bluuebunny/crossref_metadata_2025 huggingface dataset:
Allograft transplantation provokes miRNA 297–669 cluster that reduces T regulatory cell generation through targeting TGF-β2
Authors: Becker William J. , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

T regulatory cells (Tregs) are anti-inflammatory cells capable of quelling the harshest immune responses. Accordingly, Treg therapy is rapidly becoming a viable treatment option for patients stricken with autoimmune diseases or those in need of an organ transplant. In a model of allotransplantation, via microarray, our work identified miRNA cluster 297–669 is induced in CD4+ cells in the lymph nodes draining the allograft in C57BL/6 mice given a C3H skin transplant. One member of this cluster, miR 466a-3p, is induced in a co-culture of lymph node cells stimulated with alloantigen in vitro. We have shown that this miRNA specifically targets transforming growth factor beta 2 (TGF-β2). The reduction in TGF-β2 levels in draining lymph node CD4+ T cells caused a resultant decrease in the generation of FoxP3+ Tregs, both within the graft and in the draining lymph node. Furthermore, blocking the effect of this miRNA using a specific locked nucleic acid (LNA) reversed this effect. Generation of Tregs using transforming growth factor beta1 (TGF-β1), interleukin-2 and CD3/CD28 is the most common method of induction. Here we show that replacing TGF-β1 with TGF-β2 has no detrimental effect on Treg induction. Indeed, Tregs induced with TGF-β2 have equal in vivo suppressive capability as TGF-β1 Tregs in a skin graft model when adoptively transferred. Moreover, these TGF-β2 Tregs have increased inducible T-cell costimulatory (ICOS) expression. The ability of TGF-β2 to induce Tregs was reversed when a TGF-β2 neutralizing antibody was used, while this antibody had no effect on TGF-β1 induced Tregs. The work herein describes, for the first time, a novel isoform of TGF-β that may be involved in the generation of Tregs with implications in all areas of immunology.
Tolerogenic synthetic nanoparticles for the prevention of murine acute graft-versus-host disease
Authors: Ferrari Daniel , LaMothe Robert , Kolte Pallavi , Griset Aaron , O’Neil Conlin , Leland Thomas , Fu Fen-ni , Viseux Nelly , Johnston Lloyd , Kishimoto Takashi K. , Maldonado Roberto
Published: January 2023

We have shown that treatment with biodegradable tolerogenic nanoparticles carrying rapamycin (synthetic vaccine particles, SVP) and antigen (inside the particles or admixed) induces durable, antigen-specific immune tolerance even in the presence of potent Toll-like receptor agonists. Treatment with SVP and antigen results in durable B cell tolerance, the differentiation of T and B regulatory cells, the inhibition of T and B cell activation, inhibition of hypersensitivity reactions, and the treatment of relapsing experimental autoimmune encephalomyelitis and celiac disease.

Here we describe the use of SVP to promote graft tolerance and prevent acute Graft-versus-host disease (GvHD). GvHD occurs when donor T cells contained in bone marrow transplants become activated against host cells and subsequently destroy them. Current mitigation strategies involve chronic immune suppression regimens. However, prolonged immunosuppression has many side effects, including susceptibility to opportunistic infections. A favorable strategy would be to tolerize the graft to the host. To evaluate SVP we utilized two mouse models of acute GvHD: (1) B6-to-F1 and (2) B6-to-Balb/c. A single SVP administration provided at the time of the graft improved disease score, blunted inflammatory cytokines and controlled the activation of donor cells. Importantly, this resulted in a significant increase in survival compared to mock treated animals.

SVP therapy represents a novel approach for the induction of immunological tolerance and the prevention of acute GvHD.
Targeting Chemokine Receptors to Modulate MDSCs for Cancer Therapy
Authors: Mittal Payal , Leach Craig , Clemente Jose , Sender Mathew , Hancock Wayne W. , Turunen Brandon
Published: January 2023

Host anti-tumor responses, including the actions of cytotoxic T cells, play a key role in targeting tumors, but can be hindered by multiple mechanisms active within the tumor microenvironment. One such mechanism involves the recruitment and expansion of immunosuppressive cells such as myeloid derived suppressor cells (MDSCs) in the tumor microenvironment. We have utilized two transplantable lung tumor cell lines (TC1 and LLC) to address the role of certain chemokine receptors in MDSC associated tumor progression. In addition, we used a thioglycolate-induced peritonitis model of inflammation to validate the ability of chemokine receptor antagonists to inhibit trafficking of MDSCs to the site of inflammation. Finally, we tested the compounds for their ability to impair chemokine signaling within established tumors and promote antitumor immunity. In summary, genetic and pharmacologic data indicate that targeting chemokine receptors to modulate MDSCs may be an important new component of immuno-oncology based therapies.
Disruption of Mammalian Target of Rapamycin Complex 1 in Myeloid cells Promotes Lung Cancer Metastasis
Authors: Ding Chuanlin , Yan Jun
Published: January 2023

