T-cell Lines Research Articles

Heterogeneous Formation of DNA Double-Strand Breaks and Cell-Free DNA in Leukemia T-Cell Line and Human Peripheral Blood Mononuclear Cells in Response to Topoisomerase II Inhibitors

Background: Improving precision medicine in chemotherapy requires highly sensitive and easily applicable diagnostic tools. In addition, non-invasive molecular real-time monitoring of cytotoxic response is highly desirable. Here, we employed the kinetics of DNA double-strand breaks (DSB) and cell-free DNA (cfDNA) in a cell model of topoisomerase II-inhibitors in T cell leukemia (Jurkat cells) compared to normal cells (peripheral blood mononuclear cells, PBMCs). Methods: We applied automated microscopy to quantify immuno-stained phosphorylated H2AX (γH2AX) as a marker for either DNA damage response (DDR) or cell death and quantitative PCR-based analysis of nuclear and mitochondrial cfDNA concentrations. Results: Jurkat cells displayed a DDR to cytotoxic drug treatment significantly earlier than PBMCs, and etoposide (ETP) induced DSB formation faster than doxorubicin (DOX) in both Jurkat and PBMCs. Jurkat cells exhibited an earlier cytotoxic response compared to PBMC, with a significantly increased mitochondrial cfDNA formation after 2 h of DOX application. In PBMCs, increased cell death was detected after 4 h of incubation with ETP, whereas DOX treatment was less effective. Conclusions: Both automated microscopy and mitochondrial cfDNA quantification analysis indicate that (malignant) Jurkat cells are more sensitive to DOX than (healthy) PBMC. Our real-time approach can improve DDR inducing drug selection and adaptation in cancer therapy and aids in decisions for optimal patient biosampling.

Characterization of HTLV-1 Infectious Molecular Clone Isolated from Patient with HAM/TSP and Immortalization of Human Primary T-Cell Lines

Human T-cell leukemia virus (HTLV-1) is the etiological agent of lymphoproliferative diseases such as adult T-cell leukemia and T-cell lymphoma (ATL) and a neurodegenerative disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). While several molecular clones of HTLV-1 have been published, all were isolated from samples derived from patients with adult T-cell leukemia. Here, we report the characterization of an HTLV-1 infectious molecular clone isolated from a sample of a patient with HAM/TSP disease. Genetic comparative analyses of the HAM/TSP molecular clone (pBST) revealed unique genetic alterations and specific viral mRNA expression patterns. Interestingly, our clone also harbors characteristics previously published to favor the development of HAM/TSP disease. The molecular clone is capable of infection and immortalization of human primary T cells in vitro. Our studies further demonstrate that the HTLV-1 virus produced from primary T cells transfected with pBST or ACH molecular clones cannot sustain long-term expansion, and cells cease to proliferate after 3–4 months in culture. In contrast, long-term proliferation and immortalization were achieved if the virus was transmitted from dendritic cells to primary T cells, and secondary infection of 729B cells in vitro was demonstrated. In both primary T cells and 729B cells, pBST and ACH were latent, and only hbz viral RNA was detected. This study suggests that HTLV-1 transmission from DC to T cells favors the immortalization of latently infected cells.

A minority of proliferating human CD4+ T cells in antigen-driven proliferation assays are antigen specific.

