bluuebunny/crossref_metadata_2025 huggingface dataset:
Batf Stabilizes the Th17 Cell Developmental Program through Impairment of Stat5-dependent Recruitment of Ets1-Runx1 Complexes
Authors: Pham Duy , Silberger Daniel J , Nguyen Kim , Gao Min , Hatton Robin D , Weaver Casey Todd
Published: January 2023

The emergence of alternative CD4 T cell fates is controlled by transcriptional networks activated downstream of TCR and cytokine signaling inputs. Th17 cells develop from antigen-activated naive CD4 T cells in the presence of IL-6 and TGF-b. An important facet of the Th17 cell program is its intrinsic instability; early development of Th17 cells shares overlapping features with that of Treg cells and Th17 cells are prone to transdifferentiation into Th1-like cells. The basic leucine zipper transcription factor ATF-like (Batf) contributes to the transcriptional programming of multiple effector T cells but is indispensable for Th17 cell development. Although Batf is required for development of Th17 cells, its mechanisms of action to underpin the Th17 program are incompletely understood. Here, we find that in addition to activating core genes of the Th17 program, Batf also acts to restrict expression of genes of the Treg and Th1 programs through its actions to restrain IL-2-induced Stat5 activation, thereby limiting Stat5-dependent recruitment of Ets1-Runx1 factors to Th1 and Treg cell-specific gene loci. Accordingly, Batf acts both to pioneer regulatory elements in Th17-specific genes and limit assembly of a Stat5-Ets1-Runx1 enhanceosome that appears to be required for optimal expression of Th1 and Treg cell-specific genes. Our findings define a new role for Batf in T cell differentiation and unveil an important role for Stat5-Ets1-Runx1 interactions in transcriptional networks that define alternative T cell developmental fates.

Supported by NIH R01 DK115172 and T32 AI007051-37
Cell-intrinsic expression of the hemichannel Pannexin-1 promotes effector and memory CD8+ T cells via distinct metabolic pathways
Authors: da Silva Henrique Borges , Vardam-Kaur Trupti , Zhou Maggie , de Gois Macedo Bruna , Van Dijk Sarah , Jameson Stephen C
Published: January 2023

Tissue-derived extracellular ATP (eATP) is a “danger signal” sensed by immune cells via purinergic receptors (e.g. P2RX7). In addition, healthy cells can export eATP through transmembrane channels, such as the hemichannel Pannexin-1 (Panx1). CD8+ T cells express Panx1 constitutively throughout developmental and immune response stages. However, the biological role of Panx1 in CD8+ T cells is undefined. Here, we used genetic and pharmacological tools to address this question. T-cell specific Panx1 deletion did not affect CD8+ T cell thymic development. In contrast, CD8+ T cell responses to viral infection (LCMV) are impaired by Panx1 inhibition or deletion. This is true for effector expansion of CD8+ T cells and for the survival of memory CD8+ T cells. Using adoptive transfer of tamoxifen-conditional Panx1 knockout LCMV-specific (P14) cells into LCMV-infected mice, we showed that memory CD8+ T cell survival requires sustained Panx1 – except for small intestinal-resident cells. Mechanistically, Panx1 promotes memory CD8+ T cell survival via P2RX7, which also favors memory CD8+ T cells - as suggested by our data with Panx1/P2RX7-double knockout P14 cells. The role of Panx1 for effector CD8+ T cell expansion, however, is unlikely to be via P2RX7 since this receptor does not have a role in effector CD8+ T cells. Our transcriptional and metabolic data suggests that, while memory CD8+ T cells require Panx1 for optimal mitochondrial function and expression of pro-memory genes, effector CD8+ T cells lacking Panx1 had decreased activation of the glycolytic pathway. Collectively, these results suggest that, in response to acute virus, Panx1 induces CD8+ T cell effector expansion and memory survival through distinct metabolic pathways.

Supported by grants from NIH (R00 AI139381, NIAID).
Defining the extra-thymic role of HEB in the development of CD8+ T stem cell-like immunological memory
Authors: Leung Joanne , Campbell Kieran , Anderson Michele K
Published: January 2023

The transcription factor HEB is involved in multiple stages of intrathymic T cell development, complexing with stage-specific partners to regulate different genes in a context-specific manner. Remarkably, little is known about how HEB influences the differentiation of peripheral T cells, and even less so of how the transcriptional circuitry in early T cell development may be re-invoked to generate long-lasting immunity. While T cells are detected in the periphery of HEB-deficient mice (HEB cKO), the contribution of HEB to the function of T cells upon pathogen encounter is obscure. Thus, HEB cKO mice were challenged with LCMV to determine the impact of HEB in the activation and differentiation of CD8+ T cells into effector and memory lineages. Throughout the course of infection, HEB cKO mice exhibited critical differences in their antigen-specific CD8+ response and had enhanced levels of stem cell-like memory T cells (Tscm). This implicated a role for HEB downstream of T cell activation, which we further confirmed using well-established in vitro cultures supplemented with or without Tscm-inducing cytokines. Consistently, HEB transcripts were detected in publicly available scRNA datasets from P14 transgenic mice infected with LCMV. To greater define the transcriptional pattern of HEB, and of others that orchestrate thymic T cell development in the context of peripheral T cell immunity, we will conduct a scRNA-seq comparative bioinformatic analysis of developing thymocytes to splenic LCMV-specific CD8+ T cells. This will advance our understanding of how early transcriptional cascades, like those initiated by HEB, are propagated in downstream immunological reactions, which may lead to therapies that can revitalize T cell immunity.