The mTOR inhibitors are used in clinics for treating multiple cancers. However, this assertion does not fully address the effects on the tumor microenvironment (TME) which plays critical roles in cancer development and metastasis. Moreover, a controversy exists in the literature regarding the roles of mTOR in regulating myeloid cell activation in response to different environmental factors. In this study, conditional Raptor KO mice were generated to disrupt mTORC1 signaling in myeloid cells and LLC tumor cells were implanted either s.c. or i.v. in syngeneic mice. Although depletion of mTORC1 signaling promoted M2 macrophages and TAM toward an M1-like phenotype with decreased immunosuppressive activity, tumor progression in the s.c. model didn’t show difference between Raptor KO and control mice. Flow cytometric analysis demonstrated decreased Th1 T cell response in the TME of Raptor KO mice. The depletion of mTORC1 signaling decreased TAM trafficking and increased Ly6C+accumulation in LLC TME, which was correlated with lower levels of CD115 expression on Raptor-deficient macrophages. Cytokine profile analysis revealed that TNF-a secretion by myeloid cells was significantly decreased in Raptor-deficient TME. Further, disruption of mTORC1 in myeloid cells promoted lung cancer metastasis, which was associated with decreased Th1 and NK cell response. Accordingly, more CD11b+F4/80high macrophages were accumulated in the lungs of Raptor KO mice in LLC metastasis model. Taken together, our results demonstrate that mTORC1signaling in myeloid cells may have multiple roles in regulating anti-tumor immunity dependent on tumor environment, specifically with pro- metastatic effects in mTORC1-deficient lung myeloid cells.
Antibody-dependent cellular phagocytosis is responsible for efficacy of anti-CD20 monoclonal antibody therapy in chronic lymphocytic leukemia
Authors: Chu Charles C. , VanDerMeid Karl R. , Elliott Michael Rusty , Baran Andrea M. , Barr Paul M. , Zent Clive S.
Published: January 2023

Anti-CD20 (αCD20) monoclonal antibodies (mAbs) are important drugs in the management of B cell malignancies, such as chronic lymphocytic leukemia (CLL), and autoimmune diseases, such as systemic lupus erythematosus. However, the mechanisms of αCD20 mAb-induced cytotoxicity of B-cells are not fully understood. The major reported mechanisms are those highly effective at activating innate immune cytotoxic mechanisms, particularly antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC). To determine the role of these mechanisms, we tested αCD20 mAb-mediated cellular cytotoxicity of primary CLL cells by autologous or allogeneic (healthy donor) effector cells (human monocyte-derived macrophages for ADCP, or natural killer cells for ADCC) using flow cytometry or time-lapse video microscopy with fluorescent dye-labeled cells. We found that ADCP is the primary mechanism of cellular cytotoxicity by αCD20 mAb and that ADCC does not reliably predict the ADCP induced by that mAb. Furthermore, we compared ADCP using different αCD20 mAbs and found increased phagocytosis by those mAbs with higher affinity for CD20 or FcγR and by those mAbs with higher activation of complement. Finally, the effect on ADCP of inhibitors of BTK and PI3Kδ, molecules that are targeted by current CLL therapy and also found in the FcγR signaling pathway, were tested. We found that the most specific inhibitors of BTK and PI3Kδ had the least effect on ADCP. These data provide meaningful data to design more effective mAb and inhibitor regimens to best use these targeted agents to improve CLL patient outcome.
Unbiased testing of several hundred tumor-specific single nucleotide variants of a tumor for protective immunogenicity and CD8+ response reveals surprises.
Authors: Brennick Cory A. , George Mariam M. , Hagymasi Adam T. , Shcheglova Tatiana V. , Seesi Sahar Al , Mandoiu Ion I. , Srivastava Pramod K.
Published: January 2023

Somatic mutations in cancer cells can give rise to new MHC epitopes referred to as neoepitopes. Neoepitopes have been clearly demonstrated to elicit an antigen specific T cell response, which are capable of mediating targeted killing of cancer cells. Despite recent advancements, exactly what criteria make a good neoepitope for personalized therapy is currently unknown. Here, using a syngeneic tumor of C57BL/6 mice, named FABF, we present results of an exhaustive and exhausting study where we have tested hundreds of long peptides each containing a single nucleotide variant (SNV) (with the mutation in the center of the peptide) for their ability to elicit tumor rejection and independently, CD8+ T cell response. We observe that (i) about 2% of all SNVs lead to generation of peptides that can mediate tumor rejection to any significant degree; (ii) each peptide alone elicits a modest protection, and a combination elicits stronger protective immunity, (iii) all the neoepitopes that elicit tumor rejection have poor binding affinity for MHC I, and have positive values for Differential Agretopic Index (1). (iv) Even though the protective responses are CD8-mediated, there is no correlation between a neoepitope’s ability to elicit a CD8 response and tumor rejection. Some of these observations are inconsistent with some aspects of the current consensus about the defining characteristics of good neoepitopes.
Bortezomib impacts Notch—miR-155 mediated augmentation of CD8+T Cell antitumor immunity
Authors: Renrick Ariana N. , Thounaojam Menaka C. , Thomas Portia L. , Shanker Anil
Published: January 2023

The immunosuppressive tumor microenvironment dampens host antitumor immunity by multiple mechanisms including interference with the Notch system, which is important for various cell fate decisions and hematopoietic cell differentiation and function. We observed that treatment with bortezomib, a proteasome inhibitor, in mice bearing subcutaneous tumors resulted in an upregulated expression of various Notch signaling components in lymphoid tissues and increased CD8+T lymphocyte IFNγ secretion and expression of effector molecules, perforin and granzyme-B, as well as the T-box transcription factor eomesodermin. Of note, bortezomib reversed tumor-induced downregulation of Notch receptors, Notch1 and Notch2, as well as increased the levels of cleaved Notch intracellular domain (NICD) and downstream targets Hes1 and Hey1 in tumor-draining CD8+T cells. These data suggest that bortezomib can reverse tumor-induced dysfunction of CD8+T cells by its intrinsic stimulatory effects. Our preliminary data also suggest that bortezomib can positively regulate miR-155 expression in CD8+ T cells from mice bearing tumor. Further, miR-155 suppression was found to downregulate bortezomib-induced increase in Notch target genes in T cells. We are currently elucidating how bortezomib affects the expression of miR-155 and its target genes, such as suppressor of cytokine signaling 1 (SOCS1) and inositol polyphosphate-5-phosphatase (SHIP1) that are associated with T cell function. These data provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death, but also to provide lymphocyte-stimulatory effects, thereby overcoming immunosuppressive actions of tumor on antitumor T cell functions.
The role of thymocyte selection-associated HMG box protein (TOX) in CD8 T cell differentiation and dysfunction
Authors: Scott Andrew Crosbie , Philip Mary , Camara Steven , Shakiba Mojdeh , Levy Olivier , Glickman Michael , Chang Kenneth , Schietinger Andrea
Published: January 2023