Antigen-driven T-cell proliferation is often measured using fluorescent dye dilution assays, such as the CFSE-based proliferation assay. Dye dilution assays have been powerful tools to detect human CD4+ T-cell responses, particularly against autoantigens. However, it is not known how many cells within the proliferating population are specific for the stimulating antigen. Here we determined the frequency of CD4+ T cells specific for the stimulating antigen within the antigen-responsive population of CFSE-based proliferation assays. We compared CD4+ T-cell responses to a type 1 diabetes autoantigen (proinsulin C-peptide) and to a vaccine antigen (tetanus toxoid). The TCRs expressed by antigen-responsive CD4+ T cells were sequenced, and their antigen specificity was tested functionally by expressing them in a reporter T-cell line. Responses to C-peptide were weak, but detectable, in PBMC from individuals with T1D, whereas responses to tetanus toxoid were much stronger. The frequency of antigen-specific CD4+ T cells correlated with the strength of the response to antigen in the proliferation assay. However, antigen-specific CD4+ T cells were rare among antigen-responsive CD4+ T cells. For C-peptide, an average frequency of 7.5% (1%-11%, n = 4) of antigen-responsive CD4+ T cells were confirmed to be antigen specific. In the tetanus-toxoid-stimulated cultures, on average, 45% (16%-78%, n = 5) of the antigen-responsive CD4+ T cells were tetanus toxoid specific. These data show that antigen-specific CD4+ T cells are a minority of the cells that proliferate in response to antigen and have important implications for in vitro CD4+ T-cell proliferation assays.

A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens.

Cancer cells effectively evade immune surveillance, not only through the well-known PD-1/PD-L1 pathway but also via alternative mechanisms that impair patient response to immune checkpoint inhibitors. We present a novel co-culture model that pairs a reporter T-cell line with different melanoma cell lines that have varying immune evasion characteristics. We developed a scalable high-throughput lentiviral arrayed CRISPR interference (CRISPRi) screening protocol to conduct gene perturbations in both T-cells and melanoma cells, enabling the identification of genes that modulate tumor immune evasion. Our study functionally validates the co-culture model system and demonstrates the performance of the CRISPRi-screening protocol by modulating the expression of known regulators of tumor immunity. Together, our work provides a robust framework for future research aimed at systematically exploring mechanisms of tumor immune evasion.

In silico advancements in Peptide-MHC interaction: A molecular dynamics study of predicted glypican-3 peptides and HLA-A*11:01

Our study employed molecular dynamics (MD) simulations to assess the binding affinity between short peptides derived from the tumor-associated antigen glypican 3 (GPC3) and the major histocompatibility complex (MHC) molecule HLA-A*11:01 in hepatocellular carcinoma. We aimed to improve the reliability of in silico predictions of peptide-MHC interactions, which are crucial for developing targeted cancer therapies. We used five algorithms to discover four peptides (TTDHLKFSK, VINTTDHLK, KLIMTQVSK, and STIHDSIQY), demonstrating the substantial potential for HLA-A11:01 presentation. The Anchored Peptide-MHC Ensemble Generator (APE-Gen) was used to create the initial structure of the peptide-MHC complex. This was followed by a 200 ns molecular dynamics (MD) simulation using AMBER22, which verified the precise positioning of the peptides in the binding groove of HLA-A*11:01, specifically at the A and F pockets. Notably, the 2nd residue, which serves as a critical anchor within the 2nd pocket, played a pivotal role in stabilising the binding interactions.VINTTDHLK (ΔGSIE = −14.46 ± 0.53 kcal/mol and ΔGMM/GBSA = −30.79 ± 0.49 kcal/mol) and STIHDSIQY (ΔGSIE and ΔGMM/GBSA = −14.55 ± 0.16 and −23.21 ± 2.23 kcal/mol) exhibited the most effective binding potential among the examined peptides, as indicated by both their binding free energies and its binding affinity on the T2 cell line (VINTTDHLK: IC50 = 0.45 nM; STIHDSIQY: IC50 = 0.35 nM). The remarkable concordance between in silico and in vitro binding affinity results was of particular significance, indicating that MD simulation is a potent instrument capable of bolstering confidence in in silico peptide predictions. By employing MD simulation as a method, our study provides a promising avenue for improving the prediction of potential peptide-MHC interactions, thereby facilitating the development of more effective and targeted cancer therapies.

Genome-Wide Methylation Profiling of Peripheral T-Cell Lymphomas Identifies TRIP13 as a Critical Driver of Tumor Proliferation and Survival.

