bluuebunny/crossref_metadata_2025_split huggingface dataset:
Common trajectories of highly effective CD19-specific CAR T cells identified by endogenous T cell receptor lineages
Authors: Crawford Jeremy Chase , Wilson Taylor Leigh , Kim Hyunjin , Chou Ching-Heng , Langfitt Deanna , Allen E Kaitlynn , Metais Jean-Yves , Pogorelyy Mikhail , Kottapalli Pratibha , Trivedi Sanchit , Olsen Scott , Lockey Timothy , Willis Catherine , Meagher Michael M , Triplett Brandon , Talleur Aimee C , Gottschalk Stephen , Thomas Paul Glyndwr 
Published: January 2023

Abstract
Current chimeric antigen receptor-modified (CAR) T cell therapy products are evaluated in bulk, without assessment of the possible heterogeneity in effector potential between cells. Conceivably, only a subset of the pre-infusion product differentiates into optimal effectors. We generated a comprehensive single-cell gene expression and T cell receptor (TCR) sequencing dataset using both pre- and post-infusion CD19-CAR T cells from peripheral blood and bone marrow of pediatric patients with B cell acute lymphoblastic leukemia (B-ALL). We identified potent effector post-infusion cells with identical TCRs to a subset of pre-infusion CAR T cells. Effector precursor CAR T cells exhibited a unique transcriptional profile compared to other pre-infusion cells, and the number of effector precursor cells infused correlated with peak CAR T cell expansion. Additionally, we identified an unexpected cell surface phenotype (TIGIT+, CD62Llo, CD27−), conventionally associated with inhibiting effective T cell responses, that we used to successfully enrich for subsequent effector potential. Collectively, these results demonstrate that highly diverse effector potentials are present among cells in pre-infusion cell products, which can be exploited for diagnostic and therapeutic applications. Furthermore, we provide an integrative experimental and analytical framework for elucidating the biological mechanisms underlying effector development in other CAR T cell therapy products.
This work was supported by the National Institutes of Health (NIH)/National Cancer Institute grant P30CA021765, NIH grants U01AI150747 and R01AI136514 (PGT), the American Society of Transplantation and Cellular Therapy (AT), the American Society of Hematology (AT), the Key for a Cure Foundation (PGT), the Mark Foundation ASPIRE Award (PGT), and the American Lebanese Syrian Associated Charities (SG, PGT). Part of the laboratory studies were performed by the Center for Translational Immunology and Immunotherapy (CeTI2), which is supported by SJCRH. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Chimeric Cytokine Targeting NKG2D and IL-2 Receptor Augments CD8+ T Cell-Metabolic Fitness and Improves Immunotherapy
Authors: Banerjee Anirban , Li Dongge , Guo Yizhan , Mei Zhongcheng , Chen Kelly , Schrum Adam , Krupnick Alexander 
Published: January 2023

Abstract
T cell mediated immunotherapy has gained credible traction for cancer treatment. To date experimental and clinical strategies have relied on antibody-mediated co-stimulatory blockade, or the administration of high doses of naturally occurring or engineered cytokines to increase T cell activation. Cytokine therapy is by limited undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here we describe functionality of a unique chimeric cytokine by design, called OMCPmutIL-2, binds with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signals through the common γ-chain cytokine receptor, thus eliminating IL-2Ra mediated off-target side effects. In addition to precise activation of cytotoxic T cells due to redirected binding we describe that OMCPmutIL-2 results in superior activation of both human and murine CD8+ T cells by improving their survival, decreasing exhaustion, and improving memory cell generation. This functional improvement is the result altered signal transduction based on the reorganization of surface membrane lipid rafts resulting in JAK-mediated phosphorylation of the T cell receptor (TCR) rather than canonical STAT/AKT signaling intermediates activated by other common γ-chain cytokines. This altered signaling pathway increases CD8+ T cell response to low affinity antigens, activates Nuclear Factor of Activated T Cells (NFAT) transcription factors, and enhances mitochondrial biogenesis with improved T cell survival, cytotoxicity, and memory cell generation. OMCPmutIL-2 thus outperforms other common γ-chain cytokines as both a reagent for T cell expansion and in vivo CD8+ T cell-based immunotherapy.
Supported by grant I01 BX002299
CD8 T cell activation in cancer is comprised of two distinct phases
Authors: Prokhnevska Nataliya , Cardenas Maria A , Valanparambil Rajesh , Sobierajska Ewelina , Jansen Caroline , Master Viraj , Sanda Martin , Kissick Haydn 
Published: January 2023

