cluster contained most cytokines with significant increases in peak levels (Figure 1). Conclusions IEC-HS is a potentially life-threatening toxicity associated with CAR22 across age and underlying malignancy. The high rates of non-relapse mortality and severe infections in this study highlight the importance of understanding the immune dysregulation underlying IEC-HS. Higher CAR22 dose and expansion were associated with IEC-HS, indicating optimal dosing as a potential preventive strategy. The observed IFN-γ cytokine signature is consistent with myeloid cell activation and Th1 T-cell skewing that likely contributes to IEC-HS pathogenesis. These cytokines may be amenable to therapeutic intervention and further study of approaches targeting JAK/STAT and IFN-γ are warranted.

Graft-versus-host disease is associated with skewed γδ T-cell clonality after umbilical cord blood transplantation in children with nonmalignant diseases

Background aimsThe γδ T-cells (GDT) are a subpopulation of lymphocytes expressing a distinct T-cell receptor coded by the TRG and TRD genes. GDTs may have immunoregulatory function after stem cell transplantation (SCT), but the relationship between GDT clonality and acute graft-versus-host disease (aGVHD) is not known. MethodsWe prospectively studied spectratype complex complexity of TCR Vγ (γ) and TCR Vδ (δ) pre-SCT and at approximately day 100 and day 180 post-SCT in a cohort of immunocompetent children receiving allogeneic umbilical cord blood SCT for nonmalignant diseases, with identical reduced-intensity conditioning and aGVHD prophylaxis. ResultsWe studied 13 children undergoing SCT at a median age of 0.9 years (total range 0.4–16.6). In those with grade 0–1 aGVHD (N = 10), the spectratype complexity of most γ and δ genes was not significantly different from baseline at day 100 or day 180 post-SCT, and there was balanced expression of genes at the γ and δ loci. In those with grade 3 aGVHD (N = 3), spectratype complexity was significantly below baseline at day 100 and day 180, and there was relative overexpression of δ2. CD3+ cell counts were also lower in participants with grade 3 aGVHD. ConclusionsRecovery of a polyclonal GDT repertoire is an early part of immunological recovery after SCT. γ and δ gene expression is balanced in young children before and after SCT. Severe aGVHD is associated with GDT oligoclonality post-SCT and with skewed expression of δ2, which has not been previously reported. This association may reflect aGVHD therapy or aGVHD-associated immune dysregulation. Further studies of GDT clonality during the early post-SCT period may establish whether abnormal GDT spectratype precedes the clinical manifestations of aGVHD.

Repercussions of high extracellular K +on T-cell antitumor responses

Abstract Tumours create an immunosuppressive microenvironment that limits the antitumor function of infiltrating T-cells. Dying/necrotic tumor cells extrude intracellular potassium (K +) into the local extracellular milieu, raising the extracellular K +([K +] e) by 8–15 fold, which then accumulates in tumor-infiltrating T-cells. Here, we determined the functional impact of high-[K +] e(50 mM) on human T-cells. Using molecular assays, we demonstrated that prolonged exposure to high-[K +] ereduced CD3ɛ and CD25 (intermediate activation marker) surface expression, suggesting weakened T-cell activation. High-[K +] eperturbed T-cell receptor signalling and the downstream glycolysis pathway. Using metabolomics analysis, we identified dysregulation in pathways involving fatty acid oxidation, TCA cycle and choline metabolism under high-[K +] e. Exposure to high-[K +] eimpeded T-cell motility and skewed T-cell polarization towards T-helper 2 (Th2) and regulatory T-cell (T reg) subsets. A similar Th2 subset trend, although not significant, was seen towards the core of patient-derived tumor biopsies (n=7) along with the amassment of T regcells intratumorally. Collectively, our findings substantiate the immunosuppressive effects of high-[K +] eon T-cell functions, which underlines the potential use of K +efflux channel activators in reversing dampened T-cell antitumor responses. Supported by Singapore Ministry of Education (MOE) under its MOE Academic Research Fund (AcRF) Tier 1 Grant (2020-T1-001-062) and Tier 2 (MOE2017-T2-2-004) grants. #LINK# #ENDLINK#

Clonal Hematopoiesis and the Heart: a Toxic Relationship.

