Patient PBMCs Research Articles

CXCL12-loaded-hydrogel (CLG): A new device for metastatic circulating tumor cells (CTCs) capturing and characterization

BackgroundCirculating Tumor Cells (CTCs) represent a small, heterogeneous population that comprise the minority of cells able to develop metastasis. To trap and characterize CTCs with metastatic attitude, a CXCL12-loaded hyaluronic-gel (CLG) was developed. CXCR4+cells with invasive capability would infiltrate CLG. MethodsHuman colon, renal, lung and ovarian cancer cells (HT29, A498, H460 and OVCAR8 respectively) were seeded on 150 μl Empty Gels (EG) or 300 ng/ml CXCL12 loaded gel (CLG) and allowed to infiltrate for 16 h. Gels were then digested and fixed with 2 % FA-HAse for human cancer cell enumeration or digested with HAse and cancer cells recovered. CLG-recovered cells migrated toward CXCL12 and were tested for colonies/spheres formation. Moreover, CXCR4, E-Cadherin and Vimentin expression was assessed through flow cytometry and RT-PCR. The clinical trial “TRAP4MET” recruited 48 metastatic/advanced cancer patients (8 OC, 8 LC, 8 GBM, 8 EC, 8 RCC and 8 EC). 10 cc whole blood were devoted to PBMCs extraction (7 cc) and ScreenCell™ filters (3 cc) CTCs evaluation. Ficoll-isolated patient's PBMCs were seeded over CLG and allowed to infiltrate for 16 h; gels were digested and fixed with 2 % FA-HAse, cells stained and DAPI+/CD45-/pan-CK + cells enumerated as CTCs. ResultsHuman cancer cells infiltrate CLG more efficiently than EG (CLG/EG ratio 1.25 for HT29/1.58 for A498/1.71 for H460 and 2.83 for OVCAR8). CLG-recovered HT29 cells display hybrid-mesenchymal features [low E-cadherin (40 %) and high vimentin (235 %) as compared to HT29], CXCR4 two-fold higher than HT29, efficiently migrate toward CXCL12 (two-fold higher than HT29) and developed higher number of colonies (171 ± 21 for HT29-CLG vs 131 ± 8 colonies for HT29)/larger spheres (spheroid area: 26561 ± 6142 μm2 for HT29-CLG vs 20297 ± 7238 for HT29). In TRAP4MET clinical trial, CLG-CTCs were isolated in 8/8 patients with OC, 6/8 with LC, 6/8 with CRC, 8/8 with EC, 8/8 with RCC cancer and 5/8 with GBM. Interestingly, in OC, LC and GBM, CLG isolated higher number of CTCs as compared to the conventional ScreenCell™ (CLG/SC ratio = 1.88 for OC, 2.47 for LC and 11.89 for GBM). Bland and Altman blot analysis and Passing and Bablok regression analysis showed concordance between the methodological approaches but indicate that SC and CLG are not superimposable suggesting that the two systems select cells with different features. ConclusionCLG might represent a new and easy tool to isolate invasive CTCs in multiple cancers such as OC, LC and GBM at today orphan of reliable methods to consistently detect CTCs.

Inflammatory risk contributes to post-COVID endothelial dysfunction through anti-ACKR1 autoantibody.

Subclinical vascular impairment can be exacerbated in individuals who experience sustained inflammation after COVID-19 infection. Our study explores the prevalence and impact of autoantibodies on vascular dysfunction in healthy COVID-19 survivors, an area that remains inadequately investigated. Focusing on autoantibodies against the atypical chemokine receptor 1 (ACKR1), COVID-19 survivors demonstrated significantly elevated anti-ACKR1 autoantibodies, correlating with systemic cytokines, circulating damaged endothelial cells, and endothelial dysfunction. An independent cohort linked these autoantibodies to increased vascular disease outcomes during a median 6.7-yr follow-up. We analyzed a single-cell transcriptome atlas of endothelial cells from diverse mouse tissues, identifying enriched Ackr1 expressions in venous regions of the brain and soleus muscle vasculatures, which holds intriguing implications for tissue-specific venous thromboembolism manifestations reported in COVID-19. Functionally, purified immunoglobulin G (IgG) extracted from patient plasma did not trigger cell apoptosis or increase barrier permeability in human vein endothelial cells. Instead, plasma IgG enhanced antibody-dependent cellular cytotoxicity mediated by patient PBMCs, a phenomenon alleviated by blocking peptide or liposome ACKR1 recombinant protein. The blocking peptide uncovered that purified IgG from COVID-19 survivors possessed potential epitopes in the N-terminal extracellular domain of ACKR1, which effectively averted antibody-dependent cellular cytotoxicity. Our findings offer insights into therapeutic development to mitigate autoantibody reactivity in blood vessels in chronic inflammation.

