bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the aryl hydrocarbon receptor positively regulates gut migration markers on mouse B cells
Authors: Ortiz Linda F , Costa Alex B , Dekrey Gregory K
Published: January 2023

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that links diet and environmental factors to intestinal immune function. Activation of the AhR has been shown to enhance migration to the gut by some murine CD4+ T cells with upregulated expression of CCR9. In B cells, AhR activation has been shown to suppress B cell activation and class switch recombination (CSR), but an impact on B cell migration has not been explored. Because AhR activation can enhance fecal over serum IgA levels in mice, we hypothesized that AhR activation increases B cell migration to the gut. To test this hypothesis, we examined the expression of α4β7 and CCR9 on mouse CD19+ spleen cells in vitro. Cells were activated under conditions to stimulate CSR to IgA (poly-IgD-Dextran + LPS + TGF-β), cultured with AhR ligands for 96 hours, and analyzed by flow cytometry. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (50 nM) did not change α4β7 surface expression (mean fluorescence intensity, MFI), but the MFI for CCR9 was significantly increased by 96.7% (P < 0.05) when compared to CH-223191 exposure (AhR antagonist, 100 μM). Similarly, when comparing exposure to TCDD versus CH-223191, TCDD exposure increased the percentage of α4β7+ and CCR9+ cells by 3.8% and 23.3%, respectively (P < 0.05). These results suggest that enhanced migration of B cells to the gut may be positively regulated by AhR activation.

Supported by University of Northern Colorado
Defective Humoral Immunity Disrupts Bile Acid Homeostasis which Promotes Inflammatory Disease of the Small Bowel
Authors: Kubinak Jason L , Mohammed Ahmed Dawood , Mohammed Zahraa , Roland Mary , Chatzistamou Ioulia , Jolly Amy , Schoettmer Lillian , Arroyo Mireya , Kakar Khadija , Tian Yuan , Patterson Andrew , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Mucosal antibodies maintain gut homeostasis by promoting spatial segregation between host tissues and luminal microbes. Whether and how mucosal antibody responses influence gut health through modulation of microbiota composition is unclear. Here, we use a CD19−/− mouse model of antibody-deficiency to demonstrate that a relationship exists between dysbiosis, defects in bile acid homeostasis, and gluten-sensitive enteropathy of the small intestine. The gluten-sensitive small intestine enteropathy that develops in CD19−/− mice is associated with alterations to luminal bile acid composition in the SI, marked by significant reductions in the abundance of conjugated bile acids. Manipulation of bile acid availability, adoptive transfer of functional B cells, and ablation of bacterial bile salt hydrolase activity all influence the severity of small intestine enteropathy in CD19−/− mice. Collectively, results from our experiments support a model whereby mucosal humoral immune responses limit inflammatory disease of the small bowel by regulating bacterial BA metabolism.

J.L.K. was supported by a NIAID K22 Award (AI123481), a Jeffrey Modell Network Specific Diseases Research Award, a University of South Carolina ASPIRE-I Award, a pilot project award through the University of South Carolina Center for Alternative Medicine COBRE program (P20GM103641; awarded to Drs. Mitzi and Prakash Nagarkatti), an R21 (AI142409), and 1R01AI155887.
The miseducation of T cells in autoimmunity
Authors: Kouame Elaine , Brigleb Pamela , Sangani Kishan , Dermody Terence S , Jabri Bana
Published: January 2023