Cytosine methylation contributes to the regulation of gene expression and normal hematopoiesis in mammals. It is catalyzed by the family of DNA methyltransferases that include DNMT1, DNMT3A, and DNMT3B. Peripheral T-cell lymphomas (PTCLs) represent aggressive mature T-cell malignancies exhibiting a broad spectrum of clinical features with poor prognosis and inadequately understood molecular pathobiology. To better understand the molecular landscape and identify candidate genes involved in disease maintenance, we profiled DNA methylation and gene expression of PTCLs. We found that the methylation patterns in PTCLs are deregulated and heterogeneous but share 767 hypo- and 567 hypermethylated differentially methylated regions (DMRs) along with 231 genes up- and 91 genes downregulated in all samples, suggesting a potential association with tumor development. We further identified 39 hypomethylated promoters associated with increased gene expression in the majority of PTCLs. This putative oncogenic signature included the TRIP13 (thyroid hormone receptor interactor 13) gene whose genetic and pharmacologic inactivation inhibited the proliferation of T-cell lines by inducing G2-M arrest and apoptosis. Our data thus show that human PTCLs have a significant number of recurrent methylation alterations that may affect the expression of genes critical for proliferation whose targeting might be beneficial in anti-lymphoma treatments.

Treponema pallidum Periplasmic and Membrane Proteins Are Recognized by Circulating and Skin CD4+ T Cells.

Histologic and serologic studies suggest the induction of local and systemic Treponema pallidum-specific CD4+ T-cell responses to T. pallidum infection. We hypothesized that T. pallidum-specific CD4+ T cells are detectable in blood and in the skin rash of secondary syphilis and persist in both compartments after treatment. Peripheral blood mononuclear cells collected from 67 participants were screened by interferon-γ (IFN-γ) ELISPOT response to T. pallidum sonicate. T. pallidum-reactive T-cell lines from blood and skin were probed for responses to 89 recombinant T. pallidum antigens. Peptide epitopes and HLA class II restriction were defined for selected antigens. We detected CD4+ T-cell responses to T. pallidum sonicate ex vivo. Using T. pallidum-reactive T-cell lines we observed recognition of 14 discrete proteins, 13 of which localize to bacterial membranes or the periplasmic space. After therapy, T. pallidum-specific T cells persisted for at least 6 months in skin and 10 years in blood. T. pallidum infection elicits an antigen-specific CD4+ T-cell response in blood and skin. T. pallidum-specific CD4+ T cells persist as memory in both compartments long after curative therapy. The T. pallidum antigenic targets we identified may be high-priority vaccine candidates.

Activation of the CDK7 Gene, Coding for the Catalytic Subunit of the Cyclin-Dependent Kinase (CDK)-Activating Kinase (CAK) and General Transcription Factor II H, by the Trans-Activator Protein Tax of Human T-Cell Leukemia Virus Type-1.

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The trans-activator protein Tax of HTLV-1 plays crucial roles in leukemogenesis by promoting proliferation of virus-infected cells through activation of growth-promoting genes. However, critical target genes are yet to be elucidated. We show here that Tax activates the gene coding for cyclin-dependent kinase 7 (CDK7), the essential component of both CDK-activating kinase (CAK) and general transcription factor TFIIH. CAK and TFIIH play essential roles in cell cycle progression and transcription by activating CDKs and facilitating transcriptional initiation, respectively. Tax induced CDK7 gene expression not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs) along with increased protein expression. Tax stimulated phosphorylation of CDK2 and RNA polymerase II at sites reported to be mediated by CDK7. Tax activated the CDK7 promoter through the NF-κB pathway, which mainly mediates cell growth promotion by Tax. Knockdown of CDK7 expression reduced Tax-mediated induction of target gene expression and cell cycle progression. These results suggest that the CDK7 gene is a crucial target of Tax-mediated trans-activation to promote cell proliferation by activating CDKs and transcription.