HBV immune checkpoint receptor CD33 (Siglec-3) is a promising therapeutic target for chronic hepatitis B infection

Abstract Approximately 300 million people globally suffer from chronic hepatitis B (CHB), leading to complications like hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. Despite a significant reduction in new HBV infections through vaccination, a definitive CHB cure remains elusive. Current treatments rely on antiviral nucleos(t)ide analogues, often failing to induce crucial anti-HBsAg antibody (HBsAb) production. Our prior study unveiled myeloid CD33 (Siglec-3) as an immune checkpoint receptor for HBV, contributing to HBV-induced tolerance. Blocking CD33 with anti-hCD33 mAb enhances antigen-presenting molecule expression and activation markers in dendritic cells, restoring TLR7 agonist-induced cytokine production in CHB patient PBMCs. We recently identified a novel CD19+ B cell population expressing abundant CD33 in CHB patient PBMCs (5-20% of CD19+ B cells), contrasting with rare detection in healthy donors (0.5-3%). Anti-hCD33 mAb treatment induces HBsAb production in PBMCs and CD19+ B cells. Furthermore, establishing a CHB mouse model via pAAV-HBV-1.2 hydrodynamic injection in human CD33 transgenic mice (hCD33 Tg) demonstrated prolonged HBsAg persistence compared to wild-type mice, suggesting human CD33 impedes HBV clearance. Remarkably, anti-hCD33 mAb administration accelerates HBsAg clearance in hCD33 Tg mice, highlighting CD33 as a promising CHB therapeutic target, with anti-hCD33 mAb potential to reactivate host immunity for HBV clearance in CHB patients.

KLRB1 defines an activated phenotype of CD4+ T cells and shows significant upregulation in patients with primary Sjögren's syndrome

ObjectiveThis study aimed to investigate the role of KLRB1 (CD161) in human CD4+ T cells and elucidate its significance in primary Sjögren's syndrome (pSS). MethodsPeripheral blood samples from 37 healthy controls and 44 pSS patients were collected. The publicly available single-cell RNA-Seq data from pSS patient PBMCs were utilized to analyse KLRB1 expression in T cells. KLRB1-expressing T lymphocyte subset proportions in pSS patients and healthy controls were determined by flow cytometry. CD25, Ki-67, cytokine secretion, and chemokine receptor expression in CD4+ KLRB1+ T cells were detected and compared with those in CD4+ KLRB1- T cells. Correlation analysis was conducted between KLRB1-related T-cell subsets and clinical indicators. ROC curves were generated to explore the diagnostic potential of KLRB1 for pSS. ResultsKLRB1 was significantly upregulated following T-cell activation, and Ki-67 and CD25 expression was significantly greater in CD4+ KLRB1+ T cells than in CD4+ KLRB1- T cells. KLRB1+ CD4+ T cells exhibited greater IL-17A, IL-21, IL-22, and IFN-γ secretion upon stimulation, and there were significantly greater proportions of CCR5+, CCR2+, CX3CR1+, CCR6+, and CXCR3+ cells among CD4+ KLRB1+ T cells than among CD4+ KLRB1- T cells. Compared with that in HCs, KLRB1 expression in CD4+ T cells was markedly elevated in pSS patients and significantly correlated with clinical disease indicators. ConclusionKLRB1 is a characteristic molecule of the CD4+ T-cell activation phenotype. The increased expression of KLRB1 in the CD4+ T cells of pSS patients suggests its potential involvement in the pathogenesis of pSS and its utility as an auxiliary diagnostic marker for pSS.

Multiple inborn errors of type I IFN immunity in a 33-year-old male with a fatal case of COVID-19

The host genetic inborn errors of immunity (IEIs) have been shown to contribute to susceptibility to life-threatening coronavirus disease 2019 (COVID-19), as it had been associated previously with other viral infections. Most genetic association studies have described IEIs as a monogenic defect, while there have been no reports of patients with multiple inherited immune deficiencies. This is a complex case of IEIs predisposing to severe viral infections in an unvaccinated 33-year-old male patient. The patient was admitted with no respiratory symptoms, showed a SARS-CoV-2 PCR positive test on the second day of admission, started developing progressive lung consolidation within three days of hospitalization, and was moved from non-invasive to mechanical ventilation within 12 days of hospitalization. Impaired production of type I IFN was detected in patient PBMCs treated with poly(I:C), at both mRNA and protein levels. Whole exome sequencing revealed three mutations across type I IFN production pathway, which were predicted to be loss-of-function (pLOF). The three mutations were predicted to predispose to severe viral infections: monoallelic R488X TLR3, monoallelic His684Arg TLR3, and biallelic Val363Met IRF3. Functional analysis confirmed that all these mutations dysregulated the type I IFN pathway. Evaluation of TLR3 and IRF3 IFN-β1 luciferase reporter activity showed a hypomorphic suppression of function. TOPO TA cloning was used to ascertain the positioning of both TLR3 variants, indicating that both variants were on the same allele. We have described a unique complex IEI patient with multiple mutations, particularly along type I IFN production pathway.

