bluuebunny/crossref_metadata_2025 huggingface dataset:
Voluntary physical exercise decreases MDSC (myeloid derived suppressor cell) populations in the spleen and circulation in a rat model of mammary adenocarcinoma
Authors: Hirsch Aspen L. , Brenner Cassandra L. , Costa Alex B. , Haughian James M. , Pullen Nicholas A. , Hayward Reid
Published: January 2023

Myeloid derived suppressor cells (MDSCs) are a heterogenous population of myeloid progenitor cells that suppress T cells and other potentially anti-cancer immune cells. MDSC presence in cancer patients can diminish immunotherapy response and worsen treatment outcomes. Here we examine a low-risk, low-cost approach to MDSC modulation by examining the impact of voluntary wheel-running exercise on MDSC populations in a Fisher-344 rat, MATBIII mammary adenocarcinoma model. Rat MDSCs in blood, spleen, bone marrow, and tumor tissue were identified by dual expression of surface markers His48 and CD11b and confirmed to be immunosuppressive upon sorting and interrogation in an in-vitro T cell suppression assay. We find that the percentage of MDSCs (as a proportion of total cells) is reduced in whole spleen isolates (2.55% vs. 19.68%, P≤0.005) and in the circulating white blood cell fraction (1.33% vs. 23.21%, P≤0.05) of exercised versus sedentary animals. In contrast, we find no difference between the proportion of MDSCs as a population of total cells in bone marrow (23.57% vs. 25.52%, P≥0.05) or tumor tissue (11.68% vs. 7.10%, P≥0.05). We conclude that exercise decreases splenic and circulating MDSC populations in tumor bearing animals. Further studies are necessary to establish the molecular mechanisms of this suppression to leverage potential interactions between exercise and MDSC-directed immune therapies.
TFEB regulates tumor-associated macrophages in breast cancer through autophagy-dependent and independent pathways
Authors: Fan Daping , Hodge Johnie , Fang Liang
Published: January 2023

Tumor-associated macrophages (TAMs) play a key role in tumor development in breast cancer, as they are educated in the tumor microenvironment to assist tumor growth and metastasis. We identified an important role of TFEB, a master regulator of autophagy and lysosome biogenesis, in phenotypic regulation of TAMs. In human breast cancer and mouse orthotopic breast tumors, TFEB expression and activation are down-regulated in TAMs. Previously we showed that TFEB inhibits macrophage M2 polarization through a SOCS3-STAT3 pathway. Our new microarray analysis and qPCR confirmation demonstrate that TFEB overexpression in macrophages not only significantly suppresses the induction of typical M2 genes Arg1 and IL-10 by breast cancer cell conditioned medium, but also significantly inhibits the induction of inflammatory response axis NLRP3/IL1β/IL6 and PGE2 generation axis HIF-1α/MIF/cPLA2/COX2. These two axes are known to be tumor-promoting. Further analyses indicate that TFEB regulates these two axes through both autophagy-dependent and autophagy-independent pathways. Animal studies show that macrophage-specific TFEB overexpression inhibits, while macrophage-specific TFEB deficiency enhances breast tumor growth and metastasis. Furthermore, activation of TFEB using two natural compounds halts breast cancer development in mice. Taken together, our data suggest that activation of macrophage TFEB could be developed as a therapeutic strategy against breast cancer.
Development of melittin-based anti-cancer drug for targeting tumor-associated macrophages
Authors: Lee Chanju , Jeong Hyunju , Bae Hyunsu
Published: January 2023

Tumor-associated macrophages (TAMs) are the major component of tumor infiltrating immune cells. Macrophages are broadly categorized as M1 or M2 types and TAMs have been known to express M2-like phenotype. TAMs promote tumor progression and contribute to resistant to chemotherapies. Therefore, M2-like TAMs are potential targets for the treatment of cancers. In this study, we targeted M2-like TAMs using a hybrid peptide MEL-dKLA composed of melittin (MEL) peptide, which binds preferentially to M2-like TAMs and d-KLAKLAKKLAKLAK (dKLA) peptide, which induces apoptotic cell death after cell membrane penetration. We showed that the MEL-dKLA induced selective cell death of M2 macrophages by disrupting mitochondrial membrane in vitro while MEL did not show the correlation with mitochondrial apoptosis. We also showed that MEL-dKLA selectively targeted the M2-like TAMs without affecting other leukocytes such as T cells and dendritic cells in vivo. It resulted in the inhibition of tumor growth rate and tumor weight, and angiogenesis in vivo. Importantly, although both MEL and MEL-dKLA reduced the CD206+ M2-like TAMs in the tumor, only MEL-dKLA induced the apoptosis of CD206+ M2-like TAMs whereas the MEL did not show the cell death. Taken together, our study describes MEL-dKLA can be used as the promising therapeutic agent for targeting M2-like TAMs.
Cross Reactivity of CD8+ T Cell Neo-epitopes to Bifidobacterium Boosts the Tumor Specific Population
Authors: Bessell Catherine Ami , Isser Ariel , Havel Jonathan , Chan Timothy , Schneck Jonathan P.
Published: January 2023