Supported by a grant from NIH (1P01AI102853-06) and the The AAI Intersect Fellowship Program for Computational Scientists and Immunologists
Normal microbial experiences accelerate memory T cell compartment maturation in infants
Authors: Stenger Terran D , Burger Sarah , Pierson Mark , Huggins Matt , Hamilton Sara , Schuldt Nathan J
Published: January 2023

Memory T cells cause faster and more effective elimination of pathogens during reinfection by the rapid production of effector T cells. Worldwide, the leading cause of death among children and infants is infection, partly due to under-developed T cell memory populations commonly seen in infants. In the context of biomedical research, the specific pathogen-free (SPF) facilities research animals are housed in further the deficit of memory T cells, as it is known that adult mice housed in SPF facilities have few memory T cells, but that exposure to normal microbial experiences (NME) replenishes this population of cells. This phenomenon, though, has not been investigated in infants. Therefore, we aimed to investigate how NME influences infant memory T cell commitment to reveal novel insights into natural mammalian immune development. At various time points, the trajectory of infant immune maturation of age-matched SPF and NME (pups birthed and raised in an environment exposed to NME) mice were examined using flow cytometry. We observed significant increases in both the number and frequency of memory T cell subsets (CD8+ and CD4+ Tcm and Tem cells) at all time points in pups exposed to NME. We also observed increased serum concentrations of cytokines required for memory T cell differentiation and survival (IL-7 and IL-15) in NME pups. These data support the hypothesis that NME accelerates the maturation of the memory T cell compartment in infant mice. We predict this more mature memory T cell compartment may contribute to improved immunity to novel infections. Our ongoing experiments test this hypothesis using a Listeria monocytogenes challenge model.
Dendritic cells instruct differentiation of tissue resident memory T cells in the skin to promote durable tumor immunity
Authors: Mohamed Asmaa , Vella Jennifer , Turk Mary Jo , Huang Yina H
Published: January 2023

A subset of melanoma patients treated with immune checkpoint inhibitors develops vitiligo, a CD8 T cell-mediated autoimmune disease associated with improved patient survival. Using a melanoma-associated vitiligo (MAV) mouse model, CD8 tissue resident memory (TRM) T cells in vitiligo skin were found to be necessary and sufficient for durable tumor immunity. Using immunofluorescence microscopy, we found that skin TRM cells formed large aggregates with CD11c myeloid cells proximal to hair follicles. CD11c depletion resulted in TRM cell loss, revealing an unexpected requirement for continued T cell-dendritic cell (DC) interactions in TRM cell maintenance. We hypothesize that DCs provide instructive signals that are required for continued in situ maturation of CD8 TRM cells. To identify these signals, we disrupted Toll Like Receptor (TLR) signaling in DCs by ablating the MyD88 adapter protein and observed a reduction in skin TRM cell accumulation. This prompted us to explore a role for the microbiota in CD8 TRM formation. Treatment with broad spectrum antibiotics resulted in a 50% reduction in vitiligo incidence. B6 mice from vendors with microbiota differences, exhibited divergent MAV incidence, but upon cohousing or fecal transplantation, mice from both sources exhibited high MAV incidence. Collectively, these findings indicates that the gut-skin microbiota axis plays a critical role in generating tumor protective TRM cells. Future studies seek to identify microbial antigens responsible for promoting TRM differentiation.

Supported by T32-AI007363 P30-CA023108
T cell intrinsic Nod2 controls Th17 immunity to Candida albicans infection
Authors: Koney Kylie , Lashley Sydney J , Lee Ellen J , Vance Emily E , Philpott Dana J , Dugan Jae W , Davey Michael M , Rosenzweig Holly L , Napier Ruth J
Published: January 2023

We recently discovered a role for Nod2 within T cells in controlling Th17-immunity, such that deletion of Nod2 exacerbated experimental autoimmune arthritis and uveitis. Protection against opportunistic Candida albicans infection relies on intact Th17-responses. Thus, we considered whether the T cell intrinsic Nod2 might control antifungal Th17 responses at the cost of increasing host susceptibility to autoimmunity. Nod2−/− mice infected with 105 (LD50) Candida albicans had increased survival and reduced fungal burden in the kidney within 24–72 h post-infection, suggesting a critical role for Nod2 in C. albicans immunity. Rag−/− mice reconstituted with Nod2fl/fl/CD4-cre CD4+ T cells and infected with C. albicans had decreased fungal burden compared to control CD4-Cre T cell recipients, and IL-17 depletion reverted the phenotype. After 48h infection, Nod2fl/fl/CD4-cre CD4+ T cells in the kidney had increased activation (CD69), increased proportion of T effector cells, and decreased Tregs compared to controls, suggesting endogenous Nod2 negatively regulates fungal-triggered Teff/Th17 cell responses. We next asked how Nod2 might function in SKG mice that are genetically susceptible to arthritis. Whereas Nod2−/− SKG mice developed an exacerbated form of arthritis compared to Nod2+/+ SKG controls, these same mice cleared C. albicans infection better than Nod2+/+ SKG mice. Cumulatively our data indicate a critical role for T cell intrinsic Nod2 in antifungal Th17 immunity. Given these studies we posit that human NOD2 genetic variants may offer enhanced ability to fight fungal infection via a T cell intrinsic mechanism, at the cost of triggering autoimmunity.

