Primary Lymphocytes Research Articles

Immunosuppressive activity of non-psychoactive Cannabis sativa L. extract on the function of human T lymphocytes

BackgroundCannabis sativa L. extracts (CSE) are used for treating inflammatory conditions, but little is known about their immunomodulatory effects. We investigated a novel CSE with high (14%) CBD and low (0.2%) THC concentration in comparison with pure CBD on primary human lymphocytes. MethodsProliferation, cell cycle distribution, apoptosis/necrosis and viability were analysed with standard methods. Genotoxicity was evaluated with the comet-assay. The effect on T lymphocyte activation was evaluated via CD25/CD69 marker expression, degranulation assays and the production of cytokines. The influence on the transcription factors was analysed using Jurkat reporter cell lines. Specific CB2 receptor antagonist SR144528 and TRPV1 receptor antagonist A78416B were used to study the involvement of CB2 or TRPV1 receptors. ResultsCSE inhibited the proliferation of activated T lymphocytes in a dose-dependent manner without inducing apoptosis, necrosis, or affecting cell viability and DNA integrity. The inhibitory effect was mediated via the suppression of T lymphocytes activation, particularly by the suppression of CD25 surface marker expression. Furthermore, CSE interferes with the functionality of the T lymphocytes, as indicated by inhibition of degranulation, IL-2, and IFN-γ production. AP-1-and-NFAT-reporter activation was reduced implicating an AP-1-and-NFAT-mediated mode of action. The effects were in part reversed by SR144528 and A78416B, showing that the effects were mainly mediated by CB2 and TRPV1 receptors. ConclusionCSE and CBD have immunomodulatory effects and interfere with the activation and functionality of T lymphocytes. A comparison between CSE and CBD suggests that the immunosuppressive effect of CSE is mostly due to the effect of CBD.

Method for Identifying Galectin Ligands on Lymphocyte Membrane Glycoproteins.

Glycosylation is one of the most common protein posttranslational modifications. Most human lymphocyte membrane receptors are modified by diverse glycan structures, and functional studies have indicated that a family of glycan-binding proteins, galectins, can significantly modulate lymphocyte development and function by interacting with these glycans. Several galectins have a varying degree of affinity for the N-acetyllactosamine (LacNAc) disaccharide, and some critical lymphocyte receptors can be modified by glycan structures carrying this motif. However, the site-specific glycan composition on primary lymphocyte membrane receptors in healthy individuals is largely limited. The main reason for the limitation is low abundance of available material from a single donor and compositional heterogeneity in glycan structures that can potentially modify a protein. Donor-dependent variability in N-glycan structures on CD16a isolated from primary NK cells of healthy human donors was recently reported. NK cell CD16a is glycosylated at five N-glycosylation sites, and two of the five sites are modified, almost exclusively, by N-glycans with multiple LacNAc repeats which can serve as ligands for endogenous galectins. Thus, the protocol described in this section can be utilized to identify galectin ligands at specific glycosylation sites of endogenous membrane receptor from circulating primary human lymphocytes.

Possible mechanism of CHI3L1 promoting tonsil lymphocytes proliferation in children with obstructive sleep apnea syndrome.

The typical characteristic of pediatric obstructive sleep apnea syndrome (OSAS) is systemic inflammation and adenotonsillar hypertrophy (ATH), but the inflammatory markers and mechanism of adenotonsillar proliferation are unclear. IHC, qPCR, and western blotting were used to identify the expression of CHI3L1 in the tonsils of children with OSAS. The primary tonsil lymphocytes (PTLCs) from children with OSAS were cultured and recombinant human CHI3L1 protein was added to culture media. After the stimulation with CHI3L1 protein of different concentrations and time points, lymphocyte proliferation was assessed by CCK-8 kits and flow cytometry. The activation of ERK1/2 and the effects on the proliferation of PTLCs were observed by western blotting. The expression of CHI3L1 was higher in the OSAS group than in the PS group. CHI3L1 (100 ng/mmol for 24 h) resulted in a significant increase in the proliferation rate. The ERK1/2 activator (PMA) promoted the proliferation of PTLCs and inhibitor AG126 significantly inhibited proliferation. CHI3L1 can promote the proliferation of tonsil lymphocytes via ERK1/2 pathways. This result indicates that CHI3L1 may play an important role in the pathogenesis of OSAS in children. Inhibition of CHI3L1 or ERK1/2 may be potential therapeutic targets for CHI3L1-induced proliferation in childhood OSAS. CHI3L1 may be an inflammatory marker in childhood OSAS. CHI3L1 can promote the proliferation of PTLCs in a concentration and time-dependent condition. CHI3L1 can promote the proliferation of tonsil lymphocytes via ERK1/2 pathways.