CD8 T cells are powerful adaptive immune cells that specifically recognize infected cells and eliminate them. However, in the context of tumors, antigen-specific T cells enter a state of dysfunction allowing tumors to progress. By employing genome-wide transcriptome analysis of dysfunctional tumor-specific T cells (TST) we found that a nuclear factor, thymocyte selection-associated HMG box protein (TOX), was uniquely upregulated during T cell differentiation/dysfunction in progressing tumors, but not during normal CD8 T cell differentiation (e.g. acute infections). TOX is a nuclear DNA-binding factor and member of the high-motility group box superfamily. TOX has been shown to be required for CD4 T cell and NK cell development but its role in peripheral CD8 T cells is not known. We set out to investigate the role of TOX in tumor-specific CD8 T cell differentiation. Using various murine tumor models we found that TOX was uniquely overexpressed in TST within the tumor (TIL) and its overexpression was driven by chronic TCR/antigen stimulation and not by the tumor microenvironment. To understand the role of TOX in CD8 T cell differentiation and dysfunction we are currently conducting TOX genetic knock-out and overexpression studies in vitro and in vivo. The identification and characterization of novel transcriptional regulators such as TOX will provide important insights into the molecular underpinnings defining CD8 T cell dysfunction in tumors.
Circadian Clock disruption promotes TH17 Inflammation and Colon Cancer
Authors: Osman Abu , Saadalla Abdulrahman , Gounari Fotini , Turek Fred , Keshavarzian Ali , Rakshit Kuntol , Matveyenko Aleksey , Khazaie Khashayarsha
Published: January 2023

Circadian clocks regulate numerous physiological processes that vary across the day-night (diurnal) cycle, but whether the circadian clock regulates the adaptive immune response to cancer is mostly unclear. In mouse models of polyposis, and human colorectal cancer patients, we showed that tumor infiltrating Tregs overexpressed b-catenin and RORgt, and secreted IL-17. These Tregs suppressed tumor reactive cytotoxic T cells, and promoted tumor growth. Therefore, we examined how the expression of a dominant negative ClockΔ19 gene affects inflammation and susceptibility to cancer in the APCΔ468mouse model of hereditary polyposis. ClockΔ19 mutant mice had high expression of RORgt+ and b-catenin+ Tregs. These mice showed a notable expansion of Th17 inflammation in vivo upon injection of aCD3. Functionally these Tregs showed pro-inflammatory properties. In addition to these effects, APCΔ468ClockΔ19 mice had increased numbers of CD11b+ cell, Ly6C+CXCR1− monocytes, and Ly6C+Ly6G+ myeloid derived suppressor cells (MDSC) as compared with APCΔ468 mice. APCΔ468ClockΔ19 mice had higher tumor burden in the small intestine and colon than the APCΔ468 mice. Both T-cells and tumors had intense stabilization of nuclear b-catenin and were more aggressive as indicated by abundance of stromal mMCP6+ mast cells (MC), which typically appear at the invasive borders of tumors. Our finding indicating increase in tumor load and worsening inflammation caused by direct ClockΔ19 effects in the epithelium and enhanced TH17 bias in the immune system.
Generation of a therapeutic monoclonal antibody for acute myeloid leukemia by employing the U937 cell line as a surrogate antigen
Authors: Chen Bethany , Kandov Evelyn , Ghebrehiwet Berhane
Published: January 2023

The prognosis of acute myeloid leukemia (AML) has improved for younger patients, but it still remains grim for patients over 65 years old with a likelihood of death within a year of diagnosis. Here, we evaluated the effects of novel anti-gC1qR monoclonal antibodies (mAbs) that were generated against intact U937 cells, which are used here as a model for AML. The U937 cell line is known to overexpress gC1qR, which in turn serves not only as a proliferative molecular signal but also provides a platform for the generation of angiogenic and metastatic factors. Cell viability results reveal that all of the mAbs significantly decrease (p<0.05) the percentage of viable AML cells although the mixture of those mAbs was not significant when compared to the individual mAbs themselves. The anti-U937 antibodies, monoclonal and polyclonal, showed a significant difference (p<0.05) against the anti-purified recombinant gC1qR antibodies. The anti-U937 mAb methodology of immunizing with the mammalian cell instead of the purified protein allowed the mAbs to better target the native endogenous protein present on human cells. Our study not only provided novel and potent mAbs against AML, but also implicated a potentially more efficient technique of creating therapeutic mAbs against cancer cells.
Cryptotanshinone has curative dual anti-proliferative and immunotherapeutic effects on Lewis lung carcinoma
Authors: Yang De , Liu Shuo , Han Zhen , Trivett Anna , Lin Hongsheng , Oppenheim Joost J.
Published: January 2023