Clonal hematopoiesis (CH) refers to the expansion of hematopoietic stem cell clones and their cellular progeny due to somatic mutations, mosaic chromosomal alterations (mCAs), or copy number variants which naturally accumulate with age. CH has been linked to increased risk of blood cancers, but CH has also been linked to adverse cardiovascular outcomes. A combination of clinical outcome studies and mouse models have offered strong evidence that CH mutations either correlate with or cause atherosclerosis, diabetes mellitus, chronic kidney disease, heart failure, pulmonary hypertension, aortic aneurysm, myocardial infarction, stroke, aortic stenosis, poor outcomes following transcatheter aortic valve replacement (TAVR) or orthotopic heart transplant, death or need of renal replacement therapy secondary to cardiogenic shock, death from cardiovascular causes at large, and enhance anthracycline cardiac toxicity. Mechanistically, some adverse outcomes are caused by macrophage secretion of IL-1β and IL-6, neutrophil invasion of injured myocardium, and T-cell skewing towards inflammatory phenotypes. CH mutations lead to harmful inflammation and arterial wall invasion by bone marrow-derived cells resulting in poor cardiovascular health and outcomes. Blockade of IL-1β or JAK2 signaling are potential avenues for preventing CH-caused cardiovascular morbidity and mortality.

Use of a Novel High-Dimensional Single-Cell Analytical Method for Defining T-cell Skewing Induced by a Molecular Variant of Der p 2

Successful allergen-specific immunotherapy (AIT) induces T-cell responses that suppress Th2 inflammation. However, quantifying T-cell modulatory effects of candidate AIT molecules is challenging using standard in vitro assays, owing to the rarity of allergen-specific T-cells. We describe a high-dimensional single-cell method for quantifying T-cell skewing induced by a variant of Der p 2 (2F10) containing 3 amino acid mutations within a major IgE epitope.

Perinatal Clinical Course of Vici Syndrome Associated with Novel EPG5 Variants: Unique Clinical Course of Fetal Cardiomyopathy and Skewed T-Cell Receptor Diversity

Perinatal Clinical Course of Vici Syndrome Associated with Novel EPG5 Variants: Unique Clinical Course of Fetal Cardiomyopathy and Skewed T-Cell Receptor Diversity

CagA-specific Gastric CD8+ Tissue-Resident T Cells Control Helicobacter pylori During the Early Infection Phase

Background & AimsInfection with Helicobacter pylori strongly affects global health by causing chronic gastritis, ulcer disease, and gastric cancer. Although extensive research into the strong immune response against this persistently colonizing bacterium exists, the specific role of CD8+ T cells remains elusive. MethodsWe comprehensively characterize gastric H pylori–specific CD8+ T-cell responses in mice and humans by flow cytometry, RNA-sequencing, immunohistochemistry, and ChipCytometry, applying functional analyses including T-cell depletion, H pylori eradication, and ex vivo restimulation. ResultsWe define CD8+ T-cell populations bearing a tissue-resident memory (TRM) phenotype, which infiltrate the gastric mucosa shortly after infection and mediate pathogen control by executing antigen-specific effector properties. These induced CD8+ tissue-resident memory T cells (TRM cells) show a skewed T-cell receptor beta chain usage and are mostly specific for cytotoxin-associated gene A, the distinctive oncoprotein injected by H pylori into host cells. As the infection progresses, we observe a loss of the TRM phenotype and replacement of CD8+ by CD4+ T cells, indicating a shift in the immune response during the chronic infection phase. ConclusionsOur results point toward a hitherto unknown role of CD8+ T-cell response in this bacterial infection, which may have important clinical implications for treatment and vaccination strategies against H pylori.

Nickel allergy is associated with a broad spectrum cytokine response.