Immunosuppressive effect of Cannabis sativa L. extract on human primary lymphocytes

Immunosuppressive effect of Cannabis sativa L. extract on human primary lymphocytes

Taraxasterol mitigates Con A-induced hepatitis in mice by suppressing interleukin-2 expression and its signaling in T lymphocytes

Discovery of anti-inflammatory drugs that can suppress T lymphocyte activation and proliferation by inhibiting TCR/CD3 and IL-2/IL-2R signaling is still needed in clinic, though rapamycin and other related reagents have made great success. Taraxasterol (TAS) is an active ingredient of dandelion, an anti-inflammatory medicinal herb with low in vivo toxicity that has long been used in China. Yet the action mechanism of TAS on lymphocytes remains elusive. The anti-inflammatory effects of TAS were evaluated in C57BL/6 mouse primary lymphocytes stimulated with concanavalin A (Con A) in vitro and in mouse model of Con A-induced acute hepatitis in vivo. Our results showed that TAS significantly suppressed Con A-induced acute hepatitis in a mouse model, reducing the hepatic necrosis areas, the release of aminotransferases, and the production of IL-2 and other inflammatory cytokines. Supporting this, in vitro study also showed that TAS reduced the production of IL-2 and the expression of IL-2 receptor subunit α (CD25) upon the stimulation of Con A, which was likely mediated by suppressing NF-κB activation. The downstream pathways of IL-2/IL-2R signaling, including the activation of PI3K/PDK1/mTOR, STAT3 and STAT5, were also suppressed by TAS. Consistently, Con A-induced T cell proliferation was also inhibited by TAS in vitro. Our data indicate that TAS can suppress both T lymphocyte activation and cell proliferation by down-regulating IL-2 expression and its signaling pathway thereby ameliorating Con A-induced acute hepatitis, highlighting TAS as a potential drug candidate for treating inflammatory diseases including autoimmune hepatitis.

Molecular characterization, expression analysis and cellular location of IL-4/13 receptors in large yellow croaker (Larimichthys crocea)

Interleukin (IL)-4 and IL-13 are closely related class I cytokines that play key roles in the T helper (Th)-2 immune response via heterodimeric receptors. IL-4 signals via both the type I (IL-4Rα/γc) and type II (IL-4Rα/IL-13Rα1) receptor complexes, while IL-13 signals only via the type II receptor complex. IL-13Rα2 is traditionally considered a “decoy” receptor for IL-13. However, the IL-4/13 system and its response to pathogenic infection are still not fully understood in fish. In this study, we identified four IL-4/13 receptor subunit genes in the large yellow croaker (Larimichthys crocea): LcIL-4Rα1, LcIL-4Rα2, LcIL-13Rα1, and LcIL-13Rα2. Sequence analysis showed that these receptors possessed typical characteristic domains, including a signal peptide, two fibronectin type III (FN III)-like domains, and a transmembrane domain, but their cytoplasmic regions were not well conserved. The mRNA and protein of the four IL-4/13 receptors were constitutively expressed in all examined tissues of large yellow croaker. Their mRNAs were also detected in primary head kidney macrophages (PKMs), primary head kidney granulocytes (PKGs), and primary head kidney lymphocytes (PKLs). Immunofluorescence assay further showed that LcIL-4Rα and LcIL-13Rα1 were expressed on the membrane of IgM + B cells. After stimulation by Vibrio alginolyticus and poly (I:C) (a viral dsRNA mimic), the mRNA levels of LcIL-4/13 receptors were significantly upregulated in the head kidney and spleen. Their mRNA levels were also upregulated in head kidney leukocytes in response to poly (I:C) and lipopolysaccharide (LPS) treatment. Moreover, both recombinant LcIL-4/13A and LcIL-4/13B upregulated LcIL-4Rα1 and LcIL-4Rα2 in primary leukocytes, but only recombinant LcIL-4/13A upregulated LcIL-13Rα1 and LcIL-13Rα2. These results indicated that LcIL-4/13 receptors, containing conserved functional domains, may be involved in the IL-4/13-mediated immune response to pathogenic infections in the large yellow croaker.

