bluuebunny/crossref_metadata_2025 huggingface dataset:
GITR agonism triggers antitumor immune responses through IL-21–expressing follicular helper T cells
Authors: Koh Choong-Hyun , Kim Il-Kyu , Kang Tae-Seung , Shin Kwang-Soo , Jeon Insu , Song Boyeong , Lee Jeong-Mi , Im Jiyoung , Kang Yejin , Kang Chang-Yuil
Published: January 2023

Treatment with glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) agonistic antibody (DTA-1) elicits potent antitumor immune responses in various tumor models. In this study, we demonstrate the crucial role of interleukin (IL)-21–producing follicular helper T (Tfh) cells in DTA-1-induced tumor inhibition. Administration of DTA-1 increased IL-21 expression from CD4 T cells, which is mainly made up of Tfh cells, in an antigen specific manner. Furthermore, mice treated with a neutralizing antibody to IL-21 receptor and Bcl6fl/flCD4cre mice exhibited abrogated antitumor activity induced by DTA-1. Mechanistically, IL-4 plays critical roles for the induction of IL-21–expressing Th cells by GITR co-stimulation, which is mediated by Bcl6 and c-Maf. Thus, our findings identify GITR co-stimulation as an inducer of IL-21–expressing Tfh cells and provide a novel mechanism for its antitumor activity.
CD73-dependent tumor resistance to agonistic anti-4-1BB therapy through TGF-β
Authors: Chen Siqi , Fan Jie , Zhang Minghui , Qin Lei , Dominguez Donye , Long Alan , Zhang Bin
Published: January 2023

Agonist antibodies (Ab) directed against costimulatory molecules on the surface of antigen-primed T cells are in various stages of pre-clinical and clinical trials, albeit with limited therapeutic benefit as single agents. The underlying immune suppressive mechanisms remain incompletely understood. In our study, we demonstrate an inhibitory role of ecto-enzyme CD73 for agonistic anti-4-1BB (anti-CD137) Ab therapy. In particular, anti-4-1BB treatment can downregulate the expression of CD73 on T cells by preferentially driving CD73− effector T cell response and achieve tumor inhibition. Anti-CD73 neutralizing Ab further improves anti-4-1BB therapy associated with enhanced anti-tumor T cell immunity. Furthermore, we find the same observation in the combination of anti-CD73 and anti-GITR, another agonist antibody. However, the TGF-β-rich tumor milieu confers resistance to anti-4-1BB and anti-GITR therapy by sustaining CD73 expression primarily on infiltrating CD8+T cells across several tumor models. TGF-β blockade results in downregulation of CD73 expression on infiltrating T cells and sensitizes resistant tumors to agonistic anti-4-1BB therapy with increased number and function of T cells. Thus, our findings identify a mechanism of action for more effective clinical targeting of 4-1BB or likely other costimulatory molecules.
Defining the mechanisms of B cell initiated autoimmune disease
Authors: Bazile Cassandra A , Wright Jacqueline A , Clark Emily S , Carlesso Gianluca , Gao Linlin , Mahmoud Tamer , Boucher Justin , Kleiman Eden , Satterthwaite Anne B , Harhaj Edward , Khan Wasif N
Published: January 2023

Systemic lupus erythematosus (SLE) afflicts more than 1.5 million individuals in the United States with very limited and debilitating therapeutic options. The etiology of the disease remains unclear. It is hypothesized that in SLE, peripheral B cell tolerance is breached by inappropriate survival of autoreactive cells. Consistently, overexpression of B cell activating factor (BAFF), a key regulator of B cell survival can rescue autoreactive B cells and contribute to autoimmune disease. BAFF counters apoptosis in part through decreasing BH3-only pro-apoptotic protein Bim. Notably, both BAFF and Bim are physiological regulators of B cell tolerance. Therefore, to define the role of B cells and B cell tolerance in the initiation and progression of autoimmune disease we created a mouse model wherein gene encoding Bim is selectively deleted in B cells (B.Bimf/f). Initial analyses of these mice suggest that B cells can initiate an autoimmune pathogenesis phenotypically similar to SLE, with mice exhibiting splenomegaly and increased CD21loCD23lo B cells. Given that persistent activity of NF-kB in B cells can also result in autoimmune diseases, we are now investigating whether the persistent NF-kB and dysregulated apoptosis can synergize in SLE pathogenesis. To that end, we have crossed B.Bimf/f mice with a mouse line that lacks a recently discovered potential negative regulator of NF-kB, ZFAND6 (B.Bimf/f x ZfanD6−/−). Preliminary findings suggest that the compound mutant mice display exacerbated autoimmune symptoms with higher incidence of splenomegaly and kidney pathology. These preliminary results suggest that a breach in tolerance (B.Bimf/f ) and persistent NF-kB activity cooperate in the initiation and severity of SLE pathogenesis.
Multiple tolerance checkpoints suppress low-affinity germline precursor B cells of high affinity anti-dsDNA antibody-producing cells in BCR knock-in mice
Authors: Mohammed Marwa Ali El Hussien , Sakakibara Shuhei , Tsai Chau-Yuan , Kikutani Hitoshi
Published: January 2023

Previously, we revealed that disease-associated dsDNA-reactive high-affinity antibodies evolve from ssDNA-reactive low-affinity germline precursors through clonal expansion and affinity maturation in acute systemic lupus erythematosus (SLE) patients. Our findings raise questions whether low-affinity precursors of anti-dsDNA antibody producing cells are subjected to immune tolerance, and under what conditions they expand and undergo affinity maturation in vivo. To address these questions, we generated G9gl, a site-directed knock-in (KI) mouse line carrying reverse mutated forms of VH-DH-JH and Vk-Jk regions of an acute SLE patient-derived high-affinity anti-dsDNA antibody. G9gl heterozygous mice had reduced numbers of B cells in their periphery, and half of peripheral B cells expressed endogenous IgH from non-KI allele due to receptor editing.

