bluuebunny/crossref_metadata_2025 huggingface dataset:
TCR signal strength regulates Akt substrate specificity to induce alternate Th and Treg differentiation programs
Authors: Morel Penelope A , Hawse William F , Sheehan Robert P , Boggess William C , Faeder James R
Published: January 2023

T regulatory (Treg) cells are induced following stimulation of naïve CD4 T cells with low dose antigen to an extent that is negatively correlated with signaling via the Akt/mTOR pathway. Strong TCR signals induce high levels of Akt activity that inhibit development of Treg by poorly understood mechanisms. Here, we show that high dose stimulation of T cells results in the phosphorylation of Akt on two regulatory sites, Serine (S) 473 and Threonine (T) 308, whereas low dose stimulation results in only T308 phosphorylation. Mathematical modeling shows that the phosphorylation of Akt on both S473 and T308 is controlled by a feedback loop involving PTEN, mTORC2 and the transcription factor FoxO1 that creates a sharp activation threshold with respect to antigen dose and stimulus duration. Using mass spectrometry to analyze phosphorylated Akt substrates at different levels of stimulation, we find profound differences in the substrates phosphorylated, suggesting that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates. Proteins differentially phosphorylated by these two states of Akt include RNA splicing factors, and we find changes in the splice variant expression levels of key TCR signaling proteins, such as CD3ζ and CD45 that correlate with the observed differences in cell fate. Knockdown of specific splicing factors impacted the ratios of Th versus Treg cells induced. Together, this work demonstrates that alternative splicing can affect the outcome of T cell fate decisions and identifies alternate Akt-mediated signaling networks that drive CD4+ T cell differentiation.
Early signalling changes in T cells with aging
Authors: Fulop Tamas , Le Page Aurélie , Dupuis Gilles , Larbi Anis
Published: January 2023

Aging is accompanied by many physiological changes and the immune system is not escaping this. This effect of time is affecting most cells of the immune system. Numerous changes have been described but most of them are phenomenological, a typical example is the change of phenotypes in T cells. We are interested since years to uncover the intracellular signaling associated with phenotypic and functional changes of various immune cells. We identified several signaling alterations starting from the early to the most downstream events in several cells like neutrophils, monocytes and T lymphocytes. We not only were interested in the forward signaling driven by kinases but also by the feedback or regulatory signaling mediated by phosphatases. We found profound alterations of regulatory phosphatases activity and phosphorylation (e.g. SHP-1). The question was always whether these changes are occurring also uniformly in T cell or there are differences between the various subpopulations (naive to the continuum of memory T cells). Our studies indicate that there are changes according to the subpopulations and that basal hyper-phosphorylation status we observe may be explained by signaling crosstalks. We also observed that there is a resemblance between naïve and TEMRA T cells, which differ much from CM and RM cells. In conclusion the different T cell subpopulations may have very specific changes distinguishing them from each other. This needs further investigations in relation to their functions.
Inhibition of T cell activation by Borrelia burgdorferi
Authors: Whiteside Sarah , Snook Jeremy P , Ma Ying , Weis John H , Williams Matthew A , Weis Janis J
Published: January 2023

Infection with the spirochete B. burgdorferi induces both innate and adaptive immune responses, which coordinate to control disease progression. Previous studies have demonstrated that Borrelia specific antibodies are critical to the control and resolution of infection. The antibody response is characterized by T cell-independent recognition of numerous B. burgdorferi lipoproteins, which contribute to protection from subsequent challenge. In contrast, the role of T cells in protective vs. disease modulating responses to B. burgdorferi infection is poorly understood. The failure to identify protective T cell epitopes of B. burgdorferi in both mice and humans has limited this line of investigation. We have identified bystander activation of CD4+ and CD8+ T cells during infection, a feature that may obscure the identification of antigen-specific T cell responses. Paradoxically, we have also identified a novel modulatory activity in B. burgdorferi that suppresses activation and proliferation of both CD4+ and CD8+ T cells. Suppression can be demonstrated in naïve cells from wild type and SMARTA TCR transgenic mice, indicating the ability to override antigen specific and potentially protective immune responses. Our results are consistent with those of Elsner et al., indicating that B. burgdorferi infection results in temporary immunosuppression of the host. Experiments are ongoing to identify potential molecular mechanisms of suppression.
Quantitative proteomics to predict functional consequences of ubiquitylation events during T cell stimulation
Authors: O’Leary Claire E , Ding Hua , Spruce Lynn , Fazelinia Hossein , Seeholzer Steven , Oliver Paula M
Published: January 2023

The post-translational attachment of ubiquitin to cellular proteins can alter protein function and half-life, thereby profoundly impacting cell signaling and fate decisions. New proteomics-based techniques have been developed that have the potential to reveal relationships between ubiquitylation and protein fate. Here, we assess the feasibility and scalability of using quantitative proteomic approaches to identify ubiquitylated proteins in primary T cells in a high throughput manner. Furthermore, we sought to identify TCR-dependent changes in protein ubiquitylation. We utilized immunoprecipitation of ubiquitin remnant peptides (peptides with modified lysine residues) and quantitative mass spectrometry to quantify changes in protein-specific ubiquitylation in CD4+ T cells stimulated via the TCR compared to resting T cells. Our analysis identifies thousands of modified lysines in primary T cells, and reveals dynamic changes in ubiquitylation of hundred of proteins, including key signaling transducers, during TCR stimulation. We then performed whole cell proteomic analysis to quantify changes in relative protein abundance during T cell activation. By comparing changes in ubiquitylation with changes in protein abundance in stimulated CD4+ T cells, these data can be used as a resource to predict the consequences of TCR-induced protein ubiquitylation. This work establishes ubiquitin remnant profiling and whole proteome analysis as important techniques for global analysis of ubiquitylation in primary T cells.
The transcription factor IRF8 regulates natural killer cell development and response to cytomegalovirus infection
Authors: Adams Nicholas M , Rapp Moritz , Sun Joseph Chai-Yuen
Published: January 2023