Supported by grants from VA (CDA-2 IK2BX004523 and Merit I01BX002180) and NIH (R01 EY025250).
IL-27 signaling promotes Th1 responses and is required to inhibit fungal growth in the lungs during repeated exposure to Aspergillus fumigatus
Authors: Strickland Ashley , Sun Donglei , Sun Peng , Chen Yanli , Liu Gongguan , Shi Meiqing
Published: January 2023

Aspergillus fumigatus is an opportunistic fungal pathogen that causes a wide spectrum of diseases in humans, including life-threatening invasive infections as well as a number of hypersensitivity respiratory disorders. Disease prevention is predicated on the host’s ability to clear A. fumigatus from the lungs while also limiting inflammation and preventing allergic responses. IL-27 is an important immunoregulatory cytokine, but its role during A. fumigatus infection remains poorly understood. In contrast to most infection settings showing that IL-27 is anti-inflammatory, here we report that this cytokine plays a proinflammatory role in mice repeatedly infected with A. fumigatus. We found that repeated exposure of mice to A. fumigatus led to significantly enhanced secretion of IL-27 in the lung. Genetic ablation of IL-27 receptor alpha (IL-27Rα) in mice resulted in significantly higher fungal burdens in the lung during repeated exposure to A. fumigatus. The increased fungal growth in IL-27Rα−/− mice was associated with reduced secretion of IL-12, TNF-α and IFN-γ, diminished T-bet expression, as well as a reduction in CD4+ T cells and their activation in the lung, demonstrating that IL-27 signaling promotes Th1 immune responses during repeated exposure to A. fumigatus. In addition, infected IL-27Rα−/− mice displayed reduced accumulation of dendritic cells and exudate macrophages in the lung with lower expression of MHC class II on these cells and had less M1 macrophage activation. Taken together, this study suggests that IL-27 drives type 1 immunity and is indispensable for inhibiting fugal growth in the lungs of mice repeatedly exposed to A. fumigatus, highlighting a protective role for this cytokine during fungal infection.

Supported by NIH (AI131219, AI131905)
Gut microbiota-reactive IgG regulates gut microbiota development and immunity against enteric pathogens in early life
Authors: Amir Mohammed , Sanidad Katherine , Ananthanarayanan Aparna , Zhang Lingzhi , Shiland Nicholas , Inohara Naohiro , Nunez Gabriel , Zeng Melody
Published: January 2023

The gut microbiota elicits antigen-specific IgG at steady state that cross-reacts to pathogenic Gram-negative Enterobacteriaceae to confer protection against systemic infection. The role of these IgG antibodies in the development of gut microbiome and immunity against enteric pathogens in early life, however, remains largely undefined. In this study, we demonstrate that gut microbiota-induced maternal IgG is transferred to the neonatal gut through maternal milk via the neonatal Fc receptor and confers critical protection by inhibiting colonization and attachment of C. rodentium to the mucosa in the neonatal gut. Enhanced neonatal immunity against oral C. rodentium infection was observed following maternal immunization with a gut microbiota-derived IgG antigen, outer-membrane protein A (OMP-A), or induction of protective IgG antibodies in germ-free mice after reconstitution with IgG-inducing gut bacteria. Furthermore, we generated a mouse model with complete deficiency in all IgG isotypes. IgG-deficient neonatal mice exhibited increased susceptibility to C. rodentium infection, altered gut microbiome and increased numbers of intestinal IL-17-producing γδ T cells driven by the gut microbiome at steady state. The altered gut microbiome persisted into adulthood in IgG KO mice and contributed to increased disease severity in DSS-induced colitis. Taken together, our studies have defined critical roles for gut microbiome-induced IgG antibodies in shaping the gut microbiome and immunity against Gram-negative enteric pathogens from early life to adulthood.
Education of uterine natural killer cells by maternal MHC drives fetal growth
Authors: Depierreux Delphine M , Kiekbush Jens , Shreeve Norman , Sharkey Andrew , Hawkes Delia , Colucci Francesco
Published: January 2023

Maternal uterine natural killer (uNK) cells, in partnership with fetal trophoblast cells, regulate physiological vascular changes in the uterus of pregnant women and mice. These vascular changes are necessary to build the placenta and sustain fetal growth. NK cell functions, in the uterus and elsewhere, are modulated by NK cell education, a quantifiable process that determines cellular activation thresholds. This process relies largely on interactions between self-MHC class I molecules and inhibitory NK cell receptors but its physiological importance has remained largely elusive.

NK cell education during pregnancy is unique because uNK cells are exposed to both maternal and fetal MHC-I molecules. Here, we set out to answer the question of how uNK cell education or missing-self recognition affect their function and, ultimately, fetal growth.

Using combinations of MHC-sufficient and -deficient mice, we show that maternal and not fetal MHC regulates uterine NK cell education in mice. MHC-deficient dams produce more growth-restricted fetuses, even when the fetuses themselves express self-MHC. We also show that, while peripheral NK cells reject bone marrow cells according to the established rules of missing-self recognition, uNK cells educated by maternal MHC do not reject fetuses that miss self-MHC and these fetuses grow to their full potential.

Our data shed light on the physiological importance of uNK cell education: by getting to know self, the maternal immune system sets up uNK cells to participate to the womb tissue homeostasis and to contribute to reproductive success.