G9gl-expressing B cells exhibited several anergic phenotypes, including reduced surface BCR and shorter life span. Yet, they underwent class-switching and produced antibodies following CD40 and IL-4 in vitro stimulation. G9gl B cells responded to ssDNA, but not dsDNA in vitro, and were selectively excluded from germinal center reaction in several conditions such as Treg depletion and DNA-conjugated mBSA immunization in vivo. However, some of G9gl-expressing B cells differentiated to somatically mutated IgG+ memory B cells when immunized with bacterial DNA-conjugated mBSA.

Therefore, multiple tolerance checkpoints prevent low-affinity anti-DNA precursor B cells from undergoing clonal expansion and affinity maturation in germinal centers. However, G9gl-expressing B cell might undergo class-switching and somatic hypermutation through the extra-follicular pathway.
Distinct chromatin modifications at the Il33 locus in mast cells contribute to sex dimorphic susceptibility to EAE, an autoimmune CNS demyelinating disease
Authors: Brown Melissa Ann , Yang Yuchen
Published: January 2023

Many autoimmune diseases, including multiple sclerosis (MS) exhibit a striking female bias, but the underlying mechanisms remain poorly understood. Using the SJL mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which recapitulates this sex dimorphism, we previously demonstrated that preferential IL-33 expression contributes to male protection. IL-33 expression by mast cells in PLP139–151-immunized males activates type 2 innate lymphoid cells, which in turn drives a non-pathogenic Th2 response to myelin peptide. Testosterone directly activates Il33 in male but not female mast cells, but this male-specific response is also maintained with other modes of activation, including FcɛR1-crosslinking, suggesting there are constraints on the chromatin landscape that limit Il33 expression in females. Here we use ChIP and ATAC-seq to show that the dynamics of chromatin modifications at the Il33 locus is sex-dimorphic. At steady state, ChIP assays reveal activating H3K4me3 and H3K9ac histone modifications at the promoter and conserved non-coding sequences within the Il33 gene are most prevalent in male-derived cells, while H3K27me3 repressive marks dominate in females. This pattern is similar in IgE-DNP-activated mast cells. Increased basal Il33 chromatin accessibility assessed by ATAC-seq is evident in males. Together, these results indicate that while testosterone can directly stimulate IL-33 production, it also exerts effects on Il33 during development conferring a higher potential for expression in males.
Effects of mechanical stress on cells derived from human oral mucosa
Authors: Yamamoto Toshiro , Sato Yoshiki , Yamamoto Kenta , Asai Taku , Oseko Fumishige , Kanamura Narisato
Published: January 2023

Objective

When a denture is applied, the oral mucosa is exposed to mechanical stress (MS) including occlusal force via the denture. Clinically, inflammation is occasionally observed in the oral mucosa immediately below the denture despite a proper fit. In this study, we applied MS to cells derived from human oral mucosa and evaluated its effects.

Materials and Methods

Subconfluent human oral mucosal epithelial cells (HO-1-N-1) and human gingival fibroblasts (HGF) were used to seed 1´105 cells/dish and, after 24 hours, subjected to MS (1 and 3 MPa, 60 min) using a hydrostatic pressurizer. Morphological changes of the cells were examined by Giemsa stain, and changes in cell activity were measured by the WST assay. In addition, mRNA was collected from the cells, and I Inflammatory cytokines and growth factors genes were measured by real-time RT-PCR assay and ELISA.

Results

HO-1-N-1 showed no morphological change by Giemsa stain but exhibited a significant decrease in cell activity by WST assay. Next, in HO-1-N-1, the expression of Inflammatory cytokines, and ICAM mRNA was significantly increased after MS at 1 MPa but was attenuated at 3 MPa. In HGF, the expression of IL-6 mRNA was enhanced. In HO-1-N-1 and HGF, the IL-6 and IL-8 protein levels increased significantly after MS. Furthermore, in HGF, the EGF and FGF-2 protein levels increased significantly by stimulation at 3 MPa.

Discussion

MS applied to the oral mucosa immediately below the denture caused a decrease in the activity of oral mucosal epithelial cells and induced enhancement of inflammatory cytokine production. Also, excessive MS was suggested to promote the cure of oral mucosa wounds by promoting the production of growth factors in the oral mucosa.
Implementing participatory immunology education in a diverse Latin American student population
Authors: Ferreira Leonardo , Carosso Giovanni A. , Duran Natalia Montellano , Bohorquez-Massud Soad , Vaca-Diez Gustavo , Rivera-Betancourt Laura Ines , Rodriguez Yara , Ordonez Dalila G. , Alatriste-Gonzalez Diana K. , Vacaflores Aldo Fabian , Auza Lilian Gonzalez , Schuetz Christian , Alvarado-Arnez Lucia Elena , Alexander-Savino Carolina , Gandarilla Omar , Mostajo-Radji Mohammed A.
Published: January 2023