Nickel-induced proliferation or cytokine release by peripheral blood mononuclear cells may be used for in vitro diagnosis of nickel allergy. Aim of this study was to explore the nickel-specific cytokine profile to further elucidate the pathogenesis of nickel allergic contact dermatitis (ACD) and to identify potential new biomarkers for nickel ACD. Peripheral blood mononuclear cells from patients and controls were cultured with T-cell skewing cytokine cocktails and/or nickel. Cytokine and chemokine concentrations were assessed in culture supernatants using validated multiplex assays. Specific cytokine production was related to history of nickel allergy and patch-test results. Twenty-one of the 33 analytes included in the analysis were associated with nickel allergy and included type1 (TNF-α, IFN-γ, TNF-β), type 2 (IL-3, IL-4, IL-5, IL-13), type 1/2 (IL-2, IL-10), type 9 (IL-9), type 17/1 (IL-17A[F], GM-CSF, IL-21) and type 22 (IL-22) derived cytokines as well as the T-cell/antigen presentation cell derived factors Thymus and activation regulated chemokine (TARC), IL-27 and IP-10. Receiver operator characteristics (ROC) analysis showed that IL-5 was the strongest biomarker for nickel allergy. A broad spectrum of 33 cytokines and chemokines is involved in the allergen-specific immune response in nickel allergic patients. IL-5 remains, next to the lymphocyte proliferation test, the strongest biomarker for nickel allergy.

Combined ibrutinib and venetoclax treatment vs single agents in the TCL1 mouse model of chronic lymphocytic leukemia

AbstractThe covalent inhibitor of Bruton's tyrosine kinase ibrutinib and the specific Bcl-2 inhibitor venetoclax are both highly efficacious single-agent drugs in the treatment of chronic lymphocytic leukemia (CLL). Based on their complementary modes of action, ibrutinib and venetoclax are hypothesized to act in a synergistic fashion. Currently, it is unclear whether combined treatment is indeed superior to continuous single-agent treatment and what mechanisms underlie the resistance to combination treatment. In addition, the effects of such treatment on the skewed T-cell compartment characteristic of CLL are as yet unknown. In the murine Eµ-TCL1 adoptive transfer model resembling aggressive CLL, we found that combined treatment resulted in the deepest responses, with the longest duration related to a combination of decreased proliferation and increased induction of apoptosis. In addition, alterations in T-cell subsets were most prominent after combination treatment, with increased naive cells and reduced effector memory cells. Remarkably, effects of single agents but also combination treatment were eventually interrupted by relapse, and we found downregulation of BIM expression as a plausible cause of acquired drug resistance. Nevertheless, in this murine model, the combination of venetoclax and ibrutinib has increased efficacy over single agents, accompanied by a restoration of the T-cell compartment.

Abstract 1719: OR2805, an anti-CD163 antibody derived from an elite responder to checkpoint inhibitor therapy relieves immunosuppression caused by tumor associated macrophages

Abstract Background: OR2805 is a fully human IgG1 antibody that binds to CD163, an immune-suppressive receptor highly expressed on tumor associated macrophages (TAMs). High numbers of CD163-expressing TAMs generally predict an unfavorable prognosis in solid tumors. CD163-expressing TAMs contribute to an immune-suppressive tumor microenvironment (TME) and inhibit an anti-tumor T-cell response by engaging immune checkpoints, producing immune-suppressive cytokines, and promoting T-cell skewing towards a pro-cancer Th2 phenotype. Relieving the immune suppression of CD163-expressing TAMs in the TME to improve T-cell-mediated responses is a rational adjunct to immune checkpoint inhibitor (CPI) therapy. Methods: OR2805 was discovered using OncoResponse's discovery platform by cell-based functional and phenotypic assays. B cells derived from CPI elite responders were cultured at clonal density, and IgG antibodies in supernatants were evaluated for binding to myeloid-derived suppressor cells. Variable-regions from positive hits were sequenced, cloned, and expressed as recombinant IgG1. OR2805 was identified as one of the top hits that relieved myeloid cell-mediated immune suppression. Co-cultures of immunosuppressive primary human M2 macrophages and autologous T cells were used to interrogate OR2805-dependent immunomodulatory responses in vitro. The anti-tumor activity of OR2805 in vivo was evaluated in a humanized mouse model. Results: OR2805 binds to CD163 expressed on TAMs and M2-like macrophages. OR2805 does not bind to other hematopoietic cells nor to a panel of human primary non-immune cells. OR2805 treatment reduces expression of cell-surface markers associated with tumor-promoting M2c-like macrophages. In co-culture assays, OR2805 relieves the suppressive effect of M2 macrophages and results in increased T-cell activation and proliferation, upregulation of T-cell activation markers, and enhanced T-cell-mediated tumor cell killing. Administration of OR2805 in humanized NSG-SGM3 mouse tumor models resulted in approximately 55% and 75% reduction in A549 tumor growth and NCI-H1975 tumor growth, respectively. In this model, OR2805 treatment significantly increased the proportions of human CD8+ T cells and human CD11b+ myeloid cells, as well as significantly enhanced expression of activation markers by human CD8+ T cells. Conclusions: OR2805 reduces M2 macrophage-mediated immunosuppression and enhances anti-tumor immune responses. OR2805 treatment induces anti-tumor activity in lung cancer xenograft models in humanized mice. These data support further development of OR2805 as an anti-cancer therapy, both as a monotherapy and an addition to current CPI therapy. Citation Format: Peter Probst, Randi Simmons, Valerie Wall, Meghan Zuck, Myriam Bouchlaka, Sam Lam, Raymond Fox, Darbie Whitman, Tom Graddis, Kamal D. Puri. OR2805, an anti-CD163 antibody derived from an elite responder to checkpoint inhibitor therapy relieves immunosuppression caused by tumor associated macrophages [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 1719.