Microfluidic transfection of mRNA into human primary lymphocytes and hematopoietic stem and progenitor cells using ultra-fast physical deformations

Messenger RNA (mRNA) delivery provides gene therapy with the potential to achieve transient therapeutic efficacy without risk of insertional mutagenesis. Amongst other applications, mRNA can be employed as a platform to deliver gene editing molecules, to achieve protein expression as an alternative to enzyme replacement therapies, and to express chimeric antigen receptors (CARs) on immune cells for the treatment of cancer. We designed a novel microfluidic device that allows for efficient mRNA delivery via volume exchange for convective transfection (VECT). In the device, cells flow through a ridged channel that enforces a series of ultra-fast and large intensity deformations able to transiently open pores and induce convective transport of mRNA into the cell. Here, we describe efficient delivery of mRNA into T cells, natural killer (NK) cells and hematopoietic stem and progenitor cells (HSPCs), three human primary cell types widely used for ex vivo gene therapy applications. Results demonstrate that the device can operate at a wide range of cell and payload concentrations and that ultra-fast compressions do not have a negative impact on T cell function, making this a novel and competitive platform for the development of ex vivo mRNA-based gene therapies and other cell products engineered with mRNA.

ATG12 is involved in the antiviral immune response in large yellow croaker (Larimichthys crocea)

ATG12, a core autophagy protein, forms a conjugate with ATG5 to promote the formation of autophagosome membrane, and plays an important role in antiviral immunity. However, little is known about the function of ATG12 in fish. Here, we cloned the open reading frame (ORF) of large yellow croaker (Larimichthys crocea) ATG12 (LcATG12), which is 354 nucleotides long and encodes a protein of 117 amino acids. The deduced LcATG12 possesses a conserved APG12 domain (residues 31 to 117), and shares 91.45% identities with ATG12 in orange-spotted grouper (Epinephelus coioides). LcATG12 was constitutively expressed in all examined tissues, with the highest level in intestine. Its transcript was also detected in primary head kidney granulocytes (PKG), primary head kidney macrophages (PKM), primary head kidney lymphocytes (PKL), and large yellow croaker head kidney (LYCK) cell line, and was significantly up-regulated by poly(I:C). LcATG12 was regularly distributed in both cytoplasm and nucleus of LYCK and epithelioma papulosum cyprinid (EPC) cells. Overexpression of LcATG12 in EPC cells significantly inhibited the replication of spring viremia of carp virus (SVCV). Further studies reveled that LcATG12 could induce the occurrence of autophagy in LYCK cells. Furthermore, overexpression of LcATG12 in LYCK cells increased the expression levels of large yellow croaker type I interferons (IFNs, IFNc, IFNd, and IFNh), IFN regulatory factors (IRF3 and IRF7), and IFN-stimulated genes (PKR, Mx, and Viperin). All these data indicated that LcATG12 plays a role in the antiviral immunity possibly by inducing both autophagy and type I IFN response in large yellow croaker.