PD-1/LAG-3 co-signaling profiling uncovers CBL ubiquitin ligases as key immunotherapy targets

Many cancer patients do not benefit from PD-L1/PD-1 blockade immunotherapies. PD-1 and LAG-3 co-upregulation in T-cells is one of the major mechanisms of resistance by establishing a highly dysfunctional state in T-cells. To identify shared features associated to PD-1/LAG-3 dysfunctionality in human cancers and T-cells, multiomic expression profiles were obtained for all TCGA cancers immune infiltrates. A PD-1/LAG-3 dysfunctional signature was found which regulated immune, metabolic, genetic, and epigenetic pathways, but especially a reinforced negative regulation of the TCR signalosome. These results were validated in T-cell lines with constitutively active PD-1, LAG-3 pathways and their combination. A differential analysis of the proteome of PD-1/LAG-3 T-cells showed a specific enrichment in ubiquitin ligases participating in E3 ubiquitination pathways. PD-1/LAG-3 co-blockade inhibited CBL-B expression, while the use of a bispecific drug in clinical development also repressed C-CBL expression, which reverted T-cell dysfunctionality in lung cancer patients resistant to PD-L1/PD-1 blockade. The combination of CBL-B-specific small molecule inhibitors with anti-PD-1/anti-LAG-3 immunotherapies demonstrated notable therapeutic efficacy in models of lung cancer refractory to immunotherapies, overcoming PD-1/LAG-3 mediated resistance.

Evaluating tocilizumab safety and immunomodulatory effects under ocular HTLV-1 infection in vitro

There is growing interest in evaluating the safety and therapeutic potential of existing treatments such as tocilizumab (TCZ), an IL-6 receptor antagonist used to treat inflammatory diseases. However, there have been reports of increased inflammation in patients with HTLV-1 uveitis after TCZ treatment, and its ocular safety in the HTLV-1 infected state remains unknown. This study focused on assessing the impact of TCZ on HTLV-1-infected ocular cells using an in vitro model in which retinal pigment epithelial cells were cocultured with irradiated HTLV-1-infected T-cell lines. TCZ did not significantly affect cellular viability, inflammatory markers, or HTLV-1 proviral loads at various concentrations (25/50/100 µg/ml), indicating no increased risk of HTLV-1 viral infection and no exacerbation of the inflammatory aspects of HTLV-1 infection in the ocular cells. These promising results support the potential of TCZ as a safe treatment option for HTLV-1-infected patients, particularly those with eye infections.

Novel paired CD13-negative (MT-50.1) and CD13-positive (MT-50.4) HTLV-1-infected T-cell lines with differential regulatory T cell-like activity

Adult T-cell leukemia/lymphoma (ATL) occurs after human T-cell leukemia virus type-1 (HTLV-1) infection with a long latency period exceeding several decades. This implies the presence of immune evasion mechanisms for HTLV-1-infected T cells. Although ATL cells have a CD4+CD25+ phenotype similar to that of regulatory T cells (Tregs), they do not always possess the immunosuppressive functions of Tregs. Factors that impart effective immunosuppressive functions to HTLV-1-infected cells may exist. A previous study identified a new CD13+ Treg subpopulation with enhanced immunosuppressive activity. We, herein, describe the paired CD13− (designated as MT-50.1) and CD13+ (MT-50.4) HTLV-1-infected T-cell lines with Treg-like phenotype, derived from the peripheral blood of a single patient with lymphoma-type ATL. The cell lines were found to be derived from HTLV-1-infected non-leukemic cells. MT-50.4 cells secreted higher levels of immunosuppressive cytokines, IL-10 and TGF-β, expressed higher levels of Foxp3, and showed stronger suppression of CD4+CD25− T cell proliferation than MT-50.1 cells. Furthermore, the CD13 inhibitor bestatin significantly attenuated MT-50.4 cell growth, while it did not for MT-50.1 cells. These findings suggest that CD13 expression may be involved in the increased Treg-like activity of MT-50.4 cells. Hence, MT-50.4 cells will be useful for in-depth studies of CD13+Foxp3+ HTLV-1-infected cells.

