bluuebunny/crossref_metadata_2025 huggingface dataset:
Association between bovine viral diarrhea virus load in subsets of peripheral blood mononuclear cells (PBMC) in persistently infected animals and health outcome
Authors: Falkenberg Shollie M. , Dassanayake Rohana , Walz Paul , Neill John , Ridpath Julia
Published: January 2023

Bovine viral diarrhea virus (BVDV) can cause acute and persistent infections. BVDV acute infection results in generalized immunosuppression characterized by lymphocytopenia typically associated with depletion of CD4+ and CD8+ T cells. BVDV persistent infection is the result of immune tolerance and is not associated with lymphocytopenia. The health outcome of persistently infected (PI) calves varies widely; some die of mucosal disease, some succumb to ill thrift and others survive to adulthood. Detection of BVDV at the single lymphoid cell level is important to the study of subsets of PBMC in acute and persistent infections, however there are few methods available for the detection and quantification of BVDV at the single cell level. To circumvent this difficulty, a novel PrimeFlow RNA assay using in-situ detection of BVDV was developed. This assay was used to evaluate differences in viral distribution within subsets of PBMC over time in PI calves carrying one of two different species of BVDV (type 1 and type 2). Calves were sampled at 3 different time points at least one month apart. During the course of the study, a subset of the calves died from ill thrift. Mucosal disease was not indicated in any of the deaths. Using RNA probes specific for the BVDV Npro-Erns coding region for each respective virus, BVDV RNA was detected in all PBMC of PI that appeared clinically healthy. Calves that succumbed to ill thrift were found to have no or little virus in T cells. The clearance of virus from T cells suggests a breakdown in immune tolerance in these calves. This is the first report of an association between viral load in the T cell populations and survival in PI calves.
Dietary supplementation of Allium hookeri improved intestinal immune response on necrotic enteritis of young broiler chickens
Authors: Lee Sung Hyen , Lee Youngsub , Lillehoj Hyun S. , Kim Jung-Bong , Jang Hwan-Hee , Hwang Kyung-Ah , Choe Jung-Suk , Kim Dong-Wook
Published: January 2023

This study was conducted to evaluate the effects of dietary Allium hookeri on intestinal immune response of young broiler chickens induced with necrotic enteritis (NE). One hundred fifty birds (1 day old) were randomly assigned to 6 groups (n =25 birds/treatment) and fed a basal diet (control) or basal diet supplemented with Allium hookeri (AH) root at 1 or 3% of the basal diet; AH1 and AH3, respectively. Birds were orally challenged with E. maxima 41A (1 × 104 cells/chicken) on day14 and followed by C. perfringens infection (1 × 109 CFU/chicken) on day18. NE-associated infection and intestinal immune response were assessed by average body weight gain, lesion score and oocyst shedding. The effect of dietary supplementation with AH on transcript levels of pro-inflammatory cytokines, tight junction and mucin protein in the jejunum were quantified by qRT-PCR. At day 20, the birds fed 3 % of AH in diet (AH3) significantly weighted more than the control group though the NE-challenge significantly reduced average body weight gain compared to the uninfected control group. Among the NE-challenged groups, gut lesion score and oocyst shedding significantly decreased in the birds given with AH (1 or 3%) compared to the control group. Gene expression levels of pro-inflammatory cytokines IL-8, IL-17α, and LITAF decreased in AH3 groups compared to the infected control group though the levels significantly increased in the NE infected groups. Interestingly AH significantly increased transcript levels of intestinal tight junction proteins and mucin at 1% of diet, not 3% of diet. These results suggest that dietary supplementation of AH can improve chicken health by controling immune response against NE and it depends on the dietary level of AH.
Lactation stage impacts the glycolytic function of CD4+ T cells during ex vivo activation
Authors: Eder Jordan M. , Gorden Patrick J. , Lippolis John D. , Reinhardt Timothy A. , Sacco Randy E.
Published: January 2023

Dairy cattle undergo dynamic physiological changes during pregnancy and lactation. The impact of these physiological changes on the immune system can, at times, leave the animal more susceptible to infection with specific pathogens. While research has shed light on periods of increased susceptibility to infection, the impact of the stage of lactation on immune cell function remains to be completely elucidated. We have examined the impact of stage of lactation on CD4+T cells. We focused on how their metabolic function may be altered during this period. During activation T lymphocytes increase the uptake of glucose and metabolically reprogram to support aerobic glycolysis instead of oxidative phosphorylation, the pathway favored during their resting state. Our ex vivo studies compare the effects of activation on metabolic function of CD4+T cells from dairy cattle across multiple stages of lactation. Results show dampened glycolytic function in early lactation stages. Glycolytic and mitochondrial function were analyzed using the Seahorse Extracellular Flux Analyzer. Cattle in early and mid-lactation, defined as 21–70 days in milk (DIM) and 71–150 DIM, respectively, have a decreased rate of glycolysis and reduced maximum glycolytic capacity in contrast to cattle in late lactation (151–330 DIM) and those that have been dried off and are pre-parturient. Mitochondrial results were comparable between the groups, suggesting no compensation of energy via the mitochondria in early and mid-lactation cows. CD4+T cells from dairy cattle in early and mid-lactation have decreased metabolic function as measured by glycolytic and mitochondrial respiration in contrast to those in late lactation or in the dry period.
The Florida manatee (Trichechus manatus latirostris) T cell receptor loci exhibit V segment locus synteny and chain-specific evolution
Authors: Breaux Breanna Laine , Hunter Margaret E. , Cruz-Schneider Maria Paula , Sena Leonardo , Bonde Robert K. , Criscitiello Michael F.
Published: January 2023

