bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell-intrinsic MAVS promotes antigen specific CD8+ T cell proliferation and mitochondrial metabolism
Authors: Vanderheiden Abigail , Valenzuela Renan Aguliar , Verkerke Hans , Jin Fengzhi , Price Madeline , Boss Jeremy M , Grakoui Arash , Suthar Mehul S
Published: February 2023

Mitochondrial antiviral signaling protein (MAVS), the central adaptor protein for RIG-I like receptor (RLR) signaling, is essential for promoting innate immunity against RNA virus infections. MAVS is ubiquitously expressed, however its function in T cells is less clear. Using a mouse model of West Nile virus (WNV) infection, we find that MAVS is required in a cell-intrinsic manner for promoting antigen-specific CD8+ T cell accumulation. Through adoptive transfer experiments, we find that MAVS positively regulates cell cycle genes and promotes entry into proliferation in antigen-specific CD8+ T cells. Mechanistically, knockout of MAVS leads to dysregulated metabolic transcriptional profiles and decreased mitochondrial potential in vivo. Furthermore, we determined that CD8+ T cell receptor stimulation promotes oxidative phosphorylation in a MAVS-dependent manner to facilitate entry into proliferation. Our findings identify a non-canonical role for MAVS as a positive regulator of mitochondrial respiration and proliferation in CD8+ T cells during viral infection.
Frequency and phenotype of SARS-CoV-2-specific CD8+ T cells in healthy and convalescent COVID-19 individuals
Authors: Choy Cecily , Lu Jian , Li Jiangyuan , Dunn Christopher , Wallace Tonya , Nguyen Cuong , McKelvey Julia , Melvin Denise , Egan Josephine , Weng Nan-Ping
Published: February 2023

COVID-19, an infectious disease caused by SARS-CoV-2, has caused the current global pandemic. Despite the number of infections nearing 100 million people worldwide, the CD8+ T cells against SARS-CoV-2 in healthy adults as well as in convalescent COVID-19 patients are not fully characterized. Here, we report an analysis of the frequency and phenotype of circulating CD8+ T cells recognizing seventeen epitopes from three SARS-CoV-2 proteins (S, N, and M) in HLA-A2+ healthy adults and COVID-19 convalescent patients by multi-color flow cytometry with antigen-specific tetramers. Through the analysis of 65 healthy adults ranging between 17 to 98 years of age, we found that the frequencies of CD8+ T cells recognizing SARS-CoV-2 ranged from 10−2 to undetectable. We did not find a generalized age-related change in the frequency of SARS-CoV-2-recognizing CD8+ T cells. Interestingly, we also did not find increased frequencies of SARS-CoV-2-recognizing CD8+ T cells from convalescent patients compared to the healthy controls. Phenotypical analysis showed that SARS-CoV-2-recognizing CD8+ T cells had a mixed naive and memory phenotype in healthy adults whereas convalescent patients mainly had a memory phenotype. Currently, we are identifying the TCRs of these SARS-CoV-2-recognizing CD8+ T cells. Taken together, our findings have identified the frequency and phenotype of CD8+ T cells which recognize SARS-CoV-2-specific epitopes in humans, and future TCR research will further reveal the binding specificity and functionality of these SARS-CoV-2-specific CD8+ T cells.
Concurrent microglial activation in CD8 T cell-mediated CNS vasculature permeability during Theiler’s virus infection
Authors: Khadka Roman , Zheng Jiaying , Ayasoufi Katayoun , Fain Cori E. , Jin Fang , Hansen Michael , Wu LongJun , Johnson Aaron J
Published: February 2023

Immune-mediated blood brain barrier (BBB) disruption is a prominent feature of various neurological conditions for which an emerging role of CD8 T cells is being realized. Our group has developed a unique model of CD8 T cell-mediated BBB disruption which employs Theiler’s murine encephalomyelitis virus (TMEV) infection. Upon intravenous administration of VP2121–130 viral peptide during the peak antiviral adaptive response, Db:VP2121–130 epitope specific CD8 T cells induce BBB disruption in a perforin dependent manner. In this model, we addressed the role of microglia in relation to antigen specific CD8+ T cells and permeable cerebral vessels. Using real time two-photon in vivo imaging, we demonstrate that microglia adopt distinct morphological features, including enlarged cell body and fewer ramified processes as early as 6 hours post administration of VP2 peptide. Notably, microglial population exhibit robust down-regulation of homeostatic markers including CX3CR1 during BBB disruption. This study demonstrates CD8 T cells can promote microglia activation in a perforin-dependent manner during BBB disruption. Importantly, microglial activation occurs concurrently with the onset of vascular permeability and could serve as a critical cell type in this process.
Skin-derived alternatively spliced IL-15 isoform-producing cells impair the generation of anti-viral effector CD8+ T cells in HSV-1 zosteriform mouse model
Authors: Ku ChiaChi , Wu Kwan-Lun , Chien Po-Ya
Published: February 2023