Abstract 4092: Characterizing the endogenous retroviral envelope protein ERVMER34-1 as a target for a therapeutic cancer vaccine

Abstract Endogenous retroviruses (ERVs) are remnants of retroviral germline infections which occurred over the course of evolution and constitute between 5-8% of the human genome. Although ERV protein expression is typically silenced epigenetically in normal adult human tissues, previous studies have shown that some ERVSs, can be aberrantly expressed in multiple cancer types. This atypical expression of ERVs in tumors may represent a class of immunologically relevant tumor-associated antigens that can potentially be targeted clinically using therapeutic cancer vaccines. For the first time, this study evaluates the ERV envelope protein ERVMER34-1 as a potential target for a therapeutic cancer vaccine. We first assessed the expression of ERVMER34-1 in multiple tissues and found that ERVMER34-1 protein is absent in most healthy adult tissues and over expressed in multiple types of human carcinomas. ERVMER34-1 specific T-cells were detected in PBMCs of cancer patients but not healthy donors following an overnight stimulation. However, reactive T-cells are readily expanded from both healthy donor and cancer patient PBMCs following a 7-day in-vitro stimulation. Importantly, reactive CD8+ T cells selectively killed human carcinoma cells expressing the target antigen. Based on these findings, we rationally designed a ERVMER34-1 therapeutic cancer vaccine and assessed its immunogenicity and anti-tumor efficacy in established syngeneic murine tumors expressing the full-length ERVMER34-1 protein. Our rationally designed ERVMER34-1 cancer vaccine significantly mediated tumor regression and extended survival as a monotherapy and in combination with checkpoint blockade. Altogether, our work supports the clinical development of a therapeutic cancer vaccine targeting the retroviral envelope protein ERVMER34-1 as a potential first-in-class immunotherapeutic target for the therapy of several carcinoma types. Citation Format: Maria Del Mar Maldonado, Masafumi Iida, Maria Del Mar Gracia Hernandez, Loc Le, Renee N. Donahue, Claudia Palena, Jeffrey Schlom, Duane H. Hamilton. Characterizing the endogenous retroviral envelope protein ERVMER34-1 as a target for a therapeutic cancer vaccine [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 4092.

A Novel Homozygous Germline Mutation in Transferrin Receptor 1 (TfR1) Leads to Combined Immunodeficiency and Provides New Insights into Iron-Immunity Axis

A homozygous missense mutation in the transferrin receptor 1 (TfR1), also known as CD71, leads to a rare inborn error of immunity (IEI) characterized by the impaired lymphocyte activation and proliferation due to defective iron uptake of cells. However, only one causative mutation (c.58T > C, p.Y20H) in the TFRC gene coding for TfR1 has been reported so far. We herein identified a new disease-causing homozygous germline mutation in the TFRC gene (c.64C > T, p.R22W) (referred to as TfR1R22W from now on) in a Turkish patient with combined immunodeficiency (CID). TfR1R22W results in impaired TfR1 internalization similar to previously defined TfR1Y20H mutation. We found that TfR1R22W is associated with severely restricted B and T lymphocyte clonal diversity and impaired T cell activation and cytokine production as well as defective mitochondrial oxidative phosphorylation in helper T cells. In addition, circulating NK, Treg, and MAIT cell populations were significantly decreased in the patient. Using whole transcriptome analysis, we found dysregulated immune homeostasis and novel biological processes associated with TfR1R22W. We also identified a considerable expansion of circulating low-density neutrophils (LDNs) in patient’s PBMCs. Overall, TfR1R22W mutation expands the current understanding of the IEI associated with TfR1 dysfunction and provides new insights underlying impaired immune function, lymphocyte diversity, and granulocyte homeostasis.

Organochlorine pesticides disrupt T helper cell regulation and reduce IL-2 and IFN-γ favoring infection and production of autoantibodies among pemphigus patients

The list of environmental factors that trigger autoimmune diseases in genetically susceptible individuals has grown in the recent years and is far from complete. The possible intervention of the environment in triggering these diseases is ever more perceived by the clinicians. This study investigated the effect of environmental factors like organochlorine pesticides (OCPs) on proportions of different T lymphocyte subsets and their cytokine secretion in-vitro among pemphigus patients, before and after specific immunosuppressive therapy. Higher levels of OCPs like β-HCH (isoform of hexachlorohexane), α-endosulfan (a form of endosulfan) and p,p΄-DDE (a metabolite of o,p’-dichlorodiphenyltrichloroethane) were observed in the blood of pemphigus patients as compared to healthy controls. HCH and DDT exposure caused specific reduction in CD8+CD45RA+ and CD4+CD25+ T lymphocyte subpopulations in these patient PBMCs. A strong reduction in Th1 (IL-2 and IFN-γ) cytokines upon exposure to these OCPs in-vitro was also observed. These findings indicate that HCH and DDT have a significant impact on Th1 lymphocytes. Impaired production of these cytokines might favor infections and production of autoantibodies. We therefore speculate that the systemic absorption of the pesticide after the topical contact may be one of the factors triggering the immunological mechanism among pemphigus patients.

