Browsing by Author "Zhang, Weiguo"

LAT is a transmembrane adaptor protein that is critical for the emanation of signals downstream of the TCR. Following TCR engagement, LAT is phosphorylated on multiple tyrosine residues, allowing it to serve as a scaffold for a multi-protein signaling complex. Mutation of tyrosine 136 on LAT abrogates binding of PLC-γ1. The disruption of this interaction has severe consequences on TCR-mediated calcium signaling and MAPK activation. Mice harboring a mutation at this tyrosine, LATY136F (LATm/m) mice, have drastically impaired thymocyte development; however, CD4+ T cells in the periphery rapidly expand and instigate a fatal lymphoproliferative syndrome. In order to bypass the severe developmental defects exhibited in LATm/m mice, our laboratory previously developed a conditional knock-in mouse line in which the mutated LAT allele is expressed in mature T cells following deletion of a floxed wildtype LAT allele (ERCre+LATf/m mice). LATf/m mice develop a similar lymphoproliferative syndrome as LATm/m mice. We used both of these mouse models to analyze the contribution of two other proteins that are essential for TCR-mediated signaling, RasGRP1 and Gads, in LAT-mediated autoimmunity.

Analysis of LATm/mRasGRP1-/- mice demonstrated that the additional deletion of RasGRP1 increased the thymocyte development block and, as a result, young mice contained markedly reduced T cell populations. However, by four months of age, a lymphoproliferative disease had developed in these mice. To bypass the severe developmental block, we analyzed LATf/mRasGRP1-/- mice and observed that they developed disease similarly to LATf/m mice. We also assessed the effect of Gads deletion in both mouse models of LAT disease. LATm/mGads-/- mice had an even more dramatic block in the DN stage of thymocyte development compared to LATm/m controls, although by four months of age CD4+ T cells had expanded. Following deletion of the wildtype LAT allele, LATf/mGads-/- mice also developed disease. Our results indicated that LAT-mediated autoimmunity can occur independently of the critical T cell signaling components RasGRP1 and Gads.

In addition, we more closely examined RasGRP1-mediated Erk activation in T cells. RasGRP1 is a Ras-guanyl nucleotide exchange factor that is required for positive selection of thymocytes, activation of T cells, and control of T cell mediated-autoimmunity. While the importance of various RasGRP1 structural domains has previously been explored, RasGRP1 also contains a tail domain of unknown function. To elucidate the physiological role of this domain, we generated knock-in mice expressing RasGRP1 without the tail domain, RasGRP1d/d mice. Analysis of these mice demonstrated that deletion of the tail domain led to impaired T cell development but, with age, CD4+ T cells expanded and auto-antibodies were produced. RasGRP1d/d thymocytes were unable to activate Erk and underwent aberrant thymic selection processes. Mechanistically, the tail-deleted form of RasGRP1 was not able to traffic to the cell membrane following stimulation, indicating a potential reason for its inability to activate Erk. While the DAG-binding C1 domain of RasGRP1 has long been recognized as an important factor mediating Erk activation, our data revealed the physiological relevance of the tail domain of RasGRP1 in the control of Erk signaling.

Helminth exposure, allergy and asthma each induce cellular responses in lymphoid and peripheral tissues that give rise to type-2 inflammation. Essential molecular mediators of this response are type-2 cytokines interleukin(IL)-4 and IL-13 derived from various subsets of immune cells. In lymphoid tissues, CD4+ Tfh cells make IL-4 to elicit IgE and high-affinity IgG1 production. In peripheral sites of infection, group 2 innate lymphoid (ILC2) cells make IL-13 and Th2 cells make both IL-13 and IL-4. Together, these cells mediate smooth muscle contraction, mucus production and recruitment of other innate effector cells, all of which are hallmarks of type-2 inflammation. As central mediators of type-2 inflammation, understanding the cell-specific expression and molecular regulation of type-2 cytokines in CD4+ T cells and ILC2 cells may lead to new therapies that ameliorate allergic disease and helminth infections.