The interplay between the microbiome and the anti-tumor T cell specific response is a rising field in cancer immunotherapy. The effect of antigen homology between commensal bacteria and neo-antigen specific responses in tumors has, in concept, a high degree of targeted application. The neo-antigen response is a tumor specific antigen rising from mutation in the tumor cells has been associated with an increased anti-tumor ability with immunotherapy. Using a model of neo-antigen specificity with Kb-SIY in B16-SIY melanoma and commensal bacteria Bifidobacterium we identified a model of antigen mimicry for neo-antigen. Commensal bacteria antigen “Kb-SVY” is not inhibited from binding into the MHC molecule and leads to stable peptide MHC interactions. T cell populations for Kb-SIY and Kb-SVY show cross-reactivity for homologous antigens. And the impact of Bifidobacterium on the developing response show an in vivo priming to create a robust commensal bacteria response that contributes to the anti-tumor activity.
Targeting EZH2 Selectively Alters Intratumoral Regulatory T Cells to Enhance Cancer Immunity
Authors: DuPage Michel , Wang David , Quiros Jason , Mahuron Kelly , Pai Chien-Chun , Ranzani Valeria , Young Arabella , Silveria Stephanie , Harwin Tory , Abnousian Arbi , Pagani Massimiliano , Rosenblum Michael , Van Gool Frederic , Fong Lawrence , Bluestone Jeffrey A.
Published: January 2023

Immunosuppressive regulatory T cells (Tregs) are critical for maintaining immune homeostasis, but their presence in tumor tissues impairs anti-tumor immunity and portends poor prognoses in cancer patients. Targeting Tregs may be a powerful means to unleash more potent immune responses against cancer, but targeting these cells is challenging because their generalized inactivation may incite severe autoimmune toxicities. To selectively target Tregs in tumors, we investigated the role of the H3K27 methyltransferase EZH2 in Tregs and determined that its enhanced activity at tumor sites in mice and humans leads to more robust and stable Tregs. We demonstrate that blocking EZH2 activity, both pharmacologically and genetically, selectively reprograms the function of tumor-infiltrating Tregs without systemically altering Treg function. Genetic disruption of EZH2 in tumor-resident Tregs led to their acquisition of pro-inflammatory functions that remodeled the tumor microenvironment and enhanced the recruitment and function of effector T cells, leading to the complete elimination of tumors. Moreover, abolishing EZH2 function in Tregs was mechanistically distinct from, more potent than, and less toxic than a generalized Treg depletion approach. This study reveals a novel strategy to target Tregs in cancer that mitigates autoimmunity by reprogramming their function in tumors to enhance anti-cancer immunity.
Generation of murine tumor cell lines to assess in vivo anti-tumor immune responses to melanoma.
Authors: Hope Jennifer L. , Henriquez Monique L. , Tinoco Roberto , Bradley Linda M.
Published: January 2023

Mouse models of cancer remain the preferred means to assess the efficacy of anti-cancer therapies, and several models have been used to address the effect of cancer drugs or in the discovery of biomarkers. The Yale University Mutant melanoma lines, YUMM1.5 and YUMMER1.7 murine tumor models were developed from the BrafV600E/Pten/−/−Cdkn2a−/− engineered mouse model to more closely reflect poorly immunogenic and highly mutated melanomas, respectively, that are driven by human disease-relevant mutations; however, the dominant T cell-recognized epitopes specific for these tumors remains unknown. We therefore sought to engineer OVA-expressing versions of the YUMM1.5 and YUMMER1.7 tumor lines, hereafter referred to as YUMM1.5-OVA and YUMMER1.7-OVA. Using stable transfection to insert a plasmid expressing full-length secretory ovalbumin, we have generated polyclonal and monoclonal cell lines that generate an in vivo OVA-specific response as validated by MHC class I tetramer staining and cytokine production following OVA peptide stimulation. Together, these two new models for melanoma tumors will allow us to assess intrinsic and extrinsic mechanisms regulating the generation of effective and recovery of exhausted T cell responses to melanoma in vivo.
Using anti-CD38 immunotherapy to enhance anti-tumor T-cell immunity in chronic lymphocytic leukemia (CLL)
Authors: Manna Alak , Lewis-Tuffin Laura J. , Ailawadhi Sikander , Chanan-Khan Asher A. , Paulus Aneel
Published: January 2023

Increased CD38 expression on chronic lymphocytic leukemia (CLL) cells correlates with a negative prognosis, however, its utility as a therapeutic target in this malignancy remains unknown. Our investigations show that the anti-CD38 therapeutic mAb, daratumumab (Dara) induces direct apoptosis of CLL cells and promotes their immune-mediated clearance (through ADCC). Notably, CD38 expression is quite high on a subset of CLL-cells (CD19+/CD38hi/CD24+/IL-10+; B-regulatory-like phenotype), which promote Treg expansion while prohibiting CD8+ cytotoxic T-cells (cTLs). Indeed, immunosuppression is a hallmark of CLL and patients often present with increased number of Tregs and Th2 cells but decreased effector T cells and cTLs. It has recently been reported that CD38hi Tregs are significantly immunosuppressive and when examining this population in peripheral blood mononuclear cells (PBMCs) from CLL patients (n=13), we noted that >50% of CD4+/CD25+/FoxP3+ cells were CD38hi (median MFI ~600). We hypothesized that Dara would kill CD38hi CLL cells à decreased CD38hi Treg formation/direct killing à expansion of cTLs. Indeed, ex-vivo treatment of CLL-patient derived PBMCs showed that Dara was highly lethal to both CD38hi “Breg-like” CLL cells and Tregs; associated with decrease in IL-10, increase in IFN-γ, effector T cells, Th17 subsets and proliferation of cTLs. Altogether this pointed towards recovery of anti-tumor immunity which was further evidenced by enhanced tumor-specific cytolytic activity of cTLs. Our pre-clinical data demonstrates that targeting CD38 in CLL has an anti-tumor effect via 1.) lethal effect on CLL-Bregs and Tregs and 2.) tumor-specific expansion of cTLs, which we will test clinically in an upcoming trial.
Intrapulmonary delivery of TLR agonists associated with systemic chemotherapy to treat metastatic cancer.
Authors: Goguet Emilie , Klinman Dennis M. , Tross Debra
Published: January 2023

Introduction

TLR7/8 agonist (3M-052) and TLR9 agonist (CpG ODN) both trigger innate immune responses supporting the induction of tumor-specific immunity. Previous studies showed that this specific combination of TLR agonists synergistically promoted the elimination of large established tumors (>500 mm3). This effect was optimized when delivered directly into the tumor, leading to a conversion from immunosuppressive to inflammatory milieu. The current work explores the effect of a combination of a localized TLR agonists therapy and a systemic chemotherapy on lung metastases.

