bluuebunny/crossref_metadata_2025 huggingface dataset:
Constitutive Activation of Stat3 in Thymic Epithelial Cells Drives Medullary Formation in the Thymus
Authors: Lomada Dakshayani , DiGiovanni John , Richie Ellen R.
Published: June 2021

It is well established that medullary thymic epithelial cells (mTECs) play an essential role in establishing central tolerance due to their unique ability to present a diverse array of tissue restricted Ags (TSA) that induce clonal deletion of self reactive thymocytes. However, the molecular signals that drive the development of mTECs have yet to be completely defined. A previous study using epithelial specific signal transducer and activator of transcription 3 (Stat3) revealed the importance of Stat3 in postnatal maintenance of thymic architecture and thymocyte survival (Sano et al Immunity, 2001). To determine the effect of constitutive Stat3 signaling in mTECs, we examined thymic development in mice that express a constitutively active Stat3 transgene regulated by a keratin 5 promoter (K5 Stat3C). The thymus of adult K5.Stat3C mice was hypo‐plastic, thymocyte cellularity was reduced by 80% and mTEC compartment was markedly expanded in 4‐week‐old mice. In Rag 2−/− mice, mTEC development is severely impaired due to an early block in thymocyte differentiation. Introduction of the K5.Stat3C transgene in Rag 2−/−mice rescues mTEC development and restores TSA and chemokine expression. These findings implicate a role for Stat3 signaling in development of the mTEC compartment. This research is supported by Rothman‐Stevenson Foundation.
Regulation of CD4 cell memory by PSGL‐1
Authors: Lin Melissa Michelle , Bradley Linda M
Published: June 2021

Long‐term immunity against infections depends upon the generation and maintenance of memory T cells. CD4 cells play a pivotal role in the induction of immunity to influenza viruses by regulating the responses of both CD8 cells and B cells. A fundamental feature of CD4 cells is their capacity to generate functionally distinct subsets that confer different protective functions. Recently, we found that the capacity to generate glycosylated, biologically active selectin ligands is acquired by a subset of CD4 effectors after influenza virus infection and that P‐selectin is induced in the lungs. In this study, we used CD4 cells from OT‐II TCR transgenic mice that lack the fucosyl transferases necessary for glycosylation (FucT IV/IV) of selectins to investigate the role of those receptors in the CD4 cell response to influenza. We demonstrate that selectin binding is not required to generate a normal primary response to PR8 influenza virus. However, FucT IV/VII −/− OT‐II cells exhibit elevated production of IFN‐gamma, increased homeostatic turnover, and a failure to undergo secondary expansion as memory cells. Our data support the overall hypothesis that mechanisms regulated by adhesion molecules can be essential for the delivery of signals which control CD4 cell responses and their homeostatic maintenance, thereby sanctioning the development and persistence of memory.

This research is supported by NIH Grant P01 AI046530.
Massive expansion of antigen‐specific CD4 T cells without memory differentiation following acute infection
Authors: Williams Matthew A , Ravkov Eugene
Published: June 2021

While recent efforts have delineated some of the signals required for CD8 memory T cell differentiation, the signals that lead to the differentiation of CD4 memory T cells are less clear. We analyzed the requirements for CD4 memory differentiation using adoptively transferred CD4 T‐cell receptor transgenic cells (SMARTA) specific for the immunodominant CD4 epitope of lymphocytic choriomeningitis virus (LCMV). Following LCMV infection, effector and memory differentiation of SMARTA cells mimicked the endogenous CD4 response throughout the effector and memory phases. In contrast, infection with a recombinant Listeria expressing the same epitope (Lm‐gp61) resulted in massive initial expansion of SMARTA cells but rapid loss of effector function and failure to form memory. Defective memory differentiation was seen only after stimulation of naive SMARTA cells, was independent of precursor frequency and associated with a gene expression profile indicative of Bim‐mediated apoptosis. Furthermore, their inability to form memory corresponded to a lower functional avidity as compared to endogenous responders in the same host, and long‐lived endogenous CD4+ memory T cells skewed to a higher functional avidity over time. These results support a model in which CD4 memory differentiation and longevity depend on the strength of the antigen‐driven signal during the primary response.
IL‐7 Receptor alpha expression marks CD8 T cells with increased proliferative capacity and ability to transduce AKT signals from common gamma receptor cytokines
Authors: Hand Timothy W , Kaech Susan M
Published: June 2021

Virus‐specific CD8 T cells expand greatly in response to viral infection and then contract to restore T cell homeostasis. How the death of these cells is regulated is not well understood. A population of CD8 T cells that maintains the expression of IL‐7 receptor (IL‐7R) during infection has been shown to preferentially survive contraction and differentiate into long‐lived memory T cells. Our previous findings using ectopic IL‐7R expression in all virus‐specific CD8 T cells showed that IL‐7R down regulation was not the cause of death of effector CD8 T cells following infection, and indicated that this process occurred largely in an IL‐7 independent manner. We have expanded this finding to show that compared to IL‐7Rhi effector CD8 T cells, IL‐7Rlo cellsare unable to proliferate substantially to the homeostatic cytokines IL‐2, 7 and 15, lymphopenic conditions or to specific antigen. Interestingly, this phenotype of IL‐7Rlo CD8 T cells correlates with an impaired ability to signal via the AKT pathway as shown by a reduced phosphorylation of the AKT, GSK‐3beta and FOXO transcription factors in response to cytokine stimulation. These results indicate that even as effectors, those T cells destined to become memory CD8 T cells have increased proliferative potential, possibly related to the function of the AKT pathway. Additionally, the death of IL‐7Rlo CD8 T cells is likely tied to their reduced signaling through this pathway.
Dual roles of IL‐15 in maintaining IL‐7 receptor alpha low CCR7− memory CD8+ T cells in humans via recovering the PI3K/AKT pathway
Authors: Kang Insoo , Kim Hang‐Rae , Hwang Kyung‐A
Published: June 2021