We previously reported a Pedigree 191 (P191) of N-ethyl-N-nitrosourea (ENU) mutagenized mice that highly expressed an alternatively spliced IL-15 isoform with deletion of the first 16 amino acids in exon 7 (IL-15ΔE7). Infection of P191 mice with herpes simplex virus-1 (HSV-1) via flank skin generated a prolonged and early re-emerged HSV-1 zosteriform rash compared to wild type (WT) C57BL/6 mice. Using Kb/gB498–505 MHC Class I tetramer provided by NIH Tetramer Core Facility, we found that the numbers of gB-specific CD8+ T cells generated in draining lymph nodes (dLN) of P191 mice were greatly reduced over 10-day course after HSV-1 flank infection compared with WT mice. As expressions of IFN-γ, granzyme B and perforin were induced in WT CD8+ T cells by in vitro restimulation with gB peptides, they were poorly expressed in P191 CD8+ T cells. While HSV-1 skin infection induced migration of Langerhans cells into dLNs accompanied with increased IL-15 expression in WT mice, the number of migrated Langerhans cells was less in P191 and they preferentially expressed IL-15ΔE7 rather than IL-15. The reverse relationship between the cell number of Langerhans cells and the increased expression of IL-15ΔE7 mRNA in dLN of P191 mice suggested that the influx of skin-derived IL-15ΔE7 expressing cells plays an important role in influencing the quantity and quality of anti-HSV-1 CD8+ T cells generated in P191 mice. More experiments will be carried out to understand how the alternatively cytokine splicing mechanism regulates a long term and effective CD8+ T cell immunity against virus infection.
Defining cellular correlates of protection and vaccine failure to influenza across two human cohorts
Authors: Mettelman Robert C , Souquette Aisha , Van de Velde Lee-Ann , Allen E. Kaitlynn , Wood Timothy , Krammer Florian , Petrie Joshua G , Martin Emily T , Monto Arnold S , Huang Q. Sue , Thomas Paul Glyndwr
Published: February 2023

Influenza viruses are endemic viral pathogens causing mild to severe respiratory illness in humans. Immunologic protection against influenza is determined by immune correlates of protection– factors associated with reduced infection or severe disease. While antibodies specific to viral surface proteins are known correlates, waning seasonal vaccine efficacy and reported infection of patients despite elevated antibody titers suggest that humoral responses alone do not provide complete protective immunity. Indeed, evidence points to a larger role for cell-mediated immunity (CMI; innate cells and antigen-specific T cells) in conferring protection. CMI correlates, which act independently from humoral responses, have yet to be identified. Here, we analyzed samples from adult human subjects across two influenza infection and vaccination cohorts to identify distinct CMI correlates of protection to influenza. We profiled CMI responses using high-dimension flow cytometry from PBMCs collected pre- and post-exposure to influenza infection or vaccination. Statistical comparison of cell frequency and infection status identified candidate CMI correlates, which were validated using logistic regression accounting for vaccination, demographic (age, sex, BMI), and serologic (antibody) covariates. We identified 9 individual and 6 cell clusters across myeloid and lymphoid compartments associated with protection. The strongest correlations were observed in Th17, cTfh, and subsets of NK and dendritic cells. Further, AUROC models identified 3 baseline CMI infection classifiers. Lastly, our study identified correlates of vaccine failure– pre-exposure CMI profiles correlated with positive infection status despite vaccination.
Analyses of the interactions of tapasin and ERp57-tapasin proteins with PaSTa 1 and PaSTa 2 antibodies and MHC-I molecules
Authors: Taylor Daniel Kyle , Boyd Lisa F , Jiang Jiansheng , Mage Mike M , Cresswell Peter , Margulies David H , Natarajan Kannan
Published: February 2023

The intracellular loading of major histocompatibility complex class I (MHC-I) molecules with high affinity peptides is a pre-requisite for their cell surface display as ligands for T and NK cells. Peptide acquisition occurs in the ER within the peptide loading complex (PLC) and is accomplished through the concerted action of several proteins, most notably tapasin. In the absence of tapasin, peptide loading is compromised, resulting in decreased cell surface expression of some, though not all, MHC-I allelomorphs. In order to gain a mechanistic understanding of tapasin function as well as its apparent allelomorph specificity we have produced recombinant Fab fragments of two anti-tapasin antibodies, PaSTa 1 and PaSTa 2, and investigated their binding to human recombinant soluble tapasin and ERp57/tapasin by SPR methods. Both antibody Fabs bind with nanomolar affinities characterized by slow off rates. Based on these results we developed an MHC-I binding assay employing PaSTa 1-captured tapasin or ERp57/tapasin which reveals affinities in the micromolar range for selected MHC-I alleles. These affinities are notably weaker than those obtained for the structurally related but PLC-independent chaperone, TAPBPR (TAP-binding protein, related), and may reflect distinct evolutionarily derived functions for the two related molecules.
Penalized regression analysis identifies features of the peptide:MHC class I interaction and mRNA expression as key to prioritizing neoantigen immunogenicity
Authors: Borden Elizabeth S , Phung Tanya N , Buetow Kenneth H , Lafleur Bonnie , Wilson Melissa A , Hastings Karen Taraszka
Published: February 2023

Current approaches to prioritization of tumor-specific neoantigens have low sensitivity and specificity, directly impacting the development of personalized vaccines and the prediction of immunotherapy efficacy. Many biological features have been suggested to contribute to accurate neoantigen prioritization. We calculated predictors for a comprehensive set of biological features for presentation and recognition of neoantigens on a validated neoantigen dataset. One thousand random, evenly-split combinations of immunogenic and non-immunogenic neoantigens were analyzed using penalized regression (lasso) for variable selection. This analysis isolated the peptide:MHC class I dissociation constant, binding stability, and mRNA expression as the key predictive variables of tumor-specific neoantigens. Based on these results, we fit a logistic regression model and tested its performance on three independent datasets. We compared our results to the models by Wells et al., Łuksza et al., and Zhou et al. Our model has improved performance as measured by the area under the receiver operator curve (AUC) compared to Łuksza and Zhou et al. Our model selects the same terms as Wells et al. and performs equivalently. However, it has the advantage of providing a predicted immunogenicity score for each individual neoantigen. Individual antigen approaches are useful for prioritizing vaccine candidates and predicting response to immunotherapy. Future work will focus on the application of this predictor to patient response to immunotherapy.
Kidney function, age, and education as contributors to depression and anxiety in juvenile systemic lupus erythematosus
Authors: Poole Brian D , Figueiredo-Braga Margarida , Silva Beatriz , Ganhão Sara , Aguiar Francisca , Cornaby Caleb , Brito Iva
Published: February 2023

Background:

Juvenile systemic lupus erythematosus (JSLE) is diagnosed in children younger than 18 years of age. Depression and anxiety are common, but not well understood in JSLE. We investigated the clinical and psychological factors associated with the psychological manifestations of JSLE.

