Patient-derived Peripheral Blood Mononuclear Cells Research Articles

A Novel Transmembrane CXCR4 Variant That Expands the WHIM Genotype-Phenotype Paradigm

Warts, Hypogammglobulinemia, Infections, Myelokathexis (WHIM) syndrome is a rare immunodeficiency disease that results from impaired leukocyte trafficking (myelokathexis) predominately caused by gain-of-function variants in C-X-C chemokine receptor type 4 (CXCR4). Clinical manifestations of WHIM syndrome can differ in familial forms or in people harboring identical CXCR4 variants. All known pathogenic CXCR4 variants associated with WHIM syndrome (CXCR4WHIM) to date are localized in the intracellular C-terminus of CXCR4. We identified 4 unrelated patients with variable WHIM-like clinical presentations harboring a novel heterozygous CXCR4 variant (c.250G>C; p.D84H) localized at a highly conserved position in the transmembrane domain of the receptor outside the C-terminus. Functional characterization of the CXCR4D84Hvariant (CXCR4D84H) using patient-derived peripheral blood mononuclear cells and in vitro cellular assaysshow decreased CXCR4 internalization and increased chemotaxis in response to CXCL12, similar to known CXCR4WHIM, but also revealed unique features of CXCR4D84H signaling to cAMP, Ca2+ mobilization and AKT/ERK pathways. These findings are consistent with molecular dynamics simulations that show disruption of the Na+ binding pocket by D84H, resulting in collapse of the hydrophobic gate above and destabilization of the inactive state of CXCR4. Mavorixafor, a CXCR4 antagonist being evaluated in clinical trials for chronic neutropenia and WHIM syndrome, normalized CXCL12-mediated chemotaxis of CXCR4D84H patient lymphocytes ex vivo and improved WBC and subset counts in 1 patient with CXCR4D84H enrolled in the chronic neutropenia phase 1b clinical trial (NCT04154488). The present study expands the current understanding of CXCR4 function and genotype-phenotype correlations in WHIM syndrome and in people with WHIM-like phenotypes.

Investigating cryopreserved PBMC functionality in an antigen-induced model of sarcoidosis granuloma formation

Sarcoidosis, a systemic inflammatory disease, poses challenges in understanding its etiology and variable clinical courses. Despite ongoing uncertainty about causative agents and genetic predisposition, granuloma formation remains its hallmark feature. To address this, we developed a validated in vitro human granuloma model using patient-derived peripheral blood mononuclear cells (PBMCs), offering a dynamic platform for studying early granuloma formation and sarcoidosis pathogenesis. However, a current limitation of this model is its dependence on freshly isolated PBMCs obtained from whole blood. While cryopreservation is a common method for long-term sample preservation, the biological effects of freezing and thawing PBMCs on granuloma formation remain unclear. This study aimed to assess the viability and functionality of cryopreserved sarcoidosis PBMCs within the granuloma model, revealing similar granulomatous responses to fresh cells and highlighting the potential of cryopreserved PBMCs as a valuable tool for studying sarcoidosis and related diseases.

Impact of doxorubicin-loaded ferritin nanocages (FerOX) vs. free doxorubicin on T lymphocytes: a translational clinical study on breast cancer patients undergoing neoadjuvant chemotherapy

Despite the advent of numerous targeted therapies in clinical practice, anthracyclines, including doxorubicin (DOX), continue to play a pivotal role in breast cancer (BC) treatment. DOX directly disrupts DNA replication, demonstrating remarkable efficacy against BC cells. However, its non-specificity toward cancer cells leads to significant side effects, limiting its clinical utility. Interestingly, DOX can also enhance the antitumor immune response by promoting immunogenic cell death in BC cells, thereby facilitating the presentation of tumor antigens to the adaptive immune system. However, the generation of an adaptive immune response involves highly proliferative processes, which may be adversely affected by DOX-induced cytotoxicity. Therefore, understanding the impact of DOX on dividing T cells becomes crucial, to deepen our understanding and potentially devise strategies to shield anti-tumor immunity from DOX-induced toxicity. Our investigation focused on studying DOX uptake and its effects on human lymphocytes. We collected lymphocytes from healthy donors and BC patients undergoing neoadjuvant chemotherapy (NAC). Notably, patient-derived peripheral blood mononuclear cells (PBMC) promptly internalized DOX when incubated in vitro or isolated immediately after NAC. These DOX-treated PBMCs exhibited significant proliferative impairment compared to untreated cells or those isolated before treatment initiation. Intriguingly, among diverse lymphocyte sub-populations, CD8 + T cells exhibited the highest uptake of DOX. To address this concern, we explored a novel DOX formulation encapsulated in ferritin nanocages (FerOX). FerOX specifically targets tumors and effectively eradicates BC both in vitro and in vivo. Remarkably, only T cells treated with FerOX exhibited reduced DOX internalization, potentially minimizing cytotoxic effects on adaptive immunity.Our findings underscore the importance of optimizing DOX delivery to enhance its antitumor efficacy while minimizing adverse effects, highlighting the pivotal role played by FerOX in mitigating DOX-induced toxicity towards T-cells, thereby positioning it as a promising DOX formulation. This study contributes valuable insights to modern cancer therapy and immunomodulation.