Novel Pi3kδ Inhibitor Roginolisib Synergizes with the Bcl-2 Inhibitor Venetoclax in Hematological Malignancies

Roginolisib (IOA-244) is a first in class allosteric modulator and non-ATP competitive, selective PI3Kδ inhibitor currently in a Phase 1 clinical study in lymphoma and solid tumors. Initial structural and biochemical studies identified unique chemical structure, excellent selectivity, and excellent PK properties for roginolisib as compared to earlier PI3Kδ inhibitors, while the clinical trial has demonstrated a favorable safety profile. Consistent with prior PI3Kδ inhibitors, roginolisib inhibits the in vitro growth of lymphoma cells. In contrast to other PI3Kδ inhibitors, roginolisib activity is correlated with the expression levels of PIK3CD, suggesting specific and on target cancer cell-intrinsic effects (PMID: 37066023). To identify rational combination strategies, initial pharmacological screening of 474 compounds in combination with roginolisib was carried out using a MTT assay in the lymphoma cell lines HH (cutaneous T cell lymphoma, CTCL) and SP-53 (mantle cell lymphoma, MCL). Initial screening identified that roginolisib synergized with the bcl-2 inhibitor venetoclax in the HH cell line with a combination index value (CI) of 0.003 by the Chou-Talalay method. To further validate this finding, the combination of roginolisib with venetoclax or with another bcl-2 inhibitor S55746 was tested in a broad range of lymphoma cell lines including GRANTA519, JVM2, SP49 (MCL); FARAGE, TMD8 (diffuse large B cell lymphoma); MEC1 (chronic lymphocytic leukemia); MJ (cutaneous T cell lymphoma); and YT (NK lymphoma). The combination showed synergism in all the cell lines (CI 0.81 - 0.05) except SP49 (CI- 1.3). Similarly, the combination of roginolisib with S55746 showed synergism in most of the cell lines (CI- 0.46 - 0.089), additive effect in SP49 (CI- 1) and antagonistic effect in GRANTA519 (CI- 1.3) and FARAGE (CI- 2.17). To assess the tumor-intrinsic effect of roginolisib in patient derived CLL cells, PBMCs isolated from CLL patients responding to prior BTK inhibitor (i) (n=4) or progressed on BTKi (n=6) were tested to check the effect of roginolisib on BCR signalling. Cells were treated with increasing doses of roginolisib from 0.625 μM to 5 μM in the presence of anti-IgM. Other BCRi such as idelalisib, duvelisib and acalabrutinib were used at 5 μM concentrations as positive controls. Roginolisib showed a dose dependent suppression of the activity of BCR downstream kinases such as AKT and ERK. The suppression of phosphorylation of AKT and ERK were comparable with other BCRi tested. Additionally, the apoptosis analysis by annexin/7AAD showed comparable activity of roginolisib with idelalisib, duvelisib or acalabrutinib at 5 μM concentration. To investigate the possible synergy of roginolisib with venetoclax in CLL patient samples who are BTKi responders (n=6) or BTKi progressors (n=8), apoptosis analysis was carried out in patient PBMCs. Viability of CLL cells following 48h of ex vivo treatment with increasing concentrations of roginolisib (0.625 to 5 μM) and venetoclax (1 to 8 nM) alone or in combination was tested. Both the BTKi responders and progressors showed strong synergy with the combination, with slightly better cell killing activity in BTKi progressors ( p=0.007) than responders ( p=0.019). To understand the mechanism by which roginolisib increases the sensitivity to venetoclax, changes in the expression levels of various Bcl-2 family proteins that have been implicated in venetoclax resistance such as Mcl-1, Bcl-xL, Bcl-2, A1 and BIM were measured by immunoblotting. Changes in expression were evaluated in PBMCs from both the BTKi responders (n=8) and progressors (n=7) after 24h treatment with 0.625 and 5 μM concentrations of roginolisib. A significant dose dependent downregulation of Mcl-1 was observed compared to DMSO treated cells in both BTKi responders ( p=0.0033) and progressors ( p= 0.0025). In addition, a significant increase in proapoptotic BIM EL was also observed at both the 0.625 μM ( p=0.0054) and 5 μM ( p=0.0194) concentrations compared to DMSO. In summary, the novel PI3Kδ selective inhibitor roginolisib shows tumor cell killing on various hematologic malignancies including CLL cells from patients with disease progression on BTK inhibitors. In addition, roginolisib synergized with venetoclax in lymphoma cell lines and CLL patient samples. Our data support extending this combination strategy to clinical trials in hematological malignancies.

Phase 1 Study of SC291, a Hypoimmune, Allogeneic CD19-Directed CAR T Cell Therapy for Relapsed/Refractory B-Cell Malignancies (ARDENT) - Initial Clinical Data