Methods:

Twenty-nine JSLE patients were recruited for the study. They responded to surveys testing their psychological status and perceptions about their health, including the Hospital Anxiety and Depression scale, the Fatigue Severity Scale, and a Medical outcomes questionnaire (the SF-36). Clinical lab results were obtained from the patients’ medical records. Univariate and multivariate analyses were used to compare the patient populations with adult-onset SLE (ASLE) patients and unaffected controls, finding indicators that correlated with depression and anxiety in JSLE patients.

Results:

Kidney disease was associated with depression in the JSLE patients. The BUN levels, BUN/creatinine ratio, and leukocyturia were all significantly associated with depressive symptoms. The BUN/creatinine ratio was the most predictive laboratory value for both depression and anxiety.

The JSLE patients had less depression than the ASLE, although not less anxiety. JSLE patients had comparable levels of fatigue and pain to the adult-onset SLE patients. Age and education were protective against depression in the JSLE patients.

Conclusions:

Kidney function correlated with symptoms of depression and anxiety. This may mean that symptomatology is an important indicator of whether the patient needs psychiatric care. Age and education were likely protective against depression in the JSLE population.
Androgen-associated increases in Bacteroides abundance correlate with enhanced immunoregulation and suppression of lupus in BWF1 mice.
Authors: Ma Jing , Harder James W , Alard Pascale , Kosiewicz Michele M
Published: February 2023

Similar to humans, lupus incidence is higher in female than male NZBxNZW1(BWF1) mice, and castration abolishes male protection implicating androgens in disease suppression. Gut microbiota and metabolite compositions differ between female and male BWF1 mice; males have higher abundance of Bacteroides and the metabolites, phytol and phytanic acid (RXR agonists that can increase retinoic acid, RA). Male cecal transfers into females suppress disease which correlates with an increase in Bacteroides in recipients. In addition, female gut CD103+ dendritic cells (CD103+DC) have decreased ability to induce pTreg due to an RA deficiency (decreased RALDH2) that can be restored by male cecal transfer. The goal of this project was to study androgen effects on these processes. We found that male castration altered microbiota and metabolite compositions similarly to those found in female mice, e.g., reduced Bacteroides, and phytol and phytanic acid abundance. Castrated male cecal transfer did not suppress disease or increase Bacteroides abundance in recipients. As in females, castrated male CD103+DC had decreased ability to induce pTreg (decreased RALDH2) but induced high levels of IFNγ. And although androgen receptor mRNA was undetectable in CD103+DC, male mice treated with androgen receptor inhibitor (flutamide) had the same negative effect on CD103+DC function as castration, suggesting androgens affect CD103+DC indirectly. Interestingly, flutamide-treated and castrated male microbiota looked very similar, e.g., reduced Bacteroides. These data suggest that androgens in males increase Bacteroides abundance via receptor signaling in host cells that may in turn play a role in enhancing immunoregulation and suppressing lupus.
Regulators of Type III Cytokines in Type III Innate lymphoid Cells Identified by CRISPR Activation and Inhibition Screens
Authors: Brown Rachel Anne , Dangel Andrew W , Saini Ankita , Oltz Eugene M
Published: February 2023

Type III cytokines interleukin (IL)-22 and IL-17 are expressed by both innate and adaptive immune cells to defend against extracellular pathogens, yet their dysregulation contributes to autoimmunity and malignancy. A deeper knowledge of IL-22/17 regulation is warranted to understand physiologic processes as well as disease pathogenesis, thereby elucidating druggable targets. Toward this goal, we applied genome-wide CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi) screens to identify the collection of factors that positively or negatively regulate IL-17/22 expression in a murine type III innate lymphoid cell (ILC3) model. Following lentiviral transduction of a CRISPRi or CRISPRa guide RNA (gRNA) library, cells were stimulated and sorted by levels of intracellular IL-22/17 protein expression. Relative gRNA frequencies in each sequenced population revealed enrichment and depletion of potential regulators (“hits”), which were then tested with gene-specific gRNAs to validate or exclude them from further study. These genome-wide screens have illuminated hits from several biological pathways, ranging from transmembrane channels to RNA splicing. We identify previously known regulators, including IL23R, STAT3, and ZFP36, as well as novel potential regulators, including SLC family members and a nuclear protein called SON. Our screens provide a diverse array of candidate factors that govern expression of type III cytokines by ILC3s, with potential for extrapolation to other cytokine-expressing cells. Genome-wide gain and loss of function screens are powerful, complementary tools to facilitate regulatory pathway discovery.
IL-27 enhances cytokine secretion by TIGIT+ HIVGag-specific T cells
Authors: Cheng Jie , Myers Timothy G. , Kumar Princy , Kumar Jai , Goshu Bruktawit A , Catalfamo Marta
Published: February 2023