The Association between Inefficient Repair of DNA Double-Strand Breaks and Common Polymorphisms of the HRR and NHEJ Repair Genes in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation affecting up to 2.0% of adults around the world. The molecular background of RA has not yet been fully elucidated, but RA is classified as a disease in which the genetic background is one of the most significant risk factors. One hallmark of RA is impaired DNA repair observed in patient-derived peripheral blood mononuclear cells (PBMCs). The aim of this study was to correlate the phenotype defined as the efficiency of DNA double-strand break (DSB) repair with the genotype limited to a single-nucleotide polymorphism (SNP) of DSB repair genes. We also analyzed the expression level of key DSB repair genes. The study population contained 45 RA patients and 45 healthy controls. We used a comet assay to study DSB repair after in vitro exposure to bleomycin in PBMCs from patients with rheumatoid arthritis. TaqMan SNP Genotyping Assays were used to determine the distribution of SNPs and the Taq Man gene expression assay was used to assess the RNA expression of DSB repair-related genes. PBMCs from patients with RA had significantly lower bleomycin-induced DNA lesion repair efficiency and we identified more subjects with inefficient DNA repair in RA compared with the control (84.5% vs. 24.4%; OR 41.4, 95% CI, 4.8-355.01). Furthermore, SNPs located within the RAD50 gene (rs1801321 and rs1801320) increased the OR to 53.5 (95% CI, 4.7-613.21) while rs963917 and rs3784099 (RAD51B) to 73.4 (95% CI, 5.3-1011.05). These results were confirmed by decision tree (DT) analysis (accuracy 0.84; precision 0.87, and specificity 0.86). We also found elevated expression of RAD51B, BRCA1, and BRCA2 in PBMCs isolated from RA patients. The findings indicated that impaired DSB repair in RA may be related to genetic variations in DSB repair genes as well as their expression levels. However, the mechanism of this relation, and whether it is direct or indirect, needs to be elucidated.

Development of a Same-Day Closed CAR-T Cell Manufacturing Process to Generate Lentivector Loaded Lymphocytes Ready for Subcutaneous Injection

Background: Adoptive T-cell therapies with CD19 chimeric antigen receptors (CAR) are now the standard of care for refractory/recurrent hematologic B-cell malignancies. However, challenges remain in delivering a more fit cell product that can be manufactured in a more timely and cost-effective manner. To address these challenges, a rapid same-day fully closed manufacturing process was developed to generate a more fit product for subcutaneous administration and to enable an efficient transition from early-phase clinical trials to commercial-scale production. Methods: A lentivector (LV) was designed with the capability of directly targeting and activating T cells simultaneously to enable manufacturing without prior T-cell selection. Experiments were performed to understand the kinetics of direct lentivector-induced T-cell activation, internalization, reverse transcription, and vector genome integration. After establishing critical process parameters for efficient loading of T cells with LVs, a closed manufacturing process was developed from peripheral blood mononuclear cells (PBMC) starting material derived from whole blood or apheresis. PBMCs were isolated in an automated closed system and exposed to LV for a four-hour incubation period. Cells were washed in an automated closed system to remove residual LV, formulated, and vialed with release testing for identity, potency, purity, and safety. Three full-scale engineering lots were manufactured from donor-derived apheresis in addition to three small-scale analytical lots with patient-derived PBMC. Results: For targeted gene delivery into T cells within PBMC, the CD19 CAR LV was designed to contain a CD3 targeting motif in its envelope to enable T-cell binding, activation, and viral entry into CD3+ T cells. The CD3-directed LV encoding a CD19-CAR and a novel synthetic driver co-stimulatory domain was manufactured in a chemically defined 25L clinical scale suspension-based process and vialed to a target unit potency for ease of cell loading. The purified CD3-directed LV is capable of inducing T-cell receptor internalization and activation through specific bioassays. In surrogate analytical cultures, following LV exposure and automated washing, viral entry into T cells was complete. Analytical cultures demonstrate the ability to successfully generate CAR+ cells with a unique CD3+CD56+CD45RA+CCR7+ T memory-stem cell with NK-like features and a vector copy number of less than 5 vector copies/transduced cell (1.95 +/- 0.58). In preparation for clinical investigation, full-scale runs were performed in GMP facilities with qualified methods for release testing against identity, potency, purity, and safety. Small-scale analytical processes utilizing B-cell lymphoma patient-derived PBMC from whole blood were evaluated for differences relative to donor-derived material. Conclusions: To address current challenges in the manufacturing of autologous cell products, a same-day closed manufacturing process was developed to produce cells with enhanced fitness. Qualification of this novel design included 1) completion of the production of the CD3-directed LV encoding a CD19 CAR with a novel synthetic co-stimulatory driver element at the 25L scale; 2) completion of three full-scale closed engineering runs along with the qualification of QC release testing for identity, potency, purity, and safety and 3) comparability studies utilizing patient-derived PBMC.