This work was supported by the Cambridge NIHR BRC Cell Phenotyping Hub and by grants from the Wellcome Trust (094073/Z /10/Z), the MRC (MR/P001092/1) and the Centre for Trophoblast Research.
Microbiota drive concomitant expression of IL-17 and the inhibitory receptor PD-1 to regulate effector functions of γδ17 cells in the intestine
Authors: Huang Hsin-I , Hammer Gianna
Published: January 2023

While developmental programming of innate-like γδT cells generates dedicated IFN-γ-producing (γδIFN-γ) or IL-17-producing (γδ17) subsets, emerging evidence suggests these are further shaped by environmental cues. Such cues include the intestinal microbiota, although the nature of microbiota-dependent regulation is poorly understood since some reports hold that microbiota exert strictly activating signals, while other reports show microbiota are strictly suppressive. Whether the impact of microbiota on γδT cells is indeed exclusively binary, and whether regulatory modules stemming from the microbiota can be dynamic in response to changing physiologic conditions remains unknown. Here we identify a microbiota-dependent regulatory module for γδ17 cells in which microbiota concomitantly upregulate IL-17-production and the inhibitory receptor programmed cell death protein 1 (PD-1). Concomitant expression of these two modules, the requirements for microbiota, and preferential upregulation of a PD-1high phenotype is conserved for γδ17 cells resident within multiple mucosal barriers. Importantly, microbiota-driven PD-1 inhibits IL-17 production by endogenous γδ17 effectors. We further show that these two microbiota-dependent modules are dynamic, wherein both PD-1 and IL-17 producing functions are downregulated upon depletion of microbiota, and concomitantly upregulated in response to intestinal inflammation. We also identify enhanced capacity for lipid uptake as an inflammation-associated metabolic response specific to the γδ17 subset, thus linking augmented γδ17 effector functions to dynamic subset-specific metabolic remodeling within the intestine.

Supported by NIH R01-AI088100 awarded to MLS
Multi-transgene AAV immunotherapy re-establishes immune tolerance and provides comprehensive protection in animal model of MS
Authors: Sagadevan Addelynn Sonia , Keeler Geoffrey D , Gaddie Cristina D , Cote Isabelle , Senior Kevin G , Min Daniel , Hoffman Brad E
Published: January 2023

To re-establish long-term immune tolerance in Multiple Sclerosis (MS), our lab had previously developed a pre-clinical Adeno-associated virus (AAV) gene immunotherapy that is capable of preventing and reversing Myelin Oligodendrocyte Glycoprotein (MOG) induced Experimental Autoimmune Encephalomyelitis (EAE). However, MS is a disease that involves multiple myelin proteins including Proteolipid Protein (PLP). Therefore we expanded the capability of our gene immunotherapy to restore tolerance and ameliorate disease to multiple major myelin proteins, simultaneously. In this report we demonstrate that a mixture of 2 individual vectors, AAV MOG & AAV.PLP, was capable of preventing, and more importantly reversing, preexisting EAE disease induced with a mixture of MOG35–55 & PLP139–151 peptides in (C57BL/6JxSJL)F1 mice. To minimize the total vector load we further developed this immunotherapy into a novel single AAV-dual transgene expressing vector (AAV.MOG.PLP) using specific gene linkers for independent expression. Following a single peripheral injection, western blot analysis confirmed stable and simultaneous hepatic expression of both neuroprotein transgenes. When injected into mice 2 weeks before induction of EAE with either MOG35–55 or PLP139–151, or combination of both, the dual transgene AAV immunotherapy significantly reduced or prevented disease in mice compared to controls. Overall, these results demonstrate proof of concept that a single AAV vector simultaneously expressing more than one transgene may be an effective therapeutic treatment for restoring tolerance in an autoimmune disease like MS.

Supported by NIHSarepta Pharmaceuticals NIH R01Act AI128074Project
Gut bacteria-derived isoflavone metabolites ameliorate experimental autoimmune encephalomyelitis (EAE) through estrogen receptor engagement
Authors: Peterson Stephanie R , Jensen Samantha , Dencklau Dalton , Mangalam Ashutosh K
Published: January 2023

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system which may be influenced by gut bacteria-produced metabolites. We have shown that isoflavone-metabolizing bacteria are less abundant in MS patients as compared to healthy individuals and loss of these bacteria leads to increased disease severity in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Isoflavone metabolites can modulate the immune response through binding to estrogen receptors α and β (ERα and ERβ). Thus, we aim to investigate the role of ERα and ERβ in isoflavone-mediated disease protection in EAE by determining ER expression on immune and intestinal epithelial cells as well as receptor specificity for isoflavone-mediated protection in EAE. We hypothesize that isoflavone metabolites induce an anti-inflammatory state via engagement of ERs and that reduction/absence of these molecules may predispose patients to MS development and/or severity. To test this, we administered an isoflavone-containing diet to mice and induced EAE. Mice fed an isoflavone-containing diet developed milder disease than mice fed an isoflavone-free diet while also exhibiting higher expression of ERα, but not ERβ, in the gut. Surprisingly, there was no change in ERβ expression in splenic CD4+ or CD8+ T-cells of mice fed an isoflavone-free versus an isoflavone-containing diet. Mice administered equol also exhibited milder disease than controls. Ongoing experiments using chemical or genetic inhibition of ERα and ERβ signaling will determine the role of these receptors in EAE disease amelioration. Our results suggest that bacterial metabolism of isoflavones leads to EAE disease suppression through utilization of estrogen receptors.

Supported by grants from NIH (T32AI007485, 1R01AI137075-01) and a generous gift from Margaret Heppelmann and Michael Wacek.
Characterization of ARTS-011, a potent and selective TYK2 inhibitor for the treatment of inflammatory disease
Authors: Liang Jiaqi , Liu Yiting , Zhang Xiaobin , Zhang Tao , Liu Xipan , Fu Jia , Cheng Dai , Xiong Wen , Ding Peter , Zhu Cong , Li Fang , Chen Yaoyu
Published: January 2023