In chronic HIV infection, HIV-specific T cells have diminished effector function and fail to control/eliminate the viral reservoirs. HIV-specific T cells and latently HIV infected T cells express checkpoint receptors suggesting that therapeutic strategies improving T cell function and targeting the reservoir will be required to treat HIV infection. IL-27, a member of the IL-6/IL-12 cytokine superfamily, can be considered as such agent because of its anti-viral properties and immune modulatory functions. IL-27 signaling activates mainly Signal Transducer and Activator of Transcription (STAT) 1 and 3. IL-27 can inhibit HIV replication, however, its immunomodulatory functions in T cells are largely unknown. In this study, we hypothesized that IL-27 promotes pro-inflammatory properties enhancing HIV-specific T cell immunity. We found that IL-27 enhanced STAT1 activation in T cell subsets from people living with HIV (PLWH). Moreover, the RNA-Seq analysis showed that IL-27 stimulation induced global transcriptional changes that overlapped with both IFNα and IL-6 signaling. We also determined that the T cells response to IL-27 stimulation were enriched in genes associated with IFN/STAT1 signaling pathways in PLWH and healthy donors. In addition, overnight in vitro stimulation with IL-27 upregulated expression of CD69 and T-bet in CD4 and CD8 T cells. Accordingly, IL-27 increased IFN-ɣ and TNF-αsecretion by TIGIT+ HIVGag specific CD4 and CD8 T cells. The previously described anti-viral properties and these newly immunomodulatory features of IL-27 in HIV-specific T cell function suggest its potential therapeutic use in HIV cure strategies.
Systematic dissection of cytokine responses in vivo at single-cell resolution
Authors: Cui Ang , Huang Teddy , Li Shuqiang , Ma Aileen , Perez Jorge , Keskin Derin , Sander Chris , Wu Catherine J. , Fraenkel Ernest , Hacohen Nir
Published: February 2023

Cytokines mediate a highly complex intercellular signaling network in health and disease. While many individual cytokines have been studied in-depth, we do not have a systems-level view of cell-type-specific responses to each cytokine and do not yet understand how cytokines orchestrate cell-cell communication networks in a complex immune response. To address these two gaps in cytokine biology, we performed single-cell RNA sequencing to measure gene expression profiles of over 20 mouse lymph node cell populations in response to over 80 cytokines in vivo. We first investigated how distinct cell types respond to the same cytokine and found that while some pathways are activated across multiple cell types, many cytokines induced cell-type-specific gene expression signatures. Next, we compared responses across cytokines and found that cytokines induced both unique and shared biological processes. For example, in NK cells, we found that IL-2/7/12/15/27/33/36a, IFN-a1/b/g/e/k, LIF, CT-1, and NP each induced a cytolytic program, but also cytokine-specific gene expression programs involved in antigen presentation, NF-kB activation, and mRNA splicing. Finally, based on our compendium of cytokine signatures, we created an algorithm to assess the level of cytokine responses and reconstruct cell-cell communication networks, and used this approach to uncover cytokine mediators in checkpoint blockade cancer immunotherapy treatment. Our study provides a global view of cell-type specific cytokine responses in vivo. The framework can be readily applied to other transcriptomic datasets for assessing the roles of cytokines and cell-cell communication networks in any immune response.
BATF regulates innate lymphoid cell hematopoiesis and homeostasis
Authors: Liu Qingyang , Kim Myung H. , Friesen Leon , Kim Chang H
Published: February 2023

Early hematopoietic progenitors undergo sophisticated developmental processes to become committed progenitors for innate lymphoid cells (ILC) in hematopoietic tissues, which ultimately become mature ILC subsets in the periphery. Basic leucine zipper ATF-like transcription factor (Batf) plays important roles in lymphocyte biology. We report that Batf regulates the production of bone marrow ILC progenitors and maintenance of peripheral ILCs. Batf is most highly expressed in ILCs among all lymphocytes. Data from in vivo and in vitro ILC differentiation experiments revealed that the expression of Batf is induced in early ILC progenitors at the α-lymphoid progenitor stage in response to the cytokine IL-7. Our Chromatin immunoprecipitation and retroviral gene transfer experiments revealed that up-regulated Batf binds and activates transcription of the Nfil3 gene to promote ILC hematopoiesis. Data from bone marrow chimera and parabiosis experiments support that Batf is intrinsically necessary to maintain normal numbers of early and late ILC progenitors in the bone marrow and mature ILC1, ILC2, ILC3, and NK cells in most peripheral tissues. Batf-deficient mice have ILC3 lymphopenia and fail to control Citrobacter rodentium infection. Moreover, ILC2 responses to cytokines were defective in Batf-deficient mice. Thus, Batf is required for normal ILC responses to inflammatory cytokines and bacterial infection. These data suggest that Batf plays fairly comprehensive roles in supporting bone marrow hematopoiesis, maintenance, and effector functions of ILCs.
Exploring the B cell microenvironment in von-Hippel Lindau (Vhl) Dmp1-specific knockout mice
Authors: Chicana Betsabel J , Abbasizadeh Nastaran , Burns Christian , Spencer Joel , Manilay Jennifer
Published: February 2023