Comparison of Tumor Microenvironments Between Primary Tumors and Brain Metastases in Patients With NSCLC

IntroductionThis study investigates the immune profile of the primary lung tumors and the corresponding brain metastasis from patients with NSCLC using multiplex fluorescence immunohistochemistry. MethodsThe study evaluated 34 patients who underwent autopsy or surgical resection for brain metastasis and autopsy, surgical resection, or core biopsy for primary lung cancer. We compared the densities of various immune cells in the primary tumors and the brain metastases by multiplex fluorescence immunohistochemical analysis. ResultsThe density of CD4-positive (CD4+) T-cells, CD8-positive T-cells, and CD4+ Foxp3-positive T-cells were statistically higher in both tumor and stromal areas in primary lung cancer specimens when compared with brain metastases samples (p < 0.0001). Only CD204-positive cells were statistically higher in the tumor areas of the brain metastases (p = 0.0118). Tumor-infiltrating lymphocytes associated with brain metastases positively correlated with overall survival, but primary lung tumor-infiltrating lymphocytes did not. The density of CD4+ and CD4+ Foxp3-positive T-cells in brain metastases with radiation was statistically higher in the carcinoma and stromal areas compared with those without radiation (p = 0.0343, p = 0.0173). ConclusionsOur findings that CD204-positive cells were higher in brain metastases may have broader implications for treatment as these macrophages may be immunosuppressive and make the immune environment less reactive. Furthermore, the finding that the density of CD4+ T-cells was higher in cancer and stroma areas of brain metastases after radiotherapy supports the addition of immunotherapy to radiation therapy in the treatment of brain metastases in NSCLC.

Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes

Adeno-associated virus serotype 6 (AAV6) is a valuable reagent for genome editing of hematopoietic cells due to its ability to serve as a homology donor template. However, a comprehensive study of AAV6 transduction of hematopoietic cells in culture, with the goal of maximizing ex vivo genome editing, has not been reported. Here, we evaluated how the presence of serum, culture volume, transduction time, and electroporation parameters could influence AAV6 transduction. Based on these results, we identified an optimized protocol for genome editing of human lymphocytes based on a short, highly concentrated AAV6 transduction in the absence of serum, followed by electroporation with a targeted nuclease. In human CD4+ T cells and B cells, this protocol improved editing rates up to 7-fold and 21-fold, respectively, when compared to standard AAV6 transduction protocols described in the literature. As a result, editing frequencies could be maintained using 50- to 100-fold less AAV6, which also reduced cellular toxicity. Our results highlight the important contribution of cell culture conditions for ex vivo genome editing with AAV6 vectors and provide a blueprint for improving AAV6-mediated homology-directed editing of human T and B cells.

Increased DNA-incorporated thiopurine metabolite as a possible mechanism for leukocytopenia through cell apoptosis in inflammatory bowel disease patients with NUDT15 mutation

Polymorphisms in the nucleotide diphosphate-linked moiety X-type motif 15 (NUDT15) gene are associated with thiopurine-induced leukopenia in patients with inflammatory bowel disease (IBD). NUDT15-associated subcellular thiopurine metabolism has not been investigated in primary lymphocytes. We hypothesized that NUDT15 mutation increases DNA-incorporated deoxythioguanosine (dTG) and induces apoptosis in lymphocytes. DNA-incorporated dTG in peripheral blood mononuclear cells (PBMCs) and 6-thioguanine nucleotides (6-TGN) in red blood cells were measured in patients with IBD undergoing thiopurine treatment. The association of a single nucleotide polymorphism for NUDT15 (rs116855232) with dTGPBMC was examined. The pro-apoptotic effect of DNA-incorporated dTG was examined ex vivo in association with NUDT15 genotypes by co-culturing patient-derived peripheral CD4+ T lymphocytes with 6-thioguanine (6-TG). dTGPBMC was significantly higher in NUDT15 variants than in non-variants. dTGPBMC, but not 6-TGNRBC, negatively correlated with peripheral lymphocyte counts (r = - 0.31 and - 0.12, p = 0.012 and 0.173, respectively). DNA-incorporated dTG significantly accumulated to a greater extent in lymphocytes from NUDT15 variants when co-cultured with 6-TG ex vivo than in those from non-variants and was associated with decreased proliferation and increased apoptosis. Increased DNA-incorporated dTG may be responsible for thiopurine-induced leukocytopenia through cell apoptosis in IBD patients with NUDT15 mutation.