Immunology is typically first encountered by students in advanced college or graduate courses. Additional challenges to teaching immunology exist in the developing world, where limited resources require curricula balancing state-of-the-art knowledge with region-specific practical considerations. Project-based teaching is especially difficult to execute due to lack of infrastructure and teacher training. Here, we report the results of implementing short hands-on low-cost microbiology and immunology courses for high school and college students in Bolivia. This initiative, “Clubes de Ciencia Bolivia”, brings graduate students and postdoctoral fellows from research institutions in developed nations to teach weeklong intensive courses in Bolivia. These courses (“clubes”) feature project-based and active learning components and are designed and taught with local Bolivian science instructors. Our approach was effective at transmitting concepts in microbiology and immunology, as measured by subject-specific test scores before and after course completion. Importantly, student learning was not significantly affected by their background, education level, socioeconomic status, or initial interest in the specific course taken. Moreover, participants reported a heightened interest in scientific careers after course completion. Strikingly, participants scored higher in subject-specific tests than Bolivian college students who had taken related semester-long college courses. Altogether, these data indicate that participatory learning for immunology in a developing nation can be used to effectively transmit knowledge. Similar approaches are likely to drive scientific engagement in other developing economies.
In for the long haul: teaching immunology in a longitudinal medical curriculum
Authors: Bottaro Andrea
Published: January 2023

In recent years, undergraduate medical education has undergone significant transformations, including stronger integration of clinical and basic science content in both early and late phases of the curriculum; frequent replacement of traditional topic-specific courses with system-specific blocks in which all basic sciences can be weaved in longitudinally; and an increasing utilization of active learning approaches to replace traditional lectures. At the same time, immunology has greatly expanded its relevance to clinical care, impacting an increasing number of medical subspecialties. As a consequence, medical graduates now require a higher degree of immunology literacy than ever before.

Here, I will discuss the experience of building (and sustaining) a longitudinally integrated immunology curriculum at a new (now 7-year-old) medical school. I will first highlight a number of key challenges, including the ballooning amount of information that medical students are required to learn in increasingly time-constrained curricula; the balancing of the competing needs of updating content for scientific advancements vs maximizing student success in the slower-moving but ever-important medical board exams; collaborating with faculty colleagues whose training in immunology often happened decades in the past; and dealing with content gaps and redundancies in shifting course curricula. Lastly, I will provide some examples of how we have addressed some of these challenges locally, while others may require more concerted, global solutions.
Open Discovery in an Undergraduate Immunology Course Lab: Testing Anti-Inflammatory Drugs
Authors: Brennan Catherine , Roshandell Mobina
Published: January 2023

I will present the design and outcomes from a course laboratory module in which undergraduates apply techniques learned earlier in the semester (cell culture, spectrophotometry, standard curves, ELISA) towards the testing of an anti-inflammatory drug of their choice. Students rationalize their dose and assay selections, calculate all dilutions, perform the assays, and analyse and interpret all results. Student-directed course research experiences have been shown to solidify student understanding of advanced scientific concepts. Additionally, we observed that this exercise, in which students analyse the function of medications used by themselves or loved ones, leads to enthusiastic engagement with and mastery of complex concepts.
Role of GILT and MHC class II in melanoma cells on regulating the anti-tumor immune response
Authors: Macy Anne , Adams Anngela , Hastings Karen Taraszka
Published: January 2023

The MHC class I antigen presentation pathway in melanoma cells has a well-established role in immune-mediated destruction of melanoma. However, the role of the MHC class II pathway in melanoma cells is not fully understood. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens by antigen presenting cells. While GILT and MHC class II expression is typically limited to antigen presenting cells, GILT and MHC class II can be expressed constitutively or induced by IFN-γ in melanoma cells. In human melanoma specimens, high GILT expression and an active and intact MHC class II pathway are associated with improved survival. The goal of this project is to investigate a causal role for GILT and MHC class II in melanoma cells, using Yale University Mouse Melanoma (YUMM) lines YUMM2.1, YUMMER1.7, and YUMMERG. These cell lines constitutively expressed GILT, and MHC class II expression was IFN-γ-inducible in a subset of cells under serum-free conditions. We have genetically engineered GILT−/− YUMM2.1 cells and MHC class II (H2Ab1)−/− YUMM2.1 cells via CRISPR/Cas9 and verified the deletion. We have generated lentiviral constructs expressing GILT alone, CIITA alone, and dual expression of H2Ab1 and CIITA, to allow for the generation of melanoma cell lines with and without GILT and MHC class II. Transduction of GILT−/− YUMM2.1 cells with GILT resulted in high transduction efficiency and overexpression. Thus, we have developed models to determine the effect of GILT and MHC class II expression in melanoma cells on regulating in vivo tumor growth and the anti-tumor immune response.

This work was supported by a Basic Science Award from the Harry J. Lloyd Charitable Trust and a Merit Review Award I01-BX005336 from the US Department of Veterans Affairs (VA), Biomedical Laboratory Research and Development Service (K.T.H.). The contents do not represent the views of the VA or the US Government.
Distinct myeloid antigen presenting cells dictate differential fates of tumor-specific CD8 T cells in pancreatic cancer
Authors: Burrack Adam L , Schmiechen Zoe , Patterson Michael , Miller Ebony , Spartz Ellen J , Rollins Meagan , Raynor Jackson , Mitchell Jason , Kaisho Tsuneyasu , Fife Brian T , Stromnes Ingunn
Published: January 2023

Pancreatic ductal adenocarcinoma (PDA) contains numerous protumor myeloid cells at the expense of antitumor dendritic cells (cDC1s). We identify that tumor antigenicity is a major determinant of myeloid composition and functionality. Neoantigen-tumors contained pro-tumor macrophages and a paucity of cDC1s whereas neoantigen+ tumors accumulate cDC1s via Xcr1 signaling and macrophages exhibit a diminished role. Effective immunotherapies increased splenic cDC1s, which were required for endogenous T cell expansion following α-PD-L1 and transferred effector and memory T cells. Batf3−/− mice, which lack cDC1s, failed to spontaneously generate tumor-specific CD8 T cells and were resistant to T cell therapy and α-PD-L1. In contrast, agonistic α-CD40 exhibited partial benefit in Batf3−/− mice and expanded atypical tumor-specific CD8 T cells. Monocyte depletion abrogated atypical tumor-specific CD8 T cell priming yet enhanced α-CD40-mediated antitumor activity in Batf3−/− mice. In contrast, α-Gr1 abrogated the therapeutic benefit of CD40 agonist in Batf3−/− mice. In sum, our study supports that CD40 agonist promotes a cDC1-dependent antitumor immunity and a monocyte-dependent protumor arm of immune system. These results further underscore an essential antigen presenting role for cDC1s in the expansion and differentiation of naïve, effector, and memory T cells into Klrg1+ potent cytotoxic effector T cells capable of targeting pancreatic cancer.