Osteoblasts (OBs) and osteocytes (OCYs) have been shown to support B cell development. The von-Hippel Lindau protein (VHL) regulates hypoxia-inducible factor degradation and is involved in responses to low oxygen environments. We utilized Dmp1-Cre;Vhl conditional knockout mice (cKO), in which Vhl is deleted in OBs and OCYs. The cKO display dysregulated bone growth, high bone density, smaller bone marrow (BM) cavity volume, and overall decrease in BM cellularity compared to wild-type (WT) mice. B cell progenitors were significantly increased in frequency and numbers whereas Hardy Fractions B, C, D, E and F were significantly decreased in the cKO BM. cKO Fraction B-C cells showed an increase in apoptosis and decreased proliferation. Reciprocal BM chimeras showed significant reduction in Fractions B-C and later Fractions in the WT→cKO, while B cell development was normal in the cKO→WT chimeras, indicating a cell-extrinsic effect of Vhl on B cells within the cKO BM microenvironment. We hypothesize that B cell development is not supported due to reduction of key niche cells such as stromal cells, OBs, mesenchymal stem cells and decreased production of key cytokines. In preliminary studies, we found that CXCL12 and SCF, key cytokines for B cell development, were reduced in the cKO BM serum. We have begun longitudinal studies using intravital and ex vivo imaging of the cKO BM to identify temporally dependent changes in oxygenation, blood vessel type, structure, permeability, and location of stromal cells. Ex vivo long bone imaging of cKO BM in 6-month-old mice revealed an increase in blood vessel diameter compared to WT. These studies will reveal details and novel molecular mechanisms by which Vhl in Dmp1 expressing cells may serve as a B cell niche.
IFNs induce prolonged hematopoietic suppression via IL-18-induced cell death during shock-like Ehrlichial infection
Authors: Howard Jennifer E , Cruz Jose Luis , MacNamara Katherine C
Published: February 2023

The long-term effects of shock-like infection on hematopoietic stem and progenitor cells (HSC/HSPC) has not been carefully investigated. Using Ixodes ovatus ehrlichia (IOE), a tick-born pathogen that causes severe shock-like illness and bone marrow (BM) aplasia, we explored mechanisms that cause hematopoietic suppression. During IOE infection, type I IFNs promote loss of HSPCs via direct (cell death) and indirect mechanisms (quiescence). Mice lacking IFNαβ receptor (IFNαR−/−) had improved survival relative to WT mice. IFNγR−/− mice had profound BM aplasia and mortality, like WT mice, however mice lacking both type I and II IFN receptors (IFNαR;IFNγR−/−) had protected HSPCs and improved survival, even relative to IFNαR−/− mice. WT and IFNγR−/− mice had reduced HSPCs and increased levels of IL-18 relative to IFNαR−/− and IFNαR;IFNγR−/− mice, while IL-18R expression induced during IOE required IFNγ signaling. IL-18-deficiency prevented IOE-induced BM aplasia and reduced HSC quiescence and necrotic (7AAD+) cells. To test whether IL-18R-signaling acted on HSPCs directly, we generated WT: IL-18R−/− mixed BM chimeras. IL-18R−/− cells had an advantage over WT cells and IL-18R was intrinsically required for increased HSPC cell death. In mixed BM chimeras given antibiotics to allow recovery from acute infection, we found that two weeks post-infection IL-18R−/− HSPCs were present at higher numbers and IL-18R−/− cells had greater functional output in colony-forming unit assays. Our data support IFN driven hematopoietic suppression through IFNαR-dependent induction of IL-18 and IFNγR-dependent IL-18R expression, and that direct effects of IL-18 on HSPCs during infection-induced shock may have long-lasting effects on HSCs.
B7-CD28 co-stimulation enforces central tolerance by thymic clonal deletion and Treg cell generation through distinct mechanisms
Authors: Watanabe Masashi , Lu Ying , Breen Michael , Hodes Richard J
Published: February 2023

To establish central T cell tolerance in the thymus, thymocytes expressing TCR strongly reactive to self-Ag-MHC complex are eliminated from the conventional T cell (Tconv) repertoire by two distinct processes, clonal deletion or clonal diversion to non-conventional T cell lineages such as Foxp3+ regulatory T (Treg) cells. We analyzed the requirements for B7-CD28 co-stimulation for thymic clonal deletion and Treg cell generation of endogenous tissue-restricted-antigen (TRA)-specific thymocytes. We found that: 1) both clonal deletion and Treg cell generation of TRA-specific thymocytes were B7-CD28-dependent, and that clonal deletion had a greater effect than Treg diversion in eliminating TRA-specific T cells from the Tconv repertoire; 2) B7-expressing APC requirements differed for clonal deletion and Treg generation; and 3) although B7-CD28 mediated both clonal deletion and Treg cell generation, CD28 cytoplasmic domain requirements for these two processes were different. These results indicate that qualitatively distinct CD28 signaling determines fates of strongly self-reactive thymocytes. To further characterize the CD28 signaling pathways involved in clonal deletion and Treg cell generation, single cell transcriptomic analyses of TRA-specific thymocyte are underway.
CCR4 and CCR7 differentially regulate thymocyte subset localization with distinct outcomes for central tolerance
Authors: Li Yu , Lancaster Jessica N , Nam Seohee , Seo Hyemin , Ehrlich Lauren Ilyse Richie
Published: February 2023