Patient-derived stem cell line UKMi005-A (hiPSC) harboring a non-synonymous heterozygous KCNJ5 gene variant

A published heterozygous gain-of-function variant in the KCNJ5 gene (p.Trp101Cys) encoding the G-protein-activated inward-rectifier potassium channel 4 subunit of the IK,ACh channel is associated with human sinus node dysfunction (SND). Differentiated hiPSC-cardiomyocytes may serve as an in-vitro model to study SND and to develop pharmacological rescue strategies. Therefore, a mutant hiPSCs line from patient-derived peripheral blood mononuclear cells (PBMCs) were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit. The hiPSC line (KCNJ5 K8) showed a regular karyotype, a typical hiPSC morphology, expressed pluripotency-associated markers in immunofluorescence stainings and RT-qPCR analysis. The ability for differentiation into all three germ layers was shown.

In vitro vascularized immunocompetent patient-derived model to test cancer therapies

This work describes a patient-derived tumoroid model (PDTs) to support precision medicine in lung oncology. The use of human adipose tissue-derived microvasculature and patient-derived peripheral blood mononuclear cells (PBMCs) permits to achieve a physiologically relevant tumor microenvironment. This study involved ten patients at various stages of tumor progression. The vascularized, immune-infiltrated PDT model could be obtained within two weeks, matching the requirements of the therapeutic decision. Histological and transcriptomic analyses confirmed that the main features from the original tumor were reproduced. The 3D tumor model could be used to determine the dynamics of response to antiangiogenic therapy and platinum-based chemotherapy. Antiangiogenic therapy showed a significant decrease in vascular endothelial growth factor (VEGF)-A expression, reflecting its therapeutic effect in the model. In an immune-infiltrated PDT model, chemotherapy showed the ability to decrease the levels of lymphocyte activation gene-3 protein (LAG-3), B and T lymphocyte attenuator (BTLA), and inhibitory receptors of Tcells functions.

Comprehensive Genetic Analysis Unraveled the Missing Heritability and a Founder Variant of BEST1 in a Chinese Cohort With Autosomal Recessive Bestrophinopathy.

To describe the genetic landscape of BEST1 for a large Chinese cohort with autosomal recessive bestrophinopathy (ARB), identify the missing heritability, and report a common Chinese founder variant. We recruited 65 patients from 63 families with a clinical diagnosis of ARB. All patients underwent ophthalmic examinations and comprehensive genetic analyses, including Sanger DNA sequencing of BEST1 and whole genome sequencing (WGS). The effects of deep intronic variants (DIVs) on splicing were assessed using in vitro splicing assays in HEK293T cells and patient-derived peripheral blood mononuclear cells. Haplotype mapping was performed for 17 unrelated patients harboring variant c.867+97G>A. We identified 54 distinct disease-causing variants of BEST1 in 63 pedigrees, 62probands with biallelic variants, and one family with monoallelic variants. Sanger DNA sequencing of BEST1 initially detected 51 variants in 61 pedigrees, including 19probands with one heterozygous variant. Subsequent WGS, combined with supplementary Sanger sequencing, revealed three missing DIVs (c.1101-491A>G, c.867+97G>A, and c.867+97G>T) in 20 families. The novel DIV c.1101-491A>G caused an abnormal splicing resulting in a 204-nt pseudoexon (PE) insertion, whereas c.867+97G>A/T relatively strengthened an alternative donor site, resulting in a 203-nt intron retention (IR). The PE and IR generated a premature termination codon downstream. Haplotype analysis identified c.867+97G>A as a common founder variant with an allele frequency of 16%. Our results expand the pathogenic variant spectrum of BEST1, and DIVs can explain almost all of the missing heritability. The c.867+97G>A DIV is a common founder variant for Chinese patients with ARB.

Alterations in immune cell phenotype and cytotoxic capacity in HER2+ breast cancer patients receiving HER2-targeted neo-adjuvant therapy.

