bluuebunny/crossref_metadata_2025 huggingface dataset:
Ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 inhibits IL-6 production by regulating Nur77 stability
Authors: Baek Suk-Hwan , Pei Han Zhong
Published: January 2023

Nur77 (NR4A1) plays an important role in various cellular responses such as apoptosis and inflammation. Nur77 is rapidly degraded in cells and its protein level is critically controlled. Although few ubiquitin E3 ligases regulating the Nur77 protein have been defined, the deubiquitinase (DUB) responsible for Nur77 stability has not been reported to date. We identified ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 (OTUB1) as a DUB that stabilizes Nur77 by preventing its proteasomal degradation. We found that OTUB1 interacted with Nur77 to deubiquitinate it, thereby stabilizing Nur77 in an Asp88-dependent manner. This suggests that OTUB1 targets Nur77 for deubiquitination via a non-canonical mechanism. Moreover, we confirmed that OTUB1 reverses ubiquitination of the K48-linked chains in Nur77. Functionally, OTUB1 inhibited TNFα-induced IL-6 production by promoting Nur77 protein stability. OTUB1 modulated the stability of Nur77 as a counterpart of tripartite motif 13 (Trim13). That is, OTUB1 reduced the ubiquitination and degradation of Nur77 potentiated by Trim13. In addition, this DUB also inhibited IL-6 production, which was further amplified by Trim13 in TNFα-induced IL-6 production. These findings suggest that OTUB1 is an important regulator of Nur77 stability and plays a role in controlling the inflammatory response.
Modulation of Id3 induces a fetal-specific HEB-dependent gamma delta T cell developmental pathway in the adult mouse thymus
Authors: Selvaratnam Johanna Samantha , Anderson Michele K
Published: January 2023

Gamma delta T cells (γδ T cells) are key players in tissue barrier homeostasis and immunity. Understanding the molecular mechanisms by which γδ T cells are programmed to develop into the γδT17 lineage is essential to develop strategies for controlling IL-17 activity during pathogenic conditions. Previously we showed that the E protein transcription factor HEB is an essential regulator of the γδT17 lineage, and that the generation of fetal γδT17 cells occurs through a HEB-dependent fetal-restricted pathway that does not involve upregulation of CD73 (Pathway 2). Id3, an antagonist of E proteins, inhibits key regulators of γδT17 development. Therefore, we hypothesized that the loss of Id3 would enhance γδT17 development. We characterized adult Id3-RFP knock-in mice, and found that the Id3−/− recapitulated the previously established expansion of Vγ1+ γδ T cells. However, the adult Id3+/− thymus showed a re-emergence of Pathway 2, suggesting that reduced Id3 levels promotes the fetal HEB-dependent γδT17 program. Moreover, there was an increase in Vγ1-Vγ4- γδ T cells, which suggests the appearance of Vγ6+ cells that are normally restricted to the fetal thymus. Interestingly, Vγ4+ cells and Vγ1-Vγ4-cells in Id3+/− mice displayed Pathway 2 profiles, while Vγ1+ cells exhibited the normal adult thymic developmental profile leading to mature CD73+ γδ T cells in both Id3+/− and Id3−/− mice. Future studies will examine whether reduced HEB in the Id3+/− mice inhibits Pathway 2 in the adult thymus, and obtain more definitive evidence as to whether the Id3+/− γδ T cells in the adult thymus develop toward the IL-17 fate. In conclusion, our studies indicate that the balance between E proteins and Id3 regulates the developmental programming pathway of γδ T cells.
A critical role for the Slam/SAP signaling pathway in the developmental programming of γδ T cell function
Authors: Boyson Jonathan E , DeVault Victoria L , Mistri Somen K , Mei Linda , Musial Shawn C , Spear Camarie , Dragon Julie A. , Veillette Andre , Dienz Oliver
Published: January 2023

Tissue-resident γδ T cells constitute a prominent source of IL-17 and IFN-γ in mucosal tissues where they provide an important first line of defense against pathogens. Unlike conventional αβ T cells, most γδ T cells are programmed during thymic development to produce either IFN-γ or IL-17. The mechanisms, however, that direct γδ T cell functional programming in the thymus remain unclear. Slam receptors are a family of nine (SLAMf1 to SLAMf9) cell surface receptors that are expressed only on hematopoietic cells and which play a central role in the host immune response. Most Slam receptors transduce signals via the small adapter protein SAP, and mice deficient in SAP exhibit numerous immunological defects. Relatively little is known, however, of the function of this signaling pathway in γδ T cells. Here, we report that distinct Slam receptor expression profiles mark functional γδ T cell subsets. Whereas SLAMf1 expression was associated with IL-17-producing CD27− RORγt+ γδ T cells, SLAMf6 expression was associated with IFNγ-producing CD27+ cells. We found that Slam receptor expression profiles were established on discrete thymic γδ T cell subsets as early as embryonic day 17 (E.17), as was the association of SLAMf1 with RORγt expression. Disruption of Slam receptor signaling by deletion of SAP resulted in a significant loss of E.17 thymic CD44hiRORγt+ γδ T cells, and a significant shift in the ratio of IFN-γ/IL-17-producing γδ T cells in both thymus and peripheral tissues. These results demonstrate for the first time that the Slam/SAP signaling pathway plays a significant role in the functional programming that regulates the γδ T cell IFN-γ/IL-17 axis during thymic development.
Conditional deletion of the V-ATPase a2-subunit disrupts intrathymic T cell development
Authors: Peterson Theodore V , Reynolds Joseph M
Published: January 2023