M.R. is supported by National Institutes of Health (NIH) T32 AI 007313 E.J.S. is supported by NIH T35 AI118620 I.M.S. is supported by NIH R01 CA249393, R01 CA255039, Department of Defense #PA200286, an American Association for Cancer Research (AACR) Pancreatic Cancer Action Network Career Development Award (17-20-25-STRO), an AACR Pancreatic Cancer Action Network Catalyst Award (19-35-STRO), American Cancer Society Institutional Research Grant (124166-IRG-58-001-55-IRG65).
The impact of GSK3β on the metabolism of IFNγ activated macrophages
Authors: Ankley Laurisa M , Olive Andrew J
Published: January 2023

The activation of macrophages by IFNγ induces transcriptional and metabolic changes that enable the direct control of pathogens and improves interactions with T cells. In particular, IFNγ induces the expression of antigen presentation machinery like MHCII. Despite its central role in host immunity, the regulation of IFNγ-induced MHCII is poorly understood. Using CRISPR Cas9 screens we examined regulators of distinct IFNγ-mediated mechanisms in macrophages including MHCII. One key regulator found was the multifunctional kinase, GSK3β. Not only was GSK3β required to induce transcriptional activation of MHCII, but RNAseq analysis suggests broad changes to the IFNγ response in the absence of GSK3β. Recent work in our group suggests that changes in metabolism are essential for macrophages to respond to IFNγ. While GSK3β is known to directly modulate metabolism, its role in controlling metabolic changes following IFNγ activation remains unknown. Given that GSK3β interfaces with metabolic pathways like mTORC1 and glycolysis we hypothesize that the loss of GSK3β inhibits important metabolic shifts required for macrophages to effectively respond to IFNγ. Using a Seahorse Analyzer we are now determining how genetic or chemical inhibition of GSK3β changes the rate of glycolysis or oxidative phosphorylation following IFNγ stimulation. We will then directly target metabolic networks in the presence and absence of GSK3β to determine how these impact IFNγ responses including the expression of MHCII. Given the broad impact of GSK3β on several disease states, such as inflammatory disease and tumor progression, understanding the regulation of GSK3β has the potential to contribute to several aspects of human health.
Dissecting the role of H-2Db class I molecule in the development of brain atrophy during Theiler’s murine encephalomyelitis virus (TMEV) infection
Authors: Wininger Katheryn M , Goddery Emma , Khadka Roman , Tritz Zachariah P , Fain Cori , Ayasoufi Katayoun , Jin Fang , Hansen Michael , Johnson Aaron J
Published: January 2023

Brain atrophy is a common feature of many neurological diseases as diverse as Alzheimer’s disease, cerebral palsy, Huntington’s disease, Krabbe disease, multiple sclerosis, Pick’s disease, epilepsy, encephalitis, neurosyphilis, neuroAIDS, and Covid-19 infection. We have developed a murine model of brain atrophy using the Theiler’s murine encephalomyelitis virus (TMEV) infection mouse model of multiple sclerosis. In this study, we investigated the contribution of the MHC class I molecule, H-2Db, in generating an immune response associated with the onset of brain atrophy. To define the roll of the MHC class I molecule in the cellular and molecular mechanisms of brain atrophy, we developed a novel bi-transgenic mouse lines with tamoxifen induced conditional ablation of the H-2Db class I molecule in Cx3CR1+ brain resident myeloid cells on the C57BL/6 background. Interestingly, both CX3CR1cre/Db LoxP mice and their cre- littermate controls experienced short-term memory loss using novel object recognition tests. However, Cx3CR1cre+/Db LoxP mice presented with significantly less brain atrophy as assessed by analysis of lateral ventricular volume at 45 d.p.i. using T2-weighted MRI, compared to cre- littermates. This change in ventricular volume was sustained at 6 months post infection. These results strongly imply a requirement for antigen presentation by H-2Db on myeloid cells for the development of brain atrophy and provided insight into the molecular mechanism of this poorly understood neuropathology.

Supported by NIH (RFI NS122174)
Mechanism of Peptide Loading as Revealed by Structure of tapasin/MHC-I Complex
Authors: Jiang Jiansheng , Taylor Daniel K , Kim Ellen , Boyd Lisa F , Cresswell Peter , Mage Michael G , Margulies David H , Natarajan Kannan
Published: January 2023

MHC-I molecules loaded with peptide for subsequent cell surface expression are critical for adaptive immunity. Tapasin, a component in the Peptide Loading Complex (PLC), serves as an MHC-I chaperone, and it facilitates peptide loading onto MHC-I in the endoplasmic reticulum (ER). Although the structure of TAPBPR (a homolog of tapasin) complexed with MHC-I was reported several years ago, the detailed mechanism of peptide loading by tapasin remains unclear. Here, we report the structure of a complex of tapasin with the MHC-I molecule, HLA-B* 44:05. The model of tapasin/HLA-B*44:05 compared with that of unliganded HLA-B*44:05 and to the structure of tapasin bound to ERp57 reveals dramatic changes that refect both chaperone and catalytic activities. Tapasin cradles the MHC-I molecule through contacts with the N-terminal alpha1 and alpha2 domains, as well as with alpha3 and beta-2 microglobulin interaction via its C-terminal IgC domain. Thus the MHC-I molecule is stabilized in a peptide accessible form. We compare this tapasin/HLA-B*44:05 structure with our previously determined structure of TAPBPR/H2-D(d). Although the general dispositions of tapasin and TAPBPR are the same in their complexes with MHC-I, the structural details of their interactions differ. Analyses of tapasin mutants that disrupt the interaction with MHC-I are consistent with the X-ray crystal structure. Thus, the two MHC-I dedicated chaperones, tapasin and TAPBPR, although acting at distinct stages of the MHC-I loading pathway, show broadly conserved binding modes and similar mechanisms of peptide loading in antigen presentation.

Supported by the intramural research program of the NIAID, NIH
Investigating the potential binding partners of H2-O to further elucidate its role in MHCII antigen presentation
Authors: Dobkin Julie , Golovkina Tatyana , Denzin Lisa K
Published: January 2023

The production of neutralizing antibodies in response to viral pathogens requires CD4+ T cell help. For this to occur, pathogen-derived peptides must be loaded onto class II major histocompatibility complex (MHC II) molecules in endosomal/lysosomal compartments and trafficked to the surface of antigen presenting cells. The αβ heterodimeric nonclassical MHCII molecules, H2-M and H2-O, modulate peptide loading. While H2-M directly catalyzes MHCII peptide loading, H2-O is a negative regulator of MHCII antigen presentation. Current dogma supports that H2-O is an MHCII structural mimic that exerts control over peptide loading via direct competition with MHCII for H2-M-binding. Work done in our labs, however, suggests this current model is incomplete and suggests the involvement of other, yet undefined, factors. I/LnJ mice, which are resistant to retroviruses through the production of potent neutralizing antibodies, inherit an H2-Ob allele that encodes nonfunctional H2-O. I/LnJ H2-O binds to but does not inhibit H2-M, directly challenging current dogma. Compared with H2-Oβ from retrovirus-susceptible mice, I/LnJ H2-Oβ contains three unique amino acid (AA) substitutions in the conserved immunoglobulin domain which we have found to be critical for the H2-O inhibitory activity of H2-M. We hypothesize that altered H2-O function in I/LnJ mice is due to an inability to interact with essential binding partners. We recently generated B6J mice in which we have knocked-in these three AA substitutions. This novel tool will be exploited to discover potential binding partners of variant H2-Oβ and elucidate the mechanism by which H2-O activity is modulated.
T Cell Recognition of Disulfide Modified Antigens
Authors: Dai Shaodong , Li Wei , Wang Yang
Published: January 2023

Disulfide formation is one of the most common post-translational modifications and plays important in protein structures and functions. We discovered that the disulfide bridge was the epitope of a highly diabetogenic CD4 T cells. This is a shift from the paradigm that T cells commonly recognize the linear peptide antigens and showed a novel to create neo-epitopes. We identified in vivo that the different subsets of CD4 T cells could be stimulated by both reduced and oxidized form of the same antigens, IAPP. This suggested an imbalance in redox homeostasis may be important for T cell mediated pathogenesis. A disulfide bridge specific monoclonal antibody was created and showed extraordinary ability in preventing type 1 diabetes. The disulfide bridge specific T cells were able to be generalized another protein, CGRP. The lysosomal thiol reductase knock-out experiments showed that the reductase may be important for disulfide modified antigen generation during antigen processing. Elevating GSH levels through glutamate cysteine ligase pathway inhibited CD4 T cell response in a dose dependent manner. Although NOX attributed to the importance of hydrogen peroxide production in pancreatic beta cells, a Nox inhibitor effectively attenuated the BDC5.2.9 T cell activation. Our results shed light on how thiol-disulfide exchange reaction impacts the T cell responses.

Supported by NIH R56 AI153488 NIH R21 AI149655
Lupus Susceptibility Gene Pbx1 Regulates STAT3 in T cells via JAK2/STAT3 Signaling Pathway
Authors: Roach Tracoyia A , Choi Seung-Chul , Park Yuk Pheel , Morel Laurence
Published: January 2023

Systemic Lupus Erythematosus (SLE) is an autoimmune disease in which poorly characterized genetic factors lead to the production of autoreactive or inflammatory T cells. Pre-B cell leukemia homeobox 1 (Pbx1) is a transcription factor whose dominant negative splice isoform (Pbx1-D) is overexpressed in CD4 T cells of lupus patients and lupus-prone mice as compared to the normal isoform (Pbx1-B). Pbx1-D overexpression impairs Treg cell development and function while favoring the production of follicular helper T cells. Based on previous studies showing that Pbx1 promotes cell proliferation via JAK2/STAT3 signaling pathway in a renal cell carcinoma, we hypothesized that Pbx1-D decreases T cell proliferation and viability by attenuating the JAK2/STAT3 signaling pathway. U3A STAT3 reporter cells transfected with Pbx1-B plasmid have a greater luciferase expression, and hence pSTAT3 transcriptional activity in comparison to cells transfected with Pbx1-D or control plasmids. We also observed that Jurkat T cells stably overexpressing Pbx1-B increases STAT3 gene expression as compared to Pbx1-D. Furthermore, Pbx1 silencing in Jurkat T cells leads to the attenuation of STAT3 message. These results suggest a mechanism by which Pbx1 contributes to impaired T cells in lupus pathogenesis is via the JAK2/STAT3 pathway. Current studies are conducted to dissect the mechanism by which Pbx1 regulates this pathway in primary T cells.