Daratumumab Decreases Treg-Mediated Immunosuppression and Potentiates CD8+ T-Cell-Induced Killing of Chronic Lymphocytic Leukemia (CLL) Cells Ex Vivo

Introduction: CD38 is expressed on the plasma membrane of lymphoid cells and in chronic lymphocytic leukemia (CLL) pts. increased CD38 expression is associated with a poor clinical outcome. While its role as a prognostic marker has been well established, the approval of daratumumab (Dara, first-in-class anti-CD38 mAb) offers a new role for CD38 as a potential therapeutic target in CLL. Matas-Cespedes et al have reported that Dara induces CLL cell death through antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP). While CD38 is expressed on CLL cells, it is also expressed on T-cell subsets, such as Tregs. We hypothesized that in addition to its direct immune-mediated activity, Dara modulates the immune cellular environment; skewing T-cell populations from an immunosuppressive towards immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.Methods: CD19+ cells were isolated from PBMCs of CLL pts. (n=30). Pts. with ≥30% CD38+ CLL cells were classified as CD38+ (n=22) and pts. with <30% CD38+ cells as CD38- (n=8). Dara-induced ADCC was assessed in Calcein AM labelled CLL cells cocultured with healthy donor PBMCs (E:T ratio, 50:1); CDC using 10% serum from a healthy donor and ADCP using human macrophages (E:T ratio, 2:1). Apoptosis was determined by annexin-V/PI staining followed by flow cytometry. Regulatory T-cells (Treg; CD3+CD4+CD25+CD127loFoxP3+) and cytotoxic effector T-cells (cTL, CD3+CD8+) were identified and isolated by FACS. Data are represented as mean±SEM.Results: Dara (0.1ug/mL) induced significantly greater ADCC in CD38+ (24.63±3.46%) vs. CD38- cells (14.11±1.11%) (p<0.001), whereas CDC (18.22±2.41 vs. 13.58±0.95) and ADCP (9.37±0.77% vs. 8.79±0.92%) were marginally, but insignificantly, increased in CD38+ vs. CD38- CLL cases. We also observed significantly greater apoptosis in CD38+ (36.41±3.34%) vs. CD38- CLL cells (23.39±1.70%) (p<0.005) (Fig 1A). Immune repertoire analysis revealed lower CD3+ T-cell numbers in CLL pts. (11.45±3.25%) compared to those in healthy donor PBMCs (23.64±7.36%); but with a higher percentage of Tregs (10.71±2.47 in CLL pts. vs. 1.96±0.60 in healthy donors). To determine whether Dara could decrease CLL-Tregs, we treated CLL-PBMCs withDara (10µg/ml; 5 days) ex vivo, and noted significant decrease in Treg cells from 12.75±1.33 (Isotype control) to 7.17±0.91, (n=5, p<0.001). As downregulation in Tregs can consequentially lead to a rise in other T-cell subtypes, we observed increased effector cTLs: from 50.08±5.41 to 64.53±3.19 (p<0.02) (Fig 1B). Further analysis on CFSE-labeled CLL-cTLs (primed or unprimed with Dara), showed cTLs proliferated significantly more so when co-cultured with Dara-treated apoptotic CLL (51.75±1.16) cells vs. ibrutinib (31.44±0.36) or rituximab-treated apoptotic CLL cells (36.25±3.77) or live CLL cells (7.04±3.34) (Fig 1C, representative CLL pt.). To determine if Dara-primed cTLs had enhanced cytolytic aptitude towards autologous CLL cells, we cocultured primed cTLs with CLL cells. Compared to unprimed cTLs, specific lysis of target CLL cells was greater in the primed cTL-CLL coculture (12.61±1.75% vs. 28.27±0.82%, respectively). And this effect was noted to be patient-specific as cTLs cultured with JVM13 (CD38+) cells showed lower specific lysis (Fig 1D, E).Conclusion: Here we demonstrate for the first time that Dara modulates CLL-Treg levels and increases effector cTL proliferation. Moreover, Dara-primed cTLs display heightened CLL-specific cytolytic activity than compared to Dara-naïve cTLs. Dara also induces direct apoptosis of CLL cells and exposure of Dara-killed CLL cells to cTLs increases cTL proliferation significantly more so than cTL exposure to CLL cells apoptosed using ibrutinib or rituximab. Altogether, our findings highlight the multiple mechanisms by which Dara (and potentially other anti-CD38 agents, currently being investigated by us) can induce CLL cell death and potentially revert dysfunctional cTL activity from that of a pro-CLL to an anti-CLL composure. [Display omitted] DisclosuresParikh:AstraZeneca: Honoraria; Pharmacyclics: Research Funding; Pharmacyclics: Honoraria. Ding:Merck: Research Funding. Sher:LAM Therapeutics, Inc: Research Funding. Malavasi:Jaansen Pharmaceuticals: Research Funding. Ailawadhi:Amgen: Consultancy, Honoraria; Pharmacyclics: Research Funding; Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Early HHV-6 Reactivation in CMV-Seronegative Cord Blood Transplant Recipients Is Associated with Inferior Relapse-Free and Overall Survival