Proper orchestration of T lymphocyte development is critical, as T cells underlie nearly all responses of the adaptive immune system. Developing thymocytes differentiate in response to environmental cues carried from cell surface receptors to the nucleus, shaping a distinct transcriptional program that defines their developmental outcome. Our recent work has identified a previously undescribed role for the vacuolar ATPase (V-ATPase) in facilitating the development of murine thymocytes progressing toward the CD4+ and CD8+ αβ T cell lineages. Vav-Cre recombinase-mediated deletion of the a2 isoform of the V-ATPase (a2V) in mouse hematopoietic cells leads to a specific and profound loss of peripheral CD4+ and CD8+ αβ T cells. Utilizing T cell-restricted Lck- and CD4-Cre strains, we further traced this deficiency to the thymus and found that a2V plays a cell-intrinsic role throughout intrathymic development. Loss of a2V manifests as a partial obstruction in the double negative stage of T cell development, and later, a near complete failure of positive selection. These data deepen our understanding of the biological mechanisms that orchestrate T cell development, and lend credence to the recent focus on V-ATPase as a potential chemotherapeutic target to combat proliferative potential in T cell lymphoblastic leukemias and autoimmune disease.
STAT5b dimers and tetramers are critical for Mast Cell Function
Authors: Kiwanuka Kasalina N , Lin Jianxin , Leonard Warren J , Ryan John J
Published: January 2023

Signal transducers and activators of transcription (STATs) are latent transcription factors vital for lymphoid, myeloid, and erythroid cell development, survival, proliferation, and function. Activated STATs form dimers that translocate to the nucleus and bind high affinity STAT DNA motifs to modulate gene expression. We previously found that Stat5b is required for IgE-mediated mast cell cytokine production in vitro. In this study, we found that STAT5b KO mice demonstrate decreased sensitivity to IgE-mediated passive systemic anaphylaxis (PSA) and reduced cytokine production in vivo. This is not due to differences in mast cell numbers or distribution. Interestingly, STAT5b KO mice exhibit elevated levels of serum IgE that could diminish PSA by preventing binding of antigen-specific IgE. Increased serum IgE is not tied to changes in B cell number or their ability to produce IgE in vitro. In addition to dimers, activated STAT proteins can also form tetramers through an oligomerization process once dimers bind to tandem low-affinity STAT DNA motifs. Similar to STAT5bKO, in vitro IgE-mediated cytokine production is decreased in Stat5a-Stat5b double knock-in (DKI) mice in which STAT5 proteins can form dimers but not tetramers. In vivo, DKI mice exhibit decreased PSA sensitivity, with no difference in mast cell numbers or distribution, and a modest increase in serum IgE. Collectively, these data illuminate the unique and specific roles of STAT5 dimers and tetramers in mast cell function and allergic disease.
Transient Suppression and Functional Modulation of Mucosal Mast Cells for the Treatment of Allergic Disease
Authors: Kurashima Yosuke , Takasato Yoshihiro , Kiuchi Masahiro , Hirahara Kiyoshi , Murasaki Sayuri , Kunisawa Jun , Kubo Masato , Nakayama Toshinori , Kiyono Hiroshi
Published: January 2023

Food allergy is estimated to affect about 5 percent in children and adults and causes life-threatening reactions. Especially food allergy in infant and child shows severe gastrointestinal symptoms, including diarrhea, vomiting, and abdominal pain. To control the food allergy, allergen desensitization by oral immunotherapy (OIT), has been shown to an effective therapeutic approach; however, limited information is currently available for basic understandings of OIT induced immunological events at intestinal mucosa. In this study, we found that orally desensitized intestinal mast cells acquired the regulatory function for the inhibition of food allergic response. Indeed, the gene analysis showed the increase of regulatory cytokine IL-2 and IL-10 expressions in orally-desensitized intestinal mast cells; on the contrary, decreased Th2 cytokine expressions were suppressed by epigenetic control. Thereby, mice depleting these regulatory mast cells during OIT led to the reduction of functional regulatory T cell-mediated inhibition of allergic responses. These findings newly uncovered characteristics desensitized mast cells to acquire regulatory suppressive milieu in the allergic disease mucosa and indicated the novel roles of mast cells in the effective anti-allergic therapy.
Pre-treatment with lipopolysaccharide (LPS) protects against a normally lethal respiratory viral infection
Authors: Resiliac Jenny , Rohlfing Michelle , Santoro Jennifer , Hussain Syed-Rehan A. , Grayson Mitchell H
Published: January 2023

Rationale

Severe respiratory viral infections are responsible for significant morbidity and mortality in the young and elderly. The mechanism of the mortality is not known. We hypothesized that impaired neutrophil (PMN) function might contribute. To test this, we used the Sendai virus (SeV, murine parainfluenza virus type 1) mouse model, and examined the effect in mice with deficient PMN responses (Mpo−/−). Since lipopolysaccharide (LPS) exposure leads to accumulation of PMN, we tested the effect of LPS on mortality. Because the SeV model depends on production of IgE against the virus, we used Ige−/− mice to examine whether IgE could be playing a role in development of mortality.

Methods

C57BL6 wild-type (WT) mice were intranasally (i.n.) inoculated with 2×106 pfu SeV (lethal dose) alone or given 3μg LPS i.n. 24 hours before or after SeV and survival determined. Similar experiments were performed with mice deficient in myeloperoxidase (Mpo−/−) or IgE (Ige−/−).

Results

Regardless of genetic background, all mice infected with SeV or given LPS after SeV died by day 12 post SeV. However, LPS given before SeV led to 25% survival in WT mice, compared with 88% survival of Ige−/− mice and 100% survival of Mpo−/− mice (p=0.0134 for all 3 groups, Mantel-Cox, n≥4).