Methods

66Cl4-luc cells (a luciferase-expressing variant of the 4T1 murine mammary carcinoma line) were orthotopically implanted into the fourth mammary fat pad of syngeneic Balb/c mice. The combination therapy was initiated when the primary tumor exceeded 300 mm3 and pulmonary metastases were present.

Results

Treatment delivered into both the primary tumor and the lungs, when associated with systemic chemotherapy significantly reduced tumor growth and prolonged survival.

Discussion

The combined effect of the two TLR agonists was further enhanced when associated with classic chemotherapy, providing a significant decrease of tumor growth at both sites. These results provide new leads to explore when considering lung metastases treatment.
Investigating the effect of pegylated granulocyte colony stimulating factor therapy on experimental mastitis in lactating Holsteins
Authors: Powell Ellis J. , Lippolis John D. , Reinhardt Tim A. , Casas Eduardo
Published: January 2023

Mastitis dominates dairy economic and health concerns. Neutrophils are the most prominent cellular defense against mastitis causing pathogens making neutrophil expansion and activation, prime candidates for targeted therapeutics. The granulocyte colony-stimulating factor (gCSF) cytokine targets neutrophil differentiation and causes neutrophilia. Recently pegylated (PEG) gCSF has been shown to decrease naturally-occurring cases of mastitis. To better understand the mechanisms employed by PEG-gCSF to reduces mastitis incidence we have evaluated PEG-gCSF in an experimental mastitis challenge. We challenged 11 lactating Holsteins with Escherichia coli by intra-mammary infusion. Cows received injections of PEG-gCSF 14 days and 7 days, prior to disease challenge. While somatic cell counts in the milk did not differ between treated and non-treated animals, the treated cows had lower bacterial counts in the 48 hours post infection (P < 0.05). Bovine serum albumin levels in milk, a measure of disease severity, were also lower 24 hours post challenge for PEG-gCSF treated animals. Post challenge, control cattle had reduced milk yield (P = 0.05) and a lower percent feed intake (P < 0.01) compared to treated cows. PEG-gCSF treated cattle significantly increased circulating levels of neutrophils (P < 0.01), as well as monocyte and lymphocyte responses (P < 0.05). We demonstrated that PEG-gCSF treated cattle had reduced disease severity and have begun to further elucidate the mechanism of neutrophil immune responses. This data suggests the utilization of PEG-gCSF as a potential antibiotic alternative to mastitis treatment and prevention and supports neutrophil focused therapies as an important facet of mastitis disease protection.
Characterization of the cellular immune response to Brucella abortus RB51 vaccination and boost in cattle
Authors: Boggiatto Paola M. , Olsen Steven C.
Published: January 2023

Brucellosis is a bacterial zoonosis caused by organisms belonging to the genus Brucella. In domestic species (cattle, goats, sheep) brucellosis results in infertility and reproductive loss, while in humans, brucellosis results in a chronic debilitating disease. Worldwide, brucellosis is a significant source of financial loss and a major public health concern. Bovine brucellosis, as caused primarily by Brucella abortus, is an economically important cause of reproductive loss in cattle. While vaccines are available for cattle, safe and effective vaccines for humans (and other domestic species) are not available. Currently, most of the knowledge regarding protective immune responses against Brucella have come from mouse studies. In the work presented our goal was to characterize the T cell subpopulations and effector phenotype of the immune response elicited following vaccination and boost with RB51. In order to accomplish this, we performed ex vivo analysis of peripheral blood mononuclear cells (PBMC) for the expression of surface markers associated with effector (CD45RO− CD62L−/lo CCR7−) and memory phenotypes (TEM: CD45RO+ CD62Llo/intCCR7−and TCM: CD45RO+ CD62L+ CCR7+). Additionally, we performed in vitro recall responses of PBMC and assessed cytokine production and proliferation in response to antigen stimulation. Altogether, the data from these studies will help us understand the protective, cell mediated immune response elicited by vaccination in a natural host for Brucella species.
TCR Vbeta usage differences are associated with Marek’s disease resistance in the chicken.
Authors: Hearn Cari J. , Ray Emmillie , Cheng Hans H.
Published: January 2023

Marek’s disease (MD) is a herpesvirus-induced lymphoma in chickens which is of prime importance to the poultry industry, costing over $1 billion annually. Although vaccination controls the disease, virulence shifts have prompted efforts to improve genetic resistance in the host. We studied T cell responses in two models of MD resistance in inbred layer chicken lines. The first model is MHC-matched lines which have been selected for resistance (Line 6) or susceptibility (Line 7). The second model involves MHC-congenic lines which differ in MHC alleles known to be associated with higher (Line B21) or lower (Line B19) MD resistance. We characterized the usage of Vbeta-1+ and Vbeta-2+ TCRs in the T cell populations of birds infected and uninfected with Marek’s disease virus (MDV). Vbeta-1 and Vbeta-2 are the only two Vbeta gene families present in the chicken TCR loci, and are highly homologous within family. We found that resistant lines had a higher proportion of spleen T cells expressing Vbeta-1+ TCRs than susceptible lines, and that responses to infection were visible as shifts toward increased usage of Vbeta-1+ TCRs in CD8+, but not CD4+, T cell populations. While differences between MHC-congenic lines were small, resistant Line 6 showed a significant Vbeta-1+ CD8+ response as early as 8 days post infection, while susceptible Line 7 did not. We then characterized a panel of 6C.7 recombinant congenic lines, finding that TCR usage is most likely a single-locus trait, but interacts with other loci to produce a larger Vbeta-1+ T cell pool in more MD-resistant lines. We hypothesize that the reduced receptor diversity in the chicken may have led to strong selection effects on TCR-antigen-MHC fit which impact resistance to MDV infection or tumorigenesis.
Dendritic cells influence the altered neonatal CD8 T cell immunodominance hierarchy during influenza virus infection
Authors: Heil Luke , Oliphant Samantha , Lines Louise , Hollifield Melissa , Garvy Beth A.
Published: January 2023