Ellagic Acid from Terminalia arjuna Fruits Protects Against Chromium and Cobalt Toxicity in Primary Human Lymphocytes

Increased accumulation of heavy metal ions such as Cr6+ and Co2+ due to release from prostheses and metallic implants has been reported. These metal ions have been shown to affect both resting and activated lymphocytes. Natural remedies towards mitigating the cytotoxic effects of metal ions are clearly warranted. Polyphenolic compounds which are part of hydrolysable tannins from natural plant sources are considered effective in cheating heavy metal ions in a biological system. We have isolated and characterized a polyphenolic compound (ellagic acid) from Terminalia arjuna fruits that has been tested for its ability to attenuate the metal ion toxicity in primary human lymphocytes in culture. Cr6+ and Co2+ (100μM) decreased lymphocyte viability and proliferation and increased apoptosis of resting as well as CD3 and/or CD28-stimulated lymphocytes. Metal ions markedly diminished the cytokine (interleukin-2 and interferon-γ) secretion from activated lymphocytes. Pretreatment with ellagic acid at 25, 50, and 100μM concentrations effectively improved viability and proliferative responses of both resting and activated lymphocytes, while attenuating the apoptotic index. Ellagic acid also tended to normalize the cytokine secretion from the activated lymphocytes even in the presence of metal ions, suggesting broad effects on the adaptive immune system.

Clinical and morphological portrait of tumors with microsatellite instability

Background. Microsatellites are short tandem nucleotide repeats, the change in length of which plays a key roles in the pathogenesis of various malignant neoplasms. This change is called microsatellite instability. It is caused by aberrations in the genes of DNA mismatch repair system. Tumors with microsatellite instability are a special subtype regardless of location and are characterized by high sensitivity to immune checkpoint inhibitors.Objective – determination of characteristic clinical and morphological patterns of tumors of various localizations with microsatellite instability.Materials and methods. The study included 512 patients with malignant tumors of different localizations. Of these, 359 patients were diagnosed with colorectal cancer, 57 with uterine body cancer, and 57 with stomach cancer. Determination of the status of microsatellite instability was performed by a PCR-based method using 5 mononucleotide markers: BAT-25, BAT-26, NR-21, NR-24, NR-27.Results. The prevalence of microsatellite instability in colorectal cancer, uterine neoplasm and gastric cancer was 6.4; 22.8 and 1.75 %, respectively. Patients with MSI-positive colorectal cancer are characterized by yonger age (p = 0.023), right-sided localization of the tumor (p &lt;0.0001), presence of multiple primary tumors (p = 0.0299), poorly differentiation (p = 0.0025), mucinous component (p &lt;0.0001), tumor-infiltrating lymphocytes (p &lt;0.0001) and Crohn-like reaction (p = 0.0006). Patients with uterine neoplasms with microsatellite instability are characterized by the presence of endometrial adenocarcinoma (p = 0.047), as well as the presence of tumor-infiltrating lymphocytes (p = 0.0022) and cribriform growth (p = 0.0011).Conclusion. A common pattern for colorectal cancer and uterine neoplasms is the presence of tumor-infiltrating lymphocytes. Certain clinical and morphological features of tumors of these localizations will more accurately identify candidates for microsatellite instability status determination for further immunotherapy.

CometChip analysis of human primary lymphocytes enables quantification of inter-individual differences in the kinetics of repair of oxidative DNA damage

Although DNA repair is known to impact susceptibility to cancer and other diseases, relatively few population studies have been performed to evaluate DNA repair kinetics in people due to the difficulty of assessing DNA repair in a high-throughput manner. Here we use the CometChip, a high-throughput comet assay, to explore inter-individual variation in repair of oxidative damage to DNA, a known risk factor for aging, cancer and other diseases. DNA repair capacity after H2O2-induced DNA oxidation damage was quantified in peripheral blood mononuclear cells (PBMCs). For 10 individuals, blood was drawn at several times over the course of 4–6 weeks. In addition, blood was drawn once from each of 56 individuals. DNA damage levels were quantified prior to exposure to H2O2 and at 0, 15, 30, 60, and 120-min post exposure. We found that there is significant variability in DNA repair efficiency among individuals. When subdivided into quartiles by DNA repair efficiency, we found that the average t1/2 is 81 min for the slowest group and 24 min for the fastest group. This work shows that the CometChip can be used to uncover significant differences in repair kinetics among people, pointing to its utility in future epidemiological and clinical studies.