Recently, we identified two subsets of CCR7− memory CD8+ T cells expressing high and low levels of the IL‐7 receptor alpha chain (IL‐7Rα) that is essential for memory T cell survival in human peripheral blood. IL‐7Rαlow CCR7− memory CD8+ T cells have impaired proliferation and survival in response to T cell receptor (TCR) triggering and IL‐7, respectively. These findings raise a question of how such cells are sustained at significant numbers, greater than 20% of peripheral CD8+ T cells. Here we demonstrate that IL‐7Rαlow CCR7− memory CD8+ T cells have increased expression of IL‐2/15 receptor beta chain (IL‐2/15Rβ) with enhanced gene expression of T‐bet and eomesodermin, transcriptional factors involved in IL‐2/15Rβ expression compared to IL‐7Rαhigh CCR7− memory CD8+ T cells. Such a cytokine chain is functional as IL‐7Rαlow CCR7− memory CD8+ T cells proliferate considerably in response to IL‐15. Furthermore, adding IL‐15 to TCR triggering recovers impaired TCR‐mediated proliferation of IL‐7Rαlow memory CD8+ T cells via restoring the activation of PI3K/AKT pathway. These findings indicate that IL‐15 has dual roles in maintaining IL‐7Rαlow CCR7− memory CD8+ T cells via TCR‐dependent and ‐independent mechanisms. Moreover, IL‐15 can be useful in reviving impaired proliferative function of such memory CD8+ T cells with effector functions against infections and tumors via rescuing the PI3K/AKT pathway.
Disparate roles of SLP‐76 in CD4 T cell effector function and memory generation
Authors: Bushar Nicholas David , Maltzman Jonathan S , Farber Donna L
Published: June 2021

The signaling processes coupled to the T cell receptor (TCR) for naïve T cell activation have been well‐characterized; however, the role of TCR signaling intermediates in effector function and memory development remain unknown. To evaluate the role of the linker/adapter protein SLP‐76 in these processes, we used SLP‐76 conditional knockout (SLP76 CKO) mice containing floxed slp76 and a CRE recombinase inducible yfp reporter (SLP‐76Flox/‐ R26RYFP). Control yfp reporter mice were treated in parallel as controls for these experiments (CNTRL). CD4 T cells from SLP76 CKO and CNTRL mice were activated in vitro with anti‐CD3/CD28 antibodies, treated with membrane permeable TAT‐fused CRE recombinase to down regulate SLP‐76 expression, analyzed for effector phenotype, and transferred into RAG2−/− hosts. We found that down regulation of SLP‐76 in primed CD4 T cells did not appreciably alter their activation phenotype or ability to produce IL‐2 and IFNγ in response to TCR/CD3 ligation. However, when transferred into RAG2‐/‐ hosts, slp76 deleted cells persisted in much lower frequencies relative to controls, acquired a CD44int, CD62Lhi central memory phenotype, and were hindered in their long‐term persistence in vivo. These results suggest a differential role for SLP‐76 signaling in effector T cell function relative to naïve and suggest a crucial role for TCR signaling through SLP‐76 in long‐term memory generation.
ST6Gal‐I gene expression is required for optimal Ag‐specific CD8 T cell expansion in vivo
Authors: Onami Thandi M. , Patel Jenish R. , Zeng Junwei , Baum Linda G. , Ahmed Rafi
Published: June 2021

Glycosylation can affect the localization and interactions of cell surface glycoproteins, impacting cell signaling and function. ST6Gal‐I is a glycosyltransferase expressed by T and B cells that catalyzes the addition of a2,6 sialic acid to galactose on N‐linked glycoproteins such as CD45. We show that changes in expression of ST6Gal‐I during memory CD8 T cell differentiation results in changes in cell surface sialylation as demonstrated by SNA lectin binding. Moreover, lectin binding revealed heterogeneity within the memory CD8 T cell population isolated from tissues, identifying SNAhigh and SNAlow memory CD8 T cells. To further understand the role of this modification in T cell function in vivo, we examined ST6Gal‐I null mice following LCMV infection. Our analysis revealed lower numbers of Ag‐specific CD8 T cells at day 8 post‐infection compared to WT mice. In addition, adoptive transfer of P14×ST6Gal‐I+/− or P14×ST6Gal‐I−/− cells into WT recipient mice show reduced numbers of transgenic null CD8 T cells on day 8, suggesting the defect is T cell intrinsic. Finally we demonstrate using in vivo CFSE labelling of purified P14×ST6Gal‐I+/− or P14×ST6Gal‐I−/− CD8 T cells into WT recipient mice that null transgenic CD8 T cells show substantially reduced proliferation. These studies suggest that differential expression of surface carbohydrates can significantly impair CD8 T cell activation and proliferation in vivo.
Determining Th17 lineage commitment
Authors: Stritesky Gretta Lynne , Yeh Norman , Mathur Anubhav , Kaplan Mark
Published: June 2021

Autoimmune diseases including type 1 diabetes, multiple sclerosis, and rheumatoid arthritis are thought to be diseases mediated by a newly characterized subset of CD4+ T cells, Th17 cells. Th17 cells develop in the presence of TGFβ+IL‐6, and IL‐23 is thought to play a role in maintaining the phenotype. However, the commitment of Th17 cells is still unclear. We show that Th17 cells cultured with antigen presenting cells for multiple rounds of stimulation secrete high levels of IL‐17 upon re‐stimulation. IL‐17 production in long term cultures is enhanced when Th17 cells are cultured with IL‐1β. We further show that Th17 cells are not committed to secrete IL‐17. After three rounds of stimulation Th17 cells cultured with cytokines that promote Th1 or Th2 development have decreased levels of IL‐17 and increased levels in IFN‐γ and IL‐4, respectively. Decreased IL‐17 in Th17 cells cultured in opposing cytokines correlates with a decrease in mRNA expression of Th17 specific factors including RORγt and IL‐23R. This is in contrast to the ability of Th1 and Th2 cells to maintain their phenotype under these culture conditions. Thus, our results suggest that Th17 cells are not committed to an IL‐17‐secreting phenotype.
Induction of Th17 responses by human skin dendritic cells is dependent on IL‐15 and IL‐6
Authors: Mathers Alicia R , Janelsins Brian M , Rubin Joseph P , Tkacheva Olga A , Larregina Adriana T
Published: June 2021