Neonates are more susceptible to influenza virus infection than adults, resulting in increased morbidity and mortality as well as delayed clearance of the virus. Previous work has indicated that decreased T cell and dendritic cell function underlies some of this vulnerability. We sought to understand CD8 T cell specificity and immunodominance during neonatal influenza infection as well as how any differences from the adult hierarchy might impact immunodominance and protection in subsequent infections. We found that neonatal C57BL/6 mice display an altered CD8 T cell immunodominance hierarchy, preferentially responding to an epitope in the influenza protein PA rather than the co-dominant adult response to NP and PA. Additionally, upon secondary infection, mice first infected as pups display inconsistent immunodominance and suffer increased morbidity compared to mice infected previously as adults. Finally, transfer of influenza infected adult dendritic cells to pups resulted in increased T cell activation and enhanced viral clearance as well as a slight induction of NP specific CD8 T cells. Taken together, these data suggest that infection early in life alters the specificity of memory responses to that pathogen and that dendritic cells may play a role in mediating this process. Additionally, vaccines targeting T cells should consider epitope usage and age specific dendritic cell physiology if the intended patient population includes infants as well as adults.
Virus antigen-specific CD8 T cells contribute to brain atrophy during Theiler’s murine encephalomyelitis virus (TMEV) infection of the CNS.
Authors: Mix Madison R. , Ruiz Vanessa Y. , Huseby Kelcher April M. , Macura Slobodan I. , Mishra Prasanna K. , Johnson Aaron J.
Published: January 2023

Brain atrophy is a central feature of numerous neurological diseases for which the contribution of immune cells remains poorly defined. For example, in human multiple sclerosis, clonally expanded CD8 T cells have been observed in proximity to lesions, but their role in mediating brain atrophy has yet to be elucidated. This study aims to define how antigen-specific CD8 T cells contribute to brain atrophy in the TMEV model of neuroinflammation. Specific MHC class I haplotypes have been shown to dictate neuropathology and functional outcomes in mice following intracranial infection with TMEV. In this study, we demonstrate that transgenic FVB mice which express the H-2Db class I molecule rapidly clear picornavirus infection but at the cost of a novel neurodegenerative phenotype consistent with brain atrophy. Following TMEV infection out to four months, FVB mice expressing the Db class I molecule display increased lateral ventricular volume via T2-weighted MRI and hippocampal neuron loss by hematoxylin and eosin staining. Furthermore, these transgenic FVB mice present enhanced brain infiltration of virus-specific CD8 T cells for the immunodominant Db:VP2121–130 epitope. To address these findings mechanistically, we inhibited the Db:VP2121–130 epitope-specific CD8 T cell response and monitored the effect on brain atrophy. This inhibition strategy resulted in a reduction in brain atrophy to levels comparable to wild-type FVB controls. Not confounded by persistent infection or demyelination, these studies are the first to demonstrate antigen-specific CD8 T cells have the capacity to induce brain atrophy in a murine model of neurodegeneration.
Vitamin A deficiency provokes aberrant T cell responses and exacerbates liver injury in LCMV-infected mice
Authors: Liang Yuejin , Yi Panpan , Sun Jiaren
Published: January 2023

Vitamin A deficiency (VAD) is a public health problem worldwide; however, its role and underlying mechanisms in viral infection are not entirely understood. In this study, we detected elevated LCMV-specific T cell responses in VAD mice at the acute stage (7 dpi). These VAD mice exhibited higher serum ALT levels and increased liver permeability. Consistently, enhanced antiviral T cell responses were observed in VAD mice in persistent infection (30 dpi), as evidenced by decreased PD-1 expression and increased cytokine production. To investigate whether VAD causes an intrinsic abnormality of T cells during animal development and growth, we transferred spleen cells from control and VAD mice into CD45.1 recipient mice, followed by LCMV infection, and found that antiviral T cell responses were comparable between control and VAD donor cells. However, when CD45.1 cells were transferred into control and VAD recipient mice, they showed a highly activated phenotype in VAD mice. These results suggest that vitamin A or its metabolites restrict T cell activation through an extrinsic way. Retinoic acid (RA) is considered one of the important metabolites of vitamin A. We found that RA administration inhibited excessive T cell responses in VAD mice. To dissect the mechanism of RA, we treated splenocytes and purified CD8+ T cells with RA, followed by PMA stimulation. RA treatment reduced the phosphorylation of AKT, p38, ERK, and S6, as well as the abundance of transcriptional factor NFATc1. Together, our study demonstrated that VAD caused an aberrant T cell response to viral infection, and RA may have the potential to serve as a therapeutic agent for excessive immune responses and tissue injury in VAD patients.
A Random Forest based approach to MHC class I epitope prediction and analysis
Authors: Wilson Eric A. , Krishna Sri , Anderson Karen S.
Published: January 2023

Background

Increased hydrophobicity of the contact regions between MHC class I-restricted peptides and TCR is associated with increased immunogenicity. To improve prediction of antigenic peptides and to identify additional biochemical properties associated with immunogenicity, we have created a random forest (RF)-based computational model that evaluates target epitopes using 66 parameters that cover a range of amino acid biochemical properties.