Interferon regulatory factor 8 (IRF8) is a transcription factor critical for specifying monocyte and dendritic cell lineage fate and for directing B cell development, yet its function in natural killer (NK) cells is not understood. NK cells are innate lymphocytes that develop in the bone marrow and are capable of antigen-specific clonal proliferation. We observe in mixed bone marrow chimeric mice that despite a prevalent NK progenitor (NKP) population compared to wild-type, peripheral Irf8−/− NK cells exhibit proper maturation and full functionality. However, using an adoptive transfer system, we demonstrate that IRF8 is indispensable for robust expansion of NK cells in response to mouse cytomegalovirus (MCMV). Consistent with this finding, Ly49H+ NK cells upregulate Irf8 transcript during the early stages of MCMV infection. Furthermore, the Irf8 locus is dynamically regulated upon activation, showing extensive STAT4 binding at promoter and enhancer regions, and upregulation of the activating epigenetic marks H3K4me3 and H3K4Ac27. Thus, our work elucidates the functional contribution of IRF8 during NK cell development and antiviral responses.
Regulation of cytotoxic T cell and germinal center B cell responses by the c-MYC-AP4 transcription factor cascade
Authors: Chou Chun Jim , Pinto Amelia K , Persaud Stephen P , Verbaro Daniel J , Tonc Elena , Holmgren Melanie , Cella Marina , Allen Paul M , Colonna Marco , Bhattacharya Deepta , Diamond Michael S , Egawa Takeshi
Published: January 2023

The process of amplifying immune responses by expanding a pool of antigen-specific cells, termed clonal expansion, is an important feature of the adaptive immunity. Whereas clonal expansion of cytotoxic T lymphocytes is required for complete eradication of pathogens in tissues, proliferation of B lymphocytes in the germinal centers is critical for generating a diverse immunoglobulin gene repertoire from which protective antibody carrying multiple mutations can arise. While the proto-oncogene c-MYC is absolutely required for the activation and cell cycle initiation in lymphocytes, its expression is temporally restricted. Activated lymphocytes, however, continue to proliferate after c-MYC levels decay to maximize clonal expansion. It remains unknown how lymphocytes sustain their proliferative program in the absence of c-MYC. We demonstrated that the c-MYC-inducible transcription factor, AP4, is required for sustained expansion of antigen-specific lymphocytes. Mice lacking AP4 in T cells exhibit diminished cytotoxic T cell responses and succumb to West Nile virus infection due to uncontrolled viral replication in the central nervous system. Furthermore, conditional deletion of AP4 in B lymphocytes impaired germinal center growth. These mice failed to control chronic viral infection due to blunted neutralizing antibody responses. ChIP-seq and gene expression analyses revealed a significant overlap between AP4 and c-MYC regulated genes. Thus, the c-MYC-AP4 transcription factor cascade is a general module utilized by activated lymphocytes to maximize immune responses.
The Notch signaling pathway: a new mastor regulator of effector CD8 T cell differentiation
Authors: Duval Frédéric Antoine , Mathieu Mélissa , Daudelin Jean-François , Labrecque Nathalie
Published: January 2023

Following an infection, naïve CD8 T cells expand and differentiate into effectors able to eliminate the pathogen. At the peak of the response, two major populations of effectors are distinguishable: short-lived effector cells (SLECs) meant to die by apoptosis and memory precursor effector cells (MPECs) destined to survive as memory cells that confer long-term protection. We postulated that the Notch signaling pathway, known for its role in cellular differentiation and binary cell fate choice, acts as a key player in the MPEC/SLEC differentiation choice. To elucidate the role of Notch signalling, we have analyzed CD8 T cell response in mice lacking or not the expression of Notch1 and Notch2 in mature CD8 T cells. Following infection with Listeria or vaccination with antigen-pulsed dendritic cells, Notch deficiency drastically diminished SLEC generation and affect cytokines production without impairing CD8 memory T cell generation. Interestingly, altered SLEC differentiation occurs despite normal expression of the transcription factors, T-bet and Blimp-1, known to be necessary for SLEC differentiation. Moreover, the reduced generation of SLECs in absence of Notch signaling correlated with defective expression of CD25 by effectors. However, restoration of CD25 expression along with daily IL-2 complementation in Notch1/2-deficient T cells did not correct SLEC differentiation. These results suggest either that Notch regulates the expression of new players involved in SLEC differentiation or Notch signaling collaborates with already known actors of SLEC differentiation such as T-bet or Blimp-1. The identification of the genes regulated by Notch should help to reveal its role in CD8 T cell response.
Deciphering the IFN-γ-mediated immunopathology during Plasmodium yoelii N67C infection
Authors: Lacerda-Queiroz Norinne , Riteau Nicolas , Eastman Richard , Moore Ian , Sher Alan , Jankovic Dragana , Su Xin-zhuan
Published: January 2023