ARDENT is a phase 1 study evaluating safety and tolerability of SC291, a hypoimmune (HIP), allogeneic, CD19-directed CAR T cell therapy in subjects with Non-Hodgkin's Lymphoma and Chronic Lymphocytic Leukemia (CLL). SC291 is derived from healthy donor CD4+ and CD8+ T cells that are genetically engineered to disrupt function of CD3 and HLA class I/II, overexpress CD47, and express a CD19-directed CAR. A primary obstacle for allogeneic CAR T cell approaches remains their immune recognition and rejection. Patient immune responses prevent durable allogeneic CAR T clinical responses despite the use of enhanced lymphodepletion (LD) regimens. We therefore developed hypoimmune CAR T cells that avoid immune recognition and have the potential to deliver clinical efficacy while using a standard LD chemotherapy regimen (as defined by approved autologous CAR T products). The HIP engineering approach involves the depletion of HLA class I/II to prevent adaptive immune rejection and the overexpression of CD47 to prevent innate immune rejection, which has previously demonstrated alloimmune protection in preclinical models ( Nat Biotechnol 2019;37(3):252-258; Proc Natl Acad Sci U S A 2021;118(28):e2022091118; J Exp Med. 2021;218(3):e20200839; Nat Biotechnol. 2023; doi: 10.1038/s41587-023-01784-x, and Nat Commun. 2023;14(1):2020). The initial patient, a 74-year-old male with CLL (unmutated IGHV, del(11q), BTK C481S mutation) and 3 prior lines of therapy (FCR, ibrutinib, venetoclax + rituximab), received a LD regimen of cyclophosphamide 500 mg/m 2 and fludarabine 24 mg/m 2 (daily for 3 days) followed by a starting dose of 60 million CAR+ SC291 cells (of which approximately 80% are fully HIP engineered cells). SC291 was well tolerated with no observed CRS or ICANS. The patient developed non-neutropenic fever after LD (prior to SC291 infusion). At the Day 28 visit post-SC291 infusion, the patient had a partial response (per iwCLL 2018 criteria), along with significant (99%) B-cell reduction and improvements in platelet levels. Analysis of cellular kinetics is ongoing. SC291 is a mixture of T cells, in which portions are partially or fully HIP-engineered. As such, we evaluated the initial patient's immune response against SC291 subpopulations using a panel of previously described in vitro assays. Blood samples from the patient were taken pre-treatment (Day -5) and at the Day 13 and Day 28 visits following SC291 treatment and were assessed for immune evasion. The patient's PBMCs were sorted into CD3+ T cells and CD3-CD56+ NK cells and incubated with the following subpopulations sorted from SC291 drug product (DP): (i) fully HIP engineered (CD19 HIP CAR T cells), (ii) HLA I/II negative T cells without CD47 overexpression (DKO), and (iii) HLA I/II positive CAR T cells overexpressing CD47 (WT CD19 CAR T cells). Patient serum from Day -5 and the Day 28 visit were used for donor-specific antibody analyses, complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC) against the DP subpopulations. At Day -5, T cell EliSpot assays showed no immune responses against any of the SC291 sorted subpopulations, but by Days 13 and 28, these assays produced high and increasing IFN-g spot frequencies against the WT CD19 CAR T cells. Similar results were shown with T cell cytotoxicity assays (Fig. 1B). In contrast, no T cell activation and no T cell killing was observed with DKO and CD19 HIP CAR T cells at any time point. As expected, patient NK cells vigorously killed DKO T cells at all time points but spared WT CD19 CAR T and CD19 HIP CAR T cells (Fig. 1B). No antibodies against any sorted subpopulation were detected at Day -5, but IgG antibody binding of the WT CD19 CAR T population was observed at Day 28. These antibodies did not bind to DKO or CD19 HIP CAR T cells, and there was no CDC or ADCC response against these subpopulations. Interestingly, with the LD regimen used, we observed a 95% and 99% reduction of CD19+ cells on Days 0 and 28, respectively, while T cells rebounded from an 88% reduction on day 0 to pretreatment levels on Day 28 (Fig. 1A). These data demonstrate the preliminary safety and tolerability of SC291 in the initial treated patient. Immune assays demonstrated that the CD19 HIP CAR T cell subpopulation effectively evades the host adaptive and innate immune responses and could overcome the allogeneic barrier in humans. Additional data from the ARDENT study will be presented at the time of the conference.

Upregulation of CX3CR1 expression in circulating T cells of systemic lupus erythematosus patients as a reflection of autoimmune status through characterization of cytotoxic capacity

ObjectiveThis study investigated CX3CR1 expression in human peripheral blood T lymphocytes and their subsets, exploring changes in SLE patients and its diagnostic potential. MethodsPeripheral blood samples from 31 healthy controls and 50 SLE patients were collected. RNA-Seq data from SLE patient PBMCs were used to analyze CX3CR1 expression in T cells. Flow cytometry determined CX3CR1-expressing T lymphocyte subset proportions in SLE patients and healthy controls. Subset composition and presence of GZMB, GPR56, and perforin in CX3CR1+ T lymphocytes were analyzed. T cell-clinical indicator correlations were assessed. ROC curves explored CX3CR1′s diagnostic potential for SLE. ResultsCX3CR1+CD8+ T cells exhibited higher GPR56, perforin, and GZMB expression than other T cell subsets. The proportion of CX3CR1+ was higher in TEMRA and lower in Tn and TCM. PMA activation reduced CX3CR1+ T cell proportions. Both RNA-Seq and flow cytometry revealed elevated CX3CR1+ T cell proportions in SLE patients. Significantly lower perforin+ and GPR56+ proportions were observed in CX3CR1+CD8+ T cells in SLE patients. CX3CR1+ T cells correlated with clinical indicators. ConclusionCX3CR1+ T cells display cytotoxic features, with heightened expression in CD8+ T cells, particularly in adult SLE patients. Increased CX3CR1 expression in SLE patient T cells suggests its potential as an adjunctive diagnostic marker for SLE.