Manatees are a vulnerable, charismatic sentinel species from the evolutionarily divergent order Afrotheria. We have previously described the immunoglobulin heavy chain locus of the Florida manatee (Trichechus manatus latirostris) and found limited antigen receptor diversity in this humoral arm of the adaptive immune system. We therefore investigated the antigen receptor loci of the cellular arm: the T cell receptor. Manatees are the first species from either Afrotheria or Xenarthra, the most basal eutherian taxonomic groups, to have an in-depth characterization of all T cell receptor loci and expressed repertoires. We manually annotated the three TR loci on the T. m. latirostris genomic scaffolds using BLAST and RSSsite, and compared the segments and overall genomic organization to the human TR loci. We identified the human V segment subgroups that are conserved in T. m. latirostris and found that TRADV are most conserved, TRBV are moderately conserved, and TRGV are the least conserved. The V segment order in the TRAD and TRB loci also showed synteny between the two species. The TRAD locus had five V segment subgroup synteny blocks that showed long-range duplications in the T. m. latirostris locus. The TRB locus had three V segment subgroup synteny blocks that showed short-range duplications. The conservation of V segment sequence and order correlated to the locus complexity, emphasizing the role of genomic organization on gene evolution. We also identified evidence for a VHδ pseudogene for the first time in a eutherian mammal. These novel findings underline the value of including species within the basal radiation of eutherian evolution in comparative studies.
GITR/GITRL interaction in the lung provides signal 4 for T cell expansion and TRM formation
Authors: Chu Kuan Lun , Wang Kuan Chung , Batista Nathalia V. , Clouthier Derek , Zhou Angela C. , Watts Tania H.
Published: January 2023

Influenza remains an important global threat and it is important to identify key mechanisms by which the immune system handles the virus. TNFR family members can play a crucial role in determining the magnitude of T cell response against viral infections. Previous studies using TCR transgenic models showed that GITR, an NF-κB activating TNFR family member, is required on CD8 T cells for maximal response against influenza virus. However, it remains unknown how GITR affects the endogenous T cell response during influenza infection. Using competitive mixed bone marrow chimeras, we found that GITR is intrinsically required for the accumulation of effector CD4 and CD8 T cells in the lung and secondary lymphoid organs during influenza infection as well as for optimal lung Trm formation. GITR affected PA224–233-specific CD8 T cells more dramatically than NP366–374-specific CD8 T cells, which exhibited a compensatory increase in TCR affinity in the absence of GITR. GITRL expression was higher on inflammatory APCs compared to classical DCs with peak expression day 3 to day 5 post-infection in the lung. We observed peak GITR expression on CD4 T cells day 7 post-infection in the lung but earlier for CD8 T cells. The finding that both the receptor and the ligand are expressed in the lung raises the possibility that GITR costimulation plays an important role in the lung. Consistently, within the same mouse, lung GITR+/+OT-II had a higher level of pS6 (downstream of GITR signaling) than GITR−/− OT-II, providing evidence of GITR costimulation (signal 4) in the lung. In sum, GITR on CD4 and CD8 T cells plays an important role in their accumulation in the lung, likely through interaction with GITRL on inflammatory rather than classical DCs.

Funded by CIHR:MOP133443
IRF4-dependent DCs regulate T cell effector and memory responses in influenza virus infection
Authors: Ainsua-Enrich Erola , Hatipoglu Ibrahim , Kadel Sapana , Turner Sean , Paul Jinny , Singh Simar , Bagavant Harini , Kovats Susan
Published: January 2023

Dendritic cells (DCs) activate adaptive immune responses during respiratory virus infection. A number of transcription factors govern the development and function of tissue DCs, including IRF4. To determine the role of IRF4-dependent lung DCs in influenza virus infection, we studied CD11c-cre-Irf4f/f (KO) mice. In this KO, one lung resident CD11b+ DC subset is missing, and the other DC subsets lack IRF4. Upon infection with A/PuertoRico/8/34 virus, KO lung DCs contained increased IL-12 and reduced IL-10 RNA. This altered IL-12/IL-10 balance in KO mice led to increased numbers of T-bet+ IFNg+ influenza antigen-specific effector CD8+ and CD4+ T cells, yet reduced numbers of Foxp3+ T regulatory cells (Tregs) and CD8+ antigen-specific memory precursor effector cells (MPECs) at later stages of infection. The remaining MPECs in KO mice showed decreased expression of Foxo1 and Bcl6, key transcription factors important for T cell memory differentiation. This suggests that factors including IL-10 produced by IRF4-dependent DCs elicit a memory-biased transcriptional program in late CD8+ T cell effectors. Accordingly, KO lungs contained reduced numbers of resident CD8+ memory T cells (TRM) forty days post-infection. Upon rechallenge with heterosubtypic virus, KO mice showed deficient expansion of virus-specific IFNγ+ CD8+ T cells. Taken together, these data show that KO mice mount a stronger antiviral T cell response upon primary infection but then elaborate a weaker CD8+ T cell memory response to heterologous virus. Our data support the hypothesis that IRF4-dependent DCs normally act to promote the differentiation of CD8+ memory T cells during influenza virus infection.
SIV-specific CD8 T cells are largely excluded from B cell follicles during early SIV infection
Authors: Li Shengbin , Connick Elizabeth , Skinner Pamela J.
Published: January 2023