Abstract
The CD8 T cell response to tumors is extremely variable with heterogenous T cell subsets, a stem-like CD8 T cell (PD1+TCF1+) that sustains the CD8 response and gives rise to a terminally differentiated (TD) cytotoxic cell (TCF1-Tim3+). Although these subsets have been described, how tumor-specific CD8 T cells are activated and differentiate in tumors are not well defined. Using a prostate cancer model that expresses the LCMV-GP (TRAMPC1-GP), we studied tumor-specific CD8 T cell activation by transferring LCMV-GP specific P14 CD8 T cells into tumor-bearing mice. We found that P14s are activated in the tumor-draining LNs (TDLNs) and acquire a stem-like phenotype. These cells migrate into the tumor as stem-like CD8 T cells and only differentiate into a TD CD8 T cell in the tumor. We found that stem-like CD8 T cells need additional co-stimulation from antigen presenting cells within the tumor to fully differentiate, even though they have been previously activated in TLDNs. Similarly, stem-like CD8s from human kidney cancer require both TCR and co-stimulatory signals to divide and differentiation ex-vivo and can differentiate when co-cultured with autologous dendritic cells. The addition of IL12 with TCR alone was not sufficient to induce differentiation, but improved differentiation when co-stimulation was present. This demonstrates the necessity of additional TCR and co-stimulation once activated stem-like CD8 T cells migrate into the tumor. Overall, these data suggest two distinct phases of CD8 T cell differentiation, the first occurs in the TDLN where they are initially activated. The second occurs in the tumor, where they require additional co-stimulation to differentiate and acquire an effector phenotype.
Functional virus-specific memory CD8+ T cells survey glioblastoma
Authors: Rosato Pamela , Ning Jianfang , Gavil Noah Veis , Wu Shaoping , Wijeyesinghe Sathi , Weyu Eyob , Ma Jun , Li Ming , Grigore Florina-Nicoleta , Dhawan Sanjay , Skorput Alexander G , Musial Shawn C , Kleist Sierra A , Isaacs Jordan F , Chen Clark C , Masopust David 
Published: January 2023

Abstract
Glioblastoma multiforme (GBM) is among the most aggressive, treatment resistant cancers, and despite standard of care surgery, radiation and chemotherapy, is invariably fatal. GBM is marked by local and systemic immunosuppression, contributing to resistance to existing immunotherapies that have had success in other tumor types. Memory T cells specific for previous infections reside in tissues throughout the host, including the brain, and are capable of rapid and potent immune activation. Here, we show that virus-specific memory CD8+ T cells expressing tissue resident markers populate the mouse and human glioblastoma microenvironment. Reactivating virus-specific memory T cells through intra-tumoral delivery of adjuvant-free virus-derived peptide triggered local immune activation. This delivery translated to anti-neoplastic effects, which improved survival in a murine glioblastoma model. Our results indicate that virus-specific memory T cells are a significant part of the glioblastoma immune microenvironment and may be leveraged to promote anti-tumoral immunity.
Supported by UMN SPORE Program Project Planning grant (DM, CCC), NCI 1R01CA238439 (DM), Humor to Fight the Tumor Foundation (JN), NCI 5P30CA023108-42 (PR)
CD19-CAR T Cells Develop Exhaustion Epigenetic Programs During a Clinical Response
Authors: Zebley Caitlin C , Brown Charmaine , Mi Tian , Fan Yiping , Alli Shanta , Boi Shannon , Galletti Giovanni , Lugli Enrico , Langfitt Deanna , Metais Jean-Yves , Lockey Timothy , Meagher Michael , Triplett Brandon , Talleur Aimee C , Gottschalk Stephen , Youngblood Ben A 
Published: January 2023