NLRC4 gain-of-function variant is identified in a patient with systemic lupus erythematosus

NLRC4 gain-of-function variants are known to cause various autoinflammatory phenotypes, including familial cold autoinflammatory syndrome (FCAS4) and NLRC4 macrophage activation syndrome (NLRC4-MAS). However, to date, no study has linked NLRC4 gain-of-function variants to systemic lupus erythematosus (SLE). In this study, we identified a novel NLRC4 W655S variant in an SLE patient and her son, who had neonatal lupus complicated with macrophage activation syndrome. Our in vitro experiments demonstrated that the W655S NLRC4 increased ASC speck formation and mature IL-1β secretion compared to the wild-type NLRC4. In addition, the patient had elevated levels of IL-1β and IL-18 in both serum and PBMCs. RNA sequencing showed that NF-κB and interferon signaling pathways were significantly activated in the patient compared to healthy controls. Furthermore, gene set enrichment analysis revealed upregulation of NLRC4-related pathways in patient PBMCs. In conclusion, our study identified the NLRC4 W655S variant in a patient with SLE. This is the first report linking inflammasomopathy to monogenic SLE. Our findings suggest that inflammasome activation may be a critical driver in the pathogenicity of lupus, and autoinflammatory pathways may play important roles in the development of the disease.

A gain-of-function variation in PLCG1 causes a new immune dysregulation disease

Phospholipase C (PLC) γ1 is a critical enzyme regulating nuclear factor-κB (NF-κB), extracellular signal-related kinase, mitogen-activated protein kinase, and nuclear factor of activated T cells signaling pathways, yet germline PLCG1 mutation in human disease has not been reported. We aimed to investigate the molecular pathogenesis of a PLCG1 activating variant in a patient with immune dysregulation. Whole exome sequencing was used to identify the patient's pathogenic variants. BulkRNA sequencing, single-cell RNA sequencing, quantitative PCR, cytometry by time of flight, immunoblotting, flow cytometry, luciferase assay, IP-One ELISA, calcium flux assay, and cytokine measurements in patient PBMCs and T cells and COS-7 and Jurkat cell lines were used to define inflammatory signatures and assess the impact of the PLCG1 variant on protein function and immune signaling. We identified a novel and de novo heterozygous PLCG1 variant, p.S1021F, in a patient presenting with early-onset immune dysregulation disease. We demonstrated that the S1021F variant is a gain-of-function variant, leading to increased inositol-1,4,5-trisphosphate production, intracellular Ca2+ release, and increased phosphorylation of extracellular signal-related kinase, p65, and p38. The transcriptome and protein expression at the single-cell level revealed exacerbated inflammatory responses in the patient's T cells and monocytes. The PLCG1 activating variant resulted in enhanced NF-κB and type II interferon pathways in T cells, and hyperactivated NF-κB and type I interferon pathways in monocytes. Treatment with either PLCγ1 inhibitor or Janus kinase inhibitor reversed the upregulated gene expression profile invitro. Our study highlights the critical role of PLCγ1 in maintaining immune homeostasis. We illustrate immune dysregulation as a consequence of PLCγ1 activation and provide insight into therapeutic targeting of PLCγ1.

Identification of an IL-1 receptor mutation driving autoinflammation directs IL-1-targeted drug design

The interleukin 1 (IL-1) pathway signals through IL-1 receptor type1 (IL-1R1) and emerges as a central mediator for systemic inflammation. Aberrant IL-1 signaling leads to a range of autoinflammatory diseases. Here, we identified a de novo missense variant in IL-1R1 (p.Lys131Glu) in a patient with chronic recurrent multifocal osteomyelitis (CRMO). Patient PBMCs showed strong inflammatory signatures, particularly in monocytes and neutrophils. The p.Lys131Glu substitution affected a critical positively charged amino acid, which disrupted the binding of the antagonist ligand, IL-1Ra, but not IL-1α or IL-1β. This resulted in unopposed IL-1 signaling. Mice with a homologous mutation exhibited similar hyperinflammation and greater susceptibility to collagen antibody-induced arthritis, accompanied with pathological osteoclastogenesis. Leveraging the biology of the mutation, we designed an IL-1 therapeutic, which traps IL-1β and IL-1α, but not IL-1Ra. Collectively, this work provides molecular insights and a potential drug for improved potency and specificity in treating IL-1-driven diseases.