The AP-1 factor basic leucine zipper transcription factor ATF-like (BATF) has been identified as a pioneer factor in in vitro-generated Th17 cells. BATF facilitates chromatin remodeling at the IL-17 locus as well as loci of key Th17-associated lineage specifying factors. It has also been deemed essential to the generation of functional humoral immunity through the development of follicular helper T (Tfh) cells and germinal center B cells. However, the role of BATF in the development and function of other CD4+ T helper subsets and innate immune cells in vivo has remained unclear. I show here that mice deficient in BATF do not develop type-2 inflammation after exposure to the parasitic helminth Nippostongylus brasiliensis. Since type-2 cytokine expression by Th2 and ILC2 cells is essential for expedient helminth expulsion, I hypothesized that BATF likely has a role in the development and/or induction of cytokine expression in CD4+ Th2 cells and ILC2 cells. Consistent with this hypothesis, I found that BATF utilizes a novel mechanism to control Th2 cytokine expression in Th2 cells. Specifically, BATF promotes permissive epigenetic modifications to alter the chromatin landscape early during Th2 cell differentiation. In addition, my data show that BATF deficiency inhibits the activation of ILC2 cells, preventing ILC2-mediated helminth clearance.

In addition to uncovering BATF-mediated regulations of type-2 inflammation, my work has revealed new insight into the role of a second bZIP transcription factor, cMaf, during type-2 immunity. As mentioned above, helminth exposure elicits IL-4 production by both CD4+ Tfh and Th2 cells. Although type-2 cytokine transcription has been well characterized in Th2 cells, Tfh cell-mediated IL-4 production has yet to be fully defined. Importantly, I show that IL-4 production by Tfh cells is sustained upon deletion of classical IL-4 regulatory factors signal transducer and activator of transcription 6 (STAT6) and STAT5 and is not dependent on high GATA-3 expression. In sum, Tfh-driven IL-4 production is induced independent of classical pathways in Th2 cells.

Presently, the non-canonical transcription factors involved in IL-4 production by Tfh cells remain unclear. C-Maf works with BCL6, the master regulator of Tfh cells, to elicit Tfh formation. However, the precise role of c-Maf in Tfh cell fate and function remains unclear. So far, it has been shown that in Th2 cells, c-Maf binds to the IL-4 promoter and in Tfh cells, c-Maf binds to the CNS2 enhancer of the IL-4 locus to regulate IL-4 expression. Therefore, I hypothesized that c-Maf is important in non-canonical, GATA-3-independent IL-4 production by Tfh cells.

Here, I show that Tfh cells lacking canonical Th2 pathways for IL-4 expression express high levels of c-Maf and IL-4 transcript. Deletion of c-Maf in CD4+ T cells resulted in normal induction of BCL6 expression. Thus the initial stages of Tfh cell generation were induced. However, cMaf-deficient CD4+ T cells did not express important molecules associated with Tfh cell migration. Immunohistochemistry also confirmed that c-Maf deficiency inhibited CD4+ T cell migration from the paracortex into the B cell follicle.

These defects did not inhibit cMaf-deficient CD4+ T cells from making IL-4 transcript; however, IL-4 protein production was significantly impaired. Together, these results demonstrate that c-Maf is essential for Tfh cell-mediated immunity by promoting CD4+ T cell migration to the B cell follicles and the production of IL-4 protein in the germinal centers.