Lung cancer is the leading cause of cancer-related mortality, with very limited effective therapeutics. Screening of a variety of Traditional Chinese Medicines (TCMs) for capacity to inhibit the proliferation of human lung cancer A549 cells and to induce the maturation of human dendritic cells (DCs) led to the identification of cryptotanshione (CT), a compound purified from the TCM Salvia miltiorrhiza Bunge. CT can be chemically synthesized. Here CT was shown to inhibit the proliferation of mouse Lewis lung carcinoma (LLC) cells by upregulating p53 and downregulating cyclin B1 and Cdc2 and consequently inducing G2/M cell-cycle arrest of LLC cells. In addition, CT promoted phenotypic maturation of mouse and human DCs with upregulation of costimulatory and MHC molecules, and stimulated DC to produce TNFa, IL-1b and IL-12p70, but not IL-10. CT-induced maturation of DCs depended on MyD88 and also involved the activation of NF-kB, p38, and JNK. CT was effective in the treatment of established LLC tumors and, when used in combination with low-dose anti-PD-L1, can cure LLC-bearing mice with the induction of subsequent anti-LLC specific immunity. Our data indicate that CT has novel unique antitumor and immunotherapeutic activities that may provide a new promising therapeutic for the treatment of human lung cancers.
Altering the cytokine profile in the pancreatic cancer microenvironment with heat shock protein-90 inhibitors to enhance immunotherapy.
Authors: Lesinski Gregory B. , Zhang Yuchen , Farren Matthew R. , Komar Hannah , Ware Brandon , Olson Brian , Zaidi Mohammad , Nagaraju Ganji P. , Akce Mehmet , Alese Olatunji B. , Shaib Walid Labib , Wu Christina Sing-Ying , El-Rayes Bassel
Published: January 2023

One feature of pancreatic ductal adenocarcinoma (PDAC) is the fibrotic stroma, which is a result of interactions between tumor cells, immune suppressive cells and pancreatic stellate cells (PSC). Stroma-derived cytokines including IL-6 represent a significant barrier restraining immunity against PDAC. Targeting pathways in the tumor microenvironment (TME) that regulate cytokine secretion can be an innovative means to improve access of effector immune cells to PDAC. We hypothesize that targeting Hsp90 can modulate the TME, through its ability to target inflammatory signaling and cytokine production by PSC and enhance the efficacy of immunotherapy. The Hsp90 inhibitor XL888 led to a concentration-dependent inhibition of IL-6 and MCP-1 production by PSC in vitro. Concurrently, PSC displayed growth inhibition as determined by MTT assay, and reduced alpha-SMA expression, a marker of cell activation. Inhibition of Jak/STAT and MAPK signaling was confirmed via immunoblot. These data compliment our published results indicating that Hsp90 inhibitors modulate survival pathways and PDAC growth in vitro and in vivo. This has led to an investigator-initiated Phase Ib/II clinical trial of XL888 (Hsp90i) and pembrolizumab (anti-PD-1) now accruing at our institution. Extensive laboratory correlative studies are ongoing to examine the impact of Hsp90 inhibition on anti-PD-1 mediated T cell proliferation, cytokine production, and impact on PSC-derived cytokine signatures. This work is being performed in paired biopsy samples (pre- and on-treatment), and peripheral blood from expansion cohorts of patients with metastatic pancreatic cancer (n=16) or colorectal cancer (n=16).
Transcriptome Responses to Respiratory Virus Infection of Pigs within the Tracheobronchial Lymphnode Following Infection with PRRSV, PCV2 or IAV
Authors: Miller Laura C. , Fleming Damarius S. , Harhay Gregory P. , Kehrli Marcus E. , Lager Kelly M.
Published: January 2023

Porcine reproductive and respiratory syndromevirus (PRRSV) is a major respiratory pathogen of swine that has become extremely costly to the swine industry worldwide, often causing losses in production and animal life due to their ease of spread. However, the intracellular changes that occur in pigs following viral respiratory infections are still scantily understood for PRRSV, as well as, other viral respiratory infections. The aim of this study was to acquire a better understanding of PRRS disease by comparing gene expression changes that occur in tracheobronchial lymph nodes (TBLN) of pigs infected with either porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), or swine influenza virus (IAV) infections. The study identified and compared gene expression changes in the TBLN of 16 pigs following infection by PRRSV, PCV2, IAV, or sham inoculation. Total RNA was pooled for each group and time-point (1, 3, 6, and14 DPI) to make 16 libraries, for analysis by Digital Gene Expression Tag Profiling (DGETP). The data underwent standard filtering to generate a list of sequence tag raw counts that were then analyzed using multidimensional and differential expression statistical tests. The results showed that PRRSV, IAV and PCV-2 infections followed a clinical course typical of experimental infection of young pigs with these viruses. Gene expression results echoed this course, as well as, uncovered genes related to shared and unique host immune responses to the 3 viruses. By testing and observing the host response to other respiratory viruses, our study has elucidated similarities and differences that can assist in development of vaccine and therapeutics that shorten or prevent a chronic PRRSV infection.
The fish adaptive immune response and its suppression by helminths
Authors: Steinel Natalie C. , Bolnick Daniel
Published: January 2023