Human skin dendritic cells (sDCs) are potent Ag presenting cells that prime and bias naïve CD4+ T cells into Th1 or Th2 effector lymphocytes. Recent data demonstrated that human monocyte derived DCs secreting IL‐12 induced Th1 responses but abrogated a Th17 bias. However, it is unknown if human sDCs that do not secrete IL‐12 and promote Th1 differentiation by secretion of IL‐23 are also capable of initiating proinflammatory IL‐17 responses. Here we analyzed the stimulatory properties of sDCs regarding their ability to promote the differentiation of both Th1 and Th17 responses. DCs purified from cultures of human skin explants, stimulated the proliferation of naïve CD4+ T cells that differentiated into two distinct effector Th cell subsets secreting IFN‐γ and IL‐17 and which expressed the skin homing molecules CCR4 and CCR10. The Th1‐biasing function of sDCs relied on the production of IL‐23. Th17 responses depended on the combined effects of IL‐15 and stabilized IL‐6:IL‐6 Ab complexes that stimulated CD4+ T cells through a molecular mechanism involving IL‐6R trans‐signaling, an effect that was inhibited by specific blockade of the IL‐6R. Additionally, IL‐17 production was independent of IL‐23 and suppressed by TGF‐β. We conclude that human sDCs induce coexistent Th1 and Th17 effector T cells with potential to home to the skin which is relevant for the development of cutaneous inflammation and autoimmunity.

Supported by NIH grants R01CA100893 to ATL and T32CA82084 fellowship to ARM.
Down‐regulation of IL‐7R on memory CD4+ T cells correlates with expression of cytotoxic granule proteins
Authors: Endsley Janice J , Hogg Alison E. , Lancaster Katrina L
Published: June 2021

Survival of activated T cells into long‐lasting memory cells has been shown to be dependent on expression of IL‐7 receptor (IL‐7R) at the peak of the immune response. The memory phenotype of cytotoxic CD4+ T cells is poorly defined compared to CD8+ T cells. The objective of these studies was to correlate IL‐7R expression on activated CD4+ T cells with expression of cytotoxic granule proteins: granulysin, perforin, and granzyme B. Our results demonstrate that TCR activated CD4+ T cells within peripheral blood down regulate IL‐7R as the cells divide and this down‐regulation of IL‐7R and CCR7 positively correlates with enhanced expression of granzyme B, granulysin, and perforin. After TCR activation of sorted CD4+ T cell memory subsets, the highest level of granulysin and perforin mRNA was observed in CD45RO+IL‐7R‐ CD4+ T cells. In contrast, granzyme B mRNA levels were most significantly increased in the CD45RO+IL‐7R+ subset and correlated with down‐regulation of IL‐7R levels. Our data suggests that although the IL‐7R+ memory CD4+ T cells lack immediate expression of cytotoxic proteins, upon secondary stimulation, these memory cells down‐regulate IL‐7R and give rise to IL‐7R−CD4+ T cells with cytotoxic potential. This research is supported by the Dept. of Microbiology and Immunology, UTMB.
Mature B cell deficiency in the A/WySnJ mouse is associated with altered TRAF interactions with BAFFR
Authors: Hildebrand Joanne , Larison Karen , Mayne Chris , Hayes Colleen , Bishop Gail
Published: June 2021

B cell activating factor (BAFF) is a member of the TNF family that is required for B cell survival and maturation. BAFF is known to bind three receptors of the TNF Receptor family of proteins; BAFF Receptor (BAFFR), TACI and BCMA. TNF receptor family proteins utilize characteristic combinations of TNF receptor associated factors (TRAFs) to bridge receptor activation with a multitude of downstream signaling pathways. BAFFR was initially discovered through studies of the A/WySnJ mutant mouse, which exhibits many characteristics of the BAFF knockout mouse including severe splenic B cell deficiency. In the A/WySnJ mouse the last eight amino acids of BAFFR are replaced with twenty one randomly encoded amino acids due to a retrotransposon insertion event. To date, BAFFR has been shown to recruit only TRAF3, via a distinct motif in the cytoplasmic portion that is analogous to the TRAF binding motif in CD40. While the distinct TRAF3 binding motif is not altered in the A/WySnJ mutant BAFFR, the insensitivity to BAFF and severe B cell phenotype of this mouse is evidence of defective signaling by this modified BAFFR. In this study, we utilized hCD40‐BAFFR chimeras stably expressed in cultured B cells to show that the mutant BAFFR of the A/WySnJ mouse exhibits different TRAF binding properties, which results in the altered activation of the alternative NF‐κB pathway (NF‐κB2) required for B cell survival and maturation.
Regulatory T cells and superantigen‐induced toxic shock. A study using HLA class II transgenic mice
Authors: Rajagopalan Govindarajan , Epstein Benjamin , Lytle Anna , David Chella
Published: June 2021