Method

A dataset of publicly available 9 mer epitopes with experimentally defined immunogenicity (n=29,874) and/or HLA binding (n=108,069) were collected from IEDB and the MassIVE database. Epitopes restricted to target HLA alleles were scored based on the biochemical parameters of each amino acid, and used to train a RF model specific to that HLA. For biochemical property analysis, redundant features were removed with a wrapper algorithm before being used to train the RF model.

Results

The RF model specific for HLA-A*02:01 (n=5500) was evaluated for HLA binding and immunogenicity prediction accuracy using a mutually-exclusive testing set of HLA-restricted peptides that were non-immunogenic, immunogenic, or non-binders. The model showed an immunogenicity prediction accuracy of 83% with an AUC of 0.88, and a HLA binding prediction accuracy of 97.4%, with a sensitivity of 95.2% and specificity of 99.9%. The RF model confirmed that hydrophobic interactions at peptide positions 4 and 8 as well as H-bonding at position 1 were important for immunogenicity.

Conclusion

RF-based algorithms can be used to identify biochemical parameters associated with immunogenic class I-restricted T cell epitopes. This model is being expanded to other HLA alleles.
A role for the nonclassical MHC molecule DO in Treg selection and function
Authors: Jurewicz Mollie M. , Nanaware Padma Pandurang , Stern Lawrence J.
Published: January 2023

Selection of CD4 T cells into the conventional and regulatory lineages is thought to be dependent on the strength of interaction of rearranged T cell receptors with MHCII-bound peptides displayed on APCs in the thymus. The nonclassical MHC molecule DO inhibits DM-mediated peptide exchange and has been shown to play a role in selection of peptides for presentation to CD4 T cells. In mice deficient in DO (H2-O), alterations in T cell responses and in antigen presentation as well as increased autoimmunity have been described. We show that DO−/− mice have increased numbers of thymic and peripheral Tregs, which also display a more activated phenotype. These DO−/− Tregs additionally exert greater suppressive capacity in vivo as compared to WT Tregs, as demonstrated by reduced T cell activation following Treg transfer into Treg-deficient mice. T cell receptor Vβ chain sequencing indicates the Treg population is less diverse and the conventional CD4 T cell population more diverse in DO−/− mice, suggesting skewing of thymic selection as a result of DO expression. To understand the mechanism behind altered T cell selection due to DO, we eluted peptides from thymic and B cell-derived I-Ab and characterized them by tandem mass spectrometry. Peptide diversity was increased in both cell types isolated from WT as compared to DO−/− mice. We speculate that through control of peptide diversity, DO tunes the number and specificity of thymocytes selected into the Treg lineage. These results highlight a previously unappreciated role for DO in controlling Treg selection and function.
Identification of a gamma IFN inducible lysosomal thiol reductase (GILT)-dependent MHC-II associated epitope that induces T cell responses in GILT−/− mice
Authors: He Xiao , Reyes-Vargas Eduardo , Hu Dai , Wang Xiaomin , Jensen Peter E.
Published: January 2023

Gamma-IFN inducible lysosomal thiol reductase (GILT) is currently the only identified thiol reductase that has optimal activity at acidic pH and localizes to lysosomes. GILT is constitutively expressed in antigen presenting cells (APC), is inducible by gamma-IFN, and participates in Ag processing by catalyzing the reduction of disulfide (S-S) bonds, thus facilitating the unfolding of native S-S bond containing Ag for further processing. Previous studies reported that T cells from GILT knock-out (GILT−/−) mice mounted a significantly reduced proliferative response to S-S containing protein Ag in vitro, GILT−/− APCs have a selective deficiency in presentation of some cysteine-containing epitopes from hen egg lysozyme, and GILT−/− mice exhibited a change in the pathogenic epitope of experimental autoimmune encephalomyelitis. Using MS/MS analysis, we identified a set of unique GILT-dependent, MHC-II associated-peptides in WT vs. GILT−/− mice, which we then validate by generating T cells specific to a unique GILT-dependent epitope from GILT−/− but not WT B6 mice. Our data suggests that GILT−/− mice are deficient in the generation of specific T cell epitopes due to a loss in GILT activity. This is consistent with our another on-going study where we find that GILT−/− mice failed to generate an Ab response to insulin due to inefficient processing of an S-S containing epitope in insulin. We hypothesize that in GILT−/− mice, GILT inactivation causes a change in the epitope repertoire that may lead to survival (escape from negative selection) of the T cells specific to GILT dependent epitopes in the thymus. The presence of these auto-reactive T cells might have implications for autoimmunity and autoimmune diseases.
Assessing non-classical antigen presentation in HIV-infected CD4+ T cells.
Authors: Sengupta Srona , Boronina Tatiana , Cole Robert N. , Siliciano Robert F. , Sadegh-Nasseri Scheherazade
Published: January 2023