Helminth infections can immunosuppress hosts, leaving them vulnerable to co-infection. This talk will present a model of helminth immunosuppression: the teleost, stickleback (Gasterosteus aculeatus), and its helminth, Schistocephalus solidus. The teleost adaptive immune response is thought to occur in the melano-macrophage center (MMC). This structure is the proposed site in which B cells proliferate and generate high affinity antibody, homologous to germinal centers in mammals. Stickleback MMCs transiently increase in size following immunization, suggesting that the MMC likely participates in the adaptive immune response. Infection of stickleback with S.solidus, however, suppresses MMC size, suggesting helminth-mediated immunosuppression. To investigate the mechanisms underlying parasite-mediated immunosuppression in teleosts, wild populations of Vancouver Island stickleback were identified that vary in their magnitude of helminth-mediated immunoregulation. Helminth-infected stickleback from Gosling lake show suppressed MMC responses, while Roberts lake fish are refractory to helminth-induced immunosuppression. The resistance of Roberts lake fish to immunosuppression, however, is helminth genotype specific, with some helminth genotypes able to suppress MMC size. F2 hybrid fish were generated from Gosling and Roberts populations and QTL analysis was performed to identify loci associated with differential immunoregulation of the MMC response by helminths. As MMCs are found in most teleosts (and reptiles and amphibians), these findings highlight a new and broadly applicable assay of immunity in both laboratory and wild animals, which does not require the development of species-specific reagents.
Galectin-3 enhances avian H5N1 influenza A virus-induced pulmonary inflammation by promoting NLRP3 inflammasome activation
Authors: Chen Huan-Yuan , Chen Yu-Jung , Wang Sheng-Fan , Lo Tzu-Han , Liu Fu-Tong
Published: January 2023

Virus-induced excessive inflammatory response contributes to severe disease and high mortality rates. Galectin-3, a β-galactoside-binding protein widely distributed in immune and epithelial cells, regulates various immune functions and modulates microbial infections. Here we describe galectin-3 upregulation in mouse lung tissue following challenges with the H5N1 influenza virus. We investigated the effects of endogenous galectin-3 on H5N1 infection and found that survival of galectin-3 knockout (Gal-3KO) mice was comparable to Wild-type (WT) mice following infections. Compared to infected WT mice, infected Gal-3KO mice exhibited less inflammation in the lungs and reduced interleukin-1 beta (IL-1β) levels in bronchoalveolar lavage fluid. We also found that bone marrow-derived macrophages (BMMs) from Gal-3KO mice exhibited reduced oligomerization of apoptosis-associated speck-like proteins containing caspase-associated recruitment domains (ASC), and secreted less IL-1β compared to BMMs from WT mice. However, we observed similar levels of the inflammasome component of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) in two genotypes of BMMs. Co-immunoprecipitation data indicate galectin-3 and NLRP3 interaction in BMMs infected with H5N1. We also observed an association between galectin-3 and NLRP3-ASC complex. Our results indicate that galectin-3 promotes host inflammatory responses and regulates IL-1β production by macrophages via interaction with NLRP3. It suggests that endogenous galectin-3 enhances the effects of H5N1 infection.
Interferon signal transduction in airway barrier epithelial cells is key to controlling human picornavirus infection in the lung
Authors: Zhang Yong , Mao Dailing , Keeler Shamus P. , Wu Kangyun , Agapov Eugene , Holtzman Michael J.
Published: January 2023

Interferon (IFN) signal transduction is required for defense against viral infection, and this signal in airway barrier epithelial cells is assumed to be responsible for controlling common respiratory viruses. However, these conventional concepts have not been rigorously tested in an experimental model given the relative lack of native human respiratory viruses that replicate efficiently in non-human models and the absence of tools to analyze IFN signal transduction specific to airway epithelial cells. To address these issues, we generated a new line of Scgb1a1-Foxj1-Cre-Stat1f/f mice to selectively target IFN signaling in airway epithelial cells and tested these mice for control of infection with the human picornavirus pathogen EV-D68 that might cause more severe infection than human rhinoviruses. Our results show significant replication of EV-D68 with a 1×105-fold increase in viral RNA in lungs of Scgb1a1-Foxj1-Cre-Stat1f/f mice compared to wild-type mice and was similar to viral titer and accompanying lung inflammation and mucous cell formation in conventional Stat1−/− mice. We also found increases in viral titer and inflammation in Scgb1a1-Foxj1-Cre-Stat1f/f mice compared to wild-type mice after the natural mouse pathogen murine parainfluenza virus aka Sendai virus (SeV) infection, but in this case the defect was significantly more pronounced in Stat1−/− mice, consistent with evidence of extra-airway spread of SeV infection. Together, these results indicate a critical requirement for airway epithelial cell capacity for IFN signaling in protection against respiratory picornavirus infection, and to our knowledge, the best evidence to date for the proposed role of airway barrier epithelial cells in host defense.
Early transcriptional responses to influenza virus infections in vivo
Authors: Langlois Ryan A. , Sjaastad Louisa , Fay Elizabeth , Maccietto Marissa , Shen Steve
Published: January 2023

Influenza A virus (IAV) infects a broad range of cell types within the respiratory tract. The cells initially targeted by the virus in a naïve host are the site of primary replication and virus spread. These cells are difficult to detect using replication competent IAV as the virus rapidly spreads to secondary cells. To overcome this obstacle we utilize a fluorescence expressing single cycle IAV (scIAV) to identify the cells initially infected in the mouse lung. Using this tool, we observed two distinct populations of epithelial cells during the early stages of infection: cells with high virus replication and cells with low virus replication. This suggests that some cells within the lung are innately permissive to virus replication while others are able to blunt replication. We have determined that this phenotype is not due to coinfection of a cell with multiple virions, and it appears to be independent of type I IFN. The level of replication is also not dependent on epithelial cell type. There are distinct cellular pathways that are significantly impacted in each population of infected cells compared to uninfected cells. Additionally, there are subsets of interferon-stimulated genes that are specifically upregulated in each population. These data indicate that different levels of virus replication may activate distinct cellular responses to infection. Fluorescent reporter scIAVs could be used to further elucidate the mechanism of cellular permissibility and the early innate immune response to IAV infection.
ZIKV-specific CD8 T cell immunity in humans is affected by DENV pre-exposure
Authors: Grifoni Alba , Pham John , Peters Bjorn , de-Oliveira-Pinto Maria Luzia , de Silva Aravinda , Durbin Anna , Diehl Sean A. , Harris Eva , Crowe James , Busch Micheal , Vivanco-Cid Hector , Graham Barney S. , Turtle Lance , Kallas Esper , Watkins David , Weiskopf Daniela , Sette Alessandro
Published: January 2023