Immune response plays an important role in controlling malaria infection; however, excessive inflammatory response can lead to severe disease. Here we investigate the mechanism of pathogenesis in mice after infection with a virulent rodent malaria parasite, Plasmodium yoelii nigeriensis strain N67C. Previous studies showed that C57BL/6-infected mice display high parasitemia, strong pro-inflammatory response, culminating in extensive splenic damage and host lethality at around 7 days post-infection. The present work aims to characterize the cellular and molecular events associated with host immune-mediated pathology in response to N67C parasite. Mice were infected with 106 infected red blood cells by intraperitoneal injection. C57BL/6-infected mice develop progressive inflammation in the spleen, characterized by intense cellular death and up-regulation of pro-inflammatory cytokines (notably IFNγ, IL-6, CCL2 and CXCL1). Interestingly, we identified splenic CD4+ and CD8+ T cells as major sources of IFN-γ as early as day 4 post-infection. Additionally, we demonstrated that IFN-γ was a major mediator of splenic pathology because its absence or blockade prolonged host survival and prevented excessive host inflammatory response. Moreover, by employing chimeric mice, we showed that the absence of IFN-γ receptor in the non-hematopoietic compartment protected mice from early death. Elucidation of this IFN-γ signaling mechanism may enable further development of interventions to decrease human morbidity and mortality associated with severe malaria.
Commensal exopolysaccharides induce M2 macrophages that prevent pathogen-induced colitis
Authors: Paynich Mallory L , Jones Sara E , Knight Katherine L
Published: January 2023

Trillions of bacteria live within the gastrointestinal tract and are critical for maintaining intestinal homeostasis; however, the mechanisms utilized by specific bacterial molecules to contribute to homeostasis are not well understood. We utilize a mouse model in which a single oral dose of the probiotic, B. subtilis, protects mice from acute colitis induced by the enteric pathogen C. rodentium. Our goal is to elucidate the mechanism by which B. subtilis prevents inflammation. We identified exopolysaccharides (EPS) to be the active molecule of B. subtilis, and a single dose of EPS protects mice from disease. EPS binds F4/80+CD11b+ peritoneal macrophages (MΦ), and following EPS treatment, MΦ increase expression of CD206, Arg1, YM-1, and IL-4Rα, and inhibit T cell activation and proliferation in vitro, indicative of M2 MΦ. Induction of M2 MΦ is TLR4-dependent, and in mice treated with clodronate liposomes to deplete MΦ, EPS no longer protects mice from disease. Addition of anti-TGFβ neutralizing antibodies removed the inhibitory effect of M2 MΦ suggesting that EPS-induced M2 MΦ produce TGFβ to inhibit T cell proliferation in vitro. CD4+ T cells drive much of the inflammation associated with C. rodentium infection, and we hypothesized that EPS inhibits CD4+ T cell responses in vivo. Accordingly, we measured levels of IFNγ (Th1), IL-17 (Th17), and IL-13 (Th2) in splenic T cells following EPS treatment and found decreased levels of these cytokines. We hypothesize that EPS-induced M2 MΦ mediate inhibition of CD4+ cell responses in vivo, preventing acute colitis induced by C. rodentium. The data suggest that EPS-induced M2 MΦ mediate protection from C. rodentium disease by inhibiting CD4+ T cell responses through production of TGFβ.
Cyclic dinucleotide detection by RECON controls innate immune responses
Authors: McFarland Adelle , Luo Shukun , Ahmed-Qadri Fariha , Thayer Elizabeth F. , Tabakh Hannah , Tong Liang , Woodward Joshua J.
Published: January 2023

Host cells have evolved a sophisticated arsenal of germ-line encoded pattern recognition receptors that detect a vast array of microorganisms in distinct tissues and cellular compartments. Cytoplasmic sensors monitor for microbes that gain access to the host cell cytosol following breach of the plasma membrane or intracellular invasion. This sensing is achieved through detection of invariant molecular patterns associated with microorganisms or cell stress responses that indicate the presence of a pathogen. Cyclic dinucleotides (cdNs) of both bacterial and host origin have emerged as important molecules that are sensed during infection. Details about the receptors cdNs engage in host cells and the responses those interactions elicit are just coming to light. We have recently discovered the first bacterial cdN-specific pattern recognition receptor, the oxidoreductase RECON. Our work on this receptor has revealed its central role in directing innate immune responses when engaged by bacterial cdNs. These findings reveal a new pattern recognition receptor specific for bacterial cdNs that orchestrates cytosolic immune surveillance by shaping downstream inflammatory gene activation.
The structural basis of IRF-3 recruitment, activation, and targeting in innate immunity
Authors: Li Pingwei
Published: January 2023

Type I interferons (IFNs) are key cytokines mediating innate antiviral immunity. cGAS, RLRs, and TLRs recognize microbial DNA, RNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of IRF-3. STING, MAVS, and TRIF mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, while residues upstream of the motif confer specificity. The structure of IRF-3 phosphomimetic mutant S386/396E bound to CBP reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 employs a pLxIS motif to target IRF-3 for degradation. However, phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune response.
CD28 co-stimulatory signaling and AP-1 transcription factor are involved in the establishment of open chromatin during T cell activation.
Authors: Yukawa Masashi , Jagannathan Sajjeev , Kartashov Andrey , Barski Artem
Published: January 2023