Celiac disease (CeD) is an immune-mediated enteropathy characterized by an inflammatory T-helper type 1 (Th1) immune response against dietary gluten. Although 40% of individuals in the U.S. have the genetic factors required for the development of CeD, less than 1% will be diagnosed with CeD. This suggests that additional environmental factors are necessary for the development of CeD. Our previous work demonstrates that viral infections can disrupt tolerance to oral antigens, leading to immune activation as seen in CeD. However, the mechanisms by which viral infections trigger inflammatory responses to dietary antigen remains unknown. Here we show that upon infection with reovirus, a subset of migratory dendritic cells, cDC1, sense and uptake virally infected and necroptotic intestinal epithelial cells and secrete type I interferon (IFN), resulting in a dietary antigen specific Th1 immune response. Our results identify a novel mechanism by which certain viruses can trigger inflammatory immune responses to dietary antigen. We also demonstrate that only a subset of enteric viruses can disrupt homeostatic response to dietary antigens. Only those that both induce type I IFN, and cause necroptosis will result in such pathology. This data identifies specific viral targets for clinical intervention via vaccine that could potentially reduce the incidence of CeD and other dietary-triggered immune enteropathies. More generally, knowledge from our work enhance our understanding of how viral-host interactions drive autoimmune disorders at the mucosal interface.

Supported by grant from NIH (RO1 5R01DK098435-06)
Sensing and alarm function of vaccine-elicited SIV-gag specific CD8 TRM in the reproductive mucosa of rhesus macaques
Authors: Joag Vineet , Quarnstrom Clare , Soerens Andrew , Stolley James Michael , Schenkel Jason M , Fraser Kathryn , Vezys Vaiva , Skinner Pamela J , Hunter Eric , Bimber Benjamin , Amara Rama Rao , Masopust David
Published: January 2023

TRM constitute a recently identified lymphocyte lineage that occupies non-lymphoid tissues (NLT) without recirculating. Experiments in mice demonstrated that TRM reactivation triggers antiviral responses in neighboring cells by promoting local stimulation of innate and adaptive immune cells, and by recruiting immune effectors. Collectively this is referred to as ‘sensing and alarm’ function. We wished to broadly identify TRM functions in non-human primates (NHP). Here we applied a prime-boost vaccine modality in rhesus macaques to generate abundant SIVgag-specific CD8 TRM in the female reproductive tract (FRT) and 14 other NLT. To assess sensing and alarm function, macaques were challenged intravaginally with CM9 peptide from the SIV gag protein and necropsied 24 or 48 hours later. CD8 TRM reactivation increased expression of CD69 and granzyme B in SIV-gag specific CD8 T cells throughout the FRT at 24h, in situ proliferation (Ki67 expression) by 48h, and rapid antiviral and IFN response gene expression in essentially all hematopoietic and non-hematopoietic cells by 24h. Upregulation of effector genes in CD8 T cells, CD4 T cells, NK cells, and ILCs peaked at 24h and persisted at 48h. Mucosal CD4 T cells expressed various HIV restriction factors, and had reduced expression of the HIV-coreceptor CCR5. Increased numbers of vaginal T and B cells coincided with increased expression of chemokines and VCAM-1 on endothelial and stromal cells and a concomitant reduction in circulating T cells, B cells and SIV-env-specific B cells. These data demonstrate that vaccine-elicited CD8 TRM rapidly trigger local activation and the recruitment of innate, cellular, and humoral immune responses to the site of antigen exposure.
Epithelial MHC class II directs microbiota-specific intestinal immune homeostasis
Authors: Eshleman Emily M , Shao Tzu-Yu , Woo Vivienne , Rice Taylor , Whitt Jordan , Engleman Laura , Way Sing Sing , Alenghat Theresa
Published: January 2023