While HIV− and SIV-specific CD8 T cell responses are critical for suppression of virus producing cells, these cells fail to fully suppress viral replication. During chronic infection, one potential mechanism for this failure is relatively low levels of virus-specific CD8 T cells in B cell follicles, where virus is most concentrated, permitting ongoing virus replication. It is not known whether this phenomenon also occurs during early infection. Here, we determined the location, abundance, and phenotype of SIV-specific CD8 T cells in lymph nodes from SIV-infected rhesus macaques at 21 days post-infection. We found that levels of SIV-specific CD8 T cells in B cell follicles were significantly lower than in extrafollicular regions. Within follicles, SIV-specific CD8 T cells were largely excluded from germinal centers. A median of 62% of follicular SIV-specific CD8 T cells expressed PD-1. In addition, a median of 12% of follicular SIV-specific CD8 T cells were in direct contact with Foxp3+ cells, and 4% were themselves Foxp3+. Ki67 was expressed by a median of 41% of follicular SIV-specific CD8 T cells and 25% expressed high levels of perforin. These data suggest that during early stages of infection, low levels of follicular SIV-specific CD8 T cells may permit ongoing viral replication, similar to what we previously reported in chronic disease. Furthermore, these data suggest that follicular SIV-specific CD8 T cells are possibly exhausted, that subsets may be inhibited by Foxp3+ Tregs, that subsets are activated and dividing, and subsets express the effector molecule perforin suggesting cytolytic potential. These findings provide important insights into SIV immunopathogenesis and may help inform future cure strategies.
Profiling TCR affinity distribution in human CD8+ T cell subsets responding to a cytomegalovirus epitope
Authors: Williams Chad Manjeot , Ma Keyue , He Chenfeng , Sun Eric , Zhang Shuqi , Jiang Ning
Published: January 2023

CD8 T cell subsets have long been distinguished using phenotypic markers like CCR7, CD45RA, and IL-7Rα. These markers have allowed for the isolation of CD8 T cell populations with distinct functional and proliferative capacities as well as those that maintain better proliferative responses in vivo without antigenic stimulation thus contributing more to immunological memory. Although phenotypic analysis has allowed for the discerning of functional, proliferative, and homeostatic proliferative potential, there is still little known about the role TCR affinity plays in recruitment of various affinity TCRs into memory and effector phenotypes that generate long term immunological memory in human infection. Using the newly developed iTAST technique, CMV specific CD8 T cells were isolated from the blood and spleen of human patients seropositive for CMV. CMV specific T cells were split into memory and effector subsets using phenotypic markers CCR7, CD45RA, and IL-7Rα before isolation and quantification of 2D TCR affinity and sequence. A roughly 100-fold difference of 2D affinity was present in CMV specific cells within the immunological memory response; however, with an enrichment for high affinity TCRs across all phenotypes assessed. This is consistent with previous studies demonstrating high avidity TCR selection in CD8 T cell responses to CMV infection, coupled with a greater clonality observed in CMV specific TCRs within healthy older individuals infected with CMV. Overall the iTAST technique allows for quick and easy isolation of antigen specific populations, quantification of TCR affinity, and TCR sequence thus giving unique insight into immunological memory responses in individuals infected with human persistent viruses.
Comparative analysis of DR1 binding with H1N1 Hemagglutinin derived peptides
Authors: Osan Jaspreet Kaur , Kuhn Thomas
Published: January 2023

Binding of antigenic peptides to class II MHC molecules (MHCII) and the activity of the “editing” molecule HLA-DM (DM) on the resulting peptide-MHCII complexes are factors critical to the antigen presentation pathway. Different algorithms are available to predict peptide-MHC binding and identify CD4+ T-cell epitopes within pathogenic proteins. Fewer algorithms are focused on MHCII as compared to MHCI., and their reliability is also less solid. Possibly one reason for such difference lies in the greater complexity of peptide binding to MHCII, of which open-ended binding site features a larger flexibility and allows interaction with longer sequences. To validate algorithm predictions, we have compared the experimental IC50 of a library of overlapping 18-mers offset by 5 amino acids spanning the sequence of H1N1 influenza virus HA protein while interacting with HLA-DR1 with the one indicated by different prediction platforms online. The IC50 values for each peptide in the presence and absence of DM were measured using competition binding assay. The ex-vivo CD4+ T cell response to these sequences has already been published, on which basis they have been categorized into immunodominant, subdominant, weak and negative. Comparative analysis shows that measured IC50 is different from predictive IC50, with measured IC50 with DM showing immunodominant peptide as a top hit. Our data also shows that presence of DM reduced the affinity of all the peptides triggering the smaller CD4+ T cell response or negative, whereas the effect on the other peptides was less consistent. Thus, currently available algorithms are still inadequate to predict epitope for MHCII and DM is one important factor that should be considered when designing algorithms for MHCII.
Butyrophilin (BTN) molecules control γδ T cells in tissue epithelia
Authors: Rhodes David A. , McCarthy Neil , Trowsdale John , Eberl Matthias
Published: January 2023

Gammadelta (γδ) T cells are a lineage of innate-like T lymphocytes with potent cytotoxic, pro-inflammatory and regulatory properties. They express, as a defining feature, a T cell receptor (TCR) composed of a γ and δ chain heterodimer, both products of V(D)J recombination, which distinguishes them from conventional αβ T cells.

Although restricted to anatomical locations including skin and intestinal epithelium, major sites of interaction with the microbiota and of autoimmune pathology, the role of γδ T cells in immune surveillance is poorly understood. It is now clear that butyrophilins (BTNs) are pivotal in the maintenance of immune homeostasis in tissue epithelium, by controlling activation of γδ T cells. Despite homology to B7 costimulatory receptors like CD80/86 and PD-L1, ligands for BTN molecules have remained elusive. Some data show a direct interaction of BTNs with the γδ TCR, although this remains to be confirmed.