The JAK family kinase TYK2 is a key regulator of IL-12 and IL-23 signaling pathways, which have been shown to be involved in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, lupus and inflammatory bowel diseases (IBD). TYK2 inhibition is an attractive therapeutic strategy for human autoimmune diseases. Here, we report a potent oral TYK2 allosteric inhibitor ARTS-011, with high selectivity and potency through optimized binding to the JH2 regulatory domain of the TYK2 protein. ARTS-011 potently binds to TYK2 JH2 domain in binding assay and inhibits IL-23 and IFNα/β induced reporter activation depending on TYK2 kinase. ARTS-011 also inhibits IFNα triggered pSTAT5 potently in human whole blood assay. In addition, ARTS-011 excellent selectivity against other JAK family kinases and panels of receptors, transporters, ion channels, CYP enzymes and the hERG channel. Furthermore, ARTS-011 was also investigated in multiple animal models including IL12/18-induced serum IFNγ mouse model, IL-23-induced mouse psoriasis model and CD40 antibody-induced mouse inflammatory bowel disease (IBD) model. In vivo pharmacodynamic study indicates that ARTS-011 significantly reverses IL12/18-induced IFNγ production in mouse. ARTS-011 also significantly reduces the ear swelling in a dose-dependent manner in IL-23-induced mouse psoriasis model. Lastly, ARTS-011 was highly efficacious in anti-CD40 antibody induced mouse IBD model. In summary, ARTS-011 is a highly selective, potent and orally available small molecule TYK2 inhibitor with suitable pharmaceutical properties as potential therapeutics in inflammatory disorders.
Hv1 proton channels control myeloid landscape and promote glioma progression
Authors: Zheng Jiaying , Wang Lingxiao , Ayasoufi Katayoun , Goddery Emma , Zhao Shunyi , Fain Cori , Johnson Aaron J , Wu Long-Jun
Published: January 2023

Myeloid cells comprising up to 50% of total tumor mass in glioblastoma (GBM) contribute to tumor progression and immunosuppression. Restraining glioma-induced myeloid cell infiltration increases functional T cells within the tumors, improves the efficacy of checkpoint blockade, and significantly extends mouse survival. Here, we focus on an ion channel, voltage-gated proton channel Hv1, which is mainly expressed in myeloid cells and shapes the physiological functions of myeloid cells. Our bioinformatics analysis demonstrated that elevated Hv1 expression in the tumor mass correlates with poor disease prognosis in patients. Using the leading immunocompetent model of GBM in mice, we showed that Hv1 knockout mice (Hv1−/−) exhibit slower glioma progression and prolonged survival. Using in vivo two-photon imaging, we found that myeloid cells infiltrate into tumor mass since early time points and have intimate interaction with tumor cells. Notably, by full-spectrum flow cytometry, we found that Hv1−/− mice have reduced monocyte/macrophage infiltration, and increased PD-1+ CD4+ T cells in the tumor-burdened hemisphere. As a result, we combined anti-PD-1 treatment with Hv1 knockout in the middle stage of glioma, and found that approximately 30% of Hv1−/− mice were cured. Together, our results demonstrated that Hv1 controls myeloid infiltration and could be a novel therapeutic target for the treatment of GBM.

Supported by R01NS110825, R01NS088627
Identifying Antigen Presenting Cells Responsible for Heat Shock Protein-Mediated Cancer Immunosurveillance
Authors: Nayak Devanshi A , Binder Robert J
Published: January 2023

The immune system detects and eliminates nascent, emerging tumor cells. This is mediated primarily by T cells and NK cells. However, the mechanisms that allow efficient T cell priming during cancer immunosurveillance, which is characterized by limited antigen availability, remain unclear. Immunogenic heat shock proteins (HSPs) such as gp96 chaperones minute levels of antigens, in the form of peptides, and allows these to be efficiently cross-presented by APCs, a process that involves the HSP receptor, CD91. Further, engagement of CD91 by HSPs initiates signals for co-stimulation and cytokine production which are necessary for priming anti-tumor responses. We previously showed that mice deficient in CD91 expression in APCs (CD91fl/fl CD11cCre) are more susceptible to chemically induced tumors compared to wildtype littermates. Tumors that emerge in these mice prime poorer T cell responses and are less infiltrated than tumors in wildtype mice. The CD91+ APC subset(s) that are responsible for HSP-mediated T cell priming have not been elucidated. This study seeks to define the APC subset(s) necessary for CD91-mediated immune responses in the tumor microenvironment and tumor-draining lymph nodes. Using genetically tagged HSPs, we track the dispersal of tumor-derived HSPs throughout the myeloid system. We show that HSPs are taken up by multiple DC and macrophage subsets. We identify APC subsets that are responsible for HSP-mediated priming of anti-tumor T cell responses, by adoptive transfer of APCs that acquire tumor-derived HSPs. These studies provide an underlying mechanism of how the immune responses to nascent tumors are initiated. They will also have significant implications for cancer prognosis and therapy.

Supported by NIH (R01 CA233803)
Mechanisms of CD40-dependent cDC1 licensing beyond co-stimulation
Authors: Wu Renee , Ohara Ray A , Jo Suin , Murphy Theresa , Murphy Kenneth M
Published: January 2023

CD40 signaling in conventional type 1 dendritic cells (cDC1s) is required for CD8 T cell-mediated tumor rejection, but the underlying mechanisms are incompletely understood. Here, we identified CD40-induced genes in cDC1 and examined their contributions to anti-tumor immunity. cDC1-specific inactivation of CD70 and COX-2, and global CD27 inactivation, only partially impaired tumor rejection or tumor-specific CD8 T-cell expansion. Loss of 4-1BB, alone or in Cd27−/− mice, did not further impair anti-tumor immunity. However, cDC1-specific CD40 inactivation reduced cDC1 mitochondrial transmembrane potential and increased apoptosis in tumor-draining lymph nodes, reducing migratory cDC1 numbers in vivo. Similar impairments occurred during in vitro antigen presentation, which were reversed by re-expression of Bcl-xL. Thus, CD40 signaling in cDC1 not only induces co-stimulatory ligands for CD8 T cells, but also induces Bcl2l1 that sustains cDC1 survival during priming of anti-tumor responses.