The intestinal microbiota is essential for instructing the host immune system, however, dysregulated immune responses to resident commensal microbes can promote pathologic inflammation. Despite this relationship, the mechanisms regulating tissue-intrinsic, microbiota-specific T cell responses remain poorly understood. Here, we find that non-hematopoietic intestinal epithelial cells (IECs) represent the dominant cells expressing major histocompatibility complex (MHC) II at the host-microbiota interface. Interestingly, epithelial MHCII and commensal-specific CD4+ T cells were simultaneously induced by post-natal microbiota colonization, prompting the hypothesis that epithelial MHCII regulates intestinal microbiota-specific CD4+ T cells. While classical MHCII-expressing antigen presenting cells promote antigen-specific T cell expansion, loss of IEC-intrinsic MHCII surprisingly resulted in elevated commensal-specific CD4+ T cells in the intestine. Further, epithelial MHCII actively limited accumulation of microbial-antigen specific CD4+ T cells in adult mice. Expansion of commensal-specific Th17 cells was restricted by epithelial MHCII, and remarkably mice lacking epithelial MHCII were highly susceptible to microbiota-triggered intestinal inflammation. Collectively, these data suggest that altered epithelial MHCII-T cell regulation in the intestine may impair tolerance to resident microbes and predispose to chronic inflammation. Future investigation into the relationship between the microbiota, IECs, and local immune cells could aid in the development of novel therapeutics to prevent or limit inflammatory conditions.

This research is supported by the National Institutes of Health (DK114123, DK116868 and F32AI147591 to E.M.E.), Kenneth Rainin Foundation, and Burroughs Wellcome Fund. This project is supported in part by PHS grant P30 DK078392.
Microfluidic gut-on chip system for reproducing the microbiome-immune cells interaction in Threespine Stickleback
Authors: Nouri Abdelmounaim , Rodgers Maria L , Bolnick Daniel L , Carrier Rebecca , Milligan-Myhre Kathryn , Scarpino Samuel , Steinel Natalie C
Published: January 2023

The development of in vitro models that mimic the immunological, mechanical, and structural properties of the gut, as well as its crucial microbial symbionts, facilitate the real-time assessment of host-microbiome interactions. Here, we describe our development of a threespine stickleback fish “gut-on-chip” biomimetic microdevice which will co-culture complex microbial communities with mucus-secreting intestinal epithelia and leukocytes. This chip was fabricated using soft lithography with liquid PDMS and consists of a middle channel coated with extracellular matrix (ECM) compounds separating two microfluidic side channels. After temperature cure, PDMS is bound on a glass slide by plasma activation, forming a closed microenvironment where cells can be grown. We have successfully isolated and independently cultured stickleback intestinal epithelial cells and leukocytes and evaluated their tolerance to various components of ECM. Our results show that fibrinogen is more suitable for culturing intestinal leukocytes compared to collagen-1 or gelatin. Leukocyte viability is more sustainable after stimulation with LPS-B5 compared to unstimulated. Unlike immune cells, stickleback epithelial cells do not survive on fibrinogen and show better viability on the collagen-1 coated medium. This study establishes the unique culture conditions necessary for teleost microfluidic gut-on-a-chip and will advance our understanding of the host-microbiome interactions in this non-model system.

Supported by Gordon and Betty More Foundation, SASI Symbiosis Model Systems #9323
Nanoparticle based depletion of Myeloid-derived Suppressor cells in Tuberculosis
Authors: Dkhar Hedwin Kitdorlang , Vantucci Casey , Enriquez Ana Beatriz , Ibegbu Chris , Vyas Shivani , Woods Nicole Alexis , Kgoadi Khanyisile , Goh Shu Ling , Roy Krishnendu , Rengarajan Jyothi
Published: January 2023