We aim to understand the function of BTNs in the regulation of γδ T cells and how their dysregulation contributes to disease. The model system we study is the human BTN3A proteins and their role in the activation of Vγ9/Vδ2 T cells, a prominent γδ T cell lineage in human blood and tissues. Since it is clear that specific BTN molecules control defined γδ T cell subsets, this work is relevant to other systems of γδ T cell activation. I will highlight how these new data on the molecular control of a major T cell compartment is relevant to human autoimmunity and cancer.
Targeted mass spectrometry identifies computationally predicted, public, leukemia associated minor histocompatibility antigens
Authors: Vincent Benjamin , Kirkpatrick christine , Lansford Jefferson , Wilkerson Emily , Herring Laura , Chai Shengjie , Hunsucker Sally , Armistead Paul
Published: January 2023

Background

Allogeneic stem cell transplantation (aSCT) remains the most effective form of immunotherapy for the treatment of high risk acute myeloid leukemia (AML). The alloreactivity that induces the so-called “graft versus leukemia” (GvL) effect is mediated by minor histocompatibility antigens (mHA) presented by recipient class I or II HLA that induce a donor derived T cell response.

Methods

To identify candidate GvL mHA, we mapped RNA-Seq data from HLA-A*02:01 transfected U937 cells to the human reference genome to identify high frequency SNPs that are expressed in hematopoietic tissue, leukemia or testis and not in GvHD target organs such as skin, colon or liver. All 8 to 11-mer amino acids containing identified SNPs were computationally evaluated for binding affinity to HLA-A*02:01, and we validated predictions using genotyping data from 101 AML patients who underwent aSCT. Peptides from highly expressed genes and with predicted high affinity to HLA-A*02:01 were synthesized. Targeted mass spectrometry using parallel reaction monitoring (PRM) compared the MS/MS spectra of a peptide epitope pool to those of the synthesized peptides.

Results

Our computational analysis predicted 63 candidate mHA, of which the 48 most promising candidates were selected for targeted MS based upon gene expression and predicted HLA-binding affinity. Of the 48 best candidates, 4 HLA-A*02:01 restricted peptide epitopes were identified by mass spectrometry: 3 different epitopes derived from CD33 and 1 derived from CD300a.

Conclusion

Combined computational prediction and targeted mass spectrometry is an efficient method for the identification of candidate GvL antigens that can be further tested for immunogenicity.
Chaperone-assisted peptide exchange on MHC-I is driven by a negative allostery release cycle: Implications for a role of peptide-editing Molecular Chaperones in scrutinizing the peptide repertoire
Authors: Sgourakis Nikolaos , McShan Andrew C. , Natarajan Kannan , Kumirov Vlad K. , Flores-Solis David , Jiang Jiansheng , Badstuebner Mareike , Kovrigin Evgenii L. , Margulies David H.
Published: January 2023

Molecular chaperones TAPBPR (TAP-binding protein related) and tapasin associate with major histocompatibility complex class I (MHC-I) to promote the loading of antigenic peptides through a poorly understood mechanism. Here, we use solution Nuclear Magnetic Resonance (NMR) spectroscopy to probe TAPBPR-induced changes on MHC-I. Dynamic motions present in the empty MHC groove become progressively dampened with increasing peptide occupancy, while allosteric communication between the A- and F-pockets regulates a conformational switch located near the TAPBPR binding site, which is crucial for chaperone release from the complex. Our analysis of NMR data recorded for a range of TAPBPR complexes prepared with both murine H2 and human HLA alleles complements the recent X-ray structures to provide atomic-resolution mechanistic insights into the selection of optimal peptide sequences for the displayed antigen repertoire. In particular, our results show that negative allosteric coupling between the MHC groove and chaperone binding sites allows TAPBPR to proofread MHC molecules containing a range of different peptides. Since the affinity of incoming peptides for the empty groove is greatly reduced in the chaperone complex, (micromolar range, relative to nanomolar for the free MHC), these interactions can provide a mechanism for optimizing the peptide repertoire, where only the highest-affinity peptides can drive chaperone release. Finally, our results suggest that TAPBPR may promote the dissociation of tightly bound peptides from MHC molecules, thereby further scrutinizing the displayed repertoire. These findings imply a similar mechanism for the specificity and editing function of tapasin in the peptide-loading complex.
Self-reactive T cells restricted by non-classical MHC Ib are associated with metabolic disease
Authors: Park Chansu , Guan Jian , Ding Tom , Gonzalez Federico , Shastri Nilabh
Published: January 2023

The normal peptide repertoire presented by classical and non-classical MHC class I molecules is regulated by ERAAP, the ER aminopeptidase associated with antigen processing. Loss of ERAAP’s peptide trimming function in cells causes dramatic changes in the peptide repertoire. The changes in the peptide repertoire enhance the immunogenicity of ERAAP-deficient cells and elicit potent immune responses in otherwise syngeneic wild-type mice. Normal ERAAP function is monitored by an unusual subset of semi-invariant CD8+ T cells that recognize the QFL ligand. The QFL ligand consists of a peptide encoded by the highly conserved FAM49a/b genes that is presented by Qa-1b, a non-classical MHC Ib molecule only on surface of ERAAP-deficient cells. We show that these QFL-specific CD8+ T cells (QFL-T cells) bear unique and semi-invariant αβ TCRs. Genetic manipulation of the expression of the self-QFL ligand and functional characteristics of QFL-T cells shows that in addition to monitoring ERAAP function, QFL-T cells may also regulate metabolic activity.
MHC class I expression by microglia is required for generating a complete antigen-specific CD8 T cell response in the CNS
Authors: Malo Courtney Sarah , Jin Fang , Hansen Michael J. , Fryer John D. , Pavelko Kevin D. , Johnson Aaron J.
Published: January 2023