Several studies have characterized humoral immunity to Zika virus (ZIKV) in humans but little is known regarding the corresponding T cell responses to ZIKV. In this study we sought to investigate the ZIKV-specific T cell response in terms of kinetics and viral epitopes targeted and whether pre-existing dengue virus (DENV) T cell immunity can affect these responses. Our results shows that memory T cell responses elicited by prior infection with DENV infection or vaccination recognize ZIKV-derived peptides. The cross-reactive response is explained by the sequence similarity of the two viruses, as the ZIKV peptides recognized are either identical or highly conserved between DENV and ZIKV. In addition, DENV exposure also influences the timing and magnitude of the ZIKV-specific T cell response. In the acute phase of infection, ZIKV-reactive T cells are detected earlier and in greater magnitude than those in DENV pre-exposed patients. Conversely, while the frequency of ZIKV-reactive T cells continues to rise in the convalescent phase in DENV-naive donors, it declines in DENV pre-exposed donors. In addition, ZIKV-specific CD8 T cells form DENV pre-exposed donors selectively up-regulated granzyme B and PD1, as compared to DENV-naïve donors. These results overall suggest a more efficient control of ZIKV replication and/or clearance of ZIKV antigen in patients with previous DENV exposure. Finally, when we focused on the viral epitopes targeted by DENV naïve donors, ZIKV structural proteins (E, prM and C) are major targets of CD8 T cell responses, whereas DENV pre-exposed individuals skew the T cell epitopes primarily in nonstructural proteins.
What have we learned about the dynamics of peptide loading from structures of TAP binding protein, related (TAPBPR)?
Authors: Margulies David H. , Jiang Jiansheng , Natarajan Kannan
Published: January 2023

Understanding the molecular details of peptide loading in the MHC-I pathway is crucial to approaches to manipulate recognition of peptide/MHC-I complexes in treating autoimmunity, tumors, and for vaccine design. Recent insights have been provided by two X-ray structures of TAPBPR, in complex with MHC-I molecules. Although structures determined in the two laboratories are remarkably similar (pdb ID: 5WER and 5OPI), the structural models based on X-ray data obtained at similar resolution, 3.4 and 3.3 Å respectively, reveal a discrepancy in the disposition of the TAPBPR 22–36 loop. This loop was not modeled in 5WER due to a lack of electron density, whereas it was modeled as a 3,10 helix in 5OPI and ascribed a significant function in peptide loading. In an attempt to resolve this structural discrepancy, we reanalyzed 5OPI based on the authors’ deposited structure factors. We calculated the refined average B-factor, an indication of positional uncertainty, as 247 Å2 and 153 Å2 for the 22–36 loop and the entire TAPBPR/MHC-I complex, respectively. Prompted by the unusually high B-factors we reexamined the 22–36 loop by calculating composite omit-simulated annealing maps from deposited structure factors of 5OPI as well as of 5WER. When model bias was eliminated in this way, continuous electron density was not visible in this loop region. Thus, we conclude that although the 22–36 loop of TAPBPR may play a role in peptide exchange, there presently is no reliable crystallographic evidence that this loop is a 3,10 helix and is positioned as modeled in 5OPI. Further structural studies of TAPBPR and of tapasin should help to resolve in detail the mechanism by which these molecules facilitate MHC-I stabilization and peptide exchange.
Investigating the MHC class-II processing and presentation “network” via high-throughput small molecule screening.
Authors: Wold Erik D. , Schultz David , Cherry Sara , Eisenlohr Laurence Crane
Published: January 2023

CD4+ T cells, activated by peptides presented in complex with MHCII, are key players in adaptive immune responses against most pathogens and cancers. Classically, MHCII presentation involves uptake of exogenous antigen, digestion in the endocytic compartment, and loading onto nascent MHCII in the late endosome. However, recent studies show viral pathogens do not exclusively adhere to the classical model. Two notable examples of non-classical MHC-II presentation are: 1) “Recycling” presentation (internalized MHCII molecules capture proteolytically unstable epitopes in the early endosome) and 2) endogenous presentation (the source of antigen is nascent viral protein). We have constructed a model in which these three MHCII processing and presentation pathways are mechanistically distinct and largely non-overlapping. To test this model, we have employed high throughput screening (HTS) using a 2240 member “known target” small molecule library with the goal of differentiating which cellular components are involved in the generation of influenza epitopes produced via the classical, recycling, and endogenous pathways. The preliminary HTS data strongly support our hypothesis, clearly identifying distinct pathways that are exclusively involved with either classical or endogenous antigen presentation. Among the 21 validated hit compounds, the screen identified 3 inhibitors of the proteasome (previously shown to figure prominently in endogenous processing). The remaining 18 compounds target pathways not previously known to contribute to MHCII antigen presentation. Compounds of particular interest are being investigated in vivo to assess their ability to bias the TCD4+ response in the context of an influenza infection.
A role for apoptotic and scavenger receptors in mediating antigen exchange between lymphatic endothelial cells and migratory dendritic cells
Authors: Caffrey Alayna Katherine , Tamburini Beth , Kedl Ross
Published: January 2023