Collectively, the objective of my thesis research is to define the roles of the bZIP transcription factors BATF and c-Maf in type-2 inflammation. My data demonstrate that BATF is essential for the differentiation and function of Tfh, Th2, and ILC2 cells during helminth infection. Additionally, I have shown that c-Maf is required for Tfh function and CD4+ T cell migration to the B cell follicle. Thus, BATF and c-Maf are central to the development of humoral and peripheral type-2 inflammatory responses against helminth infection. Given the wide spectrum of disorders associated with type-2 inflammation, the identification of factors relevant to the development and function of Th2-, ILC2- and Tfh-driven allergic pathologies is broadly relevant. A comprehensive characterization of core factors like BATF and c-Maf provide new avenues in which to explore novel therapies to modulate type-2 inflammatory responses.

Background

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Results

Conclusions

Lipids represent an important source of nutrition for infecting mycobacteria, accumulating within the necrotic core of granulomas and present in foamy macrophages associated with mycobacterial infection. In order to better understand the timing, process and importance of lipid accumulation, we developed methods for direct in vivo visualization and quantification of lipid accumulation using the zebrafish-M. marinum model of infection. We find that neutral lipids accumulate cell autonomously in mycobacterium-infected macrophages in vivo during early infection, with detectable levels of accumulation by two days post-infection. Reducing available free cholesterol and neutral lipids during early infection via treatment with ezetimibe, an FDA-approved drug, resulted in a reduction of bacterial growth in vivo. The effect of ezetimibe in reducing bacterial growth was dependent on the mce4 operon, a key bacterial determinant of lipid utilization. Thus, in vivo, lipid accumulation can occur cell autonomously at early timepoints of mycobacterial infection, and this early lipid accumulation confers a growth advantage to infecting mycobacteria.

This accumulation represents a perturbation of the normal homeostatic mechanisms by which intracellular lipids are tightly controlled. Under homeostatic conditions macrophages are central to the function of returning excess lipids to the liver where they are processed for excretion via the digestive tract. This function of macrophages, termed reverse cholesterol transport, results from the uptake of excess extracellular lipids, followed by the coordinated efflux of these lipids through the transport proteins ABCA1 and ABCG1 and packaging into HDL particles containing ApoAI and ApoAII. This process is controlled via the action of nuclear receptors including PPARγ, PPAR-α, and LXRα.

We performed RNA-seq analysis of gene expression in macrophages both uninfected and infected with mycobacteria and observed a profound down-regulation of all the major lipoproteins. The HDL associated lipoproteins ApoAI and ApoAII were the most profoundly down regulated. Based on this observation we sought to investigate the role of nuclear receptors involved in intracellular lipid sensing and control of apolipoprotein expression. We determined that agonism and antagonism of LXRα signaling decrease and increase infection burden during in vivo studies respectively. We further found that the ApoAI agonizing fibrate drugs fenofibrate and gemfibrozil reduce mycobacterial infection in vivo. This work demonstrates that lipid lowering agents already approved for use in humans might function as relevant adjuvant therapies toward treatment of mycobacterial infections.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is crucial in linking TCR engagement to downstream signaling events, such as calcium flux and Ras-MAPK pathway. Following TCR engagement, LAT is phosphorylated at its membrane-distal tyrosine residues, which mediates the binding of Grb2/Sos, PLC-1, and GADS/SLP-76 complexes. This multi-protein signaling complex initiates signaling cascades eventually leading to the activation of transcription factors that regulate the genes required for T cell proliferation and effector functions. The indispensable role of LAT in thymocyte development has been evidenced as LAT-deficient mice completely lack peripheral T cells. To study the function of LAT in mature T cells, our lab previously generated a conditional knock-in mouse line in which the lat gene can be deleted by Cre recombinase. Deletion of LAT in mature T cells revealed the critical role of LAT in T cell activation. Here, we used this inducible LAT deletion mouse line crossed with the OT-I transgenic mice to study the role of LAT in mature CD8 T cells.