Microglia are central nervous system (CNS) resident immune cells for which the impact of MHC class I antigen presentation is still being defined. To directly address if microglia contribute to CD8 T cell-mediated immunity of the CNS, we generated a novel tamoxifen inducible CX3CR1-creER H-2Kb MHC class I conditional knockout mouse. In this transgenic mouse, the H-2Kb MHC class I molecule is deleted on all CX3CR1-expressing cells following tamoxifen treatment. By two weeks post tamoxifen treatment, peripheral CX3CR1-expressing cells repopulate, restoring normal expressing of H-2Kb. This leaves only microglia as the sole MHC class I-deficient cell type. We hypothesized that deletion of H-2Kb on microglia would reduce CD8 T cell responses in the CNS. We challenged these animals and cre-negative littermates with intracranial picornavirus infection with Theiler’s murine encephalomyelitis virus expressing ovalbumin (TMEV-OVA). We also challenged mice with experimental GL261 gliomas. We found that microglia-specific H-2Kb deletion reduces the CD8 T cell response in the CNS and attenuates the activation status of brain-infiltrating CD8 T cells in both models of neuroinflammation. We conclude that microglia are functional antigen presenting cells, and are required for a complete antigen-specific CD8 T cell response in the brain. These findings are the first to definitively address the role of microglia-expressed MHC class I in CNS immunological challenges.
Oct1 and OCA-B in T cells are potent therapeutic targets for autoimmune diseases
Authors: Kim Heejoo , Shakya Arvind , Dickey Laura , Stone Colleen , Lane Thomas , Williams Matthew A , Tantin Dean R
Published: January 2023

Oct1 and OCA-B function together in CD4 T cells to promote poised gene expression states in immunomodulatory target genes (e.g. Il2, Il21, Ifng, Icos and Cd25) via the recruitment of the histone lysine demethylase Jmjd1a/Kdm3a. Oct1 and OCA-B are also critical for the generation of functional CD4 memory T cells in response to pathogen infection. Memory T cell phenotypes can promote autoimmunity, even in cases of persistent self-antigen exposure. In GWAS, single nucleotide polymorphisms in Oct1 binding sites for multiple genes are associated with T1D and MS. These findings suggest that Oct1 and OCA-B could modulate autoimmunity. Using a new T cell-specific OCA-B conditional-deleted Non-obese diabetic (NOD) mouse model, we find that T cell specific OCA-B deletion completely protects the animals from T1D. This is associated with reductions in pathogenic, pancreatic-infiltrating T cell numbers and cytokine production. In contrast, pancreatic lymph node T cell function is normal, suggesting that non-self-directed immune function is largely intact. Moreover, we have developed OCA-B inhibitor peptides as novel therapy for T1D and shown these to be efficacious in NOD mice. In EAE, which is a model for MS, Oct1 loss in T cells decreases clinical scores, T cell infiltrate and cytokine production. However, using a JHMV intracranial infection model, Oct1 loss has only minimal effects in clinical scores, viral clearance, infiltrating immune cells and cytokine production. These results suggest a therapeutic window in which deleting these proteins blunts autoimmunity while keeping normal immune function largely intact.
REGULATION OF T FOLLICULAR CELL DEVELOPMENT BY E3 UBIQUITIN LIGASE CBL-B IN SYSTEMIC LUPUS ERYTHEMATOSUS
Authors: tu sha , Tang Rong , Guo Hui , Liu Qing jun , Zhang Jian
Published: January 2023

T follicular helper cells (Tfh) are critical in providing help to B cells, and have been shown to be involved in the overproduction of pathogenic auto-Abs and tissue damage in lupus. Cbl-b, a RING finger E3 ubiquitin ligase, negatively regulates lymphocyte activation. In this study, we found that the expression of Cbl-b in CD4+ T cells from lpr mice on a C57BL/6 background (B6-lpr) was down-regulated. Introducing Cbl-b deficiency or Cbl-b C373A mutation into B6-lpr mice significantly exacerbated the onset of the disease in B6-lpr mice, which correlated with heightened serum auto-antibody titers, exaggerated Tfh, GC B cells, and plasma cells. These data indicate that Cbl-b negatively regulates lupus disease progression and Tfh development via its E3 ubiquitin ligase activity. In support of a critical role of Cbl-b in Tfh development, mice specifically deficient for Cbl-b in T cells also exhibited an increase in Tfh when they were immunized with OVA in CFA. Mechanistically, Cbl-b appeared to specifically bind to Bcl6, and target Bcl-6 for ubiquitination and degradation. In strong support of Cbl-b in exaggerated Tfh in lupus, we found that Tfh cells were increased in the peripheral blood of SLE patients. Furthermore, Cbl-b expression was significantly reduced in Tfh from patient with SLE. Therefore, our data indicate that Cbl-b suppresses Tfh development by targeting Bcl6, and that deregulated expression of Cbl-b may play a crucial in aberrant Tfh development, thus eliciting pathogenic auto-Ab production by B cells.
Single-cell transcriptome analysis reveals diverse islet-infiltrating T cell subsets and a role for BATF in promoting the diabetogenic activity of CD8 T cells
Authors: Ciecko Ashley E , Schauder David M , Foda Bardees M , Lin Chien-Wei , Cui Weiguo , Chen Yi-Guang
Published: January 2023