To understand the role of regulatory T cells in staphylococcal enterotoxin B‐induced toxic shock syndrome (TSS), we performed gene expression profiling in HLA‐DR3 transgenic mice using microarrays. We observed that at 3 hours after systemic exposure to SEB (10 μg), the splenic expression of FoxP3, a hall mark of regulatory T cells, was increased by 5 folds (p<0.005). In addition, other genes that regulate FoxP3+ Tregs were also significantly modulated by SEB. These include IL‐2R (27‐fold), IL‐2 (234‐fold), IL‐6 (689‐fold), IL‐21 (21‐fold), TGF‐B1 (0.7‐fold), IL‐17 (5.5‐fold) and IL‐22 (98‐fold). To further characterize the homeostasis of Tregs cells during superantigen‐induced TSS in vivo, HLA‐DR3 transgenic mice were first challenged with SEB and were administered BrdU in drinking water for 3 days. Mice were killed 3 days later and splenocytes analyzed by flow cytometry. We observed that in addition to FoxP3‐TCR Vβ8+ T cells, FoxP3+TCR Vβ8+ T cells also significantly expanded in vivo as evident from their BrdU staining patterns. This indicated that SEB is able to activate Tregs as well. Depletion of Tregs using anti‐CD25 antibodies (clone PC‐61, 500 ug/mouse) 2 days prior to SEB challenge did not significantly alter serum cytokine levels as compared to control mice treated with rat IgG and SEB. Thus, T regulatory cells have a minimal role in SEB‐induced TSS.
Effects of IL‐6 on Foxp3+ Treg subsets
Authors: Zheng Song Guo , Wang Juhua , Horwitz David A
Published: June 2021

TGF‐β promotes either effector or regulatory T cell differentiation depending upon other cytokines in the local environment. While IL‐2 and TGF‐β induce naive T cells to become Foxp3+ regulatory cells (iTregs), the combination of IL‐6 and TGF‐β induce pro‐inflammatory IL‐17‐producing cells (Th17). Others have reported that IL‐6 can utilize TGF‐β produced by thymus‐derived natural Treg cells (nTreg) to convert them to Th17. We report that TGF‐β‐induced Tregs were completely resistant to IL‐6, and Foxp3 expression and suppressive activity in vitro and in vivo was unaffected. We then observed that the combination of IL‐2 and TGF‐β enables nTregs to also become resistant to IL‐6 by down‐regulating IL‐6R. Thus, besides generating iTregs, IL‐2 and TGF‐β stabilizes the suppressive phenotype of nTregs. The different sensitivities of Foxp3+ nTregs and iTregs to IL‐6 may reflect different roles for these two subsets in the adaptive immune response, and emphasizes the central role of the combination of IL‐2 and TGF‐β to control immunologic homeostasis.

This work was supported by grants from the Arthritis National Research Foundation, Clinical Research Feasibility Fund, James H. Zumberge Faculty Research Innovation Fund, the Arthritis Foundation, ExCell Therapeutics, Nora Eccles Treadwell Foundation, and BD Biosciences.
Autocrine IFN‐gamma directly regulates Foxp3 expression in naive CD4+CD25− T cells
Authors: Chang Jae‐Hoon , Kang Chang‐Yuil
Published: June 2021

Regulatory T cells (Treg) can control the balance between tolerance and immunity. However, the relationship between IFN‐γ and Treg is not yet clearly clarified. Two major populations of Treg, namely natural and induced Treg, arise in the thymus and the periphery, respectively. Here we show that IFN‐γ has no relation with the development and inhibitory function of naturally occurring regulatory T cells (nTeg). On the other side, naïve CD4 T cells can develop into regulatory T cells by acquiring the transcription factor Foxp3. Combined signals from the TCR, CD28, IL‐2R, and TGF‐βR promote Foxp3 expression in primed naïve CD25−CD4+ T cells. In this case, surprisingly, autocrine IFN‐γ regulates TGF‐β‐driven Foxp3 expression in induced regulatory T cells (iTreg) and suppresses the conversion of naïve CD4 T cells into Foxp+ CD4+ T cells. These data identify autocrine IFN‐γ as a self‐regulator of Foxp3 expression and suggest that IFN‐γ can play a pivotal role in regulating the balance between effector and regulatory T cell numbers in immune responses. Therefore, autocrine IFN‐γ can regulate the induction of Treg in the periphery, not in the thymus.
An in silico model of Regulatory T cell function
Authors: Taylor Paul , von Herrath Matthias
Published: June 2021

Regulatory T (Treg) cells participate in response to infection primarily involve chronic or persistent viral infections. The findings that Treg cells influence the functional immunity during viral infections however might indicate that in some cases, virus‐specific Treg cells not only influence immune pathology or prevent pathogen elimination but also can promote a generalized state of immuno‐suppression in vivo, such that the host is more susceptible to secondary infections with other pathogens or has reduced tumour resistance. The regulatory mechanism by which Tregs act has yet to be definitively characterized with opposing evidence suggesting either a cell‐to‐cell a cytokine mediated mechanism.

We have developed a cellular model that is an in silico representation of the cells of the immune system. The model aims to faithfully reproduce these cells and their interactions with cognate rule sets derived from experimentation. This stochastic simulation of a Treg suppression assay which can be used to investigate the possible mechanisms behind their immuno‐suppressive effect. The model can rapidly simulate a large number of assays under a wide range of parameters and the detailed results allow for the analysis of the parameters at a detail level currently impossible in vitro. The insights obtained by using the automaton provide a powerful tool for directing in vitro research in to Treg function.
SHP‐1 plays a role in both conventional and CD4+CD25+Foxp3+ regulatory T cell function
Authors: Iype Tessy , Sankarshanan Mohan , Lorenz Ulrike
Published: June 2021

While the importance of regulatory T (Treg) cells for immune tolerance has been increasingly appreciated, our mechanistic and molecular understanding of their function is still very limited. We have previously shown that mice lacking the SHP‐1, so‐called motheaten (me/me) mice, have increased numbers of Treg cells. SHP‐1 is a well‐recognized negative regulator of TCR‐mediated signaling. We therefore asked whether SHP‐1 also plays a role in Treg function. A comparison of the suppressive capabilities of wild type and me/me Treg cells demonstrated increased suppressive activity of me/me Treg cells in in vitro suppression assays. While this suppression is dependent on stimulation via the TCR, our data indicate that additional factors, which are critical for an effective Treg function, are regulated by SHP‐1. Interestingly when comparing wild type and me/me conventional T cells, we observed that SHP‐1 deficient CD4+CD25‐ T cells are more resistant to Treg‐mediated suppression than wild type T cells, indicating a role for SHP‐1 both in suppression as well as in resistance to suppression. We are currently addressing at the molecular level how SHP‐1 regulates these processes. We expect from these studies to not only better understand the regulatory role of SHP‐1 in conventional and Treg T cells but to also gain further insights into the mechanism of suppression by Treg cells.