To analyze the contribution of LAT in CD8 T cells during the course of pathogen infection, we infected mice with Listeria monocytogenes-expressing Ova to elicit activation of antigen-specific CD8 T cells, and then inducibly deleted LAT in these cells at different stages of infection under the control of tamoxifen treatment. We show that LAT is important for maintaining CD8 T cell expansion during the priming phase; however, it is not required for CD8 T cell contraction. In addition, memory CD8 T cell can persist in the absence of LAT, suggesting that LAT-signaling is not necessary for memory maintenance. Nonetheless, these LAT-deficient memory T cells were unable to proliferate or produce cytokines upon secondary infection. Moreover, LAT deficiency accelerates memory differentiation during the effector-to-memory transition, leading to a higher frequency of KLRG1lowIL-7RhighCD62Lhigh memory T cells. Together, these data demonstrate that, while it is dispensable for contraction and memory maintenance, LAT-signaling regulates CD8 T cell memory differentiation and is essential for the memory response against pathogens.

The fundamental activity of CD8 T cells is to elicit cytotoxicity toward target cells that express foreign antigens, and this is mediated through granule-dependent and Fas ligand-dependent mechanisms. The signaling events that regulate these processes remain unclear. We showed that LAT-deficient cytotoxic T cells (CTLs) failed to upregulate FasL and produce IFN- after engagement with target cells. Moreover, they displayed reduced granule-mediated killing. We further dissected the effect of the LAT deletion on each step of granule exocytosis. LAT-deficiency led to altered synapse formation, subsequently causing unstable T cell:APC conjugates. MTOC polarization and granule reorientation were also impaired by LAT-deficiency, leading to reduced granule delivery. Despite these defects, granule release was still observed in LAT-deficient CTLs due to residual calcium flux and PLC activity. This revealed an unexpected finding that CTL function is not entirely dependent on LAT. Collectively, these data indicate that the signaling circuits governing CTLs are programmed to adopt multiple pathways, allowing CTLs to effectively eliminate various pathogens during adaptive immune responses.

Naturally occurring CD4+CD25+Foxp3+ regulatory T cells (TReg) are a cell lineage that develops in the thymus and exits to the periphery, where they represent 5-10% of the peripheral CD4+ T cell population. Phenotypically, they are characterized by the expression of the cell surface markers CD25, as known as the IL-2 receptor alpha chain, glucocorticoid-induced tumor necrosis factor receptor (GITR), and cytotoxic T-lymphocyte antigen-4 (CTLA-4), as well as forkhead box P3 (Foxp3), a transcription factor considered to be the most specific TReg marker. Functionally, TReg cells are defined by their ability to suppress the activation of multiple cell types including CD4+ and CD8+ T cells, B cells, natural killer (NK) cells, and dendritic cells (DCs). Suppression can be achieved by the production of immunosuppressive cytokines or direct cell-to-cell contact, with these mechanisms directly affecting suppressed cells or indirectly affecting them by modulating antigen presenting cells (APCs). The suppressive abilities of TReg cells are crucial in maintaining dominant tolerance--the active, trans-acting suppression of the immune system for the prevention of autoimmune diseases. In addition to preventing autoimmune diseases, studies have also demonstrated critical roles for TReg cells in down-modulating anti-tumor immunity, suppressing allergic diseases, such as asthma, and achieving transplant tolerance. Recent studies have also demonstrated roles for TReg cells during pathogen infection, which will be the focus of this thesis.

Studies examining TReg cells during infection have largely focused on chronic infection models. These studies have shown that TReg cells can affect responses to pathogens in various ways that can be beneficial or detrimental for either the host or the invading pathogen. In some infections, TReg cells downregulation effector responses, which can lead to pathogen persistence and, in some cases, concomitant immunity. TReg cell-mediated suppression can also reduce immunopathology at sites of infection, which can occur as a result of a vigorous anti-pathogen immune response.

In contrast to chronic infection, how TReg cells behave and function following acute infections remains largely unknown as, to date, very few studies have been conducted. Current work with acute infection models has indicated that TReg cells affect immune responses in some acute infection models, but not in all. Furthermore, the results of these studies have implicated that current approaches to examine TReg cells during acute infection by depleting the total TReg cell repertoire, as opposed to targeting pathogen-specific TReg cells, may not be ideal. Finally, it is unclear what happens to activated TReg cells following the resolution of infection.