This work was supported by grants from the NIH (R01AI150297, R01CA248919, and R21AI164142 to K.M.M., and F30CA247262 to R.W.).
Glioblastoma multiforme expresses cell surface PCNA, a potential target for NK cell-mediated immunotherapy.
Authors: Cooksey Luke C , Mathew Porunelloor A
Published: January 2023

Glioblastoma multiforme (GBM) is the most common form of primary brain cancer and carries a dreadful five-year survival rate of 9%. Current treatment options include surgery, chemotherapy, and radiation. Recently, there has been a move to pursue immunotherapy options to improve patient outcomes. These therapies often depend on the identification of molecular markers that are distinctive to the tumor cells. Some markers, such as HER2/Neu and EGFR, are overexpressed on a significant percentage of GBM tumors and are used as targets for immunotherapies. However, to address GBM tumors that do not overexpress HER2/Neu and EGFR, our lab set out to identify novel markers on GBM as future candidates for Natural Killer (NK) cell-mediated immunotherapy. Previously, our lab demonstrated that Lectin-like Transcript-1 (LLT1), membrane-bound Proliferating Cell Nuclear Antigen (PCNA), NKp44 Ligand (NKp44L) and CS1 (CD319) are targets of NK cell-mediated killing of various cancers. Based on these prior studies, we examined their expression on the well-known GBM cell lines LN-229 and LN-18 by flow cytometry using PE-labeled antibodies specific for each marker. PCNA was the lone marker identified to be highly expressed on both LN-229 and LN-18 cells. Based on our results, we concluded that cell surface PCNA is an ideal candidate for NK cell-mediated immunotherapy for GBM. Currently, we are evaluating blocking inhibitory signals to NK cells from the PCNA-NKp44 interaction to target GBM for NK-mediated killing. Recent findings demonstrating the ability to transiently open the blood-brain barrier further increase the feasibility of targeting GBM by NK cells in the future.

Supported by funding from the Osteopathic Scholars in Cancer Research Program grant from the Cancer Prevention and Research Institute of Texas (RP170301) and UNTHSC Intramural grants.
Tissue-resident memory CD4+ T cells play a dominant role in the initiation of antitumor immunity
Authors: Zhang Hui , Zhu Zhaohui , Modrak Samantha , Little Alex
Published: January 2023

Tumor immunology has been studied extensively. Tumor immunology-based cancer immunotherapy has become one of the most promising approaches for cancer treatment. However, one of the fundamental aspects of tumor immunology – the initiation of antitumor immunity – is not fully understood. Compared to that of CD8+ T cells, the effect of CD4+ T cells on antitumor immunity has not been fully appreciated. Using a gene knockout (KO) mouse model – these mice are deficient in TCRa repertoire, specifically lacking iNKT and MAIT cells - we found that the deficiency in TCRa repertoire diversity did not affect the antitumor immunity, at least to melanoma and breast cancer. However, after acquiring thymocytes or splenocytes from wild-type mice, these KO mice exhibited greatly enhanced and long-lasting antitumor immunity. This enhanced antitumor immunity depended on CD4+ T cells, especially tissue-resident memory (TRM) CD4+ T cells, but not iNKT or CD8+ T cells. We also present evidence that CD4+ TRM cells initiate antitumor immunity through IFN-g, and the process is dependent on NK cells but not macrophages. The CD4+ TRM/NK axis appears to control tumor formation and development by eliminating tumor cells and modulating the tumor microenvironment. Taken together, our results demonstrated that CD4+ TRM cells play a dominant role in the initiation of antitumor immunity.

Supported by Washington State University Start-up funds
Recombinant protein mimicking the antigenic structure of the envelope protein of dengue virus enhances antigen-specific virus-neutralizing immune response induction
Authors: Jang Yong-Suk , Kim Ju , Lim Tae-Young , Park Jisang , Kim JunYoung , Cho SooHyeon
Published: January 2023

Dengue virus (DENV) comprises four serotypes in the family Flaviviridae and infects approximately 390 million people worldwide every year. So far, there is no effective therapy available for dengue diseases other than supportive care. Therefore, the development of safe and effective vaccines is critical. Most of DENV vaccines are primarily based on the envelope proteins prM and E, which are believed to induce protective immune responses in humans. Particularly, the DENV E protein and its envelope domain III (EDIII) have been investigated as candidate antigens for recombinant subunit vaccine. However, most EDIII-based antigens do not display quaternary structure of DENV E protein which is essential to induce virus neutralizing immune responses. We developed the Recombinant of DENV-2 Envelope Domain (r2ED) as an antigen, which mimics the antigenic structure of E protein on the surface of DENV. We tested the ability of r2ED to boost antigen-specific immune response induction in vivo. As a result, we observed that r2ED elicited antigen-specific and neutralizing antibody responses and T cell-mediated immune responses higher than EDIII alone in mice. In addition, antigen-specific antibody and T cell responses were maintained in an enhanced level at 6 months after the last immunization in r2ED-immunized mice, suggesting the sustained immune memory responses for a long time. These results are helpful to develop the improved subunit vaccine for inducing an effective and long-lasting immune response against DENV infection.