The phase II neo-adjuvant clinical trial ICORG10-05 (NCT01485926) compared chemotherapy in combination with trastuzumab, lapatinib or both in patients with HER2+ breast cancer. We studied circulating immune cells looking for alterations in phenotype, genotype and cytotoxic capacity (direct and antibody-dependent cell-mediated cytotoxicity (ADCC)) in the context of treatment response. Peripheral blood mononuclear cells (PBMCs) were isolated from pre- (n = 41) and post- (n = 25) neo-adjuvant treatment blood samples. Direct/trastuzumab-ADCC cytotoxicity of patient-derived PBMCs against K562/SKBR3 cell lines was determined ex vivo. Pembrolizumab was interrogated in 21 pre-treatment PBMC ADCC assays. Thirty-nine pre-treatment and 21 post-treatment PBMC samples were immunophenotyped. Fc receptor genotype, tumour infiltrating lymphocyte (TIL) levels and oestrogen receptor (ER) status were quantified. Treatment attenuated the cytotoxicity/ADCC of PBMCs. CD3+/CD4+/CD8+ T cells increased following therapy, while CD56+ NK cells/CD14+ monocytes/CD19+ B cells decreased with significant post-treatment immune cell changes confined to patients with residual disease. Pembrolizumab-augmented ex vivo PBMC ADCC activity was associated with residual disease, but not pathological complete response. Pembrolizumab-responsive PBMCs were associated with lower baseline TIL levels and ER+ tumours. PBMCs display altered phenotype and function following completion of neo-adjuvant treatment. Anti-PD-1-responsive PBMCs in ex vivo ADCC assays may be a biomarker of treatment response.

Effect of Honokiol on culture time and survival of Alzheimer's disease iPSC-derived neurons.

Patient-derived induced pluripotent stem cells (iPSCs) have been widely used as disease models to test new therapeutic strategies. Moreover, the regenerative potential of stem cells can be improved with the use of biologically active compounds. Our study was designed to explore the effect of honokiol, a small polyphenol molecule extracted from Magnolia officinalis, on the survival and culture time of iPSC-derived neurons from a sporadic Alzheimer's disease (AD) patient. This study aimed to generate iPSCs from peripheral blood mononuclear cells (PBMCs) of an AD patient using episomal plasmids with a nucleofector system and differentiate them into neurons. These iPSC-derived neurons were used to investigate the effect of honokiol extracted from M. officinalis on their survival and long-term cultures. IPSCs were generated from PBMCs of an AD patient by introducing Oct-3/4, Sox2, Klf4, L-Myc, and Lin28 using NucleofectorTM Technology. Differentiation of neurons derived from iPSCs was carried out using inducers and recognized by biomarkers. The viability of iPSC-derived neurons with the addition of honokiol extracted from the bark of M. officinalis was determined by the MTT analytical kit. IPSCs were generated by reprogramming AD patient-derived PBMCs and subsequently converted into neurons. The survival and growth of iPSC-derived neurons were significantly enhanced by adding honokiol in the experiment conditions. AD iPSC-derived neurons had a high viability rate when cultured in the presence of honokiol. These results have shown that AD iPSC-derived neurons can be an excellent model for screening neurotrophic agents and improving the conditions for long-term cultures of human iPSC-derived neurons. Honokiol proves to be a potential candidate for cellular therapeutics against neurodegenerative disorders.

Functional analysis of a conserved site mutation in the DNA end processing enzyme PNKP leading to ataxia with oculomotor apraxia type 4 in humans

Polynucleotide kinase 3'-phosphatase (PNKP), an essential DNA end-processing enzyme in mammals with 3'-phosphatase and 5'-kinase activities, plays a pivotal role in multiple DNA repair pathways. Its functional deficiency has been etiologically linked to various neurological disorders. Recent reports have shown that mutation at a conserved glutamine (Gln) in PNKP leads to late-onset ataxia with oculomotor apraxia type 4 (AOA4) in humans and embryonic lethality in pigs. However, the molecular mechanism underlying such phenotypes remains elusive. Here, we report that the enzymatic activities of the mutant versus WT PNKP are comparable; however, cells expressing mutant PNKP and peripheral blood mononuclear cells (PBMCs) of AOA4 patients showed a significant amount of DNA double-strand break accumulation and consequent activation of the DNA damage response. Further investigation revealed that the nuclear localization of mutant PNKP is severely abrogated, and the mutant proteins remain primarily in the cytoplasm. Western blot analysis of AOA4 patient-derived PBMCs also revealed the presence of mutated PNKP predominantly in the cytoplasm. To understand the molecular determinants, we identified that mutation at a conserved Gln residue impedes the interaction of PNKP with importin alpha but not with importin beta, two highly conserved proteins that mediate the import of proteins from the cytoplasm into the nucleus. Collectively, our data suggest that the absence of PNKP in the nucleus leads to constant activation of the DNA damage response due to persistent accumulation of double-strand breaks in the mutant cells, triggering death of vulnerable brain cells-a potential cause of neurodegeneration in AOA4 patients.

Quantification of capture efficiency, purity, and single-cell isolation in the recovery of circulating melanoma cells from peripheral blood by dielectrophoresis.