Upon encountering an antigen, naïve T helper (Th) cells are activated, differentiate into several lineages and contribute to immune response. Epigenetic changes at several cytokine and other effector genes during activation has been described previously, but the mechanism behind these changes is not understood. Here, we have profiled open chromatin during human T cell activation using ATAC-Seq approach and identified the regions where epigenetic changes take place genome-wide. Open chromatin formation correlated with induction of gene expression at nearby genes. This gene set was also enriched for functions related to T cell activation and immune response in GO analysis. T cell activation requires antigen signaling via T cell receptor and co-stimulation via CD28 which in turn result in nuclear translocation of NFAT and AP-1 transcription factors. For this reason, we profiled genome-wide distribution of these proteins during activation by ChIP-Seq. Our results show that 73% of activation-induced open chromatin loci contain binding sites for AP-1, whereas 39% of them bind NFAT. Over 90 % of the binding sites for NFAT were shared with AP-1 binding sites. We and others have previously shown that the lack of co-stimulation results in reduction or delay of AP-1 translocation into the nucleus. In order to confirm the role of AP-1 in establishment of the open chromatin structure during activation we mapped open chromatin in the cells that were activated without co-stimulation. Our data showed that ATAC signal was reduced at the open chromatin sites bound by AP-1 in the absence of co-stimulation, suggesting that CD28 signaling and AP-1 transcription factor are involved in open chromatin formation during T cell activation.
Quantification of lymph node transit times reveals differences in the trafficking behavior of regulatory versus conventional CD4+ T cells
Authors: Tong Alexander , Allen Frederick , Huang Alex Yee-Chen
Published: January 2023

Regulatory T cells (Tregs) express the canonical marker Foxp3 and are critical for suppressing the immune response in homeostatic and inflammatory conditions. Despite how widely studied CD4+Foxp3+ Tregs are in mice, their physiologic trafficking and scanning behavior of antigen-presenting cells in secondary lymphoid organs (SLOs) have yet to be quantitatively analyzed in the intravital setting. Our laboratory has previously described the dynamics of T cell trafficking through SLOs and scanning of dendritic cells (DCs), revealing that CD4+ T cells and CD8+ T cells exhibit different transit kinetics through lymph nodes (LNs). On average, CD4+ T cells spend less time scanning for self-peptide/MHC (self-pMHC), dwell shorter in LNs, and egress faster compared to CD8+ T cells. Here we use flow cytometry and 2-photon laser scanning microscopy (2-PLSM) to quantitate the homeostatic trafficking behavior and surveillance strategy of Tregs in LNs and to compare their behavior to conventional T cells (Tconvs). Our data reveal surprising differences in Treg versus Tconv trafficking in mesenteric versus other peripheral LNs. Furthermore, we find that Treg LN transit time is heavily dependent on recognition of self-pMHC. Finally, we use intravital 2-PLSM to quantify the contribution of self-pMHC recognition to Treg kinetics and scanning of DCs in peripheral and mesenteric LNs. These results add quantitative data to the emerging paradigm that self-pMHC interactions dynamically control homeostatic Treg retainment and localized suppressive function in peripheral LNs.
Response Gene to Complement-32 promotes Th17 cell differentiation through TGF-β dependent and independent mechanisms
Authors: Rus Violeta , Nguyen Vinh , Tatomir Alexandru , Mekala Armugam Prasad , Rus Horea G
Published: January 2023

Response Gene to Complement (RGC)-32 is an intracytoplasmatic protein expressed in a variety of cells in response to sublytic complement activation, TGF-β and several growth factors. Upon activation it translocates into the nucleus and plays an important role in cells proliferation, migration, angiogenesis and TGF-β induced epithelial mesenchymal transition and fibrosis. A number of signaling pathways including CDC2 kinase, NF-kB, Akt, SMAD2/3, ROCK2 and PKC have been reported to mediate RGC-32 effects in different cell types. We have recently shown that RGC-32 promotes Th17 but not Treg, Th1 or Th2 differentiation in murine CD4+ T cells in vitro. The molecular mechanisms underlying the enhanced Th17 generation by RGC-32 are unknown. Under Th17 conditions, CD4+ T cells from RGC-32 knockout (KO) mice displayed significantly lower IL-6Rα and TGF-βRI mRNA expression. Smad dependent TGFβ signaling was altered in RGC-32 KO T cells as demonstrated by decreased phosphorylation of Smad2, while IL-6 induced STAT3 phosphorylation did not differ between RGC-32 KO and wild-type CD4+T cells. mRNA and protein expression of RORyt, the Th17 lineage specific transcription factor, was significantly lower in RGC-32 KO CD4+ T cells. In addition, RGC-32 KO CD4+T cells expressed significantly lower mRNA levels of the initiator transcription factors, BATF and IRF4. These results suggest that RGC-32 positively regulates the differentiation of the Th17 lineage through TGFβ dependent and independent mechanisms by enhancing the activation of Smad2, IRF4, BATF and RORyt.
O-GlcNAc regulates CD4+ T cell differentiation
Authors: Machacek Miranda E , Slawson Chad , Fields Patrick
Published: January 2023