CD4+T cells recognize peptides presented by MHC Class II (MHC-II) molecules on B cells, dendritic cells, and macrophages. Interestingly, in humans, activated CD4+ T cells themselves express MHC-II molecules (HLA-DR, -DP, -DQ), but their potential role as antigen processing cells has not been well characterized. Recent promising data in a simian immunodeficiency (SIV) model suggest that antigen presentation by CD4+ T cells may be important in immunity. In these studies, a vaccine incorporating SIV antigens into a cytomegalovirus vector induced MHC-II restricted CD8+ T cells in macaques. Notably, these non-canonical cells were also observed in HIV patients. Clones generated from these MHC-II restricted CD8+ T cells killed autologous HIV-infected CD4+ T cells, suggesting that HIV is processed and presented on MHC-II on CD4+ T cells. To investigate this potentially novel form of antigen presentation, we eluted peptides from HLA-DR on activated CD4+T cells and sequenced them by LC-MS/MS. We show for the first time that human primary CD4+ T cells indeed process and present peptides on HLA-DR. We are currently applying this approach to HIV-infected cells to detect HIV peptides. Given the low sensitivity of untargeted MS for rare viral peptides, we are also generating a library of MHC-II HIV epitopes presented on HLA-DR to help guide more targeted MS analysis. To create this library, we are using a MHC-II cell-free processing system. We have already identified several epitopes from HIV Gag, Pol, Env, Vif, Rev, and Tat, some of which we show elicit memory responses in HIV patients. Our studies reveal a novel role for CD4+ T cells in antigen presentation and show that a cell-free processing system can predict HIV epitopes that may be presented on these cells.
The role of microRNA-22 in the dysregulation of lupus Tregs and the pathogenesis of systemic lupus erythematosus
Authors: Appleton Brenna , Faust Ashley W , Michell Danielle L , Ormseth Michelle J , Vickers Kasey C , Major Amy S
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease affecting over 1.5 million Americans and at least 5 million individuals worldwide. Evidence demonstrates autoantibody producing B cells and dysfunctional CD4+T cells contribute to SLE pathology, however lack of understanding surrounding mechanisms of disease pathogenesis have prevented therapeutic advancement. Studies indicate one mechanism for dysregulated immune homeostasis in autoimmunity is through microRNAs (miRNAs). MiRNAs are short, endogenous post-transcriptional regulators of gene expression which act by degradation or translation repression of target mRNAs. Our group previously identified miR-22-3p as being increased three-fold in SLE patient plasma compared to age- and gender-matched heathy controls. MiR-22-3p levels were increased in whole CD4+T cells (four-fold) and Tregs (three-fold) from B6.SLE1.2.3 mice compared to B6 controls, and inhibition of miR-22-3p, using locked-nucleic acid (LNA)-22 ameliorated key disease pathologies including glomerulonephritis. There was also a 10% reduction in IFN-g+CD4+T cells in mice treated with LNA-22. Based on these data, we hypothesized increased miR-22-3p levels in SLE T cells propagated inflammation directly by skewing naïve CD4+T cells towards the Th1 phenotype during polarization, or indirectly by reducing the regulatory capacity of Tregs. Results show miR-22-3p deficiency is not sufficient to prevent the polarization of Th1 cells. However, inhibition of miR-22-3p in vivo increases Treg IL-10 production, suggesting heightened levels of miR-22-3p may alter their function. We conclude that miR-22-3p overexpression in Tregs indirectly contributes to autoimmune T cell dysregulation in SLE.
Choline acetyltransferase-expressing T cells are required to control chronic viral infection
Authors: Cox Maureen Ann , Mak Tak
Published: January 2023

Although widely studied as a neurotransmitter, recent advances have revealed an important role for T-cell derived acetylcholine (ACh) in regulating immunity. However, the role of lymphocyte-derived ACh in viral infection is unknown. Here, we show that the enzyme choline acetyltransferase (ChAT), which catalyzes the rate-limiting step of ACh production, is robustly induced in both CD4+ and CD8+ T cells during lymphocytic choriomeningitis virus (LCMV) infection in an IL-21-dependent manner. Deletion of chat within the T-cell compartment ablated vasodilation in response to infection, impaired the migration of anti-viral T cells into infected tissues, and ultimately compromised the control of chronic LCMV-Cl13. Our results reveal a genetic proof-of-function for ChAT in T cells during viral infection and identify a pathway of T-cell migration that sustains anti-viral immunity.
IL-9high GATA3low ILC2s are potent inducers of allergic airway inflammation
Authors: Schmitt Pauline , Duval Anaïs , Roga Stéphane , Camus Mylène , Burlet-Schiltz Odile , Gonzalez-de-Peredo Anne , Cayrol Corinne , Girard Jean-Philippe
Published: January 2023

IL-9 is key driver of chronic and allergic inflammation at mucosal surfaces, with important roles in the activation and survival of mast cells and type 2 innate lymphoid cells (ILC2s). IL-9-reporter mice have established T cells (Th9) and ILC2s as major sources of IL-9 in vivo. The discovery that TGF-β and IL-4 cooperate for induction of Th9 cells has been an important breakthrough. However, the signals that induce IL-9high ILC2s remain incompletely characterized.

Here, we show that IL-33, a critical activator of ILC2s, with important roles in type-2 innate immunity and allergic inflammation (Cayrol, Duval et al., Nature Immunology 2018) synergizes with TNF-family cytokine TL1A to induce prodigious amounts of IL-9 secretion by ILC2s. Unbiased global proteomic analyses revealed that IL-9 was the most-induced protein in ILC2s treated with IL-33 and TL1A. More than 99% of ILC2s expressed IL-9 intracellularly after co-stimulation with IL-33 and TL1A. ILC2s are known to produce very large amounts of IL-5 and IL-13, but ILC2s producing such high levels of IL-9 have not been described previously. Interestingly, IL-9 production by ILC2s was transient and associated with a phenotypic shift characterized by upregulation of IRF4, pSTAT5 and downregulation of ICOS, KLRG1 as well as master transcription factor GATA-3 and its target genes (IL-9R, ST2).