Due to the lack of knowledge about the role of pathogen-specific TReg cells during acute infection, we sought to employ a different approach to address some of the outstanding questions in the field. Here, we utilized CD4+ non-TReg and TReg cells from T cell receptor (TCR) transgenic mice that recognize a pathogen-specific epitope found in three different models of acute viral infection: recombinant vaccinia virus, recombinant adenovirus, and influenza. Using this model system, we were able to track pathogen-specific TReg cells following acute viral infection to determine their kinetics during the course of infection, as well as their influence on CD4+ non-TReg cells during different times after infection. We also employed major histocompatibility complex (MHC) Class II tetramer technology to track the fate of endogenous pathogen-specific TReg cells following infection with influenza.

Using these models systems, we show that pathogen-specific TReg cells can be activated and expand upon acute viral infections in vivo. The activated TReg cells then contract to form a "memory" pool after resolution of the infection. These "memory" TReg cells expand rapidly upon a secondary challenge, secrete large amounts of IL-10, and suppress excessive immunopathology, which is elicited by the expansion of non-TReg cells, via an IL-10-dependent mechanism. The work presented in this thesis reveals a previously unknown "memory" TReg cell population that develops after acute viral infections and may help design effective strategies to circumvent excessive immunopathology.

LAT (linker for activation of T cells) is an important transmembrane adaptor protein in TCR-mediated signaling. Upon TCR engagement, LAT associates with multiple proteins which allows for the activation of downstream signaling pathways. The interaction between LAT with phospholipase C (PLC-gamma1) is especially critical for T cell receptor (TCR)-mediated Ca2+ signaling and MAPK activation. Knock-in mice harboring a mutation at the PLC-gamma1 binding site (Y136) of LAT develop a severe lymphoproliferative syndrome. These mice have defective thymic development and selection and lack natural regulatory T cells, implicating a breakdown of both central and peripheral tolerance. The phenotype observed in LAT-/- mice is even more severe.T cells are absent in the periphery of these mice due to a complete block in thymocyte development at the DN3 stage thereby making it difficult to study the physiological role of LAT in the activation and function of mature T cells. In order to bypass the developmental defects exhibited by LAT-/- and LATY136F mice, we developed conditional knock-in lines in which only a nonfunctional (ERCreLATf/-) or a LATY136F-mutated allele (ERCreLATf/m) of LAT is expressed in mature T cells after deletion of the wildtype LAT allele.

Analysis of ERCreLATf/m T cells after LAT deletion indicated that the interaction between LAT and PLC-gamma1 plays an important role in TCR-mediated signaling, proliferation, and IL-2 production. Furthermore, the deletion of LAT induced the development of the LATY136F lymphoproliferative syndrome in these mice. Although Foxp3+ natural Treg cells were present in these mice after deletion, they were unable to suppress the proliferation of conventional T cells. Our data indicated that the binding of LAT to PLC-gamma1 is essential for the suppressive function of CD4+CD25+ regulatory T cells.

We have also performed studies using ERCreLATf/- T cells to demonstrate that total LAT deficiency reduced the expression of Foxp3, CTLA4, and CD25 in peripheral Treg cells. Interestingly, mice with LAT deleted in peripheral T cells developed a lymphoproliferative syndrome similar to that observed in LATY136F mice although the disease caused by the LATY136F mutation was more severe. These data implicate LAT in both the positive and the negative regulation of mature T cells. Moreover, our findings indicate that LAT is essential in the maintenance of the regulatory T cell profile in the periphery, thereby aiding in the prevention of lymphoproliferative autoimmune disease.