T cells infiltrate pancreatic islets and directly mediate the destruction of insulin-producing β cells during the development of type 1 diabetes (T1D). However, islet-infiltrating T cell differentiation states and functional diversity have not been completely defined. We used unbiased single-cell RNA sequencing analyses to define the phenotypic complexity of islet-infiltrating T cells in non-obese diabetic (NOD) mice. In the CD4 T cell compartment, we identified naïve, memory, and regulatory T cells, as well as multiple Il21 expressing effector subsets positive for markers indicative of Th1 and Tfh cells. In the CD8 T cell compartment, we identified naïve cells and two activated subsets defined by Slamf6 or Cxcr6 expression, respectively resembling the self-renewing progenitor cells and terminally differentiated effectors found during chronic lymphocytic choriomeningitis virus (LCMV) infection. Single-cell regulatory network inference and clustering (SCENIC) analysis revealed that regulon activity of several transcription factors with known roles in effector CD8 T cell function including Batf were turned on in the Slamf6+ and Cxcr6+ cells. Previous studies have shown that IL-21 induced BATF expression in CD8 T cells is critical to sustain their effector function against chronic LCMV infection. Similarly, we found lower BATF expression in activated islet CD8 T cells from NOD. Il21+/− compared to NOD mice. We further demonstrated that overexpression of BATF in β cell autoreactive CD8 T cells eliminated their need for IL-21 to cause T1D. Our results reveal phenotypically diverse and novel islet-infiltrating T cell subsets and suggest a model in which the IL-21-BATF pathway is critical for the diabetogenic activity of CD8 T cells.
Age-associated changes in central T cell tolerance induction
Authors: Cepeda Sergio , Hester Allison K , Yangming Xiao , Kraig Ellen B , Griffith Ann V
Published: January 2023

Susceptibility to certain autoimmune diseases increases with age; however, mechanisms linking aging and increased susceptibility are incompletely understood. One hallmark of the aging immune system is atrophy of the thymus, the primary site of T lymphocyte generation. We and others have previously shown that aging is also associated with declines in critical thymic functions, including expression of tissue restricted self-antigen (TRA) genes, many of which are regulated by the autoimmune regulator, Aire. TRA expression in the thymus allows T cells to be tolerized to antigens typically expressed in peripheral tissues, and declining TRA expression is predicted to contribute to diminished T cell tolerance in aging. Medullary thymic epithelial cells (mTECs) had been regarded as the sole source of thymic TRA expression, but it is now clear that a subset of thymic B (tB) cells also express Aire and mediate tolerance to Aire-dependent self-antigens. Recently, we found that expression of Aire and Aire-dependent self-antigens declines in tB cells in aged mice and humans, which is also predicted to diminish central T cell tolerance induction. To test these predictions, we used MHCII tetramers to detect T cells recognizing a model TRA expressed in mTECs, Apolipoprotein B (Apob, an auto-antigen involved in atherosclerosis), in the thymus and secondary lymphoid organs of aged mice. We find that the frequency of potentially auto-reactive ApoB-specific T cells increases beginning at 6 months of age. Ongoing experiments will address whether there are similar increases in T cells specific for other TRAs, including Titin (Ttn), an autoantigen involved in late onset myasthenia gravis.
Rab4A inactivation in T cells blocks mTOR activation, pro-inflammatory lineage development, and disease pathogenesis in lupus-prone mice
Authors: Huang Nick , Wyman Brandon , Cravo Emma , Winans Thomas , Choudhary Gourav , Oaks Zachary , Duarte Manuel , Lewis Josh , Lai Zhi-Wei , Banki Katalin , Perl Andras
Published: January 2023

Introduction

Expression of Rab4A is increased in T cells of patients and mice with SLE. Overexpression of Rab4A forms a positive feedback loop with mTOR that can be blocked with therapeutic efficacy in SLE. To determine the impact of this gene on disease development, the triple-congenic lupus-prone Sle1.2.3 mouse strain (TC) have been backcrossed with C57Bl/6 wild-type (WT) mice that carry floxed Rab4AQ72L (TC-FL) alleles or lack Rab4A in T cells (TC-KO).

Methods

Proteinuria was assessed by the Bradford assay. Splenocytes were examined by flow cytometry. Autoantibody production was measured by ELISA.

Results

TC mice had increased proteinuria over age and sex-matched WT controls. Deletion of Rab4A in T cells reduced proteinuria ≥ 50% in female TC-KO mice relative to TC-FL mice at 21 or 40 weeks of age. Similar trends were noted in male mice. The production of antinuclear and antiphospholipid antibodies was reduced in TC-KO mice as compared to TC-FL and parental TC controls.

Immunophenotyping unveiled a 45% depletion of CD4+ T cells and 50% expansion of CD8+ T cells in female TC-KO mice relative to TC-FL controls. CD38 expression was reduced on CD4+ T cells of TC-KO mice by 51% and 48% relative to TC and TC-FL controls. CD38 expression was also reduced on CD8+ T cells but not on CD19+ B cells. mTORC1 activity was reduced by 36% in CD4 T cells, but not in CD8 T cells or B cells of TC-KO mice. Along these lines, overexpression of Rab4A activated mTORC1, reduced expression of CD4, and increased expression of CD38 in Jurkat cells.