This work was supported by the NIH grant RO1 AI48672.
Phenotypic similarities of anergic and regulatory T cells
Authors: Zell Traci , Jenkins Marc K.
Published: June 2021

The fact that T cell mediated autoimmune disease occurs implies that self‐reactive T cells exist in the peripheral T cell repertoire. There is substantial evidence that mechanisms of self‐tolerance must be active in the periphery to quiet those cells that escape the process of central tolerance. Using a model where TCR transgenic cells are adoptively transferred into a self‐antigen expressing recipient, we show that these T cells undergo proliferation and then decline in numbers. Antigen‐specific cells that remain after the contraction phase are functionally anergic in that they proliferate poorly and fail to secrete interferon‐gamma, IL‐2, TNF‐alpha, or IL‐17 when stimulated with antigen. Anergic cells recover proliferative capacity and IL‐2 production potential when removed from the source of antigen. Gene expression analysis revealed that anergic T cells express folate receptor and CD73 which have been associated with foxp3‐expressing regulatory T cells. However, anergic cells do not express foxp3 and do not suppress other T cells. These results suggest that anergic cells are a self‐reactive population that has differentiated along a pathway that is similar to but distinct from that followed by regulatory T cells. Experiments are planned to determine if such self‐reactive anergic cells exist in the normal T cell repertoire.
CD4+CD25+ Foxp3+ Treg protect against T cell‐mediated fumilant hepatitis in a TGF‐β‐dependent manner in mice
Authors: Wei Haiming , Wei Huaxin , Tian Zhigang
Published: June 2021

The intrahepatic immune environment is associated with the induction of tolerance. Regulatory T cells (Tregs) may play a role in tolerance in the liver. In this study, we examined the role of hepatic CD4+CD25+ Tregs in the liver injury by using Concanavalin A (Con A)‐induced hepatitis model. We found that increased number of Foxp3+ Tregs was observed in the liver but not in the spleen in mice intravenously injected with sublethal dose of Con A. Moreover, the expression levels of Foxp3, cytotoxic T lymphocyte associated antigen‐4 (CTLA‐4), glucocorticoid‐induced tumor necrosis factor receptor (GITR), as well as, the frequency and expression level of CD103 of Tregs were increased after Con A injection and significantly higher in liver than in spleen. Depleting CD25+ cells aggravated liver injury and adoptively transferring of CD25+ cells reduced liver injury, these indicate a functional role of Tregs in Con A‐induced hepatitis. Finally, TGF‐β receptor II dominant‐negative (dnTGF‐βRII) mice, which T cells express a dominant negative form of TGF‐βRII and therefore cannot response to TGF‐β, had a higher mortality rate and severer liver injury than normal mice injected with the same dose of Con A. In conclusion, CD4+CD25+ Tregs are important in the protection against liver injury via a TGF‐β‐dependent mechanism.
The E3 ubiquitin ligase Itch regulates Foxp3 expression and airway inflammation via enhancing TIEG1 function
Authors: K Venuprasad , Huang Haining , Harada Yohsuke , Elly Chris , Subramanyam Malayannan , Spelsberg Thomas , Liu Yun‐Cai
Published: June 2021

Transforming growth factor (TGF)‐b signaling in naïve T cells induces expression of transcription factor Foxp3, a master regulator in the regulatory T cells (Tregs). However, the molecular mechanisms leading to Foxp3 induction remain unclear. Here we show that Itch−/− T cells are resistant to TGF‐b treatment with reduced Foxp3 expression. The E3 ubiquitin ligase Itch associates with and promotes ubiquitin conjugation to TGF‐b inducible early gene1 product (TIEG1). More importantly, Itch cooperates with TIEG1 to induce Foxp3 expression, which is reversed by TIEG1 deficiency. Functionally TGF‐b‐converted Tregs generated from TIEG1−/− mice are unable to inhibit naïve T cell proliferation in vitro and suppress airway inflammation in vivo. The results suggest an ubiquitin‐dependent non‐proteolytic pathway that is critical for Foxp3 expression and regulation of allergic responses.
Reactive Oxygen Species: Integral Components of B Cell Activation
Authors: Romer Eric J , Fernando Tharu M , Sulentic Courtney E.W.
Published: June 2021

The transcription of the immunoglobulin heavy chain (IgH) which is governed by the 3'IgH Regulatory Region (3'IgH RR) in activated B cells is a critical component of the humoral immune response. The activation of lymphocytes has been shown to be sensitive to the redox state of the cell as determined by the levels of reactive oxygen species (ROS). This study proposes that the redox state of the cell plays a role in activated B cell IgH transcription by modulating the activity of the 3'IgH RR. Utilizing the CH12.LX B cell line with an oxidizable fluorescent probe or a luciferase reporter driven by the 3'IgH RR and a variable heavy chain promoter, the treatment of activated B cells with anti‐oxidants was found to decrease ROS levels and IgH expression. The treatment of activated B cells with NADPH antagonists also modulates the activation of IgH expression by shifting the redox state of the cell. These observations suggest that 1) optimal activation of B cells and IgH expression is dependent on the redox state of the cell and 2) NADPH antagonists can modulate both the redox state of the cell and IgH expression. (Supported by NIEHS and Boonshoft School of Medicine).
STAT‐dependent transciptional regulation of inhibitory Fc gamma RIIb in human monocytes and B cells
Authors: Olferiev Mikhail , Gao Xiaoni , Blank Marissa C. , Pricop Luminita
Published: June 2021