This paper describes a dielectrophoretic method for selection of circulating melanoma cells (CMCs), which lack reliable identifying surface antigens and are extremely rare in blood. This platform captures CMCs individually by dielectrophoresis (DEP) at an array of wireless bipolar electrodes (BPEs) aligned to overlying nanoliter-scale chambers, which isolate each cell for subsequent on-chip single-cell analysis. To determine the best conditions to employ for CMC isolation in this DEP-BPE platform, the static and dynamic dielectrophoretic response of established melanoma cell lines, melanoma cells from patient-derived xenografts (PDX) and peripheral blood mononuclear cells (PBMCs) were evaluated as a function of frequency using two established DEP platforms. Further, PBMCs derived from patients with advanced melanoma were compared with those from healthy controls. The results of this evaluation reveal that each DEP method requires a distinct frequency to achieve capture of melanoma cells and that the distribution of dielectric properties of PBMCs is more broadly varied in and among patients versus healthy controls. Based on this evaluation, we conclude that 50 kHz provides the highest capture efficiency on our DEP-BPE platform while maintaining a low rate of capture of unwanted PBMCs. We further quantified the efficiency of single-cell capture on the DEP-BPE platform and found that the efficiency diminished beyond around 25% chamber occupancy, thereby informing the minimum array size that is required. Importantly, the capture efficiency of the DEP-BPE platform for melanoma cells when using optimized conditions matched the performance predicted by our analysis. Finally, isolation of melanoma cells from contrived (spike-in) and clinical samples on our platform using optimized conditions was demonstrated. The capture and individual isolation of CMCs, confirmed by post-capture labeling, from patient-derived samples suggests the potential of this platform for clinical application.

Establishment of a humanized animal model of systemic sclerosis in which T helper-17 cells from patients with systemic sclerosis infiltrate and cause fibrosis in the lungs and skin

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by inflammation, microangiopathy, and progressive fibrosis in the skin and internal organs. To evaluate the pathophysiologic mechanisms and efficacies of potential therapeutics for SSc, a preclinical model recapitulating the disease phenotypes is needed. Here, we introduce a novel animal model for SSc using immunodeficient mice injected with peripheral blood mononuclear cells (PBMCs) from SSc patients. Human PBMCs acquired from SSc patients and healthy controls were transferred into NOD.Cg-PrkdcscidIl2rgtm1Wjl (NSG) mice with concurrent bleomycin injection. Blood, skin, and lung tissues were acquired and analyzed after PBMC engraftment. In addition, we investigated whether the humanized murine model could be used to assess the efficacy of potential therapeutics for SSc. Human PBMCs from SSc patients and healthy controls were engrafted into the blood, skin, and lung tissues of NSG mice. Histological analysis of affected tissues from mice treated with SSc PBMCs (SSc hu-mice) demonstrated substantial inflammation, fibrosis and vasculopathy with human immune cell infiltration and increased expression of IL-17, TGF-β, CCL2, CCL3, and CXCL9. The proportions of circulating and tissue-infiltrating T helper 17 (Th17) cells were elevated in SSc hu-mice. These cells showed increased expression of CXCR3 and phosphorylated STAT3. SSc hu-mice treated with rebamipide and other potential Th17-cell-modulating drugs presented significantly reduced tissue fibrosis. Mice injected with patient-derived PBMCs show promise as an animal model of SSc.

Abstract 1113: NUC-3373 potentiates immune-mediated cytotoxicity of CRC cells

Abstract Background: NUC-3373 is a phosphoramidate modification of fluorodeoxyuridine-monophosphate (FUDR-MP), the active anti-cancer metabolite of fluorouracil (5-FU), which binds and inhibits thymidylate synthase (TS), disrupting DNA synthesis and repair. NUC-3373 is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, reduce generation of toxic metabolites, and deliver higher levels of active metabolite FUDR-MP to tumors. Our previous work demonstrated that NUC-3373 causes colorectal cancer (CRC) cell lines to release damage associated molecular patterns (DAMPs)- molecular signals that activate circulating immune cells leading to immunogenic cell death (ICD). NUC-3373 promotes activation of co-cultured NK-92 MI cells, a natural killer cell line, by upregulating degranulation and IFN-γ production and downregulating the immune checkpoint molecule TIGIT. This study aims to test the hypothesis that NUC-3373 induced DAMPs promote ICD in co-cultures of CRC cells with peripheral blood mononuclear cells (PBMCs). We also explore the dynamics of PD-L1 and cytokine expression and combination with a PD-1 checkpoint inhibitor. Methods: HCT116 cells were exposed to increasing concentrations of NUC-3373 or vehicle control for 24 hours prior to co-culture with healthy patient-derived PBMCs. At the time of co-culture, cells were treated with 10 υg/ml nivolumab to assess the effect of an anti-PD-1 antibody being combined with NUC-3373. CRC and PBMC co-cultures were continued for up to 72 hours. Gene expression of PD-L1 and cytokines (TNF-α, IFN-γ, and IL-2) was assessed by RT-PCR. PD-L1 surface expression was determined by flow cytometry. Confluence was determined by automated cytometry analysis (Celigo) and cell viability of CRC cells was determined by sulforhodamine-B assay (SRB). Results: NUC-3373 increased the gene expression of cytokines and PD-L1 over 48 hours of co-culture. Surface expression of PD-L1 was increased on both PBMCs and CRC cells in co-culture, as well as on CRC cells cultured without PBMCs, indicating that global increase in PD-L1 is NUC-3373 mediated. CRC cells pre-treated with NUC-3373 and co-cultured with PBMCs displayed a reduction in cell viability compared to monocultured CRC cells. Furthermore, CRC and PBMC co-cultures treated with anti-PD-1 antibody showed increased cytotoxicity when CRC cells were pre-treated with NUC-3373. Conclusions: NUC-3373 induces DAMPs and PD-L1 expression in CRC cells and promotes pro-immune cytokine production from PBMCs resulting in ICD in vitro. Furthermore, the addition of anti-PD-1 antibody to co-cultures of NUC-3373-treated CRC cells and PMBCs enhanced this ICD. With an improved clinical safety profile (NCT03428958), including less haematologic toxicity, and a more convenient dosing regimen than 5-FU, NUC-3373 is an attractive combination partner for immune checkpoint inhibitors. Citation Format: Oliver James Read, Jennifer Bré, David J. Harrison. NUC-3373 potentiates immune-mediated cytotoxicity of CRC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1113.