This work was supported by the NRF funded by the Ministry of Education No. 2017R1A6A1A03015876 and the Ministry of Science and ICT No. 2020K1A4A7A02095058. J. Park, J. Kim, and S. Cho were supported by the BK21 FOUR program in the Department of Bioactive Material Sciences.
Peripheral co-immunization with CCL27 drives robust mucosal responses to SARS-CoV-2 synDNA antigens and provides heterologous protection against Delta variant challenge
Authors: Gary Ebony Nicole , Tursi Nicholas J , Warner Bryce , Parzych Elizabeth , Ali Ali , Kobasa Darwyn , Patel Ami , Kulp Daniel , Weiner David B
Published: January 2023

SARS-CoV-2, which causes COVID-19 has caused a global pandemic which has claimed over 5 million lives to date. Emerging variants of concern and waning vaccine-induced immunity continually challenge viral control measures. We previously reported on the immunogenicity of a DNA vaccine encoding the spike glycoprotein of SARS-CoV-2, however directing vaccine-induced humoral and cell-mediated responses to mucosal surfaces such as the respiratory mucosa remains a challenge. We immunized mice with synthetic DNA (synDNA) plasmids expressing the wild-type SARS-CoV-2 spike protein (pS) alone, or with the DNA-encoded molecular adjuvant cutaneous T cell-attracting chemokine (pCTACK) Mice co-immunized with pS and pCTACK had increased spike-specific IFNy T cell responses compared to those receiving pS alone. While groups had similar levels of spike receptor binding domain (RBD)-specific IgG in their serum, in the mucosa, co-immunization with pCTACK enhanced RBD-specific IgA and only the cecal extract from pCTACK co-immunized animals neutralized pseudotyped viruses. When ACE2 transgenic mice were immunized, rested, and challenged with the heterologous SARS-CoV-2 Delta variant, co-immunization with pCTACK supported 100% survival while immunization with wild-type spike DNA antigens supported 60% survival. Importantly 100% of pCTACK co-immunized animals were protected from infection-induced weight loss. These studies demonstrate that chemokine adjuvants can direct vaccine-induced responses to specific immunological sites and that such responses can have significant impact on heterologous challenge outcomes and support the continued study of molecular chemokine adjuvants in the context of SARS-CoV-2 vaccines.

We would like to thank the Animal Facility staff at the Wistar Institute and the Public Health agency of Canada for providing care to the animals used in these studies and for additional financial support. We would also like to thank the flow cytometry core facility at the Wistar Institute. This work was supported by NIH NCI award T32 CA09171 (ENG) and NIH NIAID award T32-AI-055400 (EMP) with additional support from CEPI/Inovio Pharmaceuticals.
B cell receptor repertoire dynamics and convergent evolution following SARS-CoV-2 vaccination
Authors: Fraley Elizabeth Ross , Khanal Santosh , LeMaster Cas , Pierce Stephen , Pastinen Tomi , Bradley Todd
Published: January 2023

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel betacoronavirus causing Coronavirus disease 2019 (COVID-19). B cell receptors (BCRs) are expressed at the surface of the B cell and are secreted as soluble antibodies. These antibodies can block viral infection by neutralizing the virus, are critical for resolution of SARS-CoV-2 infection, and may be correlates of protection for COVID-19 vaccines. We have previously shown that SARS-CoV-2 immunization elicits a robust antibody response that is significantly higher in individuals who recovered from COVID-19. Moreover, recovered individuals had slower antibody decay in their levels seven months after vaccination. Here, we sequenced the BCR repertoire of individuals undergoing vaccination by SARS-CoV-2 mRNA vaccine (Pfizer, BNT162b2) with (seropositive) or without (seronegative) previous laboratory confirmed COVID-19 infection. We identified genetic differences in the BCR repertoire between groups including V gene usage, CD3R length, percentage of somatic hypermutation, and clonotype diversity. We then focused our analyses to vaccine-expanded clonotypes in both groups to further analyze the differences in BCR repertoire on candidate SARS-CoV-2-specific clonotypes. We determined the frequency of pre-existing clones present after infection that were engaged by the vaccine in the seropositive group. Moreover, in both groups we identified clonotypes that were shared among individuals that could be a result of convergent evolution. Defining the characteristics and evolution of the BCR repertoire during vaccination of individuals with different histories of viral infection will aid in understanding SARS-CoV-2 humoral response dynamics.
OX40 boosts and sustains humoral and cellular immune responses to SARS-CoV2 spike protein and RNA vaccinations
Authors: Duhen Rebekka A , Beymer Michael , Jensen Shawn , Abbina Srinivas , Abraham Suraj , Thomas Anitha , Hu Hong-Ming , Fox Bernard A , Weinberg Andrew D
Published: January 2023

To prevent SARS-CoV-2 infections and generate long-lasting immunity, vaccines need to generate both, a strong humoral immune response as well as viral-specific CD4 and CD8 T cells. Previous results from our lab have shown that immunization in the presence of an agonist antibody targeting OX40 led to higher antibody titers and increased numbers of antigen-specific CD4 and CD8 T cells. Using the same strategy, we explored the effect of OX40 co-stimulation in the prime and boost together with the SARS-CoV-2 spike protein + adjuvant in C57Bl/6 mice. Our results show that OX40 engagement led to a significant increase in long-lived antibody responses when compared to mice that did not receive additional co-stimulation. In addition, spike protein and peptide-specific proliferation were greatly increased for both CD4 and CD8 T cells, with augmented secretion of IFN-γ, TNF-α and IL-2. Booster immunizations (7 months post prime) did not lead to anergy, but instead further increased the antibody and T cell responses. In initial experiments, the self-amplifying RNA (saRNA) vaccination generated lower antibody titers, independent of OX40 co-stimulation. However, the saRNA vaccine did induce a robust expansion of antigen-specific CD8 T cells, that expressed high levels of GrzmB, which was further increased by OX40 administration. The strong immune responses and the differential effects of the protein and saRNA vaccines suggest that heterologous prime-boost approaches, as currently approved in adults, might be beneficial in boosting different arms of the anti-viral immune response against SARS-CoV2, with OX40 agonists enhancing both approaches. Further studies in animals will aid defining the effects and benefits of this approach.
Method for Increasing and Prolonging the Production of Antitoxins in Horses
Authors: Ramon G. , Lemétayer E.
Published: January 2023