IntroductionHHV-6 reactivation has been associated with transplant complications such as graft failure, acute GVHD, and CMV reactivation. However, the causal relationships and the involved mechanisms remain largely speculative. In addition, as the immunosuppressive settings that promote HHV-6 reactivation may also increase the risk of reactivation of other viruses (e.g. CMV), determining the independent effect of HHV-6 is difficult. In this retrospective study, we focused on early reactivations (until day 30 post-transplant) and their impact on relapse because of the well-established immunosuppressive effect of HHV-6 on the innate and adaptive immune system, which could theoretically weaken the graft-versus-tumor (GvT) effect, resulting in more relapse and worse survival.MethodsTo eliminate potential confounders and address the independent effect of HHV-6 reactivation, we restricted our population to CMV-seronegative recipients of UCB (2011-2017) and excluded those with DNA virus reactivations other than HHV-6 (CMV, adenovirus, EBV, and BK virus). HHV-6 reactivation was defined as ≥1 positive quantitative DNA PCR on whole blood within 30 days post-transplant. The absence of HHV-6 reactivation was defined as ≥1 negative PCR in the first 2 weeks and ≥1 negative PCR in the second 2 weeks post-transplant. A subset of patients had research blood samples collected on day 30, which we used to measure functionally relevant T-cell subsets by flow cytometry.Results54 patients were included (41 with and 13 without HHV-6 reactivation; Table 1). The groups were similar except more malignant disorders in those with HHV-6 reactivation, P= 0.02). The HHV-6 reactivation group had a lower RFS (38 [22-54]% vs. 88 [39-98]%, P= 0.03; Figure 1A), lower OS (46 [29-61]% vs. 92 [54-99]%, P= 0.01; Figure 1B), and more relapse (31 [15-47]% vs. 0%, P= 0.07; Figure 1C) at 2 years plus a trend for higher 2-year NRM (28 [14-43]% vs. 8 [0-23]%, P= 0.10; Figure 1D). For the latter two results, only patients with malignant hematological disorders were included. Neutrophil recovery, graft failure, acute GVHD grade II-IV, and chronic GVHD were similar in both groups.Next, we evaluated day 30 blood T-cell immunophenotype focusing on 3 markers with potential relevance to T-cell function: (i) CD57 (associated with T-cell replicative senescence), (ii) NKG2a (associated with resistance to immunotherapy in solid tumors), and (iii) KIR2DL2/3 (known to prematurely end the immune synapse and inhibit sustained TCR-induced gene transcription resulting in decreased IFN-γ production). The relative abundances of T cells expressing CD57, NKG2a,or KIR2KL2/3 were all significantly higher in the group with HHV-6 reactivation (P < 0.05). Interestingly, the abundance of NKG2c+CD57+ cells among NKG2a+ NK cells (so called “adaptive” NK cells) was marginally significantly higher in the group with HHV-6 reactivation (P= 0.07).ConclusionsPrevious studies have reported higher NRM in patients who reactivate more DNA viruses post-transplant (Hill J, et al. Blood 2017). We demonstrated an independent association between early HHV-6 reactivation and worse RFS/OS, attributed in part to relapse. This was consistent with the observed skewed T-cell repertoire in the group with HHV-6 reactivation, which showed markers of decreased immune effector function, possibly reflecting an attenuated GvT effect. Although the relative abundance of adaptive NK cells was somewhat higher in the group with HHV-6 reactivation (reminiscent of CMV reactivation), this did not seem to rescue the GvT effect which was apparently impaired due to T-cell defects. If confirmed in larger cohorts, rigorous prophylaxis and/or HHV-6 vaccination may be explored to decrease the risk of relapse and improve survival. [Display omitted] DisclosuresMiller:Celegene: Consultancy; Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding.