Linker for Activation of T cells, LAT, is a transmembrane adaptor protein that is vital for integrating TCR-mediated signals that modulate T cell development, activation, and proliferation. Upon engagement of the T cell receptor, LAT is phosphorylated and associates with Grb2, Gads, and PLCγ1 through its four distal tyrosine residues. Mutation of tyrosine 136 abolishes LAT binding to PLCγ1. This results in impaired TCR-mediated calcium mobilization and Erk activation. LATY136F knock-in mice have a severe but incomplete block in T cell development. Yet, CD4+ αβ T cells undergo uncontrolled expansion in the periphery, resulting in a severe autoimmune syndrome characterized by Th2 skewing and resultant B cell autoreactivity. Here, we further studied the role of LAT-PLCγ1 signaling in T cell lineage commitment, cytokine production, and autoimmunity.

First, we investigated the importance of the LAT-PLCγ1 interaction in γδ T cells by crossing LATY136F mice with TCRβ-deficient mice. Our data showed that the LATY136F mutation had no major effect on the homeostasis of epithelial γδ T cells, which could be found in the skin and small intestine. Interestingly, a population of CD4+ γδ T cells in the spleen and lymph nodes underwent continuous expansion and produced elevated amounts of IL4, resulting in an autoimmune syndrome similar to that caused by αβ T cells in LATY136F mice. Development of these hyperproliferative γδ T cells was not dependent on expression of MHC class II or CD4, and their proliferation could be partially suppressed by regulatory T cells. Our data indicated that a unique subset of CD4+ γδ T cells could hyperproliferate in LATY136F mice and suggested that LAT-PLCγ1 signaling may function differently in various subsets of γδ T cells.

In addition to examining γδ and αβ T cell development, we also were interested in further exploring the role of LAT in cytokine production. While our previous data have demonstrated that T cells in LATY136F mice are Th2 skewed, producing large amounts of IL4, we investigated other cytokines that may be important for autoimmunity and found that these CD4+ αβ T cells could also produce the proinflammatory cytokine IL6. Analysis of whole cell lysates from CD4+ αβ LATY136F T cells demonstrated that NFκB, AKT, and p38 were constitutively phosphorylated, and inhibition of these pathways resulted in reduced IL6 production. By crossing LATY136F mice with IL6 deficient mice, we demonstrated that early T cell survival was diminished in the absence of IL6. We further showed that this reduced CD4+ T cell pool was not due to further blocks in development, or an increase in FoxP3+ regulatory T cells. Finally, we demonstrated that over time, CD4+ T cells do hyperproliferate, yet B cell class switching and autoreactivity remains low. Our data uncovered a novel role for LAT-PLCγ1 signaling in regulating IL6 production by T cells during autoimmunity.

Finally, we wanted to further examine IL4 production and T helper cell differentiation in LATY136F mice. We examined IL4 production using KN2 reporter mice, where huCD2 marks T cells that have recently produced IL4 protein. We demonstrated that only a small proportion of the LATY136F T cells were actively secreting IL4. This subset of T cells were Tfh cells that expressed BCL6 and localized to B cell-rich germinal centers within the spleen. Most studies to date have examined Tfh cells in infection models, and have demonstrated that Tfh cells have very low expression of GATA3. Our results revealed in a spontaneous T cell-mediated autoimmune model system, that Tfh cells express both high levels of BCL6 and GATA3. Additionally, using an inducible deletion system, where normal development occurs, we showed that Tfh cells differentiation is the result of aberrant LAT signaling, rather than autoreactive TCRs with high affinity for self-peptide-MHC. LATY136F Tfh cells did require B cells for their development. Together, these results displayed a novel role for tonic LAT-PLCγ1 signaling in modulating Tfh cell differentiation and BCL6 expression.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that lacks any intrinsic enzymatic or transcriptional activity. Upon TCR engagement, LAT is phosphorylated at its membrane-distal tyrosine residues, which mediate the binding of Grb2/Sos, PLCγ1, and GADS/SLP-76 complexes. SLP-76 (SH2 domain-containing leukocyte protein of 76kD) is a cytosolic adaptor protein that can interact with a variety of other adaptor proteins and signaling effectors. Through its constitutive binding of GADS, SLP-76 is recruited to the plasma membrane via LAT following TCR stimulation. Together, LAT and SLP-76 nucleate a large multi-molecular signaling complex, which couples TCR proximal signaling to downstream biochemical events, including calcium mobilization and Ras-MAPK pathway activation.