Central tolerance is established as T cells mature in the thymus by the combined effects of clonally deleting autoreactive thymocytes or inducing their diversion to the Treg lineage. To ensure T cell tolerance to diverse self-antigens, thymocytes must efficiently enter the medulla and scan heterogeneous antigen presenting cells, which display the majority of the peptidome in a sparse mosaic. While it is widely accepted that CCR7 contributes to central tolerance by promoting thymocyte medullary localization and rapid migration therein, the contribution of CCR4 to these processes remains unclear. For example, it is not known whether CCR4 is simply redundant to CCR7 in promoting medullary entry or if it plays a distinct role in central tolerance. Here, we report that early post-positive selection thymocytes undergo chemotaxis towards CCR4 ligands, while progressively mature cells migrate towards CCR7 ligands. Synchronized positive selection assays reveal that CCR4 is upregulated within hours of positive selection signaling, while CCR7 upregulation is delayed. 2-photon microscopy shows that CCR4 facilitates entry of early post-positive selection cells into the thymic medulla, while CCR7 promotes medullary accumulation of mature subsets. These findings suggest that CCR4 and CCR7 have non-redundant roles in negative selection. Consistent with this possibility, flow cytometric analysis reveals that CCR4 deficiency results in a selective decline in early-stages of negative selection, while CCR7 deficiency impairs only late-stage negative selection. Our data support a new model in which CCR4 and CCR7 mediate medullary entry of different thymocyte subsets and play distinct roles in enforcing central tolerance.
Mapping the thymic neuronal connectome using rabies virus retrograde and anterograde tracers
Authors: Ayasoufi Katayoun , Yokanovich Lila T , Pfaller Christian K , Jin Fang , Johnson Aaron J
Published: February 2023

Immune organ innervation by the peripheral nervous system enables autonomic function in the development and maintenance of immunity. Specifically, the role of thymic innervation in health and following neurological insults is unknown. An in depth identification of the connectome within the thymus and the extent to which nervous impulses delivered to the thymus affect T cell development and other biological processes within the thymus is needed. We sought to determine the exact nature of innervation within the thymus. Using both flow cytometry and confocal microscopy, we determined that the thymus contains both extrinsic neuronal processes (cell bodies outside of the thymus), and surprisingly, intrinsic neurons (cells bodies within the thymus). We further verified that subsets of neurons within the thymus express tyrosine hydroxylase, BIII tubulin, and/or NeuN, and are distinct from epithelial cells (EPCAM+, UEA1+, Ly51+) and tuft cells (DLCK1+). Furthermore, neuronal cell bodies found within the thymus did not express AIRE, consistent with their identity as neurons. Subsets of intrinsic neurons within the thymus express MHCII implying a potential role in T cell development. Importantly, using rabies virus retrograde and anterograde neurotracers, we mapped a novel, previously undescribed, connectome of neurons within the thymus. Finally, we determined that both epithelial cells and neurons are significantly reduced in RAG deficient mice, indicating a possible co-regulation between thymic neurons and epithelial cells. In summary, we describe a novel network of neurons within the thymus and put forward implications of the thymic connectome’s role in T cell development and establishment of thymic architecture.
AI-guided use of balanced peroxisome proliferator-activated receptor a/g dual agonist finetunes macrophage responses in inflammatory bowel disease
Authors: Katkar Gajanan Dattatray , Sayed Ibrahim , Anandachar Mahitha Shree , Castillo Vanessa , Vidales Eleadah , Toobian Daniel , Usmani Fatima , Taheri Sahar , Dang Dharanidhar , Sawires Joseph , Leriche Geoffray , Yang Jerry , Sandborn William , Das Soumita , Sahoo Debashis , Ghosh Pradipta
Published: February 2023

A computational platform, the Boolean network explorer (BoNE) has recently been developed; it enables querying and navigating invariant Boolean Implication Networks of disease maps for prioritizing high-value targets. Here we used BoNE derived Inflammatory Bowel Disease (IBD)-map and prioritize two nuclear receptors, PPARa and PPARg. Balanced agonism of PPARa/g was predicted to impact macrophage function, ameliorate colitis in network-prioritized animal models, ‘reset’ the gene expression network from disease to health. Predictions were validated using a balanced and potent PPARa/g-dual agonist (PAR5359) in two murine models, i.e., Citrobacter rodentium- and DSS-induced colitis. Mechanistically, we show that balanced dual agonists promote bacterial clearance more efficiently than individual agonists both in vivo and in vitro, through the controlled induction of pro-inflammatory cytokines, cellular ROS and gut-barrier protection. PPARa is required and its agonism is sufficient to induce the pro-inflammatory response that is essential for bacterial clearance and immunity, but PPARg-agonism blunts these responses, delays microbial clearance. Balanced agonists achieved controlled inflammation, barrier protection and reversed the network towards the healthy side of disease. When tested on IBD-patients-derived PBMCs, PAR5359 reversed the defective bacterial clearance observed in these subjects. These findings not only deliver a macrophage modulator in IBD but also highlight the potential of BoNE to accelerate and enhance the precision of drug discovery in various diseases.
Unconjugated bilirubin modulates Th17-cell immunity by curbing glycolysis-related genes
Authors: Longhi Maria Serena , Vuerich Marta , Wang Na , Kalbasi Ahmadreza , Moss Alan C. , Cheifetz Adam S. , Robson Simon C.
Published: February 2023

Unconjugated bilirubin (UCB) controls Th17-cell function by upregulating the ectonucleotidase CD39 and through activation of aryl-hydrocarbon-receptor (AhR), a modulator of adaptive immunity. UCB-induced CD39 upregulation is defective in Th17-cells of Crohn’s disease (CD) patients. Increasing evidence suggests a role for AhR in regulating glucose metabolism. In this study we tested whether UCB modulates Th17-cell metabolism and whether impaired Th17-cell response to UCB results in metabolic alterations in CD.