Trans-Activation of the Coactivator-Associated Arginine Methyltransferase 1 (Carm1) Gene by the Oncogene Product Tax of Human T-Cell Leukemia Virus Type 1.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles in leukemogenesis by promoting proliferation of the virus-infected cells through activation of growth-promoting genes. These genes code for growth factors and their receptors, cytokines, cell adhesion molecules, growth signal transducers, transcription factors and cell cycle regulators. We show here that Tax activates the gene coding for coactivator-associated arginine methyltransferase 1 (CARM1), which epigenetically enhances gene expression through methylation of histones. Tax activated the Carm1 gene and increased protein expression, not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs). Tax increased R17-methylated histone H3 on the target gene IL-2Rα, concomitant with increased expression of CARM1. Short hairpin RNA (shRNA)-mediated knockdown of CARM1 decreased Tax-mediated induction of IL-2Rα and Cyclin D2 gene expression, reduced E2F activation and inhibited cell cycle progression. Tax acted via response elements in intron 1 of the Carm1 gene, through the NF-κB pathway. These results suggest that Tax-mediated activation of the Carm1 gene contributes to leukemogenic target-gene expression and cell cycle progression, identifying the first epigenetic target gene for Tax-mediated trans-activation in cell growth promotion.

P26 The effect of interleukin-23 stimulation on gene expression in CD4+ Jurkat cells

Abstract Introduction and aims There is now robust evidence that genetic changes along the interleukin (IL)-23 axis increase the risk of developing psoriasis, with CD4+ T cells likely to be pivotal in the disease pathway. Here, we stimulated Jurkat CD4+ T-cell lines that expressed either the risk or protective version of IL23R, identified through genetic studies in psoriasis, and measured the effect in whole-genome expression at different timepoints poststimulation, with the aim of characterizing the downstream effect on the IL-23 pathway. Methods The transformed Jurkat CD4+IL12Rβ1+ IL23Rwt/IL23Rmut-R381Q T cells were stimulated with IL-23 protein (10 ng mL−1) for 0, 2, 4, 16 and 24 h, with triplicates used for each timepoint. RNA sequencing was performed by Novogene, and statistical analyses was performed using negative binomial generalized linear models and then a likelihood-ratio test to remove condition-specific differences over time. Results Of the identified significantly expressed genes (Padj > 10−5), five are also implicated as psoriasis risk genes, i.e. HLA-B, SOCS1, MARS, HLA-C and RUNX3. Specifically, a significant increase in RUNX3 expression in wildtype (WT) vs. mutant (MuT) at 16–24 h, and in suppressor of cytokine signalling (SOCS)-1 at 2–24 h was observed, with runt-related transcription factor (RUNX)3 expression peaking at 4 h and SOCS1 showing consistently high expression beyond 0 h. Decreased expression of SOCS1 disrupts IL-23 production and signalling, whereas increased expression of RUNX3 increases T helper (Th)17 and Th22 levels, key cells downstream of IL-23 signalling Conclusions Here, we show that carriage of the WT vs. MuT IL23R has downstream effects on expression of HLA-B, SOCS1, MARS, HLA-C and RUNX3, implicating these genes in the IL-23 pathway. This is important as defining pathways that are perturbed in patients has great potential within stratified medicine, with the possibility for better annotation of personalized genetic risk scores.

Keratinocytes Present Staphylococcus aureus Enterotoxins and Promote Malignant and Nonmalignant T Cell Proliferation in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma is characterized by malignant T cells proliferating in a unique tumor microenvironment dominated by keratinocytes (KCs). Skin colonization and infection by Staphylococcus aureus are a common cause of morbidity and are suspected of fueling disease activity. In this study, we show that expression of HLA-DRs, high-affinity receptors for staphylococcal enterotoxins (SEs), by KCs correlates with IFN-γ expression in the tumor microenvironment. Importantly, IFN-γ induces HLA-DR, SE binding, and SE presentation by KCs to malignant T cells from patients with Sézary syndrome and malignant and nonmalignant T-cell lines derived from patients with Sézary syndrome and mycosis fungoides. Likewise, preincubation of KCs with supernatant from patient-derived SE-producing S aureus triggers proliferation in malignant T cells and cytokine release (including IL-2), when cultured with nonmalignant T cells. This is inhibited by pretreatment with engineered bacteriophage S aureus-specific endolysins. Furthermore, alteration in the HLA-DR-binding sites of SE type A and small interfering RNA-mediated knockdown of Jak3 and IL-2Rγ block induction of malignant T-cell proliferation. In conclusion, we show that upon exposure to patient-derived S aureus and SE, KCs stimulate IL-2Rγ/Jak3-dependent proliferation of malignant and nonmalignant T cells in an environment with nonmalignant T cells. These findings suggest that KCs in the tumor microenvironment play a key role in S aureus-mediated disease activity in cutaneous T-cell lymphoma.