Targeting CD38 is lethal to Breg-like chronic lymphocytic leukemia cells and Tregs, but restores CD8+ T-cell responses

AbstractPatients with chronic lymphocytic leukemia (CLL) are characterized by monoclonal expansion of CD5+CD23+CD27+CD19+κ/λ+ B lymphocytes and are clinically noted to have profound immune suppression. In these patients, it has been recently shown that a subset of B cells possesses regulatory functions and secretes high levels of interleukin 10 (IL-10). Our investigation identified that CLL cells with a CD19+CD24+CD38hi immunophenotype (B regulatory cell [Breg]–like CLL cells) produce high amounts of IL-10 and transforming growth factor β (TGF-β) and are capable of transforming naive T helper cells into CD4+CD25+FoxP3+ T regulatory cells (Tregs) in an IL-10/TGF-β-dependent manner. A strong correlation between the percentage of CD38+ CLL cells and Tregs was observed. CD38hi Tregs comprised more than 50% of Tregs in peripheral blood mononuclear cells (PBMCs) in patients with CLL. Anti-CD38 targeting agents resulted in lethality of both Breg-like CLL and Treg cells via apoptosis. Ex vivo, use of anti-CD38 monoclonal antibody (mAb) therapy was associated with a reduction in IL-10 and CLL patient-derived Tregs, but an increase in interferon-γ and proliferation of cytotoxic CD8+ T cells with an activated phenotype, which showed an improved ability to lyse patient-autologous CLL cells. Finally, effects of anti-CD38 mAb therapy were validated in a CLL–patient-derived xenograft model in vivo, which showed decreased percentage of Bregs, Tregs, and PD1+CD38hiCD8+ T cells, but increased Th17 and CD8+ T cells (vs vehicle). Altogether, our results demonstrate that targeting CD38 in CLL can modulate the tumor microenvironment; skewing T-cell populations from an immunosuppressive to immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.

Autoimmune Cytopenias in Chronic Lymphocytic Leukemia: Focus on Molecular Aspects.