Conclusion

LPS pretreatment provides protection from a lethal dose of SeV, but only when PMN function or IgE is deficient. Thus, PMN and IgE appear to be important for development of respiratory viral infection mortality. We are actively exploring how LPS provides protection, and the mechanism(s) underlying the PMN and IgE requirement for viral induced mortality.
Probiotic Lactobacillus GG attenuates pulmonary inflammation through interfering the crosstalk between intestinal microbiota and ILC2 in asthmatic mice
Authors: Peng Guiying , Liu Zirui , Wang Yongan , Ge Dongyu , Hao Yu
Published: January 2023

Type 2 innate lymphoid cells and microbiota play crucial roles in type 2 immune response which is associated with asthma. In this study, we applied an OVA-induced allergic asthma model to examine the treatment of LGG and antibiotics-induced microbiome depletion to analyze the relationship between the microbes and ILC2.

We found that pretreatment of LGG before sensitization in mice reduced the numbers of inflammatory cell from bronchoalveolar lavage fluid, inhibited the levels of IL-5 and IL-13 and ameliorated pulmonary inflammation as well as mucus secretion. Additionally, LGG significantly inhibited the induction of KLRG1+ILC2. 16SrDNA sequencing results indicated that asthmatic mice had a lower gut microbiome diversity and abundance compared with control mice. While LGG treatment improved microbial diversity. Notably, inflammatory cells were significantly decreased in different regions of jejunum, ileum and colon in LGG treated asthmatic mice.

On the other hand, depletion of intestinal microbiota with antibiotics obviously increased the number of KLRG1+ILC2 from intestinal lamina propria and induced the mucus hypersecretion in the lower respiratory tract. Moreover, intestinal microbial depletion strengthened the pulmonary inflammatory and induction of KLRG1+ILC2 in asthmatic mice. Furthermore, pretreatment of LGG didn’t inhibit the pulmonary inflammation in asthmatic mice with depletion of intestinal microbiota.

These findings uncovered a close crosstalk between ILC2 and pulmonary inflammatory, suggesting intestinal microbiota might be a primary determinant for the development of asthma, thus provided additional evidence for the strain treatment on allergic asthma in the future.
Dimethyl fumarate attenuates T helper type 2 (Th2) -mediated allergic airway inflammation by modulating dendritic cell function
Authors: Jaiswal Anil Kumar , Sandey Maninder , Suryawanshi Amol , Cattley Russell C , Mishra Amarjit
Published: January 2023

Dendritic cells (DCs) are primary antigen presenting cells in lungs that promote Th2 cell mediated immunity to house dust mite (HDM) allergen. DCs are attractive therapeutic targets to modulate allergen-induced airway inflammation and adaptive immune responses. Dimethyl fumarate (DMF) are methyl esters of fumaric acid and regulates immune cell differentiation and effector functions. However, the effect of DMF on lung DCs has not yet been addressed. Here, we investigated the effect of DMF treatment employing murine model of HDM-induced experimental allergic asthma and DC adoptive transfer experiment. We show that local DMF application attenuates airway inflammation, mucous cell hyperplasia, and airway resistance to increasing dosage of inhaled methacholine. Notably, DMF administration in the allergen challenge phase significantly inhibited the lung recruitment of CD11b+ conventional DCs (cDC2) and suppressed Th2 cytokine producing IL4, IL5, and IL13 CD4+ T cells. In lung draining mediastinal lymph node (mLNs), DMF treatment significantly induces CD4+T regulatory (Tregs) cells. Moreover, prior to administration of fluorescent-labeled HDM, single DMF application to naïve mice impeded the migration of CD11b+ cDC2 to the draining mLNs. Adoptive transfer of DMF treated CD11c+ HDM-pulsed DCs to naïve mice with subsequent intranasal HDM-challenge similarly attenuates the features of airway inflammation. Collectively, our findings suggest that local DMF application modulates lung DCs function, thereby attenuates airway inflammation and is a potent antiasthmatic therapeutic agent.
The role of tyrosine kinase Itk in T helper 17 and T regulatory cells in IL17 mediated hypersensitivity pneumonitis
Authors: Elmore Jessica , Carter Chavez , Huang Weishan , Koylas Nicholass , Bennett Amelia , August Avery
Published: January 2023

The balance of inflammatory and suppressive cytokines is critical in controlling inflammatory responses, and the pro- and anti-inflammatory cytokines IL17A and IL10 has been implicated in numerous pulmonary inflammatory diseases. The tyrosine kinase, Itk, plays a critical role in T cell activation. We have previously shown that Itk−/− mice do not develop allergic airway disease coupled with reduced production of IL17A in the lungs. In vitro, Itk is required for the development of Th17 cells and their production of the pro-inflammatory cytokine IL17A. Furthermore, Type I regulatory and Foxp3+ T regulatory (Tregs) cells, suppressive producers of IL10, are positively and negatively regulated by Itk respectively. Farmer’s lung is a subset of hypersensitivity pneumonitis caused by repeated exposure to the bacteria Saccharopolyspora rectivirgula (SR), and is dependent on IL17A and regulated by IL10. We find that surprisingly, exposure to SR drives robust IL17A response produced by CD4+ T cells even in the absence of Itk, accompanied by the development of pulmonary inflammation. SR also induced Itk independent development of a population of population of Foxp3+ Tregs cells that produce IL17A, accompanied by a significant decrease in Tr1 cells compared to WT mice. These studies suggest that TCR signaling through Itk differentially regulates the development of inflammatory Th17 cells and suppressive Tregs and Tr1 cells. Understanding how Itk modulates the development of Th17/Treg cytokine responses will allow us to better understand the precise role of Itk in the production of IL17A and IL10 in airway inflammation.
Augmented Co-Stimulatory Repertoire of γδ-T Cell Pool in Pediatric Peanut Allergic Patients
Authors: Watkin Levi B , Hassan Oluwatomi , Davis Carla M , Anvari Sara
Published: January 2023