Scrutiny of chimeric antigen receptor activation by the extracellular domain: experience with single domain antibodies targeting multiple myeloma cells highlights the need for case-by-case optimization.

Multiple myeloma (MM) remains incurable, despite the advent of chimeric antigen receptor (CAR)-T cell therapy. This unfulfilled potential can be attributed to two untackled issues: the lack of suitable CAR targets and formats. In relation to the former, the target should be highly expressed and reluctant to shedding; two characteristics that are attributed to the CS1-antigen. Furthermore, conventional CARs rely on scFvs for antigen recognition, yet this withholds disadvantages, mainly caused by the intrinsic instability of this format. VHHs have been proposed as valid scFv alternatives. We therefore intended to develop VHH-based CAR-T cells, targeting CS1, and to identify VHHs that induce optimal CAR-T cell activation together with the VHH parameters required to achieve this. CS1-specific VHHs were generated, identified and fully characterized, in vitro and in vivo. Next, they were incorporated into second-generation CARs that only differ in their antigen-binding moiety. Reporter T-cell lines were lentivirally transduced with the different VHH-CARs and CAR-T cell activation kinetics were evaluated side-by-side. Affinity, cell-binding capacity, epitope location, in vivo behavior, binding distance, and orientation of the CAR-T:MM cell interaction pair were investigated as predictive parameters for CAR-T cell activation. Our data show that the VHHs affinity for its target antigen is relatively predictive for its in vivo tumor-tracing capacity, as tumor uptake generally decreased with decreasing affinity in an in vivo model of MM. This does not hold true for their CAR-T cell activation potential, as some intermediate affinity-binding VHHs proved surprisingly potent, while some higher affinity VHHs failed to induce equal levels of T-cell activation. This could not be attributed to cell-binding capacity, in vivo VHH behavior, epitope location, cell-to-cell distance or binding orientation. Hence, none of the investigated parameters proved to have significant predictive value for the extent of CAR-T cell activation. We gained insight into the predictive parameters of VHHs in the CAR-context using a VHH library against CS1, a highly relevant MM antigen. As none of the studied VHH parameters had predictive value, defining VHHs for optimal CAR-T cell activation remains bound to serendipity. These findings highlight the importance of screening multiple candidates.

Pivotal role of dihydroorotate dehydrogenase as a therapeutic target in adult T-cell leukemia.

We aimed to determine the role of dihydroorotate dehydrogenase (DHODH) in pathogenesis of adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1) and the effects of its inhibition on the de novo pyrimidine biosynthesis pathway. Cell proliferation, viability, cycle, and apoptosis were analyzed using WST-8 assays, flow cytometry, and Hoechst 33342 staining. To elucidate the molecular mechanisms involved in the anti-ATL effects of DHODH knockdown and inhibition, RT-PCR and immunoblotting were conducted. HTLV-1-infected T-cell lines aberrantly expressed DHODH. Viral infection and the oncoprotein, Tax, enhanced DHODH expression, while knockdown of DHODH decreased HTLV-1-infected T-cell growth. In addition, BAY2402234, a DHODH inhibitor, exerted an anti-proliferative effect, which was reversed by uridine supplementation. BAY2402234 induced DNA damage and S phase arrest by downregulating c-Myc, CDK2, and cyclin A and upregulating p53 and cyclin E. It also induced caspase-mediated apoptosis by the upregulation of pro-apoptotic and downregulation of anti-apoptotic proteins. Furthermore, BAY2402234 induced caspase-independent ferroptosis and necroptosis. It decreased phosphorylation of IKK, IκBα, PTEN, Akt, and its downstream targets, suggesting that inhibition of NF-κB and Akt signaling is involved in its anti-ATL action. These findings highlight DHODH as a potential therapeutic target for treating ATL.