LAT is important in early thymocyte development as LAT-deficient mice have a complete block at the DN3 stage. To study the role of LAT beyond the DN3 stage, we generated mice in which the lat gene could be deleted by Cre recombinase. Deletion of LAT after the DN3 stage allowed largely normal development of DP thymocytes. However, LAT-deficient DP thymocytes were severely defective in responding to stimulation via the TCR and failed to efficiently differentiate into SP thymocytes. Moreover, deletion of LAT in peripheral mature T cells rendered these T cells completely unresponsive to CD3 crosslinking due to abolished calcium mobilization and Ras-ERK activation. Long-term survival and lymphopenia-driven homeostatic proliferation of the LAT-deficient naïve T cells were also severely impaired. Together, these data indicate that, in addition to its role in pre-TCR signaling, LAT also plays an essential role in thymocyte development during the transition from the DP to SP stage, as well as in mature T cell activation and homeostasis.

Similar to LAT, SLP-76 is also critical for T cell function and thymocyte development. While the functions of various SLP-76 domains have been extensively studied, the role of the sterile alpha motif (SAM) domain in SLP 76 function remains unknown. By generating SLP 76 knock in mice with the SAM domain deleted, we showed that the absence of the SAM domain resulted in impaired positive and negative thymic selections, leading to a partial block of thymocyte development at the DP to SP transition. TCR-mediated IP3 production, calcium flux, and ERK activation were all decreased in these ΔSAM-SLP-76 knockin T cells, leading to defective IL 2 production and proliferation. Moreover, despite normal association between GADS and SLP-76, TCR-mediated SLP 76 clustering was inhibited by the deletion of the SAM domain, likely causing the aforementioned TCR signaling defects. These data demonstrated for the first time that the SAM domain is indispensable for optimal SLP-76 signaling.

Over 1.5 billion individuals are infected with intestinal helminths worldwide, with a majority of those infected living in developing nations. In developed nations intestinal helminth infections are very rare, while incidences of allergy and asthma are common. The incidence of allergic afflictions is growing rapidly every year. Both clearance of helminth infections and propagation of allergic disease are mediated by type 2 immune responses. The cytokines interleukin-4 (IL-4) and interleukin-13 (IL-13) play major roles in the propagation of type 2 immune responses. IL-4 and IL-13 are produced by a number of immune cells, within both the innate and adaptive arms of immunity, that are important in driving allergic responses.

CD4+ T follicular helper (Tfh) cells reside in the B-cell follicle and specialize in aiding the maturation of germinal center (GC) B cells. IL-4 produced by Tfh cells is required for GC B cell Immunoglobulin (Ig) class switching to type-2 isotypes, IgE and IgG1. IgE serves as a critical mediator of type-2 immune responses. CD4+ T helper 2 (Th2) cells localize to the periphery at sites of infection and damage. Th2 cells make both IL-4 and IL-13 cytokines. Th2 cells, along with multiple innate cell types, are critical for driving the peripheral hallmarks of type-2 immune responses, including mucus production and smooth muscle contractility.