The prevalence of Atopic disease has been on a steady increase over the last 20–30 years. Of these diseases, food allergy represents a major proportion of the anaphylaxis mediated deaths, particularly to peanut. While there are presently multiple theories to explain this increase in allergy, it is clear that in food allergic individuals there is a loss of peripheral tolerance to these innocuous antigens. One of the most effective means of generating peripheral tolerance is through the gut which is home to many specialized immune cells, including intra-epithelial lymphocytes (IELs). Of these IELs, γδ-T cells represent nearly 50% of the population. Due to the large representation of the IELs in the gut, we hypothesize that alteration in the γδ-T cell pool plays a role in the loss of tolerance to these innocuous antigens. Here we examined PBMCs from peanut allergic patients and compared them to healthy controls. We find that while there is no difference within the overall frequency of total γδ-T cells between healthy and peanut allergic patients, when separated based upon CD4 and CD8 expression, we find the CD8 population decreased in the peanut allergic population. Additionally, when investigating subsets within the CD8 γδ-T cells we find altered frequencies of cells expressing costimulatory molecules both classically thought of as being expressed on T-cells (CD7 and OX40), and on antigen presenting cells (CD86). Taken together, these data suggest that γδ-T cells may contribute to the pathogenesis of peanut allergy by way of augmenting the costimulatory dynamic taking place in the gut. Further investigation is required to determine whether this observation leads to the development of peanut allergy or is a result of the disease on the individual.
Gut oxidative responses by chronic exposure to pollutants enhance IgE production via PGE2-dependent mechanism
Authors: Kim Eunsoo , Bonnegarde-Bernard Astrid , Opiyo Stephen O. , Ahmer Brain M. , Comet-Boyaka Estelle , Boyaka Prosper N
Published: January 2023

Environmental pollutants are believed to contribute to the increased incidence of allergic diseases. However, mechanisms linking exposure to pollutants with allergy are poorly understood and researches about heavy metals are mostly focused on its toxicity. Groups of mice were chronically exposed to subtoxic doses of cadmium in the drinking water. We then analyzed the impact of this exposure on gut immune homeostasis, microbiota, allergic sensitization to ingested allergen and subsequent allergic responses after allergen challenge. Chronic ingestion of subtoxic doses of cadmium increased IgE responses of mice to ingested allergen and the severity of allergy symptoms upon subsequent allergen challenge. We also found that exposure to subtoxic doses of cadmium induces dysbiosis of the gut microbiota and luminal accumulation of oxidized vitamin D3 metabolites. More importantly, we found that this microenvironment selectively enhances the activation of BAFF and the production of IgE via PGE2-dependent mechanism by intestinal epithelial cells. These findings reveal a new mechanism for the induction of IgE responses via alteration of vitamin D3 metabolism by environmental pollutants which stimulate an oxidative and inflammatory environment in the gut.
Characterization of protein allergens in a mouse model of wheat-induced anaphylaxis
Authors: Gangur Venugopal , Jorgensen Rick , Gao Haoran , Jin Yining , Salloum Jillian , Jian Dan , Ng Perry KW
Published: January 2023

Wheat allergies are among the major types of food allergies that are potentially life-threatening because of anaphylaxis. Wheat contains 3 different types of protein allergens: water/saline-soluble albumins/globulins, alcohol-soluble gliadins and acid-soluble glutenins. Although gliadins have been linked to anaphylaxis, whether or not other proteins cause anaphylaxis is not well understood. Here, we sought to identify saline-soluble wheat proteins (SSWP) involved in anaphylaxis using a mouse model. Balb/cJ mice were sensitized to SSWP by four intraperitoneal (IP) injections along with alum as an adjuvant. Sensitization was confirmed by testing for SSWP-specific IgE using ELISA. Anaphylaxis was measured by hypothermia shock response. After multiple booster injections with SSWP, hyper-IgE plasma was collected and pooled to create a mini-plasma bank. Using hyper-IgE plasma, an IgE-Western blotting (WB) method was optimized. The IgE-binding proteins were identified by sequencing using LC/MS/MS method. The hyper-IgE plasma had an IgE titer of ~2560. The optimized IgE-WB included 3 overnight incubations followed by signal detection using an HRPO-based method. Sequencing of the IgE binding bands in WB identified the following proteins: globulin 3A (66 kDa), globulin 3B (57 kDa), serpin (43 kDa), glyceraldehyde-3-phosphate dehydrogenase (37 kDa), chitinase (28 kDa) and globulin 1 (25 kDa). This is the first study characterizing salt-soluble protein allergens in a mouse model of wheat-induced anaphylaxis. Funding: USDA, NIFA, Hatch project MICL02486 (Accession Number: 1012322), Agricultural and Food Research Initiative Competitive Program, grant number: 2018-67017-27876.
The bone marrow-derived Gr-1+CXCR2+ cells are required for T cell infiltration into the skin challenge site to elicit contact hypersensitivity responses are inflammatory monocytes expressing FasL and perforin
Authors: Kish Danielle , Fairchild Robert L
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell mediated response to skin sensitization with hapten and subsequent challenge. Hapten-primed CD8 T cell infiltration into the skin challenge site during elicitation of CHS requires prior infiltration and activation of Gr-1+CXCR2+cells expressing both FasL and perforin. This study tested factors generating these myeloid cells in the bone marrow (bm) of hapten sensitized mice. The myeloid cells were enriched from the bm of DNFB sensitized mice 2 hrs after hapten challenge by depleting CD3+, B220+, NK1+, F4/80+, class II MHC+and Ly6G+cells with >90% of the remaining cells expressing Gr-1, CXCR2, CD11b, CD11c, Ly6C, CCR2, and CX3CR1, the phenotype of inflammatory monocytes (IM), plus FasL. The FasL+IM were at low numbers in the bm of naïve and hapten sensitized mice but increased to peak numbers 2–6 hrs. after hapten challenge of sensitized wild type (WT) mice. The FasL+IM were also generated in the bm of hapten sensitized and challenged Rag−/−mice and T-cell depleted WT mice, indicating that their generation can occur independently of T cells. The FasL+IM were not generated following hapten challenge of sensitized mice with targeted deletions in TLR4, IL-1β receptor or IFN-γ genes or in sensitized WT mice treated with anti-TNF-α mAb during hapten challenge. However, treatment with anti-GCSF antibody during hapten challenge increased their numbers in the bm and increased the magnitude of the CHS response elicited. Collectively, these results indicate that haptenreactive CD8 T cell infiltration into hapten challenged skin requires the TLR4-, TNF-α- and IFN-γ-dependentgeneration of bm-derived IM expressing FasL and perforin and their activation within the hapten challenged skin site.
Immune modulation caused by synthetic opioids during CD4+ T cell activation
Authors: Llorens Braulio Jose , Levine Alan D
Published: January 2023