Abstract
The goal of this study was to determine the epigenetic landscape of CD19-CAR T cells pre and post infusion in leukemia patients as an initial step to elucidate intrinsic mechanisms that limit CAR T-cell effector functions in humans. A longitudinal analysis of CD8+ CD19-CAR T cell epigenetic changes was performed by whole-genome DNA methylation profiling of CAR T cells during manufacturing and from peripheral blood mononuclear cells (PBMCs) of 15 patients enrolled on our institutional, autologous CD19-CAR T cell therapy study (NCT03573700). CAR T cell expansion and persistence were determined by measuring vector copy numbers in the PBMCs of treated patients. We had previously established novel exhaustion DNA methylation datasets that delineate between progenitor and fully exhausted T cells. These datasets served as a guide for stratifying our post-infusion CAR T cells along the exhaustion developmental trajectory. Our data show that CD19-CAR T cells lose repressive DNA methylation at effector loci (e.g. PRF1, TBET) while gaining methylation at genes associated with memory potential (e.g. LEF1, TCF7). We confirmed these epigenetic changes are coupled to endogenous human T cell effector and memory differentiation by cross-referencing our epigenetic data with publicly available transcriptional profiles for antigen-specific effector and long-lived memory CD8 T cells from individuals vaccinated for yellow fever. Furthermore, we show that CAR T cells were unable to mount an in vivo recall response after relapse of antigen-positive disease or recovery of endogenous B cells. These observations support the conclusion that CD19-CAR T cells acquire stable epigenetic exhaustion programs that limit their protective capacity.
This work was supported by the National Institutes of Health (1R01AI114442 to BY and LRP to CCZ), the National Comprehensive Cancer Network Young Investigator Award (to CZ), Alex’s Lemonade Stand Foundation Young Investigator Grant (to CZ), Stand Up to Cancer- SU2C (to BY), the American Lebanese Syrian Associated Charities (ALSAC) to BY, and Assisi foundation to BY.
SUV39H1 disruption imparts functional persistence to CD28-costimulated human CAR T cells
Authors: Jain Nayan , Zhao Zeguo , Koche Richard , Gozlan Yosi , Brocks David , Raveh-Sadka Tali , Wells Danny , Dobrin Anton , Shi Yuzhe , Lopez Michael , Gunset Gertrude , Sadelain Michel 
Published: January 2023

Abstract
Retrovirally encoded CD19-specific CARs that incorporate CD28 and CD3z signaling motifs (Rv-1928z) have induced remarkable responses in patients with refractory leukemia and lymphoma. These CARs, however, induce a strong effector differentiation program in T cells that can limit their persistence and result in T cell dysfunction. Multiple studies have shown that effector differentiation and eventual dysfunction are associated with transcriptional and epigenetic changes. In this study, we examine the effect of disrupting the histone methyl transferase, SUV39H1 on the functional persistence of human Rv-1928z CAR T cells. SUV39H1 has recently been implicated in regulating memory to effector transition in murine T cells (Pace et al. 2018).
To assess the impact of SUV39H1 in human Rv-1928z CAR T cells, we treated immune deficient mice bearing the human ALL cell line, NALM6, with limiting doses of SUV39H1-edited (SUV39H1etd) T cells. SUV39H1etd Rv-1928z CAR T cells have improved anti-tumor efficacy as compared to WT Rv-1928z CAR T cells, with 9/10 NALM6 bearing mice treated with SUV39H1etd Rv-1928z surviving as compared to 1/12 mice treated with WT Rv-1928z over the duration of observation (90 days). This enhanced tumor control in SUV39H1etd Rv-1928z CAR T cells was associated with improved CAR T cell persistence (&gt; day 50). Paired genome accessibility (ATACseq) and transcriptional analysis on SUV39H1etd and WT Rv-1928z CAR T cells that have undergone multiple rounds of tumor rechallenges in vivo revealed epigenetic changes associated with SUV39H1 loss in Rv-1928z CAR T cells that promote expression of memory associated transcription factors and curtail T cell dysfunction in Rv-1928z CAR T cells.
ITGA4 knockout prevents blood-brain-barrier migration of chimeric antigen receptor T cells
Authors: Dietze Kenneth A , Morales Erin , Luetkens Tim 
Published: January 2023