Supported by a grant from the NIH (R01 AI045050) to LM.
PKCq modulates LKB1 isoforms downstream of IL-6 signaling to mediate iTreg-Th17 cell plasticity
Authors: Mohan Deeksha , Minter Lisa
Published: January 2023

Following activation, CD4 T cells undergo metabolic and transcriptional changes responding to external cues and differentiate into T helper (Th) cells. This differentiation process was perceived to be terminal. However, it has been shown that T cells exhibit plasticity between subsets in highly inflammatory environments. This phenomenon is prevalent in autoimmune conditions, such as colitis, in which high levels of IL-6 promote plasticity between regulatory T (Treg) cells and Th17 cells. Protein Kinase C theta (PKCq) is a serine/threonine kinase that promotes Th17 differentiation while negatively regulating Treg polarization. The tumor suppressor liver kinase B1 (LKB1) encoded by Stk11, a serine/threonine kinase, enforces energy-preserving mechanisms such as fatty acid oxidation, a major energy source for Tregs. Recent studies demonstrate loss of LKB1 disrupts Treg survival and function. Stk11 can be alternatively spliced into a short variant by the inclusion of a cryptic exon. However, specificity of LKB1 isoforms influencing Th plasticity has not been elucidated. Our study shows that the expression of the Stk11 short splice variant (Stk11S) is upregulated in Th17 compared to induced (i)Tregs. We demonstrate that, in Th17 cells heterogeneous ribonucleoprotein L-like (hnRNPLL), a splicing factor promoting cryptic exon inclusion, binds to transcripts of Stk11 and correlates with increased expression of Stk11S. We further show in iTregs exposed to IL-6, Prkcq, Stk11S, and Rorc are upregulated, suggesting iTreg-Th17 plasticity is induced in response to IL-6 resulting in Stk11 splicing downstream of PKCq. Thus, PKCq may destabilize iTreg programming through its regulation of Stk11 splicing under pro-inflammatory conditions.

Supported by grants from the department of defense.
USP19 Suppresses Th17 Pathogenicity in Autoimmunity
Authors: Bouch Ronald
Published: January 2023

Th17 cells play an essential role in the pathogenesis of murine autoimmune models and human autoimmune diseases. However, Th17 cells exhibit notable plasticity in vivo, adopting pro- or anti-inflammatory effector states in a distinct milieu. The underlying mechanisms that regulate pathogenicity of Th17 cells in health and disease remain unclear. In this study, we investigate the role of the deubiquitinase USP19 in the differentiation and function of Th17 cells. We found that lineage-specific transcription factor RORγt directly binds to ubiquitination-related gene promoters, upregulating the ubiquitination pathway in pathogenic Th17 cells (pTh17). The enhanced ubiquitination activity in pTh17 cells directly correlates with autoimmune disease severity in murine models. The deubiquitinase USP19 suppresses pTh17 differentiation in vitro and Th17-mediated autoimmune disease in vivo. Mechanistically, we found that USP19 removes the K63-linked ubiquitin chain from RORγt lysine 313, resulting in the blocking of the RORγt-SRC3 interaction and ultimately, RORγt activation. Thus, these findings suggest that USP19 selectively suppresses Th17 pathogenicity in murine autoimmune disease.

Supported by P30 Award from NIH, National Cancer Institute of Health (P30CA012197) and NIH T32 Training Grant (5T32A1007401-28)
Insulin specific TCR repertoire analysis reveals functional diversity of Treg TCRs
Authors: Jing Yi , Kong Yuelin , Trout Denise , Bettini Matthew L , Bettini Maria
Published: January 2023

Regulatory T cells (Tregs) are critical in preventing autoimmunity and express a unique TCR repertoire with high affinity to self-antigens. However, whether Treg and effector T cell (Teff) TCR repertoires are similarly divergent in the context of autoimmunity, and whether Tregs are able to maintain TCR affinity advantage has not been clearly defined. Insulin tetramer positive Tregs isolated from pancreatic islets of NOD mice showed a higher level of self-reactivity (CD5) and TCR signaling (Nur77-GFP) than Teffs specific to the same epitope. We isolated and sequenced insulin tetramer+ Teffs and Tregs from islets of NOD mice expressing a fixed TCRα of an insulin specific TCR. Surprisingly, significant overlap was identified between Treg and Teff TCR repertoires, suggesting that similar TCRs can support autoimmune Treg and Teff development and recruitment into the autoimmune response. Both Tregs and Teffs exhibited TCRs with a broad range of reactivity to insulin epitope and Treg TCRs did not show increased reactivity. The disconnect between Nur77-GFP and TCR functional affinity suggests a Treg cell-intrinsic capacity to compete for antigen or to enhance downstream signaling. When re-expressed in vivo using the TCR retrogenic method, Treg derived TCRs showed lower Treg generation and collectively higher diabetogenicity. With comparable Treg generation, low affinity Treg TCR showed significantly lower protection against diabetes than high affinity Treg TCR. These findings indicate that in tissue specific autoimmunity Tregs can express a TCR repertoire with a broad range of functionality and have significant overlap with Teffs of the same specificity, which might negatively affect their suppressive capacity.