Antigen persistence following viral infection or vaccination occurs for several weeks after resolution of the inflammatory response. Lymphatic endothelial cells (LECs) capture and archive antigen, which is then transferred to migratory dendritic cells (DCs) and ultimately cross-presented to circulating memory T cells. Following vaccination, both BatF3-dependent and BatF3-independent DCs are needed for antigen exchange, while virus-associated antigen exchange is mediated only by BatF3-dependent DCs. Here, we explore the role of apoptotic and scavenger receptors in mediating this antigen capture and exchange between LECs and DCs. Cross-presentation of the DC-associated antigen to circulating T cells results in a memory pool with increased effector function and enhanced protective capacity. Overall, this work furthers our understanding of how persisting antigen impacts the CD8+ memory T cell response in both vaccine and infectious settings.
An Unbiased Determination of pMHC Repertoires for Improved Antigen Prediction
Authors: Rappazzo C. Garrett , Birnbaum Michael
Published: January 2023

Successful understanding and treatment of immune diseases spanning autoimmunity, cancer, and infectious disease relies upon understanding what is seen by the immune system. For T cell responses, this is controlled by MHCs, specialized polymorphic proteins that present a snapshot of the proteome of their parent cell or surroundings. Importantly, MHC polymorphisms cluster at their peptide-binding surface, where even single substitutions can greatly alter the peptide-MHC (pMHC) repertoire seen by T cells. Due to extreme person-to-person variability in MHC usage, data for peptide MHC specificity is largely limited to a handful of the most prevalent alleles. While algorithms that predict pMHC interactions perform well for these alleles, they can suffer from inaccuracy for less-studied alleles, due in part to biases and limited diversity in their training data.

We present a platform that provides large unbiased data sets to improve pMHC repertoire definitions and predictions. Using yeast-displayed MHC molecules displaying a random peptide library, we identified hundreds of thousands of unique peptide binders for both well- and poorly-studied human MHC alleles. Our data demonstrates strong amino acid preferences at defined “anchor” positions for each allele, but also finds preferences at auxiliary sites that are not considered by most prediction software. We also find noncanonical amino acid preferences at defined anchor positions that confound current software, suggesting alternative peptide conformations. These data show the importance of unbiased pMHC repertoires to improve existing antigen prediction software, and suggest our approach can be used to define presently under-studied MHC alleles.
The unfolded protein response alters MHC class I antigen presentation in a mouse cell line
Authors: Nagamine Brandy S. , Dolan Brian P.
Published: January 2023

Direct MHC class I antigen presentation is a necessary step to identify cells for elimination by cytotoxic CD8+ T cells. Antigenic peptides may originate from host-derived unnecessary or “retired” proteins as well as defective ribosomal products (DRiPs). DRiPs are newly synthesized polypeptides incapable of attaining a stable configuration and are thus rapidly degraded. The balance between protein synthesis and degradation, key processes to antigenic peptide generation, are likely influenced by the unfolded protein response (UPR). The UPR is a cellular response designed to alleviate the toxic accumulation of unfolded proteins in the endoplasmic reticulum. Several disease conditions requiring CD8+ T cell responses such as viral infection and tumorigenesis are known to influence the UPR. In this study, we utilized small inhibitory molecules targeting individual branches of the UPR to investigate the effect on both global MHC class I levels and the presentation of peptides from both DRiP and non-DRiP sources. Inhibition of the IRE1α signaling pathway but not the PERK pathway reduced total MHC class I levels. Presentation of peptides derived from DRiPs was unaffected by inhibiting either branch of the UPR. These data suggest that specific perturbations to the UPR has the capacity to alter host antigen presentation of peptides from particular peptide sources.
IL-23 is essential for the switching between Tfh-IFN-γ and Tfh-IL-17 in lupus
Authors: Hong Huixian , Wu Qi , Yang PingAr , Luo Bao , Li Jun , Li Hao , Cua Daniel J , Hsu Hui-Chen , Mountz John D
Published: January 2023

The role of IL-23 in promoting pathogenic autoantibody development in autoimmune disease is unclear. The purposes of this study are to use the IL-23p19 knockout in the BXD2, a lupus mouse strain, to determine: (i) if IL-23 is required for germinal center (GC) and pathogenic autoantibody development; and (ii) if IL-23 acts through follicular T-helper cells (Tfh) lineage switching to induce autoantibody formation. Sera autoantibodies were measured by ELISA. GC development and the expression of AID were determined by confocal imaging. IL-17 and IFN-γ producing Tfh cells were detected by intracellular staining. The expression of GC program genes and T cell program genes was measured by qRT-PCR. Exogenous IL-23 was administered to B6 mice using an IL-23 expressing adenovirus (AdIL-23). There was significantly increased IgM but decreased IgG autoantibodies in BXD2-p19−/− mice compared to BXD2 mice. The expression of AID was decreased in BXD2-p19−/− mice. Surprisingly, the size and number of GC were increased in BXD2-p19−/− mice. There was also similar percentage of PD1+CXCR5+Tfh cells in BXD2 and BXD2-p19−/− mice. However, the percentage of IL-17A expressing Tfh cells was suppressed but the IFN-γ producing Tfh was increased in BXD2-p19−/− mice. Consistent with this, AdIL-23 administration suppressed the expression of Tbet, Ifng, Bcl6, Il21, Gata3, and Il4 but enhanced the expression Il17a, Il17f in Tfh cells. Our data suggest Tfh-IFN-γ and Tfh-IL-17 each plays an important role to determine the development of autoantibody producing B cells. While IL-23-mediated Tfh-IL-17 does not promote the size of autoreactive GCs, it regulates the induction of AID and thereby plays an important role to determine autoantibody affinity maturation.
NKG2D signaling within the pancreas decreases NOD diabetes by enhancing CD8+ central memory T cell formation
Authors: Markiewicz Mary A , Trembath Andrew P , Sharma Neekun , Mathews Clayton E
Published: January 2023