Fc gamma receptors (FcγR) bind IgG immune complexes and play an important role in the regulation of innate and adaptive immune responses. FcγRIIb mediates negative signaling through the inhibitory motif present in the cytoplasmic domain. Altered expression of FcγRIIb on B cells and monocytes is associated with autoimmune and inflammatory conditions. We observed differential regulation of FCGR2B promoter activity and transcript expression by IL‐4 and IFN‐γ. We investigated the mechanisms involved in the transcriptional regulation of FCGR2B promoter. A putative STAT binding motif is present at position −162 to −154 in the FCGR2B promoter. EMSA experiments with nuclear extracts from human monocytic and B cell lines indicated interaction of STAT1 and STAT4 transcription factors with the FCGR2B promoter probe at this site. Mutation of the putative STAT binding motif within the FCGR2B promoter sequence altered the formation of STAT complexes and downregulated the FCGR2B promoter activity in luciferase assays. Our data suggest that STAT family factors could participate in the transcriptional regulation of the FCGR2B promoter and provide new insight into the modulation of FcγRIIb expression by cytokines during inflammation.
The role of Terminal deoxynucleotidyl Transferase (TdT) in the T‐cell‐independent antibody response to α 1–3 Dextran
Authors: Mahmoud Tamer I , Kearney John F
Published: June 2021

Understanding the antibody response to T‐cell independent (TI‐2) antigens, such as polysaccharides associated with many pathogenic microorganisms, is of importance for improving vaccine design. We sought to study the factors that influence the development of B cell clones that are specific for TI‐2 antigens. In this study, we investigated the role of the lymphoid specific enzyme TdT in generating B cell clones responsive to the polysaccharide α 1–3 Dextran (DEX). TdT is a DNA polymerase crucial for the diversity of antigen receptors during V(D)J recombination via its non‐templated nucleotide additions. By using idiotypic markers for the major B cell clone J558 involved in the anti‐DEX response, we show that TdT−/− mice have lower numbers of the J558 clone than wildtype (WT) mice. Likewise, the DEX‐specific antibody response is lower in TdT−/− mice than WT mice and J558 was not detected in the sera of TdT−/− mice. By sequencing antibody heavy chain V(D)Js of sorted plasma cells post‐immunization, we show that TdT−/− mice generate the alternative M104E clone that predominates in neonatal life rather than the predominant adult clone J558. WT sequences show diversity in only two amino acids at the D segment of the CDR3 region. These data suggest that TdT is essential for the generation of the higher affinity DEX‐responsive J558 clone. Supported by National Institutes of Health (AI007051‐31A1 to T.M. and AI045794‐09 to J.F.K.)
A MMP‐independent role for TIMP‐2 during cardiac neural crest cell migration
Authors: Brauer Philip R , Stetler‐Stevenson William G , Tran Lan Uyen , Reedy Mark V
Published: June 2021

Tissue inhibitor of metalloproteinase‐2 (TIMP‐2), a matrix metalloproteinase (MMP) inhibitor, is uniquely expressed in cardiac neural crest (NC) cells. Blocking TIMP‐2 synthesis perturbs NC migration while synthetic MMP inhibitors have little effect suggesting TIMP‐2 operates independent of MMP. TIMP‐2 can mediate growth factor‐induced responses independent of MMP inhibition by binding α3ß1 integrins. Here, we compared the effects of wild‐type TIMP‐2, a non‐MMP inhibiting form of TIMP‐2 (Ala+TIMP‐2), and a TIMP‐2/integrin blocking peptide on NC migratory behavior. Ala+TIMP‐2 increased NC cell motility, increased the distance NC cells migrated from neural tube explants, and altered NC cell shape (the cells were flatter and more arborized) compared to control cultures. Wild‐type TIMP‐2 also increased NC motility but had no effect on the distance NC cells migrated nor on their cell shape. A TIMP‐2/integrin inhibitory peptide had no effect on basal NC migration but it blocked the effects of Ala+TIMP‐2 in vitro and perturbed NC migration when injected into embryos. In addition, α3 integrin protein was expressed by the ectoderm, neural tube, and NC cells in situ. These data suggest TIMP‐2's functional role in NC migration may be mediated through α3ß1 integrin signaling rather than inhibiting MMP activity. Supported by the American Heart Association, Heartland Affiliate (#0555683Z) and NIH (#P20‐RR016469).
Epithelial‐Mesenchymal Interactions during Facial Development in Mice
Authors: Marcucio Ralph , Hu Diane
Published: June 2021

Previously, we determined that when avian neural crest cells reach the Frontonasal Process (FNP) at Hamburger and Hamilton Stage 20 (HH), Sonic hedgehog (Shh) expression is induced in the stomodeal ectoderm and forms a boundary with a Fibroblast growth factor 8 (Fgf8) expression domain. The ectoderm flanking this boundary controls FNP growth and patterning, but the mechanisms used by this tissue to pattern the face are unknown. In this work, we examined the facial ectoderm of mice to determine whether a similar signaling center exists and how it functions. Fgf8 is expressed in the distal facial ectoderm at e9.5 and continues to be expressed at e10. At e10 Shh is expressed in both median nasal processes and forms a boundary with Fgf8 in each process. Next, we grafted the mouse ectoderm from e10 embryos onto the dorsal FNP of HH 25 chick embryos. Twenty‐four hours after transplantation the graft (n=4) maintained expression of Fgf8, Shh, Bmp‐2, Bmp‐4, and Bmp‐7, and in the neural crest mesenchyme we observed up‐regulation of Bmp‐4, Bmp‐7, and Msx‐1 and ‐2. By 13 days of development the chimeras exhibited a duplication of the upper beak that was comprised of correctly patterned bone and cartilage (n=4). We observed no hair follicles, but we did observe ectopic egg teeth. These results indicate that in mice a signaling center located in the ectoderm of each median nasal process regulates development of the middle and upper face by inducing signaling molecules that govern skeletogenesis within neural crest mesenchyme. The organization of this center is different from that in the chick where a single Shh domain spans the stomodeal ectoderm and forms a continuous boundary with Fgf8 in the FNP.