PTEN Deficiency: a phenocopy phenomenon

BackgroundActivated PI3K delta syndrome 1 (APDS1) is caused by mutations in PI3KCD. The phosphatidylinositol 3-kinase (PI3K) signaling pathway is essential to cell survival, proliferation, and metabolism. PI3K plays a crucial role in the regulation of function and differentiation of immune cells. Phosphatase and tensin homolog (PTEN) serves to antagonize the PI3K pathway. Overlapping immune features of PTEN deficiency and APDS include high IgM, low IgG, increased transitional B cells, specific antibody deficiencies, autoimmunity, and lymphoproliferation. Because PTEN antagonizes PI3K activity, patients with PTEN loss-of-function mutations exhibit a similar phenotype to those with gain-of-function mutations of PI3K and can respond to mTOR inhibition like APDS patients. Overactivation of PI3K due to PTEN deficiency can lead to generalized immune dysregulation. Here we describe a patient with PTEN deficiency who presents as a phenocopy of APDS1 patients. MethodsRetrospective chart review was performed to obtain data on clinical labs of the PTEN deficient patient and APDS1 patients who presented with a similar clinical phenotype. An Epstein-barr virus (EBV) transformed B cell line (BLCL) was created from the patient PBMCs for functional studies to evaluate the PBK/AIKT/mTOR pathway. Patients were consented on an IRB approved protocol. ResultsThe patient was referred to immunology clinic for autoimmune cytopenias (Evan’s syndrome) and hypogammaglobulinemia. WES revealed De novo heterozygous c.406T>C (p.C136R) pathogenic variant in PTEN. Immune profiling showed elevated transitional B cells, increased naïve B cells and a decreased amount of class-switched memory B cells along with hyper IgM and low IgG. An abdominal ultrasound revealed significant splenomegaly with peri-splenic lymphadenopathy and hyper-echoic lesions in the spleen. Functional studies on BLCLs were reserved for confirmation of dysregulated PI3k/AKT/pS6 signaling. The patient was treated with sirolimus and IgG replacement therapy. After sirolimus therapy, lymphoproliferation and splenic findings resolved. DiscussionPTEN deficiency is a clinical and mechanistic phenocopy of APDS1 due to loss of negative regulation by PTEN to control over-activation of PI3 K. While Evan’s syndrome is reported in APDS1 more readily, it is not as prominent in PTEN. Treatment strategies employed for APDS1 and 2 are viable options for controlling the disease manifestations seen in PTEN deficiency.

A KIR x CD8 targeting bispecific modulator enhances regulatory CD8 T cell functions, and reduces inflammation in models of autoimmune disease

Abstract INTRODUCTION: We have characterized a subset of CD8 T cells (CD8 Treg) with immunosuppressive characteristics in inflammatory disease settings. CD8 Treg activation results in their oligoclonal expansion and elimination of pathogenic CD4 T cells. Here we describe a bispecific CD8 Treg modulator that activates autoimmune patient derived CD8 Treg resulting in the cytolytic elimination of pathogenic CD4 T cell in vitro, ex vivo, and in vivo. METHODS: We tested target and cell binding of a novel bispecific CD8 Treg modulator by Octet and flow cytometry. The functional impact was evaluated using cytotoxicity assays and supernatant analysis in vitro, using Celiac patient derived biopsies ex vivo, and in acute GVHD studies using human PBMCs. RESULTS: We evaluated the efficacy and mechanism of action of a novel bispecific CD8 Treg modulator directed toward CD8 and inhibitory KIR2DL1/2/3. The modulator enhanced cytolytic CD8 Treg functions in vitro using autoimmune patient PBMCs, increased activated CD4 T cell death, and decreased pro-inflammatory cytokines. In Celiac patient derived duodenal tissues, the modulator increased CD8 Treg prevalence and reduced epithelial cell death after gliadin peptide stimulation. In acute GVHD studies, CD8 Treg modulator treated mice had CD8 Treg expansion in a dose dependent fashion and reduced inflammation. CONCLUSIONS: A novel KIR x CD8 targeting bispecific modulator restores cytolytic function of CD8 Treg, resulting in a decrease of pathogenic CD4 T cells. We postulate that the bispecific CD8 T cell modulator can reduce autoreactive CD4 T cell prevalence to reestablish immune balance and may represent a selective and broadly applicable therapeutic approach for the treatment of autoimmune disease.

Abstract 6396: BET inhibition alleviates T-cell dysfunction in chronic lymphocytic leukemia

Abstract Background: The chronic lymphocytic leukemia (CLL) tumor microenvironment (TME) is laden with hyporesponsive T-cells that permit disease persistence. Yet, redundant TME immunosuppressive mechanisms and epigenetic maintenance of T-cell exhaustion limit the efficacy of T-cell targeted therapies in CLL. Bromodomain and extra-terminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T-cell function and differentiation. We hypothesize that blocking BET protein function can reverse T-cell exhaustion to yield durable tumor elimination in CLL. Methods: WT C57BL/6 mice were engrafted with Eμ-TCL1 spleen-derived lymphocytes, then treated daily with the novel pan-BET inhibitor, OPN-51107 (OPN5; 20mg/kg PO) for up to 4 weeks. Splenic gene expression was evaluated with the NanoString PanCancer iO360 panel. T-cell differentiation status, immune inhibitory receptor (IR) expression, proliferation (72 h ex vivo α-CD3/α-CD28 stimulation), and cytokine production (6 h ex vivo PMA/ionomycin stimulation) was measured via flow cytometry. CLL patient and healthy donor PBMCs were used for validation studies and to assess T-cell transcription factor (TF) expression via flow cytometry. Evaluation of BRD4 occupancy at select T-cell TFs via ChIP qPCR is ongoing. Results: OPN5 significantly increased cytotoxic cell signatures and reduced exhaustion-associated cell signatures in leukemic mice through inhibition of T-cell exhaustion signaling, as well as activation of Th1, natural killer cell, and IL-7 signaling pathways. Correspondingly, T-cells from OPN5-treated mice demonstrated greater ex vivo proliferative capacity and effector response to stimuli. A greater proportion of CD8+ T-cells from OPN5-treated mice were classified as naïve, and OPN5 significantly reduced KLRG1 expression on antigen-experienced CD8+ T-cells. Importantly, OPN5 curtailed IR co-expression (PD-1, PD-L1, VISTA, CD244, CD160, and LAG3) on splenic T-cells. These findings were confirmed with primary CLL cells ex vivo. OPN5 also impaired expression of terminal differentiation-associated TFs in CLL patient-derived T-cells, enriching for a TCF1+ progenitor T-cell population. While BTK inhibitors are known to similarly improve T-cell function in CLL, ibrutinib treatment was inadequate to revert CLL T-cell terminal differentiation. Future ATAC-sequencing analysis will inform how BET inhibition alleviates exhaustion-associated chromatin organization in CLL T-cells. Conclusion: BET inhibition dismantles immunosuppressive mechanisms in the CLL TME, alleviating CLL-induced T-cell dysfunction and terminal differentiation. These findings suggest that BET inhibition may be a useful component of combination strategies for the treatment of CLL to yield lasting anti-cancer immune memory and prevent relapsed/refractory disease. Citation Format: Audrey L. Smith, Alexandria P. Eiken, Sydney A. Skupa, Christopher R. D'Angelo, Avyakta Kallam, Matthew A. Lunning, Gregory Bociek, Julie M. Vose, Ben Powell, Gideon Bollag, Dalia El-Gamal. BET inhibition alleviates T-cell dysfunction in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6396.

Immortalized B Cells Transfected with mRNA of Antigen Fused to MITD (IBMAM): An Effective Tool for Antigen-Specific T-Cell Expansion and TCR Validation

Peripheral mononuclear blood cells (PBMCs) are the most widely used study materials for immunomonitoring and antigen-specific T-cell identification. However, limited patient PBMCs and low-frequency antigen-specific T cells remain as significant technical challenges. To address these limitations, we established a novel platform comprised of optimized HLA-matched immortalized B cells transfected with mRNA of a prototype viral or tumor antigen conjugated to MHC class-I trafficking domain protein (MITD) to increase the efficiency of epitope expression in antigen-presenting cells (APCs) essential to expanding antigen-specific T cells. When applied to CMV as a model, the IBMAM platform could successfully expand CMV-specific T cells from low-frequency CMV PBMCs from seropositive donors. Additionally, this platform can be applied to the validation of antigen specific TCRs. Together, compared to using APCs with synthesized peptides, this platform is an unlimited, highly efficient, and cost-effective resource in detecting and expanding antigen-specific T cells and validating antigen-specific TCRs.

Effect of exogenous IL-37 on immune cells from a patient carrying a potential IL37 loss-of-function variant: A case study.

Chronic inflammatory or autoimmune diseases are commonly treated with immunosuppressive medication such as NSAIDs, corticosteroids, or antibodies against specific cytokines (TNF, IL-1 IL-17, IL-23, etc.) or signalling cascades (e.g. JAK-STAT inhibitors). Using sequencing data to locate genetic mutations in relevant genes allows the identification of alternative targets in a patient-tailored therapy setting. Interleukin (IL)-37 is an anti-inflammatory cytokine with broad effects on innate and adaptive immune cell function. Dysfunctional IL-37 expression or signalling is linked to various autoinflammatory disorders. The administration of recombinant IL-37 to hyperinflammatory patients that are non-responsive to standard treatment bears the potential to alleviate symptoms. In this case study, the (hyper)responsiveness of immune cell subsets was investigated in a single patient with a seronegative autoimmune disorder who carries a heterozygous stop-gain variant in IL37 (IL37 Chr2(GRCh37):g.113670640G>A NM_014439.3:c.51G>A p.(Trp17*)). As the patient has been non-responsive to blockage of TNF or IL-1 by Etanercept or Anakinra, respectively, additional in-vitro experiments were set out to elucidate whether treatment with recombinant IL-37 could normalise observed immune cell functions. Characterisation of immune cell function showed no elevated overall production of acute-phase pro-inflammatory cytokines by patient PBMCs and neutrophils at baseline or upon stimulation. T-cell responses were elevated, as was the metabolic activity and IL-1Ra production of PBMCs at baseline. The identified stop-gain variant in IL37 does not result in the absence of the protein in circulation. In line with this, treatment with recombinant IL-37 did overall not dampen immune responses with the exception of the complete suppression of IL-17. The heterozygous stop-gain variant in IL37 (IL37 NM_014439.3:c.51G>A p.(Trp17*)) is not of functional relevance as we observed no clear pro-inflammatory phenotype in immune cells of a patient carrying this variant.

Alteration of Cellular and Cytokine Profiles in Response to Shrimp Allergens in Peripheral Blood Mononuclear Cells from Shrimp Allergic Patients

The cellular immune responses to shrimp allergen have not been completely characterized. We hypothesized different patterns of cytokines in responses to raw and cooked shrimp allergens would occur from total shrimp allergic (SA) patient PBMCs compared to healthy controls.