Conclusion

These findings reveal an opposite influence of Rab4A on endosomal recycling of CD4 and CD38 that facilitates mTORC1 activation and thus causes pro-inflammatory T-cell lineage specification and triggers autoimmunity in SLE.
Nfkbid overexpression in NOD mice elicits complete type 1 diabetes resistance
Authors: Dwyer Jennifer R , Racine Jeremy J , Quinlan Anna , Presa Maximiliano , Chapman Harold D , Serreze David V
Published: January 2023

Type 1 diabetes (T1D) in both humans and the NOD mouse model is caused by the autoimmune mediated killing of pancreatic β-cells. Increased frequency or activity of autoreactive T cells and failures of regulatory T cells (Tregs) to control autoreactive T cells have both been implicated in T1D etiology. Due to the expression of MHC-I molecules on β-cells, autoreactive CD8+ T cells are likely the ultimate mediators of this destruction. Developing CD8+ T cells undergo selection processes in the thymus whereby most autoreactive clonotypes are eliminated during their development. Fine mapping of a genetic locus associated with defective thymic deletion of autoreactive CD8+ AI4 TCR transgenic (AI4) T cells in NOD mice led to the identification of Nfkbid, a NFκB signal modulator, as contributing to this phenotype. Ablation of Nfkbid in NOD mice by CRISPR/Cas9 results in a decreased frequency of autoreactive CD8+ AI4 T cells and an unexpected acceleration of T1D incidence, associated with reductions in the frequency and functional capacity of peripheral Tregs. Transgenic overexpression of Nfkbid in NOD mice paradoxically results in further decreased levels of circulating autoreactive CD8+ AI4 T cells. However, transgenic elevation of Nfkbid expression also increased the frequency and functional capacity of peripheral Tregs with a striking complete absence of T1D development in these mice. Studies are ongoing to define the mechanisms of T1D resistance mediated by transgenic overexpression of the transcriptional co-factor Nfkbid.
Resistant effect of sodium chloride on the development and function of TGF-β-induced CD4+ Foxp3+ regulatory T cells
Authors: Zheng Song Guo , Luo Yang , Wang Julie
Published: January 2023

High-salt diets inhibit the suppressive function of thymus-derived natural regulatory T cells (tTreg). TGF-β-induced ex vivo regulatory T cells (iTreg) represent another Treg subset that displays similarities and differences with tTreg. Here, we demonstrated that iTregs are completely stable and fully functional in the high salt condition. High salt did not affect the development, differentiation and function activities of iTreg in vitro and in vivo but affected the Foxp3 stability and function of tTreg in vitro and in vivo. In addition, high salt did not significantly change the transcription profiles of iTreg signature or pro-inflammatory genes. Therefore, we conclude that iTreg, unlike tTreg, is stable and functional in the presence of high salt. Thus, our findings provide another evidence that iTreg may have different biological features from tTreg and imply a greater potential to be employed in the clinical setting for patients with autoimmune diseases in the complicated role of environmental factors, including diet.
Adenosine:Adenosine Receptor (AR) Axis in Blood Brain Barrier (BBB) Permeability Regulation
Authors: Bynoe Margaret S
Published: January 2023

Multiple sclerosis (MS) is a is a chronic, debilitating, neuroinflammatory disease that affects the central nervous system (CNS). MS affects about 2.5 million patients world-wide. MS patients experience a progressive loss in neurological function caused by immune-mediated axonal damage and demyelination in multiple areas of the brain and spinal cord. Despite years of research, the etiology of MS is still unknown. Studies using the dominant MS animal model, experimental autoimmune encephalomyelitis (EAE), demonstrate that myelin antigen-specific CD4 T cells can induce CNS inflammation, demyelination, and neurodegeneration, resulting in the loss of motor function (paralysis). Development of MS or EAE requires the entry of autoreactive immune cells into the CNS. Typically, the anatomical characteristics of the brain, such as the specialized endothelial cell tight and adherens junctions constitute the framework of the BBB, and limit the entry of lymphocytes into the CNS. We hypothesized that adenosine and/or adenosine receptors (ARs) expressed on BBB endothelial cells regulate the entry of immune cells and molecules into the brain. We showed that deletion of the adenosine-generating enzyme, CD73 (cd73−/−) expressed on BBB endothelial cells blocked immune cells entry into the brain. However, fulminant EAE could be induced in cd73(−/−) mice after the transfer of WT CD73(+)CD4(+) T cells, suggesting that CD73 must be expressed either on T cells or on BBB endothelial cells for disease induction. Next, we showed that blockade of the A2A AR block the entry of immune cells into the brain. Based on these findings we conclude that CD73 expression and adenosine receptor signaling are required for the efficient entry of lymphocytes into the CNS.
Overexpression of Pbx1-d, a novel splice isoform of Pbx1 associated with lupus susceptibility, regulates regulatory T cell plasticity
Authors: Choi Seung-Chul , Cornaby Caleb , Morel Laurence
Published: January 2023

Pbx1 controls chromatin accessibility to a large number of genes. The Pbx1-d dominant negative isoform lacking the DNA binding domain is more frequent in the CD4+ T cells from lupus patients than in healthy controls. Pbx1-d is associated with the production of autoreactive CD4+ T cells in mice carrying the Sle1a1 lupus susceptibility locus in the B6.NZM2410.Sle1.Sle2.Sle3 model. We have previously reported that Pbx1-d-Tg mice specifically expressing Pbx1-d in CD4+ T cells reproduced the phenotypes of Sle1a1 mice, and showed an impaired Foxp3+ regulatory T (Treg) cell homeostasis as well as increased inflammatory functions of their CD4+ T cells. To further study Treg cell homeostasis, we bred Pbx1-d-Tg mice with Foxp3-fate-mapping reporter constructs, and determined that Pbx1-d overexpression accelerated the loss of Foxp3 expression and the production of ex-Treg cells in unmanipulated mice. This result was confirmed by the homeostatic expansion of Fopx3-negative CD4+ T transferred into lymphopenic mice. RNAseq analyses showed that the increased ex-Treg production by Pbx1-d is associated with the expression of pro-inflammatory cytokines and an alteration of mitochondrial metabolism. Overall, these results suggest that Pbx1 regulate Foxp3 stability and that Pbx1-d promotes lupus development by decreasing the pool of effective Treg cells and promoting the production of the inflammatory ex-Treg cells.
Role of Jmjd6 in Aire expression and self-tolerance induction in the thymus
Authors: Matsubara Keisuke , Fukui Yoshinori
Published: January 2023