Blood-based untargeted metabolomics in relapsing-remitting multiple sclerosis revealed the testable therapeutic target.

Metabolic aberrations impact the pathogenesis of multiple sclerosis (MS) and possibly can provide clues for new treatment strategies. Using untargeted metabolomics, we measured serum metabolites from 35 patients with relapsing-remitting multiple sclerosis (RRMS) and 14 healthy age-matched controls. Of 632 known metabolites detected, 60 were significantly altered in RRMS. Bioinformatics analysis identified an altered metabotype in patients with RRMS, represented by four changed metabolic pathways of glycerophospholipid, citrate cycle, sphingolipid, and pyruvate metabolism. Interestingly, the common upstream metabolic pathway feeding these four pathways is the glycolysis pathway. Real-time bioenergetic analysis of the patient-derived peripheral blood mononuclear cells showed enhanced glycolysis, supporting the altered metabolic state of immune cells. Experimental autoimmune encephalomyelitis mice treated with the glycolytic inhibitor 2-deoxy-D-glucose ameliorated the disease progression and inhibited the disease pathology significantly by promoting the antiinflammatory phenotype of monocytes/macrophage in the central nervous system. Our study provided a proof of principle for how a blood-based metabolomic approach using patient samples could lead to the identification of a therapeutic target for developing potential therapy.

Bromodomain and Extra-Terminal Inhibitor BMS-986158 Reverses Latent HIV-1 Infection In Vitro and Ex Vivo by Increasing CDK9 Phosphorylation and Recruitment.

Latent reservoir persistence remains a major obstacle for curing human immunodeficiency virus type 1 (HIV-1) infection. Thus, strategies for the elimination of latent HIV-1 are urgently needed. As a bromodomain and extra-terminal (BET) inhibitor, BMS-986158 has been used in clinical trials for advanced solid tumors and hematological malignancies. Here, we found that BMS-986158 reactivated latent HIV-1 in three types of HIV-1 latency cells in vitro, and in combination antiretroviral therapy (cART)-treated patient-derived peripheral blood mononuclear cells ex vivo, without influencing global immune cell activation. BMS-986158 reactivated latent HIV-1 by increasing phosphorylation of CDK9 at Thr186 and promoting recruitment of CDK9 and RNA polymerase II to the HIV-1 long terminal repeat in J-Lat cells. Furthermore, BMS-986158 exerted strong synergism in reactivating latent HIV-1 when combined with prostratin and vorinostat and enhanced the antiviral activity of anti-HIV-1 drugs. Finally, BMS-986158 showed antiviral activity in an HIV-1 acute infection model, possibly by arresting the cell cycle in infected cells. Thus, these results suggest that BMS-986158 is a potential candidate for AIDS/HIV-1 therapy.

