Molecular Mechanisms and Antigen Receptor Requirements for Lymphocyte Adaptation to Intestinal Tissues

The intestine plays a crucial role in food digestion, nutrient absorption, water retention, and waste excretion. It contains the most populous immune cell reservoir in the body and is continuously exposed to a large and diverse number of diet- and microbiota- derived antigens. The highly stimulating luminal environment is separated from the core of the body, the lamina propria (LP), by just a single layer of epithelial cells. The intestinal immune system is thus tasked with being able to tolerate innocuous stimuli while mounting an effective response against potential pathogens in a controlled manner. To ensure appropriate balance between tolerance and resistance, T cells undergo tissue adaptation upon migrating from the gut-draining mesenteric lymph nodes (mLN) to the intestinal lamina propria and epithelium (IE). We sought to elucidate the transcriptional mechanisms and T cell receptor (TCR) signaling requirements of CD4+ T cell plasticity and adaptation in the intestinal tissues. Within the intestine, peripherally induced Foxp3+ regulatory T cells (iTregs), which are instrumental in limiting inflammatory responses to non-self antigen, are located primarily in the lamina propria. However, CD8aa-expressing intraepithelial CD4+ T cells (CD4- IELs), which also exhibit anti-inflammatory properties and depend on similar environmental cues, reside in the epithelium. Using intravital microscopy, we find distinct cell dynamics of intestinal Tregs and CD4-IELs. We addressed the molecular imprinting of the gut epithelium on T cells by integrating mouse genetics with single-cell RNAsequencing analyses. Transcriptionally, CD4+ T cells from mLN, LP and IE segregate based on the intestinal layer they occupy; trajectory analysis suggests a stepwise loss of CD4-programming and acquisition of an intraepithelial profile as CD4+ T cells adapt to the epithelium and convert to CD4-IELs. We found that upon migration to the epithelium, Tregs can lose Foxp3 expression and convert to CD4-IELs in a microbiota-dependent fashion, an effect in part attributed to loss of the CD4 lineage-defining transcription factor ThPOK. Treg fate-mapping coupled with RNA- and ATAC-sequencing revealed that the Treg program shuts down before an intraepithelial program becomes fully accessible at the epithelium. Ablation of Thpok results in premature acquisition of an IEL profile by mLN Tregs, partially recapitulating epithelium imprinting. Furthermore, we demonstrate that iTregs and CD4-IELs perform complementary roles in the regulation of intestinal inflammation in response to dietary antigen. To uncover the specific role of the T cell receptor in the process of CD4-IEL development, we combined in vivo fate-mapping and gene ablation models with single cell TCRsequencing. Single-cell TCR repertoire and transcriptomic analysis of intraepithelial CD4+ T cells revealed different extents of clonal expansion and TCR overlap between cell states; fully differentiated CD4-IELs from regulatory or conventional CD4+ T cells were the least diverse. Conditional deletion of TCR on differentiating CD4+ T cells or of MHCII on intestinