Browsing by Subject "CD4+ T cell"

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.

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.