Chronic inflammation is a feature of diabetes and obesity and enhances the risk of atherosclerosis and cancer. Specifically, proinflammatory Th1 and Th17 CD4+ T cells are increased in metabolic diseases. However, a clear molecular mechanism linking metabolic changes with proinflammatory T cells is lacking. We hypothesize that elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification of nuclear and cytoplasmic proteins, promotes proinflammatory CD4+ T cell differentiation. Since production of O-GlcNAc involves input from carbohydrate, amino acid, fatty acid, and nucleic acid metabolism, the modification acts as a general sensor of a cell’s nutritional status. To investigate O-GlcNAc’s role, we cultured mouse splenic CD4+ T cells with Thiamet-G (TMG), a selective inhibitor of the enzyme that removes O-GlcNAc (O-GlcNAcase, OGA), before activating with anti-CD3 and anti-CD28 under permissive Th0 conditions. We find a significant two-fold increase in protein and transcript levels of IL-17A, the eponymous Th17 cytokine. Intracellular cytokine staining shows two-fold increases in IFNγ+ cells, IL-17+ cells, and IFNγ+IL-17+ cells. Phosphorylation of STAT3 and STAT4, the transcriptionally active form of STATs which define the Th17 and Th1 lineage programs respectively, is increased in TMG-treated cells within 10 minutes of T cell receptor activation. Collectively, our data suggest that elevated O-GlcNAc levels prime Th17, Th1, and Th1-like Th17 CD4+ T cell differentiation by increasing activated STAT3 and STAT4. Further study into the molecular mechanism of how O-GlcNAcylation promotes proinflammatory T cells may provide insight into how nutritional excess exacerbates inflammation.
A dominant function of transcription factor Foxo1 in the development of Th9 and IL-9-producing T cells
Authors: Awasthi Amit , Malik Sakshi , Sadhu Srikanth
Published: January 2023

Interleukin 9 (IL-9), a pleiotropic cytokine of common γ-chain cytokine receptor family, plays a crucial role in allergic inflammation, autoimmunity and immunity to extracellular pathogens. Recent identification of anti-tumor function of IL-9 makes it an attractive target for anti-tumor therapy. Although Th2, Th17 cells and Foxp3+ Treg cells were shown to produce IL-9, more specialized IL-9-producing Th9 cells were identified as a distinct subset of Th cells that are proinflammatory in vivo. The master transcription factors that regulates IL-9 production in Th2, Th9 and Th17 cells has not been identified, although IRF-4, PU.1, Batf and IRF1 were shown to be essential for IL-9 induction in Th9 cells. Here we have identified Foxo1, a forkhead family transcription factor, predominantly required for the induction of IL-9 in Th9 and Th17 cells. We further identified AKT, an upstream kinase that regulates IL-9 induction in Th1, Th9 and Th17 cells via Foxo1. In addition, c-Jun N-terminal kinase (JNK), a MAPK (Mitogen Activated Protein kinase) enhanced IL-9 induction in Th9 cells via Foxo1. Chromatin Immunoprecipitation (Chip) identified a direct physical association of Foxo1 with IL-9 and IRF4 promoters. Furthermore, loss of Foxo1 suppressed IL-9 production in Th9 and Th17 cells. In fact, Foxo1 act as a transcriptional switch, which exclusively suppress IL-9 induction in Th17 cells while enhancing IL-17 and Th17 cells development. In Th9 mediated asthma model, Foxo1 inhibition substantially ameliorated allergic inflammation and IL-9 induction. Our findings thus identify Foxo1 as a master transcription factor in controlling the development of Th9 and IL-9-producing Th cells.
Optimum differentiation conditions of lactoferrin/TGFβ-caused inducible regulatory T cells
Authors: Kim Pyeung-Hyeun , Kim Sun-Jin , Seo Goo-Young , Kim Hyeon-Jin , Kwon Bo-Eun , Ko Hyun-Jeong , Rhee Ki-Jong , Kim Woan-Sub
Published: January 2023

TGF-β1 and retinoic acid (RA) are well known to stimulate naïve T cells to differentiate into regulatory T cells (Tregs). In the present study, we found that, as assessed by Foxp3 expression, lactoferrin (LF) not only promotes the TGFβ/RA-mediated Treg differentiation but it also induces Treg differentiation by itself. Furthermore, LF in combination with TGF-β1 powerfully enhanced Foxp3 expression at the transcriptional and protein levels. In this, our extensive kinetic study led the maximum Foxp3 expression within 36 h of culture. These Foxp3+ CD4+T cells strongly suppressed the proliferation of CD4+T cells activated with αCD3 and αCD28 Ab. Finally, phenotypes of these Foxp3+ CD4+T cells were CD44hi CD62Lhi, T-betlo, IFN-γlo, GATA3lo, and IL-4lo. Our optimized Treg differentiation condition would be useful when Treg adoptive transfer is required for various immunosuppressive therapies.
IL-12 and IFN-I differentially regulate secondary CD4+ T cell responses.
Authors: Meek Stephanie M , Kim Chulwoo , Williams Matthew A
Published: January 2023

Upon re-exposure to antigen, memory T cells give rise to secondary effector responses that enhance clearance and immune protection. Additionally, secondary activation often boosts the number of memory T cells. Therefore, understanding the parameters that control secondary T cell differentiation and function is critical. We report key roles for IL-12 and Type 1 I IFN (IFN-I) in controlling secondary T cell activation, differentiation and memory formation. While IL-12 was required for the development of secondary effector cells with high functional avidity, as well as the optimal formation of secondary T cell memory, IFN-I inhibited both of these features. Within 24 hours of secondary challenge we observed CD4+ memory T cell-dependent increases in serum IL-12 and IFNγ. While IL-12 neutralization resulted in a predictable reduction of IFNγ, blockade of the IFNα/β receptor (IFNAR) induced significantly higher expression of IL-12, IFNγ and IL-6. The effect was indirect, as rechallenge of WT CD4+ memory T cells in an IFNAR-deficient host resulted in increased functional avidity of secondary effector CD4+ T cells. In contrast, secondary T cell expansion was partly curtailed in these hosts, indicating both stimulatory and inhibitory effects for IFN-I in the setting of secondary challenge. Several immunoregulatory functions for IFN-I have been reported, including induction of IL-10. In support of this, secondary challenge of WT CD4+ memory T cells in IL-10-deficient hosts resulted in enhanced secondary effector function. These findings highlight a key role for IFN-I in regulating secondary CD4+ T cell differentiation and suggest potential therapeutic approaches that preferentially target its immunoregulatory functions.
Nrf2 activation skews murine and human CD4 T cell differentiation towards a Th2 phenotype
Authors: Turley Alexandra E , Zagorski Joseph Ward , Klaassen Curtis , Fields Patrick E , Rockwell Cheryl Elizabeth
Published: January 2023