Lastly, we detected an expansion of IL-9high ILC2s in the lungs of mice after IL-33/TL1A inhalation and adoptive transfer experiments revealed that IL-9high ILC2s are potent inducers of allergic airway inflammation in vivo. Thus, our study supports the rising plasticity of ILC2s and their critical role in the induction of type-2 inflammatory responses in the lungs.
T-bet expression is fine-tuned for balancing IFN-γ-producing Th1 and Tfh cell differentiation and IgG2a(c) production
Authors: Fang Difeng , Zheng Mingzhu , Mao Kairui , Reiner Steven L , Sher Alan , Zhu Jinfang (Jeff)
Published: January 2023

T follicular helper (Tfh) cells and cytokine IFN-γ are involved in Ig class switching in B cells to produce IgG2a(c). Tfh cells mainly produce IL-21 as their signature cytokine, and several studies have also shown that some Tfh cells are capable of expressing Th1 signature cytokine IFN-γ without expressing the Th1 master transcription factor T-bet. In Toxoplasma gondii peptide AS15/Complete Freund’s Adjuvant (CFA)-induced immune response, we found antigen AS15-specific IgG2a(c) were abolished in CD4-Cre-mediated T-bet-deficient mice. By using T-bet-fate-mapping mouse strains, we reported that transient expression of T-bet epigenetically imprints the Ifng locus for cytokine production in a subset of Tfh cells; In germinal centers, multi-color tissue imaging revealed that the ex-T-bet Tfh cells express IFN-γ in situ; IFN-γ-expressing Tfh cells are absent in T-bet-deficient mice. More interestingly, we noticed that IFN-γ-expressing Tfh cells were fully present in mice with T-bet deletion at late stages of T cell differentiation, and antigen AS15-specific IgG2a(c) were even further up-regulated in such “T-bet late knockout” mice. Taken together, our findings demonstrate that IFN-γ expressed by T cells plays a dominant role in antibody IgG2a(c) production; T-bet expression is fine-tuned as spatiotemporal manner for balancing Th1 and Tfh cell differentiation in vivo and regulating the strength of antibody production.
Id2 is necessary for differentiation and survival of adipose-resident Tregs
Authors: Frias Adolfo B , Hyzny Eric J , Beppu Lisa Y , Buechel Heather M , D’Cruz Louise M
Published: January 2023

Adipose regulatory T cells (aTregs) have emerged as critical cells for the control of local and systemic inflammation that is linked to health problems including type II diabetes and cardiovascular disease. While a number of factors are known to be required for aTreg survival, in this study we show a distinctive role for the transcriptional regulator Id2 in the maintenance and differentiation of aTregs. Here we show Id2 is highly expressed in aTregs compared to high Id3 expression in lymphoid Tregs. Treg-specific loss of Id2 resulted in a substantial decrease in the frequency of aTregs and a decrease in aTreg surface and intracellular markers while Tregs in the spleen and lymph nodes were unaffected. Gene expression analysis revealed that Id2 expression was essential for the survival of aTregs and loss of Id2 increased cell death in aTregs due to increased Fas expression. Surprisingly, we observed higher Hif1α expression in WT aTregs that diminished with loss of Id2. Thus, we reveal an unexpected role for Id2 in regulating survival of adipose-resident Tregs, critical cells for control of obesity-associated local and systemic inflammation.
D-amino acid oxidase contributes IgA nephropathy through regulation of IgA production in intestinal mucosa
Authors: Suzuki Masataka , Sujino Tomohisa , Goto Motohito , Takahashi Riichi , Ito Mamoru , Sasabe Jumpei , Yasui Masato
Published: January 2023

Gut mucosa protects our body against threats of bacteria using antimicrobial proteins such as defensins, cathelicidins or lysozyme. D-amino acid oxidase (DAO) is one of the antimicrobial protein that is released from goblet cells into lumen and catabolize D-amino acids, which is essential for almost all bacteria, into antibacterial agent; H2O2.

IgA is major immunoglobulin in gut. Maturation of B cells into IgA-secreting plasma cells are lead through two pathways; T cell dependent or independent mechanism. A number of reports indicated the accumulation of systemic IgA in the animals lacking antimicrobial proteins. However, the mechanism how loss of innate immunity affects acquired immunity is yet to be clarified.

We found that mouse lacking DAO activity showed accumulation of systemic IgA. Analysis of intestinal microbiota in DAO null mice revealed increased amount of specific bacteria that stimulated IgA production. Crossbreeding experiment between DAO null mice and T cell receptor double knockout (Tcrb−/−, Tcrd−/−) mice showed that the hyper production of IgA is caused by T cell dependent B cell activation.

IgA nephropathy (IgAN) occasionally causes chronic kidney impairment. Recent genome wide association analysis indicated the linkage between IgAN and genes associated with mucosal immunity. We found that high IgA (HIGA) mouse, an animal model of IgAN, lacked DAO activity, and that high plasma IgA in HIGA mice is dependent on intestinal microbiota. These results indicated that loss of DAO activity involves in pathogenesis of IgAN through disturbance of mucosal immunity.
Use of recombinant influenza vaccines expressing exogenous miRNAs for increased vaccine production
Authors: Stambas John , Ye Siying , Bakre Abhijeet , Edwards Sarah , Dlugolenski Daniel , Tripp Ralph A
Published: January 2023