The bacterial pathogen Mycobacterium tuberculosis (Mtb) successfully evades host innate immune responses to cause tuberculosis (TB) disease. Recent studies showed that myeloid-derived suppressor cells (MDSCs) are recruited to the lung after Mtb infection and harbor Mtb. While MDSC-mediated negative regulation of tumor micro-environments in cancer has been extensively studied, in-depth analyses of the phenotype, functions and transcriptional signatures of MDSCs in TB are lacking. In this study we sought to characterize MDSCs in the lungs of Mtb-infected mice and develop tools for depleting MDSCs as a strategy for host-directed therapy. We determined MDSCs in the aerosol mouse model of TB and in human blood using flow cytometry. MDSCs are recruited into the lung as early as 2 weeks post-infection and expresses immunomodulatory mediators associated with immune suppression such as iNOS, IDO and arginase. We recently reported the development of novel synthetic nanoparticle antibodies (SNAbs) that specifically target and deplete MDSCs via antibody-like mechanisms. We used SNAbs to deplete Mtb-induced MDSCs in vitro and in human PBMCs ex vivo. Interestingly, we found that SNAbs efficiently depleted MDSCs in both models. To test the efficacy of SNAb-mediated depletion of MDSCs in mouse lungs in vivo, we first used a murine lung cancer model and found that intra-tracheal delivery of SNAbs successfully depleted MDSCs in vivo. Ongoing studies include evaluating the ability of SNAbs to deplete lung MDSCs following Mtb infection of mice and to assess the impact on lung T cell functions, pathology and Mtb burden. These studies will establish a platform to investigate MDSCs as a target for host-directed therapies that improve TB treatment efficacy.

Supported by grant from NIH (1R01AI155023-01A1)
PD-1 and ICOS co-expression identifies tumor-reactive CD4 Th cells in human solid tumors
Authors: Duhen Thomas , Duhen Rebekka , Fesneau Olivier , Samson Kimberly , Frye Alexandra , Beymer Michael , Rajamanickam Venkatesh , Tran Eric , Bernard Brady , Weinberg Andrew D
Published: January 2023

CD4 T helper (Th) cells play a key role in orchestrating immune responses, but the identity of the CD4 Th cells involved in the immune response against cancer remains to be defined. We analyzed the immune cell infiltrates of head and neck squamous cell carcinoma and microsatellite stable colorectal cancers and identified a subset of CD4 Th cells distinct from FOXP3+ regulatory T cells that co-express PD-1 and ICOS. This cell population, which was present in the tumor but absent in the periphery, features of chronic stimulation and displays characteristics of tissue-resident memory T cells. PD-1+ICOS+ tumor-infiltrating (TIL) CD4 Th cells are located primarily in the tumor stroma in proximity to MHC class II+ cells and are proliferating, suggesting local antigen recognition. The T-cell receptor repertoire of the PD-1+ICOS+ CD4 Th TIL is oligoclonal, with T-cell clones expanded in the tumor, but present at low frequencies in the periphery. Finally, these PD-1+ICOS+ CD4 Th TIL were shown to recognize both tumor-associated antigens and tumor-specific neoantigens, which were distinct from the epitopes recognized by the CD8 T cells from the same patients. Our findings provide an approach for isolating tumor-reactive CD4 Th TIL directly ex vivo and imply that harnessing the cooperation between CD4 and CD8 T cells might lead to more efficient tumor recognition and control in cancer patients.

Supported by the Providence Portland Medical Foundation and the Developmental Research Program for Head and Neck Cancer.
Third generation CAR T cells form polyfunctional long-lived memory and protect from solid-tumor relapse in an immunocompetent murine model
Authors: Reed Steven James , Dunga Meenakshi , Maylor-Hagen Heather , Kalia Vandana , Sarkar Surojit
Published: January 2023

Effector CAR T cell persistence and transition to memory have been widely associated with long-term protection from hematological tumor relapse. In the context of solid tumors, anti-tumor T cell exhaustion in the tumor microenvironment (TME) leads to T cell dysfunction and impaired memory formation. However, in solid tumor models a vast number of infused CAR T cells traffic to lymphoid organs (such as spleen and nondraining lymph nodes), which are bereft of CAR-antigen. The fate of these CAR T cells which have undergone ex vivo activation, viral transduction, and expansion regimens prior to transfer back into patients remains unclear. Using a third generation CAR targeting human(h)CD19t, and syngeneic murine tumor lines expressing hCD19t tumor-associated antigen, we show that CAR T cells residing in antigen-free sites of tumor-bearing immunocompetent mice transition to fully functional memory cells. Using viral infection-induced memory T cells as a benchmark, memory CAR T cells displayed hallmark memory characteristics including homeostatic proliferation, rapid polyfunctional cytokine production upon restimulation, and secondary expansion in response to cognate antigenic stimulation. Finally, memory CAR T cells in naive mice, or in mice with primary tumor remission, were protected against lethal rechallenge with heterologous hCD19t-expressing tumors. Results from this study are among the first to directly compare canonical virus-induced memory T cells with memory CAR T cells, and provide valuable insight into the characteristics and function of memory CAR T cells in an immunocompetent model for solid tumors.

Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number F32CA265056, and the Pediatric Cancer Research Foundation
CAR T cells targeting Olfactory Receptor OR2H1 are an effective immunotherapeutic option in human epithelial tumors
Authors: Biswas Subir , Martin Alexandra , Galindo Carmen Maria Anadon , Mine Jessica , Payne Kyle K , Mandal Gunjan , Chaurio Ricardo , Powers John J , Sprenger Kimberly , Rigolizzo Kristen E , Innamarato Patrick , Harro Carly , Mehta Sumit , Perez Bradford A , Wenham Robert M , Conejo-Garcia Jose R
Published: January 2023

Chimeric antigen receptor (CAR) T cells have been successful for hematological malignancies, but so far showed limited efficiency against solid tumors. Olfactory receptors are expressed in a variety of epithelial cancers, with a limited expression in healthy tissues. We quantified olfactory receptor OR2H1 expression in ovarian cancer, non-small cell lung cancer (NSCLC), breast cancer, and many normal tissues, and we found OR2H1 is expressed in multiple solid epithelial tumors but limited to testis among healthy human tissues. CAR T cells targeting OR2H1-extracellular domain were generated, and OR2H1-specific cytotoxic killing was confirmed, both in vitro and in vivo. Correspondingly, OR2H1-CAR T cells mediate significant therapeutic effects against OR2H1+ NSCLC and high-grade serous ovarian cancer. Interestingly, OR2H1 ablation significantly delays malignant progression of at least NSCLC, but OR2H1 expression is found to be essential for the cytotoxic effects of the OR2H1-CAR T cells. In summary, T cells directed against OR2H1-expressing tumor cells could be a potential therapeutic approach in future for treating OR2H1-expressing cancers.

Supported by CA076292, R01CA157664, R01CA124515, R01CA178687, R01CA211913, U01CA232758
Inhibition of TKI-activated MIF/CXCR2 Pathway as a Novel Therapeutic Strategy in Acute Myeloid Leukemia
Authors: Hino Christopher S , Tadros Verena , Hiramoto Benjamin , Leep Benjamin , Kim Do Hyun , Reeves Mark E , Baylink David , Cao Huynh , Xu Yi
Published: January 2023

Despite significant advancements in our understanding of acute myeloid leukemia (AML), relapsed and refractory disease remains a major cause of treatment failure. Approximately 50% of patients with AML will develop relapsed disease following induction chemotherapy, which results in a dismal 5-year overall survival rate of 29%. The development of FMS-like tyrosine kinase 3 (FLT3) inhibitors have led to improved outcomes among patients with FLT3-mutated AML. However many patients eventually relapse and succumb to chemo-resistant disease, highlighting the need to characterize the molecular pathways which confer early TKI resistance. To explore the possible mechanisms responsible for the survival and proliferation of tyrosine kinase inhibitor (TKI) resistant blasts, we compared in vitro and ex vivo cytokine expression of gilteritinib (GILT)-treated and untreated blasts. Here we report that the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is significantly expressed by GILT-treated blasts when compared to untreated controls. We further demonstrate that MIF expression promotes blast proliferation through the upregulation of its receptor CXCR2. Most remarkably we found that combination of CXCR2-inhibitor plus GILT works synergistically to reduce the percentage of viable blasts. Together these findings support that targeting the TKI-activated MIF/CXCR pathway could be a novel therapeutic strategy for both newly diagnosed and relapsed/refractory AML.