Opioids are heavily prescribed medications used to treat chronic pain. Since their additional response includes euphoria, there is an exceedingly high addiction rate that has led to a country wide epidemic of opioid misuse. Opioid effects are not limited to the nervous system, since leukocytes express opioid receptors, which are G-protein coupled receptors known as μ, δ, and κ, suggesting that natural and synthetic opioids might regulate the immune system. Based on previously published reports, we hypothesized that opioids induce distinct regulatory pathways in CD4+ T cells that results in inhibition of the immune response. CD4+ T cells are isolated by negative selection from peripheral blood mononuclear cell and opioid receptor expression is assessed on resting and activated T cells using flow cytometry. Two models of opioid exposure in vitro were developed to study the effects of opioids on CD4+ T cell activation. Cells were treated with a δ opioid receptor specific agonist (DPDPE) for 24 h before or after activation and harvested 24 h after the second treatment and stained for expression of a well characterized early T cell activation marker – CD69. Cells treated with an opioid after activation showed no major shifts in CD69 intensity, suggesting opioids had no influence on a previously activated T cell. However, CD4+ T cells treated with an opioid before activation exhibited an increase in CD69 expression, suggesting an unexpected enhancement of T cell activation. In conclusion, opioids seem to play distinct biological activities depending on the activation state of the CD4+ T cell, and may function as both immunoinhibitory and immunostimulatory agents. Future studies will identify the mechanisms by which opioids regulate immunity.
Secondary stimulation of CD4 T cells generates an exhaustion phenotype
Authors: Wolf Gideon S , Singh Nevil J
Published: January 2023

Activated naïve T cells typically acquire effector functions over a 2–3 day period of differentiation in the secondary lymphoid organs, before migrating to the tissue sites to deliver effector functions. If the stimulating antigen is long lasting, as in the case of chronic infections, the T cells may undergo exhaustion, often through pathways involving co-inhibitory receptors such as PD1. Surprisingly, we find that a secondary and acute stimulation even short periods after the initial antigen encounter leads CD4+ T cells to exhibit properties of an exhausted phenotype. This is marked by the loss of proliferative potential and the upregulation of PD1. Naïve TCR-transgenic 5C.C7 T cells stimulated with their cognate peptide, proliferate rapidly between 2–4 days. Re-stimulation of these T cells on the 3rd day however, revealed a profound proliferative arrest accompanied by the rapid re-expression of PD1. The new levels of PD1 was proportional to the dose of re-stimulating antigen. This phenomenon was distinguished from Activation Induced Non-Responsiveness (AINR) as the 2nd stimulation arrested even the basal proliferation that was residual from the 1st antigen exposure. However, this is consistent with the property of effector T cell tuning that was previously described for effector T cells homing to tissue sites. We are currently examining the molecular machinery underlying this duality during T cell expansion to understand the paradoxical loss of T cell responsiveness to antigen during a primary effector response. These studies are expected to develop new approaches to not only improving primary T cell responses but also circumventing exhaustion during chronic microbial infections and tumors.
Runx1 is a key regulator of T cell anergy
Authors: Wilfahrt Drew Nicholas , Hsu Fan-Chi , McCue Shaylene , Rajcula Matthew , Shapiro Virginia Smith
Published: January 2023