Elucidating the pathways that regulate the differentiation, function, and maintenance of Tfh and Th2 cells is critically important for discovering potential therapies for allergic disease and helminth infections. Notch signaling is capable of driving Th2 IL-4 production and differentiation in vitro. However, the in vivo role for Notch signaling in Th2 populations remains unclear. The mechanisms controlling Tfh IL-4 production are largely unknown. Given that Notch signaling is required for the differentiation of Tfh cells and is known to influence cytokine production in T cells, we hypothesized that Notch signaling also plays an important role in regulating the function of Tfh cells. Nippostrongylus brasiliensis infection drives a robust type-2 immune response and allows for analysis of both Tfh and Th2 cells. Here, infection with N. brasiliensis was used to characterize whether Notch signaling is required for Th2 and Tfh differentiation and function in vivo.

Deletion of Notch receptors on T cells of infected mice results in reduced IL-4 producing Tfh, but not Th2 cells. As a result, we saw impairments in overall Tfh functionality while peripheral Th2 immunity remained intact. Notch deficient T cells had major impairments in Tfh, but not Th2, cell differentiation. Overexpression of Notch signaling in CD4+ T cells leads to increased IL-4 production by Tfh cells, but not Th2 cells. Furthermore, we identified that conventional dendritic cells (cDCs) do play a role as sources for Notch ligand early during the immune response. However, neither cDCs or follicular dendritic cells (FDCs) are essential sources of Notch ligand to drive Tfh cell differentiation.

While Notch signaling is critical for Tfh differentiation, it is not known if Notch signaling plays a continued role beyond Tfh differentiation. We used pharmacologic inhibition of Notch signaling to assess a role for Notch in Tfh maintenance. Here, we show that inhibition of Notch signaling after Tfh differentiation results in altered expression and activity of important trafficking receptors. This change was accompanied by aberrant localization of IL-4 expressing T cells in the lymph node. Additionally, late Notch inhibition resulted in an altered transcriptional program in Tfh cells. These findings suggest that Notch signaling plays a critical role in Tfh, but not Th2 driven immunity. In total, the data shown here demonstrate that Notch signaling is not only important for Tfh differentiation, but also for regulating Tfh cell fate, function, and maintenance.

T cells and B cells each express unique antigen receptors used to identify, eliminate, and remember pathogens. These receptors are generated through a process known as V(D)J recombination, in which T cell receptor and B cell receptor gene loci undergo genomic recombination. Interestingly, recombination at certain genes is regulated so that a single in-frame rearrangement is present on only one allele per cell. This phenomenon, termed allelic exclusion, requires two steps. First, recombination can occur only on one allele at a time. In the second step, additional recombination must be prevented. Though the mechanism of the second step is well-understood, the first step remains poorly understood.

The first step of recombination necessitates that alleles rearrange one at a time. This could be achieved either through inefficient recombination or by halting further recombination in the presence of recombination. To separate these mechanisms, we analyzed recombination in nuclei unable to complete recombination. We found that rearrangement events accumulated at antigen receptor loci, suggesting that the presence of recombination does not stop additional rearrangements and asynchronous recombination likely results from inefficient recombination at both alleles.

Association with repressive subnuclear compartments has been proposed to reduce the recombination efficiency of allelically excluded antigen receptor loci. Of the alleleically excluded loci, Tcrb alleles are uniquely regulated during development. Other allelically excluded alleles are positioned at the transcriptionally-repressive nuclear periphery prior to recombination, and relocate to the nuclear interior at the stage in which they recombine. However Tcrb alleles remain highly associated with the nuclear periphery during rearrangement. Here we provide evidence that this peripheral subnuclear positioning of Tcrb alleles does suppress recombination. We go on to suggest that peripheral localization mediates the first step of allelic exclusion.

In search of the mechanism by which recombination is suppressed on peripheral Tcrb alleles, we investigated the subnuclear localization of a recombinase protein. Two recombinase proteins are required for recombination, one of which is recruited to actively transcribing (and more centrally located) DNA. Here we demonstrate that one recombinase protein is unable to localize to peripheral Tcrb alleles, potentially serving as the mechanism by which recombination is suppressed on peripheral alleles.