Autoimmune cytopenias, particularly autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP), complicate up to 25% of chronic lymphocytic leukemia (CLL) cases. Their occurrence correlates with a more aggressive disease with unmutated VHIG status and unfavorable cytogenetics (17p and 11q deletions). CLL lymphocytes are thought to be responsible of a number of pathogenic mechanisms, including aberrant antigen presentation and cytokine production. Moreover, pathogenic B-cell lymphocytes may induce T-cell subsets imbalance that favors the emergence of autoreactive B-cells producing anti-red blood cells and anti-platelets autoantibodies. In the last 15 years, molecular insights into the pathogenesis of both primary and secondary AIHA/ITP has shown that autoreactive B-cells often display stereotyped B-cell receptor and that the autoantibodies themselves have restricted phenotypes. Moreover, a skewed T-cell repertoire and clonal T cells (mainly CD8+) may be present. In addition, an imbalance of T regulatory-/T helper 17-cells ratio has been involved in AIHA and ITP development, and correlates with various cytokine genes polymorphisms. Finally, altered miRNA and lnRNA profiles have been found in autoimmune cytopenias and seem to correlate with disease phase. Genomic studies are limited in these forms, except for recurrent mutations of KMT2D and CARD11 in cold agglutinin disease, which is considered a clonal B-cell lymphoproliferative disorder resulting in AIHA. In this manuscript, we review the most recent literature on AIHA and ITP secondary to CLL, focusing on available molecular evidences of pathogenic, clinical, and prognostic relevance.

Increased frequency of interleukin-4 and reduced frequency of interferon-γ and IL-17-producing CD4+ and CD8+ cells in scleromyxedema.

Little is known about the pathogenesis of scleromyxedema, a life-threatening fibromucinosis disease with immunological dysregulation. To investigate on T-cell phenotype, function and cytokine biology in search of new insights supporting the immunopathogenesis of the disease. We analysed the frequency of circulating lymphocyte subsets, the T-cell maturation stage, the generation of antigen-specific T-cell lines and T-cell cytokine secretion. The analysis of T-cell maturation stage and the TCR spectratyping findings revealed that scleromyxedema patients showed clear immunological signs of long-lasting immune system activation and stimulation leading to a skewed T-cell repertoire. Moreover, these analyses showed that both CD4+ and CD8+ T cells from scleromyxedema patients have a profound deficiency (even after stimulation) relatively to the production of IFN-γ and IL17 with respect to healthy donor control cells, while they are massively skewed towards IL4 secretion after stimulation. Our data indicate that a chronic Th2-skewed T-cell response against an unknown target antigen leading to abnormally high IL4 secretion, a pro-fibrotic cytokine, is a main immunological hallmark of scleromyxedema patients. These results, never reported before, may have a translational therapeutic value due to the availability of anti-IL4 agents such as dupilumab.

B-cell–specific IRF4 deletion accelerates chronic lymphocytic leukemia development by enhanced tumor immune evasion

AbstractChronic lymphocytic leukemia (CLL) is a heterogenous disease that is highly dependent on a cross talk of CLL cells with the microenvironment, in particular with T cells. T cells derived from CLL patients or murine CLL models are skewed to an antigen-experienced T-cell subset, indicating a certain degree of antitumor recognition, but they are also exhausted, preventing an effective antitumor immune response. Here we describe a novel mechanism of CLL tumor immune evasion that is independent of T-cell exhaustion, using B-cell–specific deletion of the transcription factor IRF4 (interferon regulatory factor 4) in Tcl-1 transgenic mice developing a murine CLL highly similar to the human disease. We show enhanced CLL disease progression in IRF4-deficient Tcl-1 tg mice, associated with a severe downregulation of genes involved in T-cell activation, including genes involved in antigen processing/presentation and T-cell costimulation, which massively reduced T-cell subset skewing and exhaustion. We found a strong analogy in the human disease, with inferior prognosis of CLL patients with low IRF4 expression in independent CLL patient cohorts, failed T-cell skewing to antigen-experienced subsets, decreased costimulation capacity, and downregulation of genes involved in T-cell activation. These results have therapeutic relevance because our findings on molecular mechanisms of immune privilege may be responsible for the failure of immune-therapeutic strategies in CLL and may lead to improved targeting in the future.