This study is supported by Loma Linda University GRASP and Loma Linda University Research Innovation Grant
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

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

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

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

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

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

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 (> 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

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

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

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

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

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.
Evolution of inflammation and immunity in a dengue virus 1 human infection model
Authors: Waickman Adam T , Lu Joseph Q , Fang HengSheng , Waldran Mitchell J , Gebo Chad , Van Wesenbeeck Liesbeth , Verpoorten Nathalie , Lenz Oliver , Tambuyzer Lotke , Herrera-Taracena Guillermo , Van Loock Marnix , Endy Timothy P , Thomas Stephen J
Published: January 2023

Dengue virus (DENV) is a significant source of morbidity and mortality throughout the tropics and subtropics. Over 400 million infections are thought to occur every year, resulting in nearly 100 million symptomatic infections and over 20,000 deaths. Early immune response kinetics to DENV infection remain unclear in large part due to the highly variable incubation period exhibited by the virus after introduction into a susceptible host. To fill this knowledge gap, we performed a comprehensive virologic and immunologic analysis of individuals experimentally infected with an under-attenuated DENV-1 strain 45AZ5. This analysis captured both the kinetics and composition of the innate, humoral, and cellular immune response elicited by experimental DENV-1 challenge, as well as the virologic and clinical feature of DENV-1 infection. Revealed in this analysis was an unexpectedly robust DENV-specific IgA antibody response that manifested between the appearance of DENV-specific IgM and IgG in all challenged individuals, as well as the presence of a non-neutralizing/NS1-specific antibody response that was delayed relative to the appearance of DENV-virion specific humoral immunity. RNAseq analysis also revealed several distinct and temporally-restricted gene modules that allowed for the identification and differentiation of the innate and adaptive immune response to DENV-infection. Our analysis provides a detailed description, in time and space, of the evolving matrix of DENV-elicited human inflammation and immunity, and reveals several previously unappreciated immunological aspects of primary DENV-1 infection.
Glycan-specific B-1 cells mediate blockade of endogenous retroviruses emergence through recognition of conserved glycan epitopes
Authors: Yang Yexin , Treger Rebecca , Hernandez-Bird Juan , Iwasaki Akiko
Published: January 2023

Endogenous retroviruses (ERVs), comprising a substantial portion of the vertebrate genome, are remnants of ancient genetic invaders. Although multiple layers of cell-intrinsic control are utilized to prevent retroviral reactivation, ERVs with intact coding potential were found to reactivate in immunodeficient mice. While previous studies have indicated that B cells are indispensable for preventing ERV reactivation, it is not yet clear which B cell population mediates the blockade of ERV emergence to prevent subsequent damage in the host. Here, we employed direct labeling of B cells reactive with emerged ERV particles to characterize the B cell population and clonal repertoire responsible for recognition of ERV, and to study the mechanism by which B cells provide protection against ERV emergence. We found that ERV-reactive B cells are enriched in innate-like B-1 cell compartment that predominantly reside in peritoneal and pleural cavities. We identified ERV-reactive antibodies in unimmunized mice, the level of which further increases upon innate sensor stimulation. B cell receptor repertoire profiling of ERV-reactive B-1 cells revealed increased usage of Igh VH genes that give rise to glycan-specific antibodies, which were further determined to target terminal N-Acetylglucosamine moieties exhibited by endogenous and exogenous viral antigens. We demonstrated that these glycan-specific natural antibodies engage complement pathway to facilitate clearance of reactivated ERV particles. In conclusion, we elucidated the role of glycan-specific B-1 cells and secreted natural antibodies in mediating blockade of ERV emergence through recognition of conserved glycan epitopes.

Supported by grants from Howard Hughes Medical Institute