NIH/NIDCR R01‐1DE018234 to R.M.
Requirements for cell lineage‐restricted expression of MHC class II in the development of CD4 T cell‐dependent immunity in the intestine
Authors: Perrigoue Jacqueline Gage , Allenspach Eric , Laufer Terri , Artis David
Published: June 2021

Many models have been proposed for how luminal antigens are sampled by immune cells within the intestinal mucosa ranging from direct sampling via specialized dendritic cell (DC) populations to intestinal epithelial cell (IEC)‐dependent transfer of antigen to the lamina propria. To test whether DC‐restricted expression of MHC class II is sufficient to promote protective CD4 T cell responses in the intestine, mice in which I‐Aβb expression is restricted to CD11c+ DC (CD11c/Aβb) were infected with the intestinal parasite Trichuris, immunity to which is dependent on Th2 cells. Following infection, littermate control mice exhibited Th2 cytokine‐dependent goblet cell hyperplasia and eradication of Trichuris. In contrast, CD11c/Aβb mice developed persistent infection associated with reduced Th2 cytokine production and increased IFN‐γ. These data suggest that in addition to DC, another population of MHC class II expressing cells is required for Th2 cytokine‐mediated immunity in the intestine. Previous studies have implicated IEC‐intrinsic MHC class II expression in intestinal immune regulation and following exposure to Trichuris, EC upregulated surface MHC class II. Identifying the functional significance of IEC‐intrinsic MHC class II expression in regulating CD4 T cell responses may have implications for the design and delivery of mucosal vaccines and treatment of T cell‐mediated inflammatory diseases.
Epithelial dendritic cells in vagina rapidly renew from bone marrow precursors
Authors: Iijima Norifumi , Linehan Melissa M. , Saeland Sem , Iwasaki Akiko
Published: June 2021

DCs represent key professional APCs capable of initiating primary immune responses. A specialized subset of DCs, the Langerhans cells (LCs), are located in the stratified squamous epithelial layer of the skin, and within the mucosal epithelial lining of the vaginal and oral cavities. The vaginal mucosa undergoes cyclic changes under the control of sex hormones, and the renewal and phenotypic characteristics of the vaginal epithelial DCs (VEDCs) remain unknown. Here, we examined the origin of VEDCs. In contrast to the skin epidermal LCs, the DCs in the epithelium of the vagina were found to be repopulated mainly by non‐monocyte bone marrow (BM)‐derived precursors, with a half‐life of 13 days under steady‐state conditions. Upon infection with herpes simplex virus 2 (HSV‐2), the Gr‐1hi monocytes were found to give rise to VEDCs. Further, flow cytometric analysis of the VEDCs revealed the presence of at least three distinct populations, namely, CD11b+F4/80hi, CD11b+F4/80int, and CD11b−F4/80neg. Importantly, these VEDC populations expressed CD207 at low levels, and had a constitutively more activated phenotype compared to the skin LCs. Collectively, our results revealed mucosa‐specific features of the VEDCs with respect to their phenotype, activation status and homeostatic renewal potential.
Lymphotoxin is critically required for the presence of IgA‐promoting iNOS+ DCs in the intestinal lamina propria
Authors: McCarthy Douglas D , deLuca Leslie Summers , Ward Lesley , Gommerman Jennifer L
Published: June 2021

Lymphotoxin‐β receptor (LTβR) signaling is essential for IgA production in the intestinal lamina propria (LP) and it has been proposed that LTβR‐signaling is needed for the accumulation of IgA‐secreting B‐1 cells in the LP. Yet, in spite of diminished numbers of B‐1 cells in the LP of LTβR−/− mice, a residual population of B‐1 cells remain and B‐2 cell numbers in the LP are normal. To further explore the mechanism for LTβR‐dependent IgA production in the gut, we hypothesized that LTβR signaling may regulate accessory cells in the LP that influence IgA production by LP‐resident B cells. The LP harbors a recently described subset of iNOS+ dendritic cells (DC) that are required for IgA responses. We characterized this DC population in the LP of WT mice as CD11c+ CD11blo Thy1+ CD8α − B220− and, interestingly, we found that these cells were essentially undetectable in LTβR−/ − mice. In mixed bone marrow chimeras, the presence of iNOS+ DC in the LP depended primarily on the expression of LTβR by radio‐resistant cells, whereas the expression of LTβR on DC themselves was not essential. We conclude that the presence of IgA‐promoting iNOS+ DCs in the LP requires intact LTβR signaling on the LP stroma, and these data provide a mechanism for defective IgA production in LTβR−/ − mice. This work was supported by the Canadian Institutes of Health Research.
Systemic delivery of a crude extract of the tapeworm, Hymenolepis diminuta, exerts an anti‐inflammatory effect in a murine model of colitis
Authors: Wang Arthur , McKay Derek
Published: June 2021