Th17-cells were differentiated from circulating CD4-cells of 31 healthy controls (HC) and 22 CD patients. Using a multiplex gene expression panel including genes of 8 metabolic pathways, we found that UCB markedly downregulated glycolysis-related genes, particularly phosphoglycerate-kinase-1 (PGK1) and aldolase-A (ALDOA); this effect being prominent in Th17-cells derived from HC but not from CD patients. Reduced Pgk1 and AldoA was also noted in mesenteric-lymph-nodes of mice exposed to dextran-sulfate-sodium-induced colitis and treated with UCB.

Functionally, UCB decreased the extracellular-acidification-rate and oxygen-consumption-rate of HC Th17-cells, while this effect was not noted in Th17-cells of CD patients. In vitro silencing of PGK1 and ALDOA further enhanced UCB immunoregulatory properties in HC Th17-cells, by boosting CD39 and Tim-3 while containing IL-17.

In conclusion, UCB modulates Th17-cell immunity by downregulating glycolysis-related genes; this property being defective in cells obtained from CD patients. Strategies aimed at blocking/inhibiting glycolytic genes might represent a potential therapeutic tool to restore immune homeostasis in CD and other inflammatory conditions.
Understanding NLRs and AIM2 cellular and molecular signalling in glioblastoma pathophysiology
Authors: Jha Sushmita , Singh Shalini , Saxena Shivanjali , Sharma Nidhi , Agrawal Ishan , Govekar Rukmini , Panchal Pranali , Sridhar Epari , Gupta Tejpal
Published: February 2023

Gliomas are the most prevalent primary brain tumors with immense clinical heterogeneity, poor prognosis and survival. Gliomas arise from glial cells and are heavily infiltrated with innate immune cells. In India high-grade gliomas or Glioblastomas (GBM) account for 59.5% of CNS tumours. A key challenge for clinical management of GBM is its highly heterogeneous nature-between patients and within a single tumour-making current therapies ineffective. The nucleotide-binding domain, and leucine-rich repeat containing receptors (NLRs) and absent-in-melanoma 2 (AIM2) are innate immune receptors crucial for initiation and progression of several cancers. There is a dearth of reports investigating NLRs and AIM2 in glioma pathology.

We are interested in studying the cellular and molecular contribution of NLRs and AIM2 in malignant gliomas in the Indian subcontinent. We previously reported a data-driven approach to identify NLRs, AIM2 and NLR-associated gene expression in gliomas using The Cancer Genome Atlas (TCGA) patient datasets. However, TCGA data is obtained from diverse populations and cellular diversity within individual tumor tissues remains unexplored. In this study we carried out tumour primary cell culture, human brain immunohistochemistry and proteomic analysis and same patient serum controls (n=100). Our results characterize novel cell specific differential expression and signalling profile of several NLR genes (including NLRP3, NLRP12 and ASC) in microglia, astrocytes, endothelial cells and the tumour cell populations in GBM patient tissue. We provide critical insights into innate immune signalling within the GBM microenvironment and identify key markers for future therapeutic interventions.
Evaluating the effect of chronic schistosomiasis on the gut microbiome at the species level
Authors: Smith Jade A , Mhanna Mariam A , Shollenberger Lisa M
Published: February 2023

Schistosomiasis is a neglected tropical disease caused by the trematode helminth Schistosoma mansoni (S. mansoni). Schistosomiasis infection, which is caused by parasitic worms, is a globally important parasitic disease, second only to malaria in its devastation. There is growing evidence that, by shifting the TH1 and TH2 responses, helminth infections can adversely affect the immune system and one expected way is by perturbing the gut microbiota. However, little is known about the effects of chronic schistosomiasis on the composition of the gut microbiota of its mammalian host. Therefore, we collected fresh fecal samples from BALB/c mice (n=3) during the 10 weeks necessary for establishment of a chronic schistosomiasis infection. We then evaluated the composition of the fecal microbiome after 16S rRNA sequencing using open-source software (Qiita). Here, we characterized fecal microbiota changes by analyzing the relative abundance of several specific operational taxonomic units (OTUs) at the species level. Our long-term goal is to deduce the potential relationship between S. mansoni and the functional aspects of these OTUs, thereby understanding the subsequent effect this alteration has on the host. These data support a modification of the mammalian gut microbiome composition by a chronic schistosomiasis infection. These functional changes serve as a foundation for the design of mechanistic studies to unravel the complex relationship between the microbial composition of the mammalian gut microbiome, chronic S. mansoni infection, and the host adaptive immune response.
Mitofusin-2 regulates leukocyte adhesion through the maturation of beta2 integrin activation in differentiation
Authors: Fan Zhichao , Liu Wei , Wang Yueyang , Hsu Alan Y , Lin Tao , Sun Hao , Pachter Joel S , Groisman Alex , Imperioli Matthew , Yungher Fernanda Wajnsztajn , Hu Liang , Wang Penghua , Deng Qing
Published: February 2023

Neutrophils are critical in inflammation and innate immunity. Neutrophil adhesion to vascular endothelium is a crucial step in neutrophil recruitment. Mitofusin-2 is required for neutrophil adhesion, but molecular details are unclear. Here, we demonstrated that β2-integrin-mediated slow-rolling and arrest, but not PSGL-1-mediated cell rolling, are defective in mitofusin-2-deficient neutrophil-like HL60 cells. This adhesion defect is associated with reduced expression of fMLP receptors and β2 integrins as well as the inhibited β2 integrin activation, as assessed by conformational-specific antibodies. Mitofusin-2 deficiency limited the maturation of β2 integrin activation during the neutrophil-directed differentiation of HL60 cells, which is indicated by identified markers CD35 and CD87. Reversed to results in overall HL60 cells, mitofusin-2 knockdown in β2-integrin-activation-matured cells (CD87high population) enhanced integrin activation. Our study illustrates the function of mitofusin-2 in leukocyte adhesion and may provide new insights into the development and treatment of mitofusin-2-deficiency-related disease.
PD-L1 reverse signaling in dermal dendritic cells promotes dendritic cell migration required for skin immunity.
Authors: Lucas Erin D , Schafer Johnathon , Tamburini Beth
Published: February 2023