CD4 T cell differentiation is a critical part of the adaptive immune response during which CD4 T cells adapt a response to a specific pathogen. Nuclear factor erythroid-derived 2 like-2 (Nrf2) is a cell-stress responsive transcription factor that regulates a battery of antioxidant, detoxification, and cytoprotective genes. Nrf2 is activated by stressors such as reactive oxygen species and electrophilic xenobiotics, including tert-butylhydroquinone (tBHQ), a commonly used food preservative. Nrf2 has been shown to be immunomodulatory: Nrf2 null mice are more susceptible to inflammatory stimuli such as sepsis, lung injury, and experimental autoimmune encephalomyelitis. In addition, Nrf2 has been shown to modulate dendritic cell and macrophage function, however, the role of Nrf2 in CD4 T cells remains only partially characterized. Thus, the goal of the present studies is to determine the role of Nrf2 in CD4 T cell differentiation in primary human and mouse CD4 T cells. Treatment with the Nrf2 activator tBHQ increased production of the Th2 cytokines IL-4, IL-5, and IL-13 in CD4 T cells isolated from wild-type, but not Nrf2-null mice, and decreased production of the Th1 cytokine IFNγ. tBHQ increased GATA-3 and decreased T-bet DNA binding in wild type but not Nrf2-null mice. In primary human CD4 T cells, tBHQ treatment increased production of the cytokines IL-4, IL-5, and IL-13, and had no effect on IFNγ production upon restimulation. Collectively, these data suggest that Nrf2 activation promotes Th2 differentiation of primary human and mouse CD4 T cells. (Funded by NIH grants ES018885 and ES007255).
High rates of cross-reactivity among apparently unrelated specificities shape antigen-specific T cell receptor repertoires
Authors: Thomas Paul Glyndwr , Dash Pradyot , Hertz Tomer , Gartland Andrew , Bradley Philip
Published: January 2023

We have analyzed several thousand antigen-specific T cell receptor repertoires from multiple animals infected with influenza virus and/or murine cytomegalovirus (mCMV). Insights into the overall structure of antigen-specific repertoire landscapes towards 7 different specificities were generated from single cell paired TCR αβ sequencing. In addition to characterization of repertoire features such as diversity, clonal structure, convergent recombination, and key deterministic features of antigen-specificity, we observed a surprisingly high rate of single or dual chain sharing between otherwise unrelated responses. These shared receptors were often observed across multiple animals in independent experiments. However, sharing appeared more likely to occur in animals where the two epitopes were presented simultaneously. Cloning and expression of these unique specificities has allowed further receptor characterization. Additional data indicate that some combinations of co-presented epitopes result in broader repertoires recruited to one or both epitopes. In summary, T cell receptors have a significant propensity for cross-reactivity across otherwise unrelated specificities, an effect that appears to be driven by co-presentation and substantially shapes the magnitude and diversity of antigen-specific repertoires.
Aging converts innate B1a cells into potentially pathogenic 4BL cells that induce cytolytic CD8+T cells.
Authors: Biragyn Arya , Lee-Chang Catalina , Bodogai Monica
Published: January 2023

Immune system becomes dysregulated in aging of mammals affecting both myeloid and lymphoid immune cell compartments. Compounded with a life-long antigenic exposure, changes in T cells and increased half-life of mature B cells, conventional B2 cells are enriched for antigen-experienced memory and mature cells at the expense of naive B cells. However, the role of aging on innate B1a cells, the key produces natural antibody to pathogens and endogenous antigens, is not known and is assumed to be negligible. Here, we question this assumption by providing evidence that B1a cells become converted into 4BL cells, the 4-1BBL+MHC class-IHiCD86Hi B cells that induce cytolytic granzyme B (GrB)-expressing CD8+T cells upon aging of humans, macaques and mice 1. This conversion is mediated by aging CD11b+ myeloid cells, which induce a chain of activating events in B1a cells. First, B1a cells express 4-1BBL and IFNgR1 to be used for up regulation of membrane TNFa (mTNFa) and CD86 expression, respectively. Then, using mTNFa, 4BL cells induce GrB expression in CD8+T cells via targeting TNFR2 while providing co-stimulation with CD86. Thus, for the first time, we report that B1a cells become dysregulated upon aging. They lose ability to support tumor growth and gain a new function – the induction of potentially antitumor and autoimmune CD8+T cells. This work was entirely supported by the Intramural Research Program, NIA/NIH.1.
The non-receptor tyrosine kinase ITK is required for type 1 regulatory T cell development
Authors: Huang Weishan , Koylass Nicholas , August Avery
Published: January 2023