The transcription factor Runx1 is critical for T cell development and maturation. CD4-Cre Runx1 conditional knockout mice have a block in T cell maturation, leading to complement deposition on recent thymic emigrants, and leaving very few CD4+T cells in peripheral lymphoid organs. Interestingly, thymic- and splenic-derived T cells from CD4-Cre Runx1 cKO mice exhibit increased expression of markers indicative of anergy including FR4 and CD73, which is not seen in other genetically modified mice with a block in T cell maturation. These data suggest Runx1 plays a role in regulating the development of T cell anergy in CD4+T cells. We have generated a 1:1 mixed bone marrow chimera (BMC) system with Estrogen Receptor-Cre (ER-Cre) Runx1 conditional knockout and wild type B6.SJL bone marrow to delete Runx1 in mature CD4+ T cells. Upon tamoxifen treatment and deletion of Runx1, the ER-Cre Runx1 cKO CD4+ T cells spontaneously develop an anergic phenotype as they fail to proliferate upon TCR/CD28 stimulation, and co-express CD73 and FR4. This cell surface phenotype is cell intrinsic since wild type B6.SJL CD4+ T cells in the mixed bone marrow chimera do not upregulate CD73 and FR4 upon tamoxifen treatment. Surprisingly, the mixed BMC system reveals both the ER-Cre Runx1 cKO and the wild type B6.SJL peripheral T cells fail to proliferate in response to TCR/CD28 stimulation, suggesting Runx1 also regulates a cell-extrinsic suppressive mechanism controlling T cell tolerance. This cell-extrinsic induction of anergy requires Runx1-deficient CD4+ T cells, but it is not dependent on Runx1-deficient Tregs. Thus, Runx1 is a key regulator of T cell anergy, and further analysis of this BMC model will serve to uncover the mechanism.
Negative feedback control of autoimmunity by a novel population of regulatory unconventional CD8+ T cells
Authors: Marrero Idania , Sheng Huiming , Maricic Igor , Fanchiang Shaohsuan S , Zhang Sai , Sant’Angelo Derek , Kumar Vipin
Published: January 2023

Immune tolerance mediated by regulatory T cells is important in the control of homeostasis. The characterization of regulatory CD8 T cells has been hampered by inability to distinguish them from conventional CD8 T cells. We have identified a novel population of unconventional cytotoxic TCRαβ+CD8+ T cells with regulatory properties, called uTr, in both naïve mice and humans. The uTr cells are innate-like, express the promyelocytic leukemia zinc finger (PLZF) transcription factor and display an activated-memory phenotype (CD122high, CD44high,CD62Llow, CD69+). They are CD8αα+, NK1.1+(CD161+ in human), express NK inhibitory receptors and are dependent upon IL-15/IL-2Rβ signaling. A significant decrease in the frequency of uTr cells in PLZF−/− mice suggests their dependency on PLZF expression. Similarly, a significant reduction of uTr cells in Qa-1b−/− mice indicates that a sizable proportion of them are restricted by Qa-1b MHC molecules. The deep-sequencing, RT-PCR and FACS analysis of sorted hepatic uTr cells indicate a polyclonal TCR repertoire in both mice and humans. Adoptive transfer of sorted hepatic uTr cells but not conventional CD8 T cells protects B6 mice from EAE as well as RAG1−/− mice from T cell-induced colitis. The uTr cells secrete IFNγ, IL-4 and IL-17 but not TGFβ and express perforin and granzyme B and the mechanism(s) of regulation is dependent upon perforin. The uTr cells are distinct from other unconventional T cells, including NKT cells, NKT-dependent innate-like CD8 T and MAIT cells. Collectively, we have identified a novel unconventional regulatory CD8 T cells that are involved in a negative feedback mechanism of regulation of autoimmunity and also have important implications for anti-cancer immunity.
LAG-3+ induced regulatory T cells confer infectious tolerance with suppression of IFN-γ response decoupled from reserved proliferation
Authors: Huang Ching-Tai , Dutta Avijit , Hung Chen-Yiu , Chen Tse-Ching , Lin Chun-Yen , Lin Yung-Chang , Chang Chia-Shiang , Chen Ting-An , Huang Yu-Lin
Published: January 2023

Induced regulatory T cells (iTreg) is an adaptive response in inflammation with disease processes such as infection, autoimmunity or cancer. A small number of iTreg may exert long-lasting regulation. Here we report the LAG-3+ iTreg transfer regulatory activity to the CD4+ T cells they suppress. This infectious propagation of regulatory activity exemplify a dominant immune regulation in vivo. Naïve hemagglutinin (HA) antigen-specific CD4+ T cells are induced to differentiate into IFN-γ competent effector T cells and then LAG-3+ Tregs in HA transgenic mice inherently expressing the cognate HA antigen. The LAG-3+ iTreg suppresses activation of effector function of cognate naïve CD4+ T cells in HA transgenic mice, with silenced IFN-γ machinery including STAT1 phosphorylation and T-bet induction. The LAG-3 iTreg suppressed, IFN-γ disarmed CD4+ T cells still proliferate and acquire LAG-3 expression on their surface in vivo. They become the next generation LAG-3+ iTregs with the ability of suppression of activation of effector function of cognate naïve CD4+ T cells in HA transgenic mice, with the same mechanism of silenced IFN-γ machinery. LAG-3+ iTreg exercises regulation in vivo by suppression of effector function decoupled from reserved proliferative response. The effector function suppressed CD4+ T cells still proliferate upon activation and acquire LAG-3+ iTreg phenotype, a reminiscence of the infectious tolerance proposed in 1970.
Construction of CAR-iTreg (Chimeric antigen receptor-inducible Treg) for inducing tolerance in transplantation
Authors: Won Hye-ji , Kim Yong-Hee , Park Chung-Gyu
Published: January 2023