While the function of the extracellular region of programmed death ligand 1 (PD-L1) through its interactions with PD-1 on T cells is well studied, little is understood regarding signaling via the intracellular domain of PD-L1, termed PD-L1 reverse signaling. The objective of this study is to determine how PD-L1 reverse signaling impacts dendritic cell (DC) function during immune responses. We outline a major role for PD-L1 reverse signaling in the control of DC migration from the skin to the draining lymph node (dLN). We find that total loss of PD-L1 on DCs impairs migration to the dLN following an innate immune stimulus. Using a mutant mouse model, we identify a TSS signaling motif within the intracellular domain of PD-L1. This TSS motif proves critical for promoting chemokine mediated DC migration to the dLN during inflammation. The loss of DC migration, in the PD-L1 TSS mutant, leads to a significant decline in T cell priming when DC trafficking is required for antigen delivery to the dLN. We found that the TSS motif is required for chemokine receptor signaling, and disruption of this motif impairs signaling downstream of the Gα subunit of the heterotrimeric G protein complex, leading to impairment in ERK phosphorylation and actin polymerization in DCs. Finally, we demonstrate that ligation of PD-L1 by one or more of the known binding partners of PD-L1, CD80 and PD-1, appears to support migration of DCs to the dLN. These studies identify a novel function for PD-L1 reverse signaling in supporting DC migration during initiation of immune responses, and raises questions about the potential impact of PD-L1 antibody blockade on DC function in patients with cancer.
Dopamine induces functional extracellular traps in microglia
Authors: Agrawal Ishan , Sharma Nidhi , Saxena Shivanjali , Arvind S. , Chakraborty Debayani , Chakraborty Debarati Bhunia , Jha Deepak , Ghatak Surajit , Epari Sridhar , Gupta Tejpal , Jha Sushmita
Published: February 2023

Dopamine (DA) is central to neurological functions like motivation, movement and reward (Klein et al. 2019). While its role in diseases like Parkinson’s, Alzheimer’s and schizophrenia are well studied, its role in regulating immune functions have just started to unfold. DA receptors are present on almost all immune cells and they regulate innate and adaptive immunity (Matt and Gaskill 2020). We have investigated the role of DA in the formation of extracellular traps (ETs) in microglia. We report that DA can induce ETs in BV2 microglia cell line and primary adult human microglia. ETs are composed of decondensed chromatin and are embedded with proteins including antimicrobial proteins like myeloperoxidase and histone. We confirmed ETs formation by staining them with DAPI, myeloperoxidase and/or DNA/Histone H1. The presence of traps in culture supernatant was confirmed by measuring SYTOX Green absorbance. To check for the mechanism of ETs formation by BV2 cells, we performed MTT assay and treated cells with N-acetyl L-cysteine or cytochalasin D. We saw that the traps were formed independent of cell death, reactive oxygen species (ROS) and actin polymerization. We further confirmed that DA induced microglia traps are functional as they were able to trap FITC tagged Escherichia coli. Lastly we checked the presence of microglia ETs in Glioblastoma multiforme tissues as recently glioma cells have been reported to secrete DA and possess DA receptors (Caragher et al. 2019). We confirmed and quantified the traps present in GBM by an unbiased algorithm. Our findings demonstrate that DA may play significant role in sterile neuro-inflammation by inducing microglia ETs.
Gut derived T cells in the joint display an activated regulatory phenotype following tissue injury
Authors: Lefferts Adam R , Norman Eric , Kantheti Uma , Kuhn Kristine A
Published: February 2023

Numerous studies have implicated a gut-joint axis in the pathogenesis of spondylarthritis (SpA), but the exact mechanisms that connect the intestine and joint remain unclear. Here we sought to identify one potential mechanism, the direct trafficking of immune cells from gut tissue to the inflamed joint enthesis, the site of injury in SpA. To track gut-derived cells in vivo, we utilized the KikGR transgenic mouse model, in which colonoscopy-guided exposure to 405nm light causes photo-conversion of the transgene and labeling of cells in situ. In this manner we selectively label the epithelium, including intraepithelial lymphocytes (IELs), while avoiding cells in the lamina propria. Within 24 hours after photo-conversion, with a maxima at 72 hours, gut-labeled T cells appear in the Achilles enthesis. This trafficking occurs constitutively, with gut-derived T cells comprising ~10% of total tissue T cells. Following the induction of inflammation by the injection of 20mg of Complete Freund’s Adjuvant (CFA) into the hind hock, we observed 25.2% of gut-derived T cells were FoxP3+ compared to 4.8% in the unlabeled population (p=0.0063). Within this Foxp3+ subpopulation we also observed significant enrichment of CD69 (p=0.0013) and a trend towards increased CD103 (p=0.065) expression, suggestive of memory residence. These enrichments were not observed in the contralateral un-injected hock, implying that inflammatory mediators are required for the recruitment of gut-derived regulatory T cells to establish residence. These data demonstrate bona-fide gut joint trafficking of T cells and suggest a regulatory role for mIELs. Ongoing work focuses on potential functional differences in mIELs derived from healthy vs inflamed mucosa.