Abstract
Chimeric Antigen Receptor (CAR) T cells have shown remarkable anti-tumor activity in various B cell malignancies. However, expanding their use to solid tumors has proven difficult due to the lack of sufficiently tumor-specific target antigens. Restricting CAR T cells from trafficking to certain healthy tissues showing shared expression of an otherwise tumor-specific target antigen may open up new avenues to the treatment of solid tumors with CAR T cells.
We developed an electroporation-based CRISPR/Cas9 approach targeting integrin a4 (ITGA4), a key component of the T cell adhesion heterodimer VLA-4 required for blood-brain-barrier migration via the endothelial adhesion protein VCAM-1. We characterize ITGA4 knockout kinetics in human T cells by flow cytometry, determine its effect on T cell adhesion to VCAM-1 using a custom in vitro adhesion assay, determine tissue distribution of ITGA4ko cells in a mouse model and characterize the anti-tumor activity of ITGA4ko CAR T cells using a luminescence-based cytotoxicity assay.
Near-complete ITGA4 knockout could be achieved during a standard 12-day CAR T cell production process and significantly decreased adhesion of human T cells to VCAM-1. ITGA4ko T cells appeared to be efficiently redirected from the brain to the bone marrow which was validated by flow cytometry showing almost a complete lack of T cells in the brain after ITGA4 knockout. Importantly, knockout did not significantly change CAR T cell expansion or anti-tumor activity.
In conclusion, we show that ITGA4 knockout is feasible, results in efficient exclusion of T cells from the brain without affecting CAR T cell function, representing a promising approach to shape CAR T cell selectivity for the treatment of solid tumors.
miRNA-target genes interactome associated with heat and immune homeostasis in evolutionarily-adapted cattle
Authors: Morenikeji Olanrewaju , Braimah Olubumi A , Memili Aylin , Murphy Thomas , Thomas Bolaji 
Published: January 2023

Abstract
Heat stress (HS) response is a direct consequence of thermal imbalance, connected to a cascade of protein activation and altered gene expression. Micro-RNAs (miRNAs) are notable for modulating gene expression, during immune and cellular response to confounding stressors. To date, the mechanism by which miRNAs regulate HS response in cattle is unknown. We hypothesize that miRNAs involved in this process are distinctly regulated between evolutionarily-adapted animals, and are important diagnostic or drug targets. We analyzed miRNA-target gene interactome in RNA-seq data from temperate and tropical cattle. A total of 3,692 genes were generated, including miRNAs that are differentially regulated. The top 10 upregulated genes, including C-C motif chemokine associated with leukocyte differentiation, APOA2 and APOH involved in HDL production, CYP1A2 and CYP2E1 involve in oxidation by cytochrome P450 were significantly upregulated in tropical animals. Conversely, GLPIR, GHSR and LOC539009, essential for homeostasis, regulation of glucose metabolism, and signaling cascades for insulin secretion were downregulated in temperate animals. Forty-one and eleven miRNAs were down- and upregulated in temperate cattle respectively. Notably, miRNA-2333 and miRNA-544b targeting GLPIR, miRNA-1248 and miRNA-6516 targeting GHSR and miRNA-2424 targeting LOC539009 were all upregulated. The downregulation of target genes in our interactome is linked to upregulation of miRNAs in temperate animals, possibly for energy conservation. This molecular interaction is significant for thermoregulation and a balanced immune response, necessities for environmental or pathogenic perturbations associated with adaptation and climate change.
Supported by None
Dietary Zinc supplementation impact on relative frequency and innate function of peripheral bovine immune cells
Authors: Diaz Fabian Esteban , Rients Emma , Franco Carlos E , Smerchek Dathan T , Hansen Stephanie L , McGill Jodi L 
Published: January 2023