Tolerance induction is ultimate goal in the field of transplantation. Cell-based therapy with regulatory T cells (Treg) has been effective and evolving, Chimeric antigen receptor (CAR) T cells to target specific antigen are new promising cell-based therapy. Engineering of CAR-Treg have many advantages that can be used in transplantation. Here, we designed development of CAR-iTreg (Chimeric antigen receptor-inducible Treg), which is engineered to become inducible Treg in vivo. DNA for single chain fragment variable (scFv) was obtained from MR1 hybridoma to target CD40L as the antigen. Cytoplasmic signaling part was constituted with CD3z and CD28 for T cell activation, and with TGFβR1 and IL-2Rβ for Treg induction. The CAR DNA construct was inserted into pLVX-IRES-ZsGreen1 expression plasmid vector. The expression vector and packaging vectors were used to produce lentiviral vector in HEK293FT cells. Harvested lentivirus has been tested in Jurkat and EL4 cells, and the expression of ZsGreen fluorescence and the CAR could be confirmed. This CAR-containing lentivirus can be used to transfect naïve CD4+ T cells and be transferred to syngeneic mice. These CAR-T cells target CD40L expressed on activated T cell and convert into inducible Treg cells in the graft environment. Thus, this uniquely engineered CAR construct allows tolerance induction in transplantation model. Furthermore, this CAR-iTreg strategy with other scFv has potential application in autoimmune diseases.
Helios deletion in CD4+T cells induces pTregs that inhibit memory recall responses
Authors: Penna Vinay , Skadow Mathias , Shevach Ethan M , Thornton Angela M
Published: January 2023

The transcription factor Helios is required for the maintenance of the suppressive phenotype of Foxp3+ regulatory T cells (Tregs) as evidenced by the spontaneous autoimmune activation that develops in mice with a Foxp3− specific Helios deficiency. Helios is also upregulated in a subset of activated CD4+Foxp3−T conventional (Tconv) cells, but its role in Tconv cells remains unknown. Mice with a CD4-Cre driven Helios deletion do not exhibit autoimmunity despite the defect in the suppressor function of their Treg population, pointing to a requisite function of Helios in the activation of Tconv cells in response to antigen. We examined the capacity of Helios-deficient, naïve Tconv cells to respond to their cognate antigen in vivo and found that Helios was not required for proliferation, differentiation into helper subsets, or the production of effector cytokines. Unexpectedly, we observed that Helios deficiency increased the susceptibility of naïve T cells to differentiate into pTregs under inflammatory conditions. These pTregs were then subsequently able to suppress the reactivation of memory T cells in an antigen-specific manner upon challenge with antigen. These findings suggest that increased pTreg differentiation in the periphery in mice with a CD4-Cre driven Helios deletion may prevent the development of autoimmunity normally associated with Helios-deficient Tregs.
Itaconate inhibits alternative activation of macrophages by targeting Janus Kinase 1
Authors: Runtsch Marah C , O’Neill Luke
Published: January 2023

In innate immune cells, TCA cycle components are not simply inert metabolites, but are key signaling molecules that can modulate inflammatory responses. Itaconate, an immunometabolite made by the enzyme aconitase decarboxylase (encoded by the gene Irg1), is highly upregulated in classically activated M1 macrophages and regulates immune responses by dampening inflammation. The role of itaconate in activation of alternative macrophage programs is of interest, as itaconate has recently been shown to be taken up by M2 macrophages. Here, we show that expression of Irg1 is specific to M1 macrophages and opposes regulators of IL-4 mediated alternative activation. Overexpression of Irg1 in murine M2 macrophages inhibited their program. Similarly, exogenous addition of a cell-permeable derivative of itaconate inhibited M2 gene expression and blocked activation of Stat6 and AKT in IL-4 activated macrophages. Itaconate-treated M2 macrophages also showed decreased oxidative phosphorylation, altogether suggesting that itaconate constrains IL-4-mediated alternative activation of macrophages. Mechanistically, this novel role of itaconate was not through previously reported pathways Nrf2 or Complex II (SDH), but rather via inhibition of the phosphorylation of Janus Kinase 1. We also show that an M2-specific microRNA, miR-378a, targets Irg1 to prevent itaconate production in these cells. Thus, we demonstrate that itaconate antagonizes M2 macrophage activation, suggesting a mechanism by which M1 macrophages inhibit alternative macrophage polarization. This may be useful in considering the therapeutic potential of itaconate as an inhibitor of M2 macrophages in such conditions as allergic asthma and fibrosis.
Targeted Deletion of CD11b+ Macrophage Subtypes alter the Acute Inflammatory Cytokine Response In Bone Healing
Authors: Cottrell Jessica , Hozain Sarah , Touma Danielle
Published: January 2023

Any immunological or inflammatory dysregulation can impair the bone healing process. Macrophages (MØ) are immune cells specialized for phagocytosis and assist in all stages of bone repair. MØs are often divided into two main subsets, MØ1, pro-inflammatory M1 macrophages and MØ2, anti-inflammatory or resolving M2 macrophages. Our preliminary data demonstrates that MØ1 to MØ2 polarization occurs following femur fracture during the first 7 days post-fracture and is associated with a specific cytokine expression profile over time. Other studies have demonstrated that dysregulated inflammation can impair fracture healing outcomes in normal mice. We hypothesize that targeted depletion of CD11b+ MØ cells will reduce the acute inflammatory response, alter the normal cytokine expression profile, and interfere with the bone healing response. Therefore, the bone marrow of mice depleted of CD11b+ MØ cells were compared to normal mice following femur fracture at days 0, 1, 2, and 4 using established methods. MØ subtypes were identified using flow cytometry and cytokine expression was quantified using Luminex. The data demonstrated that after fracture, the CD11b+ MØ depleted group had significantly less MØ1s, F480+/CD86+/MHC classII+/CD11b+ cells when compared to controls at all timepoints. MØ cytokine expression profiles were found to be altered in the control fractures when compared to the CD11b+ MØ depleted fractures. Our results demonstrate that MØ subsets contribute substantially to the signaling mechanisms that guide the acute inflammatory phase of bone healing. Proper modulation of macrophage polarization may be useful to develop novel treatment strategies to improve healing when the immune system or inflammation is dysregulated.
Forkhead box O 6 modulates immunological functions of Dendritic cells
Authors: Kim Mi Eun , Cho Jun Hwi , Lee Jun Sik
Published: January 2023