Vaccination remains the most effective means by which influenza virus infection can be controlled. Traditionally vaccine production has focused on egg-based technology. Increasingly cell culture-based vaccine production is being used to improve preparedness. Cell cultured based production methods offer significant advantages over traditional egg-based strategies by allowing (i) rapid scale-up (ii) elimination of allergic egg components and (iii) circumvention of issues relating to egg-adaption. Our aim was to improve influenza vaccines and preparedness through the establishment of mammalian cell (Vero)-based vaccines using plasmid-based recombinant flu constructs expressing exogenous miRNA19 (flu-miR19), a regulator of key host anti-viral pathways that has previously been shown to modulate influenza virus replication. We successfully rescued recombinant influenza viruses expressing miR19 using reverse genetics; validated expression of miR19 and showed increased virus replication in vitro that corresponded with increased HA titres and matrix gene expression. Knockdown of miR19 host target genes was confirmed through qRT-PCR to validate the mechanism of action. This new influenza vaccine platform is scalable and can be merged with existing production technology to offer rapid clinical translation and improve vaccine quality and production times. In summary, silencing of anti-viral host genes using a flu-miR resulted in an increase of influenza vaccine and HA antigen yield. We now plan to further augment antigen yield by propagating these viruses in enhanced Vero cell vaccine substrates.
Quantifying Immune Effector Molecules from Mouse Leukocytes with Flow-Based Multiplex Assay Panels
Authors: Sun Binggang , Rughwani Priyanka , Chow Kay , Zhao Amy , Lehmann Jason , Jiang Weiping , Ji Shaoquan
Published: January 2023

Exposure to foreign material triggers host immune responses, which are primarily mediated by leukocytes including macrophages, neutrophils, dendritic cells, NK cells, T cells and B cells. These cells produce an array of soluble factors which coordinate host defense, tissue repair, and immunoregulatory functions. In addition, these cells and the factors they produce are also critically involved in various diseases including infection, autoimmune disorders, asthma, diabetes, and cancer. Therefore, quantification of these molecules in a cell type-specific context will help achieve in-depth understanding of the immune responses and disease processes.

We have developed multiplex assay panels targeting specific mouse immune cells (CD4+ T helper cells, Macrophages/Microglia and B cells), using fluorescence–encoded beads that are suitable for use on common lab flow cytometers. Each panel allows for simultaneously quantification of 13 related analytes as shown in the table below.

Cell TypePanel TargetsT helper cellsIL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22, IFN-γ, TNF-αMacrophages/MicrogliaIL-1β, 6, 10, 12p40, 12p70, 18, 23, TNF-α, CCL22/MDC, CCL17/TARC, CXCL1/KC, G-CSF, TGF-β1B cellsIL-2, 4, 6, 10, 12p70, 13, 17A, IFN-γ, TNF-α, BAFF, BCMA, sCD40 ligand, TGF-β1

Each antibody pair was carefully optimized for assay specificity, sensitivity, accuracy and reproducibility. In addition, these panels have been validated by detecting expected expression level changes of analytes in biological samples cultured under various stimulation conditions. These high quality, low cost and easy to use panels will be useful tools to the study of specific cells involved in innate and adaptive immune responses and the relevant diseases.
20,000 layers under the model: Bayesian neural networks improve the robustness of human PBMC classifications using immunologist encoded bias
Authors: Velazquez-palafox Miguel Angel , Taylor Ian James , Spidlen Josef , Lai Janice , Halpert Richard , Stadnisky Michael
Published: January 2023

Accurate classification of immunological subsets is a cornerstone of routine assays in basic research and the clinic. This process generates terabytes of data for analysis, creating bottlenecks in reproducibility and feasibility. Advances in machine learning, specifically in deep learning, have dramatically expanded solutions to address this issue with higher accuracy and performance. Here we applied a supervised deep learning approach to guide cell classification of canonical human PBMC immune cell types from oligo-tagged antibodies and sequencing datasets. Utilizing Bayesian neural networks, we leveraged probabilities to encode scientist knowledge into the models whilst extrapolating to new datasets. The developed models produced higher F1 scores when compared to trained immunologists, data scientists, and traditional machine learning techniques. We demonstrate robust cell-type annotation over both validation and test PBMC data sets with over 95% accuracy. Furthermore, this architecture revealed the weighted relationships between the inputs and cell type predictions, giving insight into the driver genes and cell markers.
New generation CD90.1 and CD90.2 MicroBeads: fast isolation of total of adoptively transferred T cells from mouse spleen
Authors: Evaristo Cesar , Pitzka Christina , Pietz Nicole , Foerster-Marniok Anna , Dose Christian , Richter Anne
Published: January 2023

T cells play a central role in immunity to pathogens and tumors, but also in autoimmunity. T cell research has generated knowledge that led to multiple clinical breakthroughs, such as the generation of T cell vaccines and tumor immunotherapy approaches. However, additional work is required to fully understand T cell biology, harness their therapeutic potential and control immunopathologies. Cutting edge experimental protocols that interrogate and manipulate T cell biology often require prior purification of T cell populations. Strategies to simplify and accelerate T cell purification are highly desirable to save time, reduce bias and allow complex experiments to be performed.

We established workflows combining automated tissue dissociation with T cell isolation. We developed new CD90.1, and CD90.2 T cell specific enrichment reagents that significantly accelerated the isolation protocol. These reagents could be used to isolate total T cells, or rare CD90.1+ or CD90.2+ cells from adoptive transfer models. Importantly, culture of isolated T cells did not induce activation as measured by CD69 or CD25 upregulation, as compared to controls. Furthermore, magnetic isolation did not affect proliferation or cytokine expression following in vitro stimulation. Finally, T cell isolation could be fully automated and multiple samples could be processed in parallel.

Our new workflow greatly reduces time of downstream analysis while preserving cell phenotype and functional properties. We believe these innovative tools significantly shorten time-consuming experiments and can be used to increase reproducibility and the quality of data obtained in T cell research.