Abstract 3601: Improving killing of renal cell carcinoma through the combined effects of TCR engineered CD4+ and CD8+ T cells

Abstract Patients with late-stage kidney cancer have poor long-term survival rates. Our lab isolated a low affinity CD8 dependent TCR named BZ-4 from CTLs of a patient who had immune-mediated regression of renal cell carcinoma (RCC) following an allogeneic stem cell transplant and found this TCR recognized a human endogenous retrovirus type E (HERV-E) derived 10-mer peptide, CT-RCC-1. The BZ-4 clone belongs to the Vβ7.1 family and binds to CT-RCC-1 in an HLA-A11 dependent manner. Adoptive infusion of CD8+ BZ-4 transduced T cells is currently being explored in an NIH phase I trial (NCT03354390). Because CD4+ T cells have been shown to augment the anti-tumor effects of TCR-modified CD8+ T cells, we hypothesized that CD4+ T cells genetically engineered to co-express BZ-4 and CD8 would recognize and kill RCC tumors expressing the CT-RCC-1 and would improve the in vivo proliferation and tumor cytotoxicity of CD8+ BZ-4 T cells. CD4+ T cells isolated from healthy donor PBMCs were transfected via Sleeping Beauty electroporation with the control MART-1 reactive TCR (DMF5), BZ-4, or BZ-4 alongside the CD8 co-receptor (BZ-4-8). CD8+ T cells were similarly transfected with DMF5 or BZ-4. Following magnetic bead enrichment, 66% to 84% (mean 77%) of transfected T cells stained positive for Vβ7.1. TCR engineered T cells were subsequently tested for their ability to lyse a) luciferase transduced HLA A-11 expressing RCC tumors and b) a T2 cell line modified to express both HLA A-11 and luciferase that was peptide-pulsed with decreasing concentrations (5 × 10−1, 5 × 10−2, 5 × 10−3 & 5 × 10−4 ug/mL) of CT-RCC-1. CD4+ T cells expressing BZ-4 or BZ-4-8 as well as CD8+ T cells expressing BZ-4 or DMF5 were used either alone or in combination in cytotoxicity assays. For combined T cell groups, the total effector/target ratio was maintained at the same ratio as individual T cell groups. For T-2 targets, CD4+ BZ-4 T cells exhibited no cytolytic activity in contrast to both CD8+ BZ-4 T cells and CD4+ BZ-4-8 T cells, which were highly cytolytic to target cells. For RCC tumor targets, CD4+ BZ-4 T cells exhibited no cytolytic activity at 48, 72 and 96 hours in contrast to both CD8+ BZ-4 T cells and CD4+ BZ-4-8 T cells, which exhibited high levels of tumor killing at these timepoints. Remarkably, CD4+ BZ-4-8 T cells alone or in combination with CD8+ BZ-4 T cells demonstrated tumor cytotoxicity that was comparable to that observed with CD8+ BZ-4 T cells alone. In conclusion, these data show that CD4+ BZ-4 T cells transfected with the CD8 co-receptor acquire the ability to lyse RCC tumors at levels comparable to that observed in CD8+ BZ-4 T cells. Additional experiments are planned to evaluate whether mixtures of CD8+ BZ-4 T cells with CD4+ BZ-4-8 T cells will augment the ability of TCR modified T cells to proliferate in vivo and kill human RCC tumors compared to CD8+ BZ-4 T cells alone in tumor bearing NSG mice. Citation Format: Muna Igboko, Long Chen, Stefan Barisic, Elena Cherkasova, David Allan, Mala Chakraborty, Joseph Clara, Angie Parrizzi, Kyra Carney, Rosa Nadal Rios, Robert Reger, Richard Childs. Improving killing of renal cell carcinoma through the combined effects of TCR engineered CD4+ and CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3601.