Type 1 regulatory T (Tr1)cells lack the expression of Foxp3 but are potent producers of the immunosuppressive cytokine IL-10, with profound regulatory function in suppressing inflammation and promoting tolerance. Tr1 cells differentiate in response to signals engaging T cell receptor (TCR) and/or regulatory cytokine milieu. The non-receptor tyrosine kinase ITK is a key modulator downstream of TCR, playing critical role in T cell development and function. Using mouse models carrying Foxp3-RFP and IL-10-GFP dual reporters, we found that, in the absence of ITK, Foxp3− IL-10+ Tr1 cell development driven by TCR activation is severely impaired in various tissues (spleen, lymph nodes, lung, gut, and fat). Under Tr1 polarizing condition, naïve Foxp3− CD4+T cells isolated from WT mouse thymus and spleen can both give rise to Tr1cells, however, Itk−/− thymic and splenic naïve Foxp3− CD4+ T cells are deficient in Tr1 differentiation. Although Itk−/−CD4+ T cells proliferated under Tr1 differentiating conditions, they failed to up-regulate LAG3, CD49b, ICOS, PD-1, c-Maf, AHR, and IRF4 to levels observed in WT cells, suggesting that ITK is critical for Tr1 cell fate programming. Utilizing transgenic mouse model carrying an allele sensitive mutant of ITK that allows ITK kinase specific blockade by a small molecule 3MB-PP1, we also determined that the expression of the aforementioned markers is dependent on ITK kinas activity. Inhibiting ITK kinase activity also diminished Tr1 differentiation inhuman CD4+ T cells. We conclude that ITK is required for Tr1 cell development and targeting ITK signaling may be a strategy to modulate regulatory immunity for clinical benefit.
Antigen-dependent engagement of brain endothelium by CD8 T cells leads to fatal brainstem pathology during cerebral malaria
Authors: Swanson Phillip A , Hart Geoffrey T , Russo Matthew V , Pierce Susan K , McGavern Dorian B
Published: January 2023

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. Pathological studies of CM victims reveal vascular hemorrhaging and edema within the brain, yet the underlying mechanisms that lead to this fatal condition are unknown. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the factors that drive the pathogenesis of CM. We found that ECM is associated with a large influx of immune cells into the central nervous system (CNS) at the peak of infection. By depleting individual innate and adaptive immune cell populations, we discovered that CD8 T cells alone were capable of mediating the fatal pathology associated with ECM, including breakdown of the blood brain barrier, edema, and extensive neuronal death in the brainstem. Using two-photon microscopy, we observed parasite-specific CD8 T cells slowly crawling and arresting along the CNS vasculature in vivo during ECM. This behavior was facilitated by the IFN-γ-dependent upregulation of antigen presentation and adhesion molecules on CNS endothelial cells (ECs). Antibody therapy against the adhesion molecules VLA-4 and LFA-1 disrupted CD8 T cell interactions with the CNS vasculature and prevented disease. Importantly, infected mice rendered genetically unable to present antigen on ECs had reduced CD8 T cell-brain EC interactions and were resistant to ECM. These results demonstrate that parasite-specific CD8 T cells interact with brain vascular ECs in an antigen-dependent manner to induce fatal vascular and neuronal pathology during ECM.
Low affinity CD8 T cells dominate experimental cerebral malaria resulting from Plasmodium infection
Authors: Neeld Dennis Kyle , King Thayer , Lamb Tracey J , Evavold Brian D
Published: January 2023

Due to its impact on global health, gaining insight into the immune mechanisms that control Plasmodium infections and specifically the complications associated with cerebral malaria are key for developing appropriate therapeutics. For Plasmodium berghei ANKA infections of C57BL/6 mice, activated CD8 T cells mediate the development of symptoms resembling cerebral malaria (experimental cerebral malaria; ECM). We utilized the sensitivity of the two dimensional (2D) micropipette adhesion frequency assay to define the affinity and frequency of immunodominant glideosome associated protein 50 (GAP50)40-48 epitope in spleen and brain of P. berghei infected mice. Compared to tetramer staining of the spleen on day 5, which shows approximately 1.2% of the CD8 cells are antigen specific, the micropipette assay showed that nearly 30% of the CD8 T cells were specific for GAP5040-48. These differences in frequency are due to the fact the micropipette detects both high and low affinity populations of GAP50 specific cells, whereas tetramers are generally limited to detecting higher affinity cells. In P. berghei ANKA infection low affinity CD8 T cells migrate to the brain in large numbers indicating that they are important components of the pathogenic response. Adoptive transfer experiments with P14 transgenic T cells specific for the GP33 epitope of LCMV show that only Plasmodium-reactive cells migrate to the brain during infection providing further evidence that the low affinity CD8 T cells in the brain are Plasmodium-reactive. Taken together, these results demonstrate for the first time that both low and high affinity CD8 T cells participate in the polyclonal CD8 T cell response to Plasmodium infection.
Early TNFα signaling promotes classical activation of dendritic cells in lung associated lymph nodes to prime development of protective immunity against cryptococcal infection
Authors: XU JINTAO , Eastman Alison J , Flaczyk Adam , Neal Lori , Zhao Guolei , Cheng Daphne , Malachowski Antoni , Yosri Mohammed , Curtis Jeffrey L , Osterholzer John J , Olszewski Michal A
Published: January 2023

Cryptococcus neoformans is a common opportunistic pathogen that requires intact T cell-mediated immunity for protective host defenses. While Th1/Th17 responses contribute to the effective containment and elimination of C. neoformans, Th2 responses are non-protective and can worsen clearance outcomes. Previous studies showed that early tumor necrosis factor alpha (TNFα) signaling is required for optimal clearance, while its absence leads to immune dysregulation and persistent fungal infection.