Awareness of how parasites diminish the host's immune response would, in theory, allow for the development of new therapies to combat autoimmune and inflammatory disease. In an in vitro analysis, we showed that a >50kDa fraction from adult Hymenolepis diminuta blocked mitogen‐induced T cell activation. Here, we tested the hypothesis that this H. diminuta extract would block the colitis that develops in mice in response to intra‐rectal instillation of dinitrobenzene sulphonic acid (DNBS) – a system used as a model of the human inflammatory bowel disease, Crohn's disease. Balb/c mice were treated with 100μl of 50% ethanol ir (control), 3mg of DNBS (in ethanol, ir) or DNBS + worm antigen (3×1mg ip injections over 3 days). Mice were sacrificed 72h after DNBS and clinical disease scores, histological damage scores, colonic myeloperoxidase (MPO, indicates granulocyte infiltration) activity, and TNFα and IL‐10 production by splenocytes assessed. DNBS treatment caused colitis as shown by wasting, elevated MPO (14.0±3.9 vs. 2.0±0.3 U/mg; n=3) and the production of TNFα (783±27 pg/ml) but not IL‐10. In contrast, mice treated with DNBS + worm antigen showed no mortality and only minor signs of colitic disease that was accompanied by increased IL‐10 (2.0±0.9 ng/ml; n=3). Purification and synthesis of this immunosuppressive factor from H. diminuta could be used to treat inflammatory diseases. CCFC funded.
Sphingosine 1‐phosphate regulates innate and acquired intestinal IgA production
Authors: Kunisawa Jun , Gohda Masashi , Kurashima Yosuke , Higuchi Morio , Ishikawa Izumi , Miura Fumi , Kiyono Hiroshi
Published: June 2021

Intestinal IgA plays an important role in the innate and acquired phases of immunosurveillance by recognizing T‐independent (TI) and T‐dependent (TD) antigens, respectively. Sphingosine 1‐phosphate (S1P) is known to regulate lymphocyte emigration from organized lymphoid tissues, but its immunological role in the induction of intestinal IgA production remains to be elucidated. Here we show that intestinal IgA productions against TI and TD antigens are controlled by S1P. We found that disruption of S1P‐mediated signaling by the treatment of mice with FTY720, a modulator of S1P receptors, resulted in the marked reduction of intestinal IgA production against TI phosphorylcholine induced by oral immunization with heat‐killed Streptococcal pneumoniae (R36A). Similarly, FTY720 reduced production of intestinal IgA against ovalbumin (OVA), a typical TD antigen, in mice orally immunized with OVA plus mucosal adjuvant, cholera toxin. The decreased TI and TD antigen‐specific intestinal IgA productions were associated with the impaired trafficking of peritoneal B1 and Peyer s patch B2 cells into the intestinal lamina propria, respectively. These findings point to a pivotal role for S1P in innate and acquired phases of intestinal B cell immunity.
Different requirements for the RelA and RelB subunits of NF‐ κB in regulation of the polymeric immunoglobulin receptor by TNF and Toll‐like receptor signaling
Authors: Bruno Maria E.C. , Tran Thien N. , Johansen Finn‐Eirik , Kaetzel Charlotte S.
Published: June 2021

Transport of IgA antibodies across intestinal epithelial cells is mediated by the polymeric immunoglobulin receptor (pIgR), which is up‐regulated by TNF and Toll‐like receptor (TLR) signaling. We hypothesized that the RelA and RelB subunits of NF‐κB play distinct roles in the regulation of PIGR and other NF‐κB target genes. To test this hypothesis, expression of RelA or RelB was inhibited in the human IEC line HT‐29 by stable transfection of siRNA plasmids. Levels of mRNA for pIgR, the pro‐inflammatory factors TNF and IL‐8, and A20, a negative regulator of NF‐κB, were measured following stimulation with TNF, poly I:C (TLR3 ligand) or LPS (TLR4 ligand). Peak expression of pIgR was delayed until 24 h, while expression of TNF, IL‐8 and A20 peaked at 3 h and was down‐regulated by 24 h. Induction of pIgR mRNA by TNF was abrogated by siRNA‐mediated inhibition of RelA, but not RelB. Surprisingly, induction of pIgR mRNA by LPS or poly I:C was not affected by inhibition of RelA or RelB expression. By contrast, induction of IL‐8, TNF and A20 mRNA by TNF, poly I:C and LPS was dependent on RelA and largely independent of RelB expression. We conclude that the individual contributions of RelA and RelB to gene expression depend on the nature of the stimulus as well as the specific target gene. In the absence of RelA expression, NF‐κB independent signaling pathways may be involved in regulation of pIgR expression by TLR signaling. Supported by the CCFA.
Occurrence and IgE binding of mucosal mast cells in neonates
Authors: Wagner Bettina , Hillegas Julie , Flaminio Julia MB , Matychak Mary Beth , Childs Bronwen A , Thrasher Seana M , Antczak Douglas F
Published: June 2021

Maternal IgE is exclusively transferred to the neonatal foal via colostrum after birth. The primary contact site for maternal IgE is the foal's gut tissue. Our goal was to investigate neonatal tissues for the presence of mucosal mast cells (MCs), and to test whether intestinal neonatal MCs can bind maternal IgE. Tissues from the small intestine, mesenteric and peripheral lymph nodes were harvested immediately after birth (before colostrum uptake), and on days 5, 9 and 36 from healthy neonates. Mucosal MCs were detected using Alcian blue staining. At birth, most MCs were found in the crypt areas of the small intestine. On days 5 and 9, only a few mucosal MCs could be detected. On day 36, many MCs were found in the submucosa. Mucosal MCs were observed in the T‐cell areas of lymph nodes at all time points. To investigate whether intestinal MCs can bind maternal IgE directly after birth, MCs were isolated from the mucosa of the jejunum. The cells were cultured in the presence of fluorochrome conjugated IgE and measured by flow cytometry. Two populations of IgE+ cells were detected. First, an IgEhigh+/MHC class II− population; and second, cells of a IgE+/MHC class II+ phenotype. We concluded that intestinal mucosal MCs express IgE receptors to rapidly bind maternal IgE from ingested colostrum.