Differential requirement for IL-2 and IL-23 in the differentiation and effector functions of Th17/ILC3-like cells in a human T cell line.

A well-documented Achilles heel of current cancer immunotherapy approaches is T cell exhaustion within solid tumor tissues. The proinflammatory cytokine interleukin (IL)-23 has been utilized to augment chimeric antigen receptor (CAR) T cell survival and tumor immunity. However, in-depth interrogation of molecular events downstream of IL-23/IL-23 receptor signaling is hampered by a paucity of suitable cell models. The current study investigates the differential contribution of IL-2 and IL-23 to the maintenance and differentiation of the IL-23 responsive Kit225 T-cell line. We observed that IL-23 enhanced cellular fitness and survival but was insufficient to drive proliferation. IL-23 rapidly induced phosphorylation of STAT1, STAT3, and STAT4, and messenger RNA expression of IL17A, the archetypal effector cytokine of T helper 17 (Th17) cells, but not their lineage markers RORC and NCR1. These observations suggest that IL-23 endowed Th17/ILC3-like effector function but did not promote their differentiation. In contrast, spontaneous differentiation of Kit225 cells toward a Th17/ILC3-like phenotype was induced by prolonged IL-2 withdrawal. This was marked by strongly elevated basal IL17A and IL17F expression and the secretion of IL-17. Together, our data present Kit225 cells as a valuable model for studying the interplay between cytokines and their contribution to T cell survival, proliferation, and differentiation.

HIV-Infected Hepatic Stellate Cells or HCV-Infected Hepatocytes Are Unable to Promote Latency Reversal among HIV-Infected Mononuclear Cells.

Due to a common mode of transmission through infected human blood, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) co-infection is relatively prevalent. In alignment with this, HCV co-infection is associated with an increased size of the HIV reservoir in highly active antiretroviral therapy (HAART)-treated individuals. Hence, it is crucial to comprehend the physiological mechanisms governing the latency and reactivation of HIV in reservoirs. Consequently, our study delves into the interplay between HCV/HIV co-infection in liver cells and its impact on the modulation of HIV latency. We utilized the latently infected monocytic cell line (U1) and the latently infected T-cell line (J-Lat) and found that mediators produced by the infection of hepatic stellate cells and hepatocytes with HIV and HCV, respectively, were incapable of inducing latency reversal under the studied conditions. This may favor the maintenance of the HIV reservoir size among latently infected mononuclear cells in the liver. Further investigations are essential to elucidate the role of the interaction between liver cells in regulating HIV latency and/or reactivation, providing a physiologically relevant model for comprehending reservoir microenvironments in vivo.

Target genes regulated by CLEC16A intronic region associated with common variable immunodeficiency

BackgroundCLEC16A intron 19 has been identified as a candidate locus for common variable immunodeficiency (CVID). ObjectiveThe objective of this study is to elucidate the molecular mechanism by which variants at the CLEC16A intronic locus may contribute to the pathogenesis of CVID. MethodsWe performed fine-mapping of the CLEC16A locus in a CVID cohort, then deleted the candidate functional SNP in T-cell lines by the CRISPR-Cas9 technique and conducted RNA-seq to identify target gene(s). The interactions between the CLEC16A locus and its target genes were identified using Circularized Chromosome Conformation Capture. The transcription factor complexes mediating the chromatin interactions were determined by proteomic approach. The molecular pathways regulated by the CLEC16A locus were examined by RNA-seq and reverse phase protein array. ResultsOur study showed that the CLEC16A locus is an enhancer regulating expression of multiple target genes including a distant gene ATF7IP2 through chromatin interactions. Distinct transcription factor complexes mediate the chromatin interactions in an allele-specific manner. Disruption of the CLEC16A locus affects the AKT signaling pathway, as well as the molecular response of CD4+T cells to immune stimulation. ConclusionThrough multi-omics and targeted experimental approaches, we elucidated the underlying target genes and signaling pathways involved in the genetic association of CLEC16A with CVID, and highlighted plausible molecular targets for developing novel therapeutics.