Abstract PR-001: Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma

Abstract Introduction: Immune checkpoint blockade either alone or in combination with chemotherapy has been ineffective in pancreatic ductal adenocarcinoma (PDAC) likely due to underlying immunosuppressive pathways. The C-X-C chemokine receptor type 4 (CXCR4)/chemokine (C-X-C motif) ligand 12 (CXCL12) axis is a key immunosuppressive pathway where CXCL12 released by cancer-associated fibroblasts (CAF) binds to its receptor CXCR4 expressed by cytotoxic CD8+ T cells (CTL) which results in their decreased abundance within tumors. Encouraging preclinical and clinical data from mouse PDAC models and human PDAC tumor sections have shown T cell tumor accumulation after co-inhibition of CXCR4 and PD1/PDL1, however number of tumor regressions were limited. Results: To further explore the mechanism by which inhibition of the CXCR4/CXCL12 pathway modulates the immune TME in triggering an anti-tumor immune response in human PDAC, we generated an autologous co-culture system with patient-derived tumor organoids (PDTO) and peripheral blood mononuclear cells (PBMC). PDAC tumor tissue from surgical resection was used to generate lines of PDTO that recapitulate the histological and genetic characteristics of the original tumor. We demonstrate that tumor-specific reactive T cells can be obtained in PDAC by co-culture with autologous PDTO, with up to 5% of CD8+ T cells producing IFNγ after tumor organoid stimulation. No T cell reactivity was observed against normal pancreatic organoids, and blocking tumor MHC I/MHC II decreased the proportion of activated T cells. Ex vivo migration assays were established to better understand the role of CXCL12 in PDAC tumor T cell exclusion. Tumor organoids attracted autologous PBMC and addition of recombinant CXCL12 decreased PBMC migration, while treatment of PBMC with the CXCR4 inhibitor AMD3100, rescued the number of PBMCs that were able to migrate towards the tumor organoids. PBMC infiltrated PDTO-containing but not empty Matrigel domes. Addition of AMD3100 increased PBMC infiltration within the Matrigel dome containing PDTOs. Finally, we found that pre-treatment of PBMC with AMD3100 increased T cell reactivity against PDTO, and that T cell activation was further increased when stimulated with PDTO pre-treated with chemotherapy. Conclusions: We found that CXCL12 modulates the migration of T cells towards tumor organoids generated from pancreatic cancer tissue, and that inhibition of CXCR4 not only increases the migration potential, but also increases tumor-specific T cell activation. The use of ex vivo autologous co-culture and migration assays provide a unique strategy to identify modulators that may enhance T cell mediated neoplastic cell death to guide therapeutic intervention in PDAC. Citation Format: Emily Alouani, Ilenia Pellicciotta, Winston Wong, Alexander S. Thomas, Michael D. Kluger, Anna M. Chiarella, Anil K. Rustgi, Gulam A. Manji. Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-001.

A novel missense mutation in TNFAIP3 causes haploinsufficiency of A20

A20, encoded by TNFAIP3, is an effective anti-inflammatory molecule that plays a crucial role in inhibiting NF-κB signal transmission and is linked to multiple inflammatory diseases. It has been reported that the haploinsufficiency of A20 (HA20) caused by multiple base mutations in TNFAIP3 shows early-onset spontaneous Behçet-like disease. However, the mechanisms by which A20 mutations involved in inflammatory disease are incompletely defined. Herein, we reported a novel TNFAIP3 (c.1804A > T, p.T602S) variation, which has not been reported before. Summarizing the patient's immunodeficiency phenotype, we aimed to delineate the underlying mechanism for regulation of inflammation and immunity. Candidate genes associated with the Behçet-like phenotypes of the patient were screened and identified by using whole-exome and sanger sequencing. Functional studies were performed in A20(c.1804A > T, p.T602S) patient-derived peripheral blood mononuclear cells (PBMCs) and THP-1 cell lines by lentivirus mediating stable over-expression of A20 and A20(c.1804A > T, p.T602S) to analyze the activity of NF-κB signaling pathway. The clinical manifestations in patients with syndrome are Behçet-like disorder, and sequencing revealed heterozygous mutation in TNFAIP3 (c.1804A > T, p.T602S). Functional tests found that the PBMCs of the patient and his family carrying this heterozygous variant stimulated by LPS, TNF-α, or IL-1β, increased the levels of inflammatory factors and induced over-activation of the canonical NF-κB signaling pathway. Similar results were also observed in the stable transduction THP-1 (A20, c.1804A＞T) cell line stimulated by LPS, TNF-α or IL-1β. The novel loss-of-function A20 variation (c.1804A > T, p.T602S) causes over-activation of the canonical NF-κB signaling pathway and fail to terminate NF-κB signaling in response to stimulation by inflammatory cytokines. The variation triggers a dominantly-inherited Behçet-like disorder caused by haploinsufficiency of the A20 protein. Identification of the novel A20 mutation attaches great importance to prenatal diagnosis and fetal therapeutic intervention, drastically reducing the risk of newborns suffering from HA20.