Abstract
Zinc (Zn) is an essential micronutrient which is critical for optimum innate and adaptive immune function. While Zn deficiency leads to reduced growth and feed intake, the impact of Zn deficiency and supplementation on immune responses in beef cattle has not been properly addressed. To investigate to impact of dietary Zn supplementation on peripheral immune responses of growing cattle, 27 Angus crossbred steers were stratified by body weight and randomly assigned to three dietary treatments groups: 1) CON, no supplemental Zn, 2) Zn100, 100ppm of Zn or 3) Zn150, 150ppm of Zn delivered in premixes in total mixed rations. Whole blood obtained at 14 (D14) and 58 (D58) days after the start of the treatments was plated and stimulated with TLR agonists LPS (TLR4), PAM3CSK4 (TLR2/1), and Poly-I:C+Imiquimod (TLR3/7) for 48 h, then pro-inflammatory cytokine secretion was measured by ELISA on the supernatants. Additionally, whole blood was stained with surface antibodies to determine relative frequency of peripheral immune cell populations. We observed diminished IL-1ß secretion in response to Poly-I:C+Imiquimod in the CON group when compared to Zn150 (p=0.09) and Zn100 group (p=0.013) at D14 and D58, respectively. We observed no differences in IL-6 secretion upon TLR agonist stimulation between groups at any time point. Further, although no significant changes in CD4+, CD8+, TCRδ+ T cells, or CD21+MHCII+ B cells were observed, CD5+MHCII+ B cells had a higher relative frequency in the CON group when compared to Zn100 (p 0.03) and Zn150 (p 0.10) groups at D58. Future studies will address how Zn supplementation impacts the immune response to infection and disease susceptibility.
This project was supported by Agriculture and Food Research Initiative Competitive Grant no. 2020-06540 from the USDA National Institute of Food and Agriculture
Vaccination of non-human primates with self-amplifying mRNA vaccines reveal two distinct immunotypes across major SARS-CoV-2 variants.
Authors: Sullivan Brian Martin , Hastie Kathryn 
Published: January 2023

Abstract
mRNA-based vaccines have proven among the most efficacious vaccines against COVID-19. Recent SARS-CoV-2 Delta waves and the emergence of the Omicron variant have demonstrated the necessity of booster doses to a two-dose priming regimen to maintain efficacy against these variants. Global demand for primary mRNA vaccination is now competing for booster doses, thus stretching the current supply of mRNA vaccines. Arcturus Therapeutics has developed two low-dose, self-amplifying mRNA vaccines, ARCT-154 and ARCT-165, that elicits robust neutralizing antibodies in non-human primates (NHPs) as a primary vaccination series and as a booster vaccination to Comirnaty® in humans. In NHPs, we found that ARCT-154 provides better coverage against SARS-CoV-2 variants with a glutamic acid at position 484 (e.g. alpha and delta) while ARCT-165 provides better coverage against variants with a change in this position (e.g. beta, gamma, mu). When this single amino acid is reverted to a glutamic acid in either beta or gamma strains, ARCT-154 provides better neutralizing titers. However, in the context of a booster dose in humans (NCT05037097), ARCT-154 provides robust humoral responses across all variants tested. Next generation self-amplifying mRNA vaccines may provide better coverage and at booster doses that are 6–10 times lower than those of currently authorized mRNA vaccines.
Development of Quantitative Spike and Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assays (ELISAs) for Measurement of Antibodies against SARS-CoV-2
Authors: Liao Wen-Chieh , Adame Marissa , Webb Jess , Nguyen Dean , Ni Jessie 
Published: January 2023

Abstract
In the COVID-19 pandemic, serological assays will have important public health and clinical uses to monitor and respond to the pandemic. Assays to detect and quantify virus-specific antibodies are important to complement the RNA-based tests, improve our understanding of the pathogenesis of COVID-19, and inform vaccine development. The majority of commercial assays for measuring IgG to SARS-CoV-2 have been limited by a lack of quantitative information about antiviral antibodies in standardized units. Here, we have developed and commercialized several fully validated, quantitative ELISA kits using purified receptor-binding domain (RBD) or full-length of Spike S1, or Nucleocapsid proteins developed in BioLegend as the capture reagents. The SARS-CoV-2 Spike Protein antibody ELISA kits have both LEGEND MAXTM and RAPID MAXTM formats and are optimized to mitigate matrix effects and avoid false-positive results, and have a readout in standardized units (mg/mL). We have applied these sensitive and specific SARS-CoV-2 Spike Protein antibody ELISAs in an employee cohort study to monitor antibody immune response after first and second doses of 2 mRNA SARS-CoV-2 vaccines – BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna). We found that although all participants in this study are responsive to the first dose of immunization by both mRNA vaccines as evidenced by the elevation of anti-Spike S1 and RBD IgG levels above the pre-immunization baseline levels, more robust antibody responses to Spike S1 and RBD are observed after the second dose. In summary, our assays have good performance characteristics, have a quantitative readout in standardized units, and could be used by research and clinical laboratories that routinely use ELISA.