To determine mechanism(s) of early TNFα signaling in the generation of protective anti-cryptococcal immunity, we evaluated the contribution of TNFα to the priming of the innate immune system for optimal T-cell development. In CBA/J mice depleted of TNFα at the time of infection with C. neoformans, we evaluated DC accumulation, activation profile, and interaction frequencies with T cells in lung associated lymph nodes (LALN) in comparison with the infected control mice.

We found that early TNFα depletion during pulmonary C. neoformans infection resulted in: 1) lack of pulmonary fungal clearance and enhanced dissemination; 2) decreased Th1/Th17 polarization and CD4+ T cell activation in the LALN; 3) diminished DC accumulation in the LALN; 4) decreased expression of classical activation DC signatures (co-stimulatory molecules CD40 and CD86), pro-Th1 and pro-Th17 cytokines (IL-12b, TNFα, IL-23a and IL-21); and 5) increased expression of the alternative activation DC signatures (Fizz and Gal3) and pro-Th2 cytokines (IL4 and IL-10).

Thus, our data indicates early TNFα signaling promotes classical activation of DC in lung associated lymph nodes thereby priming development of protective immunity against cryptococcal infection.
Modulators and signally of Th17 axis in Chagas’ disease heart failure
Authors: Rodrigues Barbara Rocha , Rodrigues Denise Bertulucci , Tostes Sebastião , Rodrigues Virmondes
Published: January 2023

Chagas’ disease, caused by the protozoan parasite Trypanosoma cruzi, remains a leading neglected tropical disease in the Americas, associated with 546,000 disability adjusted life years and 10,300 annual deaths. The immune system plays an important role in the reduction of parasite load, but may also contribute to the development of lesions observed during the chronic phase of the disease. We analyzed in situ mRNA expression in left ventricular section from 14 subjects died with Chagas’ disease. Subjects were grouped in Heart hailure and without heart failure, using clinical and necropsies data available. Quantitative PCR were performed on cDNA copies of total RNA extracted from each ventricular sample. PCR were done using Taqman system to detected IL-6 and IL-23 receptor, Beta Actin was used as a internal gene expression. Ventricular wall obtained from subjects with heart failure presented a significant higher levels of mRNA of Interleukin-6 and Interleukin-23 receptor when compared from samples obtained from heart form subjects without heart failure. These results support the idea that a Th17 facilitators are involved in heart lesions of Chagas’ disease patients.
Immunological control of HSV by CD8+ tissue-resident memory T cells
Authors: Roychoudhury Pavitra , Schiffer Joshua T
Published: January 2023

A major challenge in the field of immunology is to generalize findings from animal models to specific human infections. Here we use a mathematical model of a single herpes simplex virus-2 (HSV-2) genital ulcer to integrate mechanistic observations of the tissue resident CD8+ T-cell (TRM) response derived from animal studies, with viral kinetic and T-cell histology data from human infection studies. Simulation with our agent-based model of HSV-2 infection demonstrates that a sufficiently high density of pathogen-specific TRM will lead to extremely rapid elimination of virally infected cells. However, at lower TRM densities, HSV-2 is likely to spread beyond the initial small core of cells. Once this occurs, our model predicts that contact mediated killing and local mucosal trafficking of TRM within mucosa is insufficient to eradicate infection due to the rapid kinetics of HSV-2 spread. Moreover, trafficking of HSV-2 specific cells from blood alone is also unlikely to occur quickly enough to curb viral expansion. We therefore propose that resident CD8+ T-cells invoke a broad antiviral cytokine response that severely limits viral replication in nearby cells. The rapidity and intensity of this response is predicted by the density of HSV-2 specific CD8+ T-cells in the microenvironment. The observed influx of cells from blood is likely a mechanism to restore TRM levels and provide protection against subsequent HSV-2 reactivation.
Trusting their gut instinct: innate lymphoid cells utilize cytolytic mechanisms to regulate IgA
Authors: Karmele Erik P , Taft Diana H , Moran Michael T , Finkelman Fred D , Waggoner Stephen N
Published: January 2023

Immunoglobulin A (IgA) is important for the maintenance of homeostasis between the host and the intestinal microbiome. Antimicrobial IgA is secreted by IgA+ plasma cells that arise from germinal center (GC) reactions within gut-associated lymphoid tissues (GALT). Dysregulation of IgA production, a common condition with an unknown etiology, can lead to the improper clearance of commensal microbes, allowing for pathogenic strains to flourish and cause disease. Thus, a better understanding of the mechanisms involved in IgA production is critical. Our lab has previously shown that natural killer (NK) cells, a type of innate lymphoid cell (ILC), suppress GC responses and long-lived humoral immunity following systemic viral infection of mice. Here we show that depletion of NK1.1-expressing ILCs (e.g. NK, ILC1, ILC3) in specific-pathogen free mice resulted in enhanced T follicular helper cell and GC B cell numbers within GALT. The numbers of lamina propria IgA+ plasma cells were increased and the expression levels of fecal IgA were elevated for weeks after depletion of NK1.1-expressing ILCs. In addition, NK1.1-depletion was associated with alterations to the intestinal microbiome. In mice with defects in cytolytic granule-mediated killing, including perforin-knockout and beige mice, depletion of NK1.1-expressing ILCs did not enhance GALT GC responses or IgA production. This suggests that a cytolytic mechanism is involved in this regulatory process, although the precise target of this killing remains undefined. Thus, innate lymphoid cells within GALT play an important role in determining the balance between host immunity and microbial commensalism, and may represent new therapeutic targets to enhance efficacy of oral vaccines.