The main drivers of type 1 diabetes are islet-specific T cells. These cells must escape a myriad of tolerance mechanisms that control their activation in healthy individuals. One pathway implicated in this process is signaling through Natural Killer Group 2 member D (NKG2D). We demonstrate that the mRNAs encoding both NKG2D and NKG2D ligands are increased in the islets of patients with type 1 diabetes, making discerning the role of NKG2D-ligand interaction in the pancreas during diabetes development of significant interest. To this end, we developed a novel mouse model to determine the role of NKG2D specifically within the pancreas. This is a NOD mouse that expresses a transgene driving expression of the NKG2D ligand RAE1ɛ in islets (RIP-RAE1ɛ NOD mice). Spontaneous diabetes development was significantly decreased in RIP-RAE1ɛ-NOD mice compared with control NOD mice, and RIP-RAE1ɛ mice contained decreased numbers of CD44hiCD62Llo(effector/effector memory, Tem, phenotype) CD8+ T cells in both the pancreas and spleen. This decrease correlated with increased numbers of CD44hiCD62Lhi(central memory T cell, Tcm, phenotype) CD8+ T cells, which is a cell population previously demonstrated to contain cells that inhibit the CD8+ Teff response. Further, we found that NKG2D signaling in both NOD and human CD8+ T cells increased CD8+ Tcm differentiation in vitro. Together, these novel results suggest that the NKG2D pathway in CD8+ T cells could be utilized to inhibit type 1 diabetes progression.
The pathogenic role of stem cell-like memory T cells in rheumatoid arthritis
Authors: Lee Ye Ji , Kim Miriam , Lee Eun Bong
Published: January 2023

Stem cell-like memory T (Tscm) cells are known as the least differentiated T cells that have ability to self-renew and differentiate into subsets of T cells. Recently, Tscm cells have been studied in various autoimmune diseases including systemic lupus erythematosus (SLE), aplastic anemia. However, the association between Tscm cells and rheumatoid arthritis (RA) remains unexplored. So, we measured the proportion of Tscm cells in peripheral blood mononuclear cells (PBMCs) of RA patients by flow cytometry using pertinent Tscm-associated fluorescent markers (CD3+ CD4+/CD8+ CCR7+ CD45RO− CD45RA+ CD62L+ CD27+ CD28+ CD127+ CD122+ CD95hi). The proportion of Tscm cells among CD4+ T cells as well as CD8+ T cells were significantly increased in RA patients than healthy controls (HCs). Tscm cells from RA patients could replicate themselves and differentiate into other T cell subsets in response to anti-CD3/CD28 stimulation. When FACS-sorted Tscm cells were stimulated with IL-6 or TNF which are key inflammatory cytokines in RA and expression of activation markers (CD69, CD25 (IL-2Rα), CD154 (CD40 ligand)) and secretion of cytokines (IFN-γ, IL-4 and IL-17A) were measured, Tscm cells from RA patients were more easily stimulated than those from HCs. In addition, RA patients in remission showed low proportion of Tscm cells among the CD4+/CD8+ T cells and responded less against cytokines than active RA patients.

Our study suggests that Tscm cells from RA patients may perpetuate RA by providing the patients with pathogenic T cells constantly.
Pathogenic follicular CD8 T cells acquire diverse function during autoimmune disease
Authors: Valentine Kristen M , Mullins Genevieve N , Lawrence Travis J , Hoyer Katrina K
Published: January 2023

CXCR5+ CD8 T cells identified within the germinal center have the capacity for diverse function in response to chronic antigen including chronic viral infection and cancer. CXCR5+ CD8 T cell pathogenic or protective function likely depends on the immunological setting. We have identified CXCR5+PD-1+ CD8 effector T cells (CD8 T follicular; Tfc) that resemble CD4 T follicular helper cells (Tfh) in multiple models of spontaneous autoantibody-mediated disease, including IL-2 deficient (IL-2-KO), scurfy and MRL/MpJ-FASlpr mice. Autoimmune CD8 T cells induce antibody class switching, and plasma cell differentiation synergistically with CD4 T cells in support of a novel function for CD8 Tfc cells during germinal center reactions. RNA-sequencing identified 478 differentially expressed genes unique to IL-2-KO CD8 Tfc compared to IL-2-KO CD4 Tfh including il21, Ifnγ, prf1. CD8 Tfc are polyfunctional and co-produce significant amounts of IL-21 and IL-4. Yet, only IL-21 produced by CD4 Tfh and not CD8 Tfc promotes total IgG class switching in in vitro cultures of activated T cell supernatant incubated with naïve B cells. CD8 Tfc supernatant deficient in IFNγ does not change antibody production. In addition to the helper profile, CD8 Tfc cells maintain the capacity to produce granzyme B, perforin, and CD107a when stimulated suggesting the possibility of direct cytolytic function within the germinal center. Our results indicate that CD8 Tfc cells contribute to autoimmune disease with CD4 Tfh cells to induce B cell responses and antibody class-switch. We will continue to investigate pathogenic CD8 Tfc cells in helper-like and cytotoxic roles during autoimmune disease.

Funding from NIH R15 and the UC President’s Dissertation Year Fellowship