Methylglyoxal induces chromosomal instability and mitotic dysfunction in lymphocytes.

Type 2 diabetes is associated with elevated levels of DNA damage, in particular micronuclei (MNi) which are formed by acentric chromosome fragments caused by double-stranded DNA breaks (DSBs), or whole chromosomes which fail to segregate during mitosis. We investigated if methylglyoxal (MGO), a reactive dicarbonyl known to be elevated in type 2 diabetes is capable of increasing chromosomal instability and DNA damage as measured by the cytokinesis block micronucleus cytome (CBMNcyt) assay in B-lymphoblastoid WIL2-NS cells and primary peripheral blood lymphocytes (PBL). We also investigated the level of various dicarbonyl stress biomarkers, including extracellular and intracellular MGO, protein and MGO modifications of DNA. WIL2-NS cells exposed to either MGO or a glyoxalase 1 inhibitor showed increases in MNi and nuclear buds, which were associated with an increase in intracellular MGO. DNA damage in the form of MNi and nucleoplasmic bridges were observed in primary PBL exposed to 10 µM MGO, suggesting low concentrations of MGO may be genotoxic. Furthermore, we showed, using fluorescent in situ hybridisation, that the majority of MNi caused by MGO in WIL2-NS cells were caused by whole chromosome loss events, rather than DSBs. Our data suggest that MGO, a reactive metabolite elevated in type 2 diabetes and other pathologies, can affect genomic integrity by impairing chromosome segregation during mitosis.

TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) Is Upregulated in Lymphocytes Stimulated with Concanavalin A.

The glycolytic modulator TP53-Inducible Glycolysis and Apoptosis Regulator (TIGAR) is overexpressed in several types of cancer and has a role in metabolic rewiring during tumor development. However, little is known about the role of this enzyme in proliferative tissues under physiological conditions. In the current work, we analysed the role of TIGAR in primary human lymphocytes stimulated with the mitotic agent Concanavalin A (ConA). We found that TIGAR expression was induced in stimulated lymphocytes through the PI3K/AKT pathway, since Akti-1/2 and LY294002 inhibitors prevented the upregulation of TIGAR in response to ConA. In addition, suppression of TIGAR expression by siRNA decreased the levels of the proliferative marker PCNA and increased cellular ROS levels. In this model, TIGAR was found to support the activity of glucose 6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentose phosphate pathway (PPP), since the inhibition of TIGAR reduced G6PDH activity and increased autophagy. In conclusion, we demonstrate here that TIGAR is upregulated in stimulated human lymphocytes through the PI3K/AKT signaling pathway, which contributes to the redirection of the carbon flux to the PPP.

Abstract 1797: Preclinical characterization of Sym024, a novel anti-CD73 antibody