CD33/CD3-Bispecific T-Cell Engaging (BiTE®) Antibody Constructs Efficiently Target Monocytic CD14+ hla-DRlow IDO+ aml-MDSCs

Acute Myeloid Leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 25%. Emerging evidence suggest that immune alterations favor leukemogenesis and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. In this study we sought to investigate whether the CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with documented pre-clinical activity against AML blasts can also target CD33+ AML-MDSCs by redirecting and activating T-cells and thereby enhance AMG 330 mediated anti-leukemic activity.First, we assessed the presence of circulating CD14+ cells that co-express CD33 but lack HLA-DR expression (HLA-DRlo) in patients with newly diagnosed AML. These monocytic cells represent one of the best-defined human MDSC subsets. Frequency of CD14+HLA-DRlo cells was significantly increased in untreated AML patients as compared to healthy controls (28,98±4,19%, n=13 versus 3,28±0,75%, n=37) but did not correlate with the number of AML blasts. Purified CD14+HLA-DRlo cells suppressed in vitro T-cell proliferation in a concentration-dependent manner allowing us their denomination as MDSCs.Interestingly, we also observed that primary AML blasts and human AML cell lines (THP, OCI-AML, and HL-60) induced HLA-DRlo cells from healthy donor-derived monocytes that were T-cell suppressive and expressed the immune regulatory indoleamine-2,3-dioxygenase (IDO). Functional and phenotypic reprogramming of monocytes was cell-contact independent.Using ex vivo co-culturing models of primary AML blasts/AML cell lines (=target cells) and T-cells (=effector cells) we observed AMG 330-triggered antibody construct-dependent cell-mediated cytotoxicity/ADCC. Short-term (three to six days) treatment of AML patient-derived peripheral blood mononuclear cells (PBMCs) with 10 or 100 pM AMG 330 led to T-cell activation based on proliferation and on increased expression/production of CD25, CD69, CD137, CD154, IL-2, and IFN-g. The initial T-cell frequency ranging from 0,16 to 14,30% had no impact on their responsiveness. Moreover, we observed a bystander activation of NK-cells. The addition of AML-MDSCs into these co-cultures did not negatively impact ADCC activity. On the contrary, T-cells formed immunological synapses with AML-MDSCs and triggered their elimination (also in presence of AML blasts) overcoming their T-cell suppressive effect. MDSC levels in AML-PBMCs had no effect on AMG 330-mediated T-cell (and bystander cell) activation, which was further corroborated in experiments where the total CD14+ cell compartment (comprising AML-MDSCs) was removed prior AMG 330 application.However, we did observe an impact of MDSCs on BiTE® mediated T-cell proliferation, which has been shown as highly relevant for clinical activity of BiTE® antibody constructs (Zugmaier et al. Blood 2015). Here we found a 1,68-fold enhanced (p=0.001) T-cell expansion in AML-PBMCs treated with 100 pM AMG 330 following CD14+ cell depletion. Given the importance of T-cell proliferation for clinical activity this finding warrants further exploration.Taken together, our results suggest that anti-CD33/CD3 bispecific BiTE® antibody constructs may achieve anti-leukemic efficacy not only through direct T cell mediated cytotoxicity against AML blasts but also through circumventing immune evasion via MDSCs targeting. Although therapeutic targeting of MDSCs in patients has not yet been successfully accomplished, bystander killing of CD33+ MDSCs via anti-CD33/CD3-bispecific BiTE® antibody constructs could represent a very promising approach to increase the anti-leukemic T-cell response in AML patients and to reverse immune evasion. DisclosuresMougiakakos:AMGEN: Research Funding. Kischel:AMGEN: Employment. Michael:AMGEN: Employment. Dos Santos:Amgen Inc.: Employment. Jitschin:AMGEN: Research Funding. Mackensen:AMGEN: Research Funding.

Transfer of PBMC From SSc Patients Induces Autoantibodies and Systemic Inflammation in Rag2-/-/IL2rg-/- Mice.

ObjectiveThe contribution of sustained autologous autoantibody production by B cells to the pathogenesis of systemic sclerosis (SSc) and granulomatosis with polyangiitis (GPA) is not fully understood. To investigate this, a humanized mouse model was generated by transferring patient-derived peripheral blood mononuclear cells (PBMC) into immunocompromised mice.MethodsPBMC derived from patients with SSc and GPA as well as healthy controls (HD) were isolated, characterized by flow cytometry, and infused into Rag2-/-/IL2rg-/- mice. In addition, PBMC from SSc patients treated with rituximab were transferred into mice. Twelve weeks later, human autoantibodies were determined in blood of the recipient mice and affected tissues were analyzed for pathological changes by histology and immunohistochemistry.ResultsMice engrafted with PBMC derived from SSc patients developed autoantibodies such as antinuclear antibodies (ANA) mimicking the pattern of the respective donors. Moreover, cellular infiltrates dominated by B cells were observed in lung, kidney and muscles of the recipient mice. By contrast, PBMC derived from HD or GPA patients survived in recipient mice after transfer, but neither human autoantibodies nor inflammatory infiltrates in tissues were detected. Furthermore, these pathological changes were absent in mice transferred with PBMC from rituximab-treated SSc patients.ConclusionThis humanized mouse model is indicative for cross-reactivity of human lymphocytes to murine autoantigens and argues for a pivotal role of B cells as well as of sustained autoimmunity in the pathogenesis of SSc. It provides a powerful tool to study interstitial lung disease and so far, under-recognized disease manifestations such as myositis and interstitial nephritis.