This work was supported by the NIH (AI125301-01A1) and The Robert and Janice McNair Foundation.
Profiling transcriptomes, TCR repertoires, and antigenic specificities of islet-infiltrating T cells in Non-Obese Diabetic mice
Authors: Zdinak Paul , Grebinoski Stephanie J , Torrey Jessica , Rathod Sanjay , Ranjan Rashi , Hicks Louise , Joglekar Alok V
Published: January 2023

Type 1 Diabetes (T1D) is an autoimmune disease caused by progressive destruction of pancreatic β-cells, resulting in reduced insulin production. The role of islet-infiltrating CD4+ T cells in mediating β-cell destruction and autoantibody formation is well established. However, many of the antigen specificities of CD4+ T cells which initiate disease are undefined. In this project, we performed single cell RNA sequencing of pancreatic islet infiltrating T cells from 6-, 8-, and 10-week-old Non-Obese Diabetic (NOD) mice. NOD mice recapitulate many immunologic features of T1D and share many autoantigens with human T1D patients. Analysis of TCR repertoires revealed clonal expansion in activated and pre-exhausted effector CD4+ T cells as well as regulatory T cells. Despite subtle differences, expanded effector CD4+ T cells showed a well-defined cell state. We cloned 40 TCRs from the top expanded T cells and expressed them in Jurkat cells. We performed antigen discovery assays on these TCRs using our lab’s Signaling and Antigen-presenting Bifunctional Receptor (SABR) platform. We constructed a SABR library of 4,075 I-Ag7-restricted epitopes based on published datasets. We identified several previously reported and novel autoantigens. Surprisingly, antigenic specificities did not influence the shared phenotype dramatically. These results identify several potential autoantigens that will be valuable for diagnostic, preventative, and therapeutic purposes. Moreover, these results suggest that the islet microenvironment is a major determinant of infiltrating CD4+ T cell states. Similar studies focused on CD8+ T cells in NOD mice and CD4+ and CD8+ T cells in humans are underway.

dkNET New investigator Pilot Program in Bioinformatics (PI: Joglekar) NIDDK New Investigator Gateway Award 1R03DK127447 (PI: Joglekar) PACER Innovative Discovery Award (PI: Joglekar)
Control of medullary thymic epithelial cell development and central tolerance by a zinc finger transcription factor
Authors: Sin Jun Hyung , Costa Juliana Sucharov , Waterfield Michael
Published: January 2023

We have identified a novel function for a zinc finger transcription factor in medullary thymic epithelial cell (mTEC) development and central tolerance. Previous studies have shown that the transcription factor has critical functions in immune cell development, however our study is one of the first to describe a role for the transcription factor in the epithelial lineage. Probing publicly available gene expression databases, we found that the transcription factor is highly expressed in mTECs. Recent studies on mTECs have shown considerable cellular heterogeneity within mTECs with at least five mTEC subtypes including a transient amplifying population (TAC-TECs), autoimmune regulator (Aire+) mTECs, Late/Post Aire mTECs, Ccl21a mTECS, and tuft cells. Using a conditional knockout mouse line, we deleted the transcription factor in mTECs which resulted in drastic changes in the cellular composition of mTECs with substantial increase in Ccl21a mTECs and tuft cells and a dramatic decrease in Aire+ mTECs and Late Aire mTECs. Furthermore, the remaining Aire+ mTECs have a significant decrease in tissue-specific antigen gene expression resulting in autoimmunity in mice. To gain mechanistic understanding of the developmental changes we observed, we performed scRNA-seq and scATAC-seq, which showed significant changes in gene expression and the chromatin landscape in mTECs and helps to explain the critical function for this transcription factor in mTEC development.
The influence of antigen recognition on Treg suppressive function
Authors: Rase Viva Jeanne , Kolawole Elizabeth M , Evavold Brian D
Published: January 2023

Tregs play an essential role in preventing autoimmunity by maintaining effector T cell balance and self-tolerance. Antigen stimulation through T cell receptor (TCR) makes Treg suppression more efficacious and as such is generally viewed as a strong predictor of Treg function. In demyelinating disease Tregs with the most effective suppressive capacity are self-antigen specific, however, it is unclear how the parameters of self-antigen stimulation (affinity, force, and bond lifetime) modulate Treg suppressive potency. Here we utilize the well-described myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide driven experimental autoimmune encephalomyelitis (EAE) to address how Treg self-antigen interactions under chronic inflammation influence suppressive potency. Recently Tregs have been shown to perform trogocytosis to remove MHC molecules from the surface of antigen presenting cells in an antigen specific manner. We hypothesize that trogocytosis correlates with optimal Treg antigen specific suppression in demyelinating disease. To test suppressive capacity of self-antigen specific Tregs we utilized a MOG knockout mouse model of EAE. In this system we show that MOG knockout Tregs are less potent suppressors when challenged with EAE despite identical affinity for MOG35–55 as wild type Tregs. In addition, we show that MOG knockout Tregs perform trogocytosis less readily than wild type Tregs demonstrating that the presence of self-antigen MOG35–55 is necessary for development of optimal Treg generation. An understanding of how Tregs respond to antigen may allow the design of therapeutic strategies to better harness Treg power in combatting autoimmune conditions.