Abstract Tumor cells are surrounded by a complex microenvironment (the tumor microenvironment, or TME) that influences each step of tumorigenesis. In the TME, increased concentrations of immune modulating factors such as adenosine help tumor cells evade host anti-tumor immune responses. Adenosine binds to adenosine receptors (ARs) expressed on various immune cells such as CD4+ and CD8+ T cells, myeloid cells and natural killer (NK) cells. These receptors exhibit distinct properties and cell and tissue distribution. Binding of adenosine to a subset of ARs suppresses the anti-tumor responses of T cells and NK cells and augments the activity of immunosuppressive cells such as regulatory T cells and myeloid-derived suppressor cells, thus promoting tumor progression. The generation of adenosine in the TME is mediated by a group of ectonucleotidases, including CD39 and CD73, which work in a common pathway converting extracellular ATP to adenosine. CD73 is frequently overexpressed on human tumor cells and mediates the hydrolysis of AMP to adenosine, particularly under hypoxic conditions. Importantly, CD73 overexpression correlates with poorer prognosis in various cancers, including lung, gastric, head and neck, pancreatic, cholangiocarcinoma, colorectal, bladder, triple negative breast, ovarian, and kidney cancer, and melanoma. Inhibition of the enzymatic activity of CD73 therefore represents an attractive way of relieving the immune suppressive activity of adenosine in the TME.Sym024 is an Fc-attenuated human monoclonal antibody that binds to human and cynomolgus monkey CD73 with sub-nanomolar avidity. Sym024 binds to a distinct epitope on the enzyme on the opposite side relative to the catalytic center and potently inhibits the activity of soluble CD73 as well as CD73 expressed on numerous cancer cell lines and primary lymphocytes. Importantly, CD73 inhibition by Sym024 alleviates AMP-mediated inhibition of T cell proliferation and activation. In vitro one-way mixed lymphocyte reaction results demonstrate the utility of combining CD73 inhibition with PD-1 blockade, as Sym024 acts to reverse the immune suppressive effect of adenosine production, thereby allowing effective T cell activation by an anti-PD-1 antibody (Sym021). Mechanistically, Sym024 inhibits cellular CD73 by direct enzymatic inhibition with moderate shedding or degradation of CD73 in vitro. In preclinical in vivo tumor models, Sym024 effectively inhibits CD73 activity and tumor growth. Sym024 was well tolerated with no observed toxicity in repeat dose studies in monkeys at dose levels up to 100 mg/kg. Dose-proportional and linear pharmacokinetics (PK) was observed at high dose levels while nonlinear PK became evident at lower exposure levels. Sym024 is currently under clinical investigation. Citation Format: Janus S. Jakobsen, Matteo Riva, Maria C. Melander, Randi W. Hansen, Klaus Kofoed, Mikkel W. Pedersen, Jutta Deckert, Lene Hansen, Niels Jorgen Skartved, Johan Lantto, Camilla Frohlich, Michael Monrad Grandal. Preclinical characterization of Sym024, a novel anti-CD73 antibody [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 1797.

Abstract 808: Functional evaluation of novel germline DNA repair variants identified in patients with early-onset renal cancer

Abstract Background: Early-onset renal cancer (eoRC) is typically associated with significant family history of cancer, and often associated with germline pathogenic variants (PVs) in ‘RC-specific' genes including VHL, MET, FLCN, TSC1, TSC2, FH, SDH, PTEN and BAP1. However, most eoRC patients lack PVs in RC-specific genes; rather, we recently identified an enrichment of likely PVs in DNA repair genes in eoRC patients. Here, using eoRC patient resources (primary and transformed lymphocytes, lymphocyte DNA, and tumor tissue), we functionally evaluated the DNA repair variants identified in eoRC patients. Methods: We performed whole-exome sequencing (WES) on lymphocyte DNA from 22 patients with genetically undefined eoRC, with extensive family history of RC and/or other cancers, provided by Risk Assessment Program and Genitourinary Group at Fox Chase Cancer Center. Prioritized variants were assessed by structural, biochemical and cellular studies. Results: 70% of patients had rare, heterozygous, missense variants in genes that suppress DNA damage. ~18% of the variants were in DNA polymerase genes (POLD1, POLE, POLH, POLK), which mediate DNA replication-repair. Increased tumor mutation burden (&amp;gt;10 mutations/Mb) was observed in tumors carrying predicted damaging polymerase variants. In functional testing, induction of DNA damage in primary lymphocytes from eoRC patients and matched cancer-free controls (n=20 each) selectively elevated γH2AX foci (indicative of DNA damage) in cells from eoRC patients (P&amp;lt;0.001, pairwise T-test). DNA fiber analysis of eoRC patient-derived lymphocyte lines carrying the polymerase variants, showed significantly slower rate of DNA replication, and replication fork restoration after DNA damage versus the matched control-derived lymphocyte lines (P&amp;lt;0.01, Mann-Whitney test). Biochemical analysis of purified polymerase proteins found that the variants differ from wild type polymerase in their ability to extend DNA substrates and bypass DNA lesion(s), and exhibit dominant negative effects. Conclusion and future work: Our results indicate that germline variants in DNA repair genes impact suppression of DNA damage in cells from patients with eoRC. Biological studies testing the impact of the identified variants on renal carcinogenesis are warranted. Overall, genetic testing for variation in DNA repair genes may provide a more comprehensive risk assessment in eoRC patients, and provide novel opportunities for targeting DNA repair vulnerabilities in RC. Citation Format: Elena V. Demidova, Waleed Iqbal, Ilya G. Serebriiskii, Andrea F. Forman, Tatiana Kent, Richard T. Pomerantz, Erica A. Golemis, Michael J. Hall, David Y. Chen, Mary B. Daly, Sanjeevani Arora. Functional evaluation of novel germline DNA repair variants identified in patients with early-onset renal cancer [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 808.