When horses are immunized by the injection of increasing doses of diphtheria toxin or anatoxin, the serum of the animals comes to contain increasing quantities of antitoxin. At the end of a period varying with different factors referring to the antigen or the animal, the antitoxic power of the serum reaches a certain degree which appears to be the highest degree which it is possible to attain under any conditions. As soon as this maximum is reached, therefore, it is considered preferable, by certain laboratories, to bleed the animals completely. In other laboratories, the practice is to allow a period of about eight days to elapse following the last injection of the antigen and then bleed the animal in two fractions of 6 to 8 liters each, done a few days apart. This double bleeding is repeated every month.
Anaphylaxis to Diphtheria Toxin
Authors: Neill James M. , Sugg John Y. , Richardson Lurline V.
Published: January 2023

Previously reported experiments (1, 2, 3) on anaphylaxis apparently due to the toxin or toxoid antigen were made upon actively sensitized (antitoxic immune) animals. The results had indicated that while other diphtheria antigens participated to some extent, the major part of the anaphylaxis could be assigned to the toxin or toxoid antigen, provided one used both for the immunization and for the intravenous test minimal amounts of unheated filtrate in which toxin or toxoid was the dominant antigen, and selected for intravenous test the individual guinea pigs that had been “earliest” in antitoxin production. Passive sensitization possesses certain advantages over active sensitization in the distinction of reactions due to toxin-antitoxin from reactions due to other diphtheria antigen-antibody combinations, for with the passive method it is possible to examine each of the sensitizing sera for antibodies other than antitoxin and, when desired, to remove the other antibodies by absorption of the antisera before using them for sensitization.
The Effect of Concentration and of Various Tissue Constituents on the Virulence of the Poliomyelitis Virus
Authors: Jungeblut Claus W.
Published: January 2023

Summary and Conclusions

Titration of poliomyelitis virus cord suspensions of varying percentages shows there is a particular concentration with maximum virulence below which the dilutions become progressively less infective and above which virulence is likewise diminished. Berkefeld-filtrates of virus cord suspensions are at times more virulent than corresponding unfiltered suspensions, although exceptions do occur. Neither normal monkey cord nor normal monkey brain when added in vitro to virus filtrates, has any appreciable effect on the virulence of the virus. The results with convalescent cord are too irregular to indicate clearly the presence of a neutralizing principle in the immune tissue. Normal monkey testicle added to virus filtrates in vitro frequently produces a conspicuous diminution of virulence of the virus in the supernatant fluid. The extent of this antagonistic effect varies with different monkey testicles. Normal rabbit testicle, under similar conditions, seems slightly to enhance the virulence of the virus.
AL008 Enhances Myeloid Antitumor Function by Inhibiting SIRPα Signaling and Activating Fc Receptors
Authors: Yang Jingping , Deresa Isaiah , Ho Wei-Hsien , Long Hua , Maslyar Daniel , Rosenthal Arnon , Liang Spencer C , Pincetic Andrew
Published: January 2023

Antagonizing the CD47–signal regulatory protein (SIRP)α pathway, a critical myeloid checkpoint, promotes antitumor immunity. In this study, we describe the development of AL008, a pan-allelic, SIRPα-specific Ab that triggers the degradation of SIRPα and, concurrently, stimulates FcγR activation of myeloid cells through an engineered Fc domain. AL008 showed superior enhancement of phagocytosis of tumor cells opsonized with antitumor Ag Abs compared with another SIRPα Ab tested. Unlike ligand-blocking SIRPα Abs, AL008 demonstrated single-agent activity by increasing tumor cell engulfment by human monocyte-derived macrophages even in the absence of opsonizing agents. This effect was due to enhanced Fc function, as blocking FcγR2A abrogated AL008-mediated phagocytic activity. AL008 also promoted human monocyte-derived dendritic cell–mediated T cell proliferation. In humanized mouse models, AL008 induced internalization of SIRPα and increased expression of CD86 and HLA-DR on human tumor-associated macrophages, confirming that the mechanism of action is retained in vivo. Monotherapy treatment with AL008 significantly reduced tumor growth in humanized mice implanted with human MDA-MB-231 tumor cells. AL008 also significantly potentiated the effects of T cell checkpoint blockade with anti–programmed death ligand-1 in syngeneic tumor models. This dual and specific mechanism of AL008, to our knowledge, provides a novel therapeutic strategy for targeting myeloid cells for immune activation.
Agglutination Reactions in Rheumatoid Arthritis
Authors: Dawson Martin H. , Olmstead Miriam , Jost Elizabeth L.
Published: January 2023

A comparative study of precipitins and agglutinins against Streptococcus hemolyticus has been made in the sera of 71 cases of rheumatoid arthritis and in 79 cases of other diseases. The results indicate that there is a close approximation, but not an absolute agreement, in the capacity of rheumatoid arthritis sera to agglutinate strains of Streptococcus hemolyticus and to precipitate various group-specific fractions of this organism. With certain exceptions, to which special reference is made, the control sera examined did not show the presence of either agglutinins or precipitins in significantly high dilutions. These findings offer suggestive evidence in favor of the hypothesis that the disease rheumatoid arthritis is associated with infection by Streptococcus hemolyticus.