Introduction

To establish immunological self-tolerance in the thymus, medullary thymic epithelial cells (mTECs) express diverse sets of tissue-specific self-antigens (TSAs). This ectopic expression of TSAs largely depends on the transcriptional regulator Aire, yet the mechanism controlling Aire expression itself remains unknown. In this study, we aimed to examine the possible roleof Jmjd6, a lysyl hydroxylase for splicing regulatory proteins, in controlling Aire expression.

Material and methods

As conventional Jmjd6 KO mice die during perinatal period, 2DG-treated fetal thymi were prepared from the KO mice and control mice, and subjected to FACS analyses, immunohistochemical analyses and RNAseq analyses. The 2DG-treated fetal thymi were also grafted into C57BL/6 nude mice to examine its effect on self-tolerance induction in vivo. In addition, the conditional Jmjd6 KO mice (Foxn1-Cre Jmjd6lox/lox mice) were developed and crossed with a diabetes mouse model (OT1+RIP-mOVA+ mice) to analyze immunological tolerance under more physiological condition.

Results

Although Jmjd6 deficiency did not affect abundance of Aire transcript in mTECs, the intron 2 of Aire gene was not effectively spliced out in the absence of Jmjd6. As a result, the expression of mature Aire protein and TSAs were markedly reduced in Jmjd6-deficient mTECs, resulting in multi-organ autoimmunity when Jmjd6-deficient thymi were grafted into C57BL/6 nude mice. In addition, we found that TEC-specific deletion of Jmjd6 exacerbated development of autoimmune diabetes in a mouse model.

Conclusion

Jmjd6 controls Aire protein expression in mTECs for induction of self-tolerance in the thymus.
Phosphorylation of CBP by IKKα links intestinal homeostasis
Authors: Miaw Shi-Chuen , Lin Yi-Ting , Hsu Yu-Hua , Liu Chi , Chen Chieh-Chang , Shun Chia-Tung , Tu Chia-Hung , Yu Jhang-Sian , Wu Ming-Shiang , Chen Ching-Chow
Published: January 2023

CBP is a transcriptional coactivator participating in the regulation of DNA accessibility to transcription factors. Phosphorylation of human CBP by IKKα at serines 1382/1386 plays a critical role in regulating cell destiny via suppressing p53-mediated gene expression. We aimed to determine whether impaired phosphorylation of CBP is involved in the pathogenesis of disease. Hence, we designed and generated mutated serine 1383/1387 to alanine knockin mice (AA mice) to study whether they exhibit any phenotype alteration. AA mice showed an IBD-like phenotype with softer, looser and hemoccult stools and shorter colon length compared to the wild type mice. Increased p53 accompanied with apoptosis in colonic tissues of AA mice was demonstrated by IHC and TUNEL staining. In addition, male AA mice were more vulnerable to DSS-induced colitis with severe crypt loss and epithelial disruption. Elevated immune cells in mesenteric lymph nodes are also more prominent in AA mice with infiltration of neutrophil and macrophage. However, the differentiation of CD4+ T cells was not affected with Cbp mutation (AA) ex vivo. Colonic biopsies show that patients with ulcerative colitis have significantly lower phosphorylated CBP (Ser1382/1386) than healthy controls. Collectively, deficiency of NF-κB signaling in AA mice did not affect immune cells but modulated apoptosis of IECs. Our findings suggested that phosphorylation of CBP by IKKα might be a critical regulator to maintain intestinal homeostasis.
Inflammasome activation in macrophages is regulated by actin-bundling protein L-plastin
Authors: Joshi Hemant , Todd Betsy Elisabeth , Szasz Taylor , Anaya Edgar , Morley Sharon Celeste
Published: January 2023

Macrophage secretion of IL-1β is crucial to combat and clear bacterial infections. IL-1β release is tightly controlled by assembly of multimolecular inflammasomes (i.e. NLRP3, NLRC4, AIM2) and irregulated production can contribute to various pathologic conditions (i.e. acute respiratory disease and ventilator-associated lung injury). Prior research suggests that mechanical stress elevates macrophage IL-1β production. Because increased lung stiffness in diseases (e.g. acute respiratory distress syndrome) exerts mechanical stress on residing alveolar macrophages (AMs). Understanding how mechanical stress modulates AMs becomes crucial to elucidating the pathophysiology of pulmonary disease. We observed that IL-1β production in AMs was abrogated in absence of L-plastin (LPL), a hematopoietic restricted actin-bundling protein. LPL is also required for podosome formation. Podosomes are integrin-based, adhesive and mechanosensitive signaling organelles. LPL-deficient macrophages showed decreased IL-1β production when cultured on varying substrate stiffness. These observations suggest that LPL may link contact-based mechanosensation to inflammasome activation. Additionally, Pneumolysin toxin in Streptococcus pneumoniae infection activates NLRP3 and may exacerbate inflammatory pathogenesis in stiffened lungs. Further, we will investigate LPL mediated mechano-sensitive inflammasome activation in pneumococcal lung infections using mouse models. Our observations indicate previously unknown mechanosensitive pathway for inflammasome activation in macrophages. Understanding the molecular mechanism will aid in developing immunomodulatory therapies to treat inflammatory disorders and diseases.