Abstract 349: Association of DNA damage response capacity with neoadjuvant chemoradiation response in locally advanced rectal cancer

Abstract Background: Locally advanced rectal cancer (RC) cases are ~60% of newly diagnosed RCs, with neoadjuvant chemoradiation therapy (nCRT) followed by surgery as the standard of care. While nCRT is highly toxic, it is also beneficial. Recent prospective trials reported that up to 59% locally advanced RC cases exhibit complete clinical response, suggesting these patients as ideal candidates for organ preservation. However, currently there is no biomarker to predict who will or will not benefit from nCRT. The goal of this study was to establish a predictive biomarker for benefit from nCRT, based on the hypothesis that inherent DNA damage response (DDR) capacity contributes to nCRT response in RC. Methods: To gain insight into inherent DDR capacity, we profiled primary peripheral blood lymphocytes (pPBLs) from RC patients by quantitative immunofluorescence, Luminex-multianalyte assay, and pPBL DNA whole exome sequencing (WES). RC patients analyzed were segregated by neoadjuvant rectal (NAR) score: NAR&amp;lt;1, complete responders (CRs), n=21; NAR&amp;gt;14, poor responders (PoRs), n=21; 1&amp;lt;NAR&amp;lt;14, partial responders (PRs), n=12. Conditional inference trees were used to establish optimal cut points for the nCRT response outcomes data. Single nucleotide variants from WES data were annotated and analyzed for variation in genes involved in DDR, and associated pathways. The variants were annotated as predicted-pathogenic based on the consensus of four variant effect prediction programs. Results: pPBLs from CRs showed significantly elevated γH2AX foci (a hallmark of double strand breaks) vs. PoRs (P&amp;lt;0.001, Kruskal-Wallis test). Multianalyte DDR panel testing revealed significantly increased expression or activation of 6 DDR proteins (ATR, MDM2, phosphorylated H2AXS139 (γ), p53S15, Chk1S345, and Chk2T68) in pPBLs from CRs versus PoRs (p&amp;lt;0.01, logistic regression model) and significantly increased protein levels of 4 DDR proteins (ATR, MDM2, p53S15, Chk2T68) in pPBLs from CRs vs. pPBLs from PRs (p&amp;lt;0.05, logistic regression model). We generated a combined DDR Score which positively and significantly correlated with CR vs. PoR (P&amp;lt;0.001, ordinal logistic regression model). From the combined DDR score, using mean score &amp;gt;0.082 as a cutpoint, we could include 90% patients with a CR. Finally, WES analysis of pPBL DNA from 15 CRs and 15 PoRs revealed that CRs were enriched in predicted-pathogenic variants in base excision repair genes vs. PoRs (p&amp;lt;0.01, q&amp;lt;0.025, FDR). Conclusion and future work: Overall, our data suggests inherent DDR capacity testing may predict the magnitude of benefit from nCRT and assist in patient selection for treatment. Validation in a larger RC patient population is needed to confirm these findings. Biological studies testing the impact of the identified variants in base excision repair on inherent DDR capacity are warranted. Citation Format: Elena V. Demidova, Randy W. Lesh, Vladimir Avkshtol, Margret B. Einarson, Erica A. Golemis, Sanjeevani Arora, Joshua E. Meyer. Association of DNA damage response capacity with neoadjuvant chemoradiation response in locally advanced rectal cancer [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 349.

Morphological profiling of human T and NK lymphocytes by high-content cell imaging.

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ Tcells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.