Supported by grants from NIH (5R01NS071518-09)
PD-1 licenses activated Treg for lymphatic migration.
Authors: Piao Wenji , Li Lushen , Saxena Vikas , Hippen Keli , Bruce Blazar , Riella Leonardo , Bromberg Jonathan
Published: January 2023

Rather than serving as fluid transport conduits, the lymphatic vessels have been proved to be critical portals for immune cell migration. As non-immune cell, the lymphatic endothelial cell (LEC) constitutively express high level of programmed death-1 (PD-1) ligand (PD-L1), known as an immune checkpoint protein. We hypothesized that LECs use PD-L1 to regulate PD-1-expressing immune cells during transendothelial migration (TEM). Activated T cells expressed substantially elevated levels of both PD-1 and PD-L1. For human and mouse Tregs PD-1 was in excess of PD-L1, while effector CD4 T cells (Teffs) expressed PD-L1 in excess of PD-1. PD-1−/− but not PD-L1−/− Tregs had reduced migration and motility compared to WT. Engagement by PD-L1 Fc induced rapid activation of Akt but suppressed classical NFkB-P65 signaling in Tregs. WT LECs constitutively expressed high level of surface PD-L1. PD-L1−/− LEC or lymphatic vessels (LV) had increased adhesion and junction proteins VCAM-1 and zonulin-1, and had more zipper junctions. PD-1 triggered PD-L1 signaling in LEC result in strong PI3K/Akt and NFkB-p65 responses that regulated endothelial structure and enhanced TEM. Similar changes to LEC junctions were observed when PD-1high Tregs migrated across LEC layers, resulting in rapidly induced NFkB-p65 translocation into the LEC nucleus. PD-1-induced PD-L1 signaling decreased VE-cadherin expression, which was restored by blocking ERK and PI3K/Akt. Importantly, PD-1-blockade of intra-tumoral Tregs reduced tumor egress and the detained Tregs converted to IFN-g-producing Th1Tregs. These data demonstrate novel roles for Treg PD-1 and LEC PD-L1 in regulation of lymphatic migration and ultimately Treg suppressive function.

Supported by grants from NIH (2R37AI062765-17A1)
cGAS-STING Mediated Inflammation In Diabetic Wound Macrophages Is Regulated By Histone Demethylase JMJD3
Authors: Audu Christopher O , Melvin William J , Wolf Sonya , Sharma Sriganesh B , Mangum Kevin D , Barrett Emily , Joshi Amrita , Gallagher Katherine
Published: January 2023

Recent work has shown that the cGAS-STING inflammatory pathway is elevated in conditions that are marked by chronic inflammation, such as obesity and diabetes. Using single cell RNA (scRNA) sequencing from human wounds, we show that the Sting gene is increased in diabetic wound macrophages. Histone demethylase JMJD3 specifically demethylates lysine 27 on histone 3 (H3K27me3) - a repressive mark for gene transcription. Using diabetic mice, we note on ChIP analysis, a decrease in the H3K27me3 repressive mark at the STING promoter in diabetic wound macrophages relative to littermate controls, with subsequently enhanced STING protein levels by western blot. Additionally, scRNA from wound macrophages obtained from a novel murine strain deficient in Jmjd3 in myeloid-cell lineages (Jmjd3f/f Lyz2Cre+), revealed significantly decreased Sting gene levels. Further, ChIP analysis of H3K27me3 levels at the STING promoter from Jmjd3f/f Lyz2Cre+ wound macrophages reveal increased H3K27me3 levels compared to littermate controls, with a concomitant decrease in STING protein as determined by western blot. Mechanistically, we use mRNA, protein and ChIP studies to reveal that in diabetic wound macrophages, IL6 is responsible for increased JMJD3 leading to increased JAK1,3/STAT3 activity and increased STING production. Wound healing curves from diabetic mice locally treated with macrophage-specific nanoparticles against JMJD3 showed significantly improved healing, and decreased STING protein production in these wounds. Taken together, this data highlights a unique epigenetic regulator of STING production in diabetic wound macrophages and provides a viable cell-specific, nanotherapeutic approach to non-healing diabetic wounds.

Supported by NIH (F32DK126471), Vascular and Endovascular Surgical Society Resident Research Award
Elucidating the mechanism of neutrophil-mediated lung injury in sepsis and the role of CD11d/CD18 integrin on this process
Authors: Koutsogiannaki Sophia , Yuki Koichi
Published: January 2023

Sepsis is the leading cause of death in intensive care unit (ICU) and the most expensive condition treated in the US, without a specific therapy yet available. Acute lung injury (ALI) is one of the most significant organ injuries in sepsis, resulting from massive migration of neutrophils to the lung, but the exact mechanism is not known. β2 (CD18) integrin family [consisting of αLβ2 (CD11a/CD18), αMβ2 (CD11b/CD18), αXβ2 (CD11c/CD18) and αDβ2 (CD11d/CD18)] and its counter-receptor on the endothelium, ICAM-1 (CD54), are critical for neutrophil migration, but their role in ALI is not yet delineated. Using a murine model of sepsis induced by cecal ligation and puncture (CLP) surgery, we showed decreased neutrophil levels and attenuated injury in the lung of β2−/− and ICAM-1−/− mice at 12h post-CLP, suggesting the importance of β2 integrins in this process. In addition, we observed decreased neutrophil levels and attenuated injury in the lung of αDβ2−/− mice but not in the lung of αLβ2−/− and αMβ2−/− mice, suggesting that αDβ2 has an orchestrating role in sepsis-induced ALI. In support, we found increased αDβ2 expression levels on neutrophils both in the lung and blood of WT mice as sepsis progressed. RNAseq analysis in neutrophils from blood and lung of WT and αDβ2−/− mice showed that αDβ2 mediates neutrophil migration to the lung through pathways associated with antigen presentation, apoptosis and NOD-like receptor signaling. αDβ2−/− neutrophils had also less Cxcr2, Ltb4r1 and Dhrs9 expression, associated with neutrophil migration and the retinoic acid pathway. Taken together, our results suggest a mechanism for ALI in sepsis in which αDβ2 integrin has a major role and could be a novel target for therapeutic intervention.

The Anesthesia Research Distinguished Trailblazer Award, Boston Children’s Hospital The William F. Milton Fund, Harvard University