Forkhead box O (FoxO) is a transcription factor which is involved in cell cycle progression, cell survival and DNA-repair. Dendritic cells (DCs) are professional antigen-presenting cells which present the antigens to T lymphocytes and regulate the adaptive immunity. Previous studies indicated that FoxO1 and 3 are plays a critical role in DC maturation and DC-T cell interaction. FoxO6, a member of FoxO family, is less attended than other FoxO families, because limited expression region of FoxO6. Also, the effect of FoxO6 is still unknown. Here, we investigated the effect of FoxO6 in dendritic cells. Out data suggest that FoxO6 is expressed in murine dendritic cells. Moreover, phosphorylated FoxO6 is increased and acetylated FoxO6 is decreased during dendritic cell differentiation. We expect that FoxO6 has important role in DC differentiation and maturation, and will further investigate the functions and the mechanisms of FoxO6 on DC. And alteration of FoxO6 expression level had effect on DCs maturation.

[This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A1A1008053), Korea.]
Analysis of regulatory elements in the HLA-A, HLA-B, and HLA-C genes provides insights into the specific role of each HLA in the immune system
Authors: Anderson Stephen , Li Hongchuan
Published: January 2023

We have conducted a detailed analysis of regulatory elements present in the upstream region of the HLA-A, HLA-B, and HLA-C genes. Significant differences in transcription factor-binding sites were identified in a tissue-specific promoter located 1.3 kb upstream, an enhancer element at −800 bp, and the core promoter region, that alter both the tissue specificity and expression level of the HLA genes. The upstream promoter has changed from macrophage-specificity in HLA-A to an unknown specificity in HLA-B, and it has gained NK specificity in HLA-C that is coupled with an elaborate post-transcriptional regulatory mechanism. The −800 enhancer region contains distinct arrays of transcription factor-binding sites in the HLA-A, HLA-B, and HLA-C genes. A comparison of the proximal promoter region of these three genes revealed multiple differences in key enhanceosome and cytokine-inducible elements, and the presence of additional trophoblast-specific elements in the HLA-C promoter. Furthermore, a comparison of HLA-C alleles reveals functional differences in transcription factor-binding sites that likely reflects allele-specific tuning of expression. A more complete understanding of the molecular evolution of regulatory elements between the HLA-A, HLA-B, and HLA-C genes will provide important clues with regard to the separate immunological functions of the three MHC class I genes present in humans.
Axl-mediated Akt phosphorylation leads to mTORC1 activation via NFk-B in renal mesangial cells
Authors: Shao Wenhai , Zhen Yuxuan
Published: January 2023

Proliferative glomerulonephritis (GN) is a critical condition in many forms of kidney diseases and characterized by the proliferation of mesangial cells with influx of inflammatory cells. Receptor tyrosine kinase Axl belongs to the TAM family, along with Mer and Tyro-3. Receptors from this family contribute to clearance of apoptotic cells and suppression of inflammation. Previously, we reported an important role of Axl-mediated mesangial proliferation in the anti-GBM induced mouse model of lupus nephritis. In this study, we investigated the signaling cascade induced by Axl activation in renal mesangial cells. We found that Axl activation led to the phosphorylation of mTORC1 complex, proceeded with the ribosomal protein S6 activation. Inhibition of NFκ-B activity diminished mTOR phosphorylation in mesangial cells, indicating that NFκ-B is a critical regulator for mTORC1 activation in the Axl/Akt pathway. Axl expression also participated in mesangial cell efferocytosis. Interestingly, mesangial efferocytosis is not mediated by Mer receptor tyrosine kinase, the canonical TAM family receptor facilitating apoptotic cell engulfment in macrophages. Taken together, data suggest that Axl mediated mTOR activation and apoptotic cell engulfment is renal mesangial cell specific, as those functions were not observed in similarly treated peritoneal macrophages.
Trib1 controls antiviral immunity by restraining CD4 and CD8 T cell effector responses during chronic infection
Authors: Rome Kelly S , Pear Warren S
Published: January 2023

During chronic infection, the magnitude of the antiviral T cell response is determined by the balance between T cell effector (TEFF) function and T cell exhaustion. While CD8 T cell exhaustion has been extensively characterized, how TEFF responses are regulated during chronic antigen exposure is less clear. We identify Tribbles Pseudokinase 1 (Trib1) as a central regulator of the TEFF response to chronic infection. We show that Trib1 expression is elevated in activated T cells and in human or mouse T cells following chronic infection. In chronically infected mice, T cell-specific deletion of Trib1 enhanced CD4 and CD8 TEFF expansion, persistence and function and reduced viral load. Mechanistically, we show that Trib1 functions as a negative regulator of signaling downstream of the T cell receptor (TCR), whereby Trib1 expression disrupts TCR signaling complexes. These data support a model of negative feedback regulation whereby Trib1 restrains TCR signaling and downstream function. Together, our work shows that Trib1 functions to restrain the magnitude and duration of the TEFF response, at least in part through the disruption of TCR signaling complexes, and reveals a new regulatory node downstream of TCR signaling that is critical during chronic infection. In light of growing interest in checkpoint blockade therapies that act to overcome exhaustion during chronic antigen exposure, our work reveals an important new avenue for enhancing the efficacy of current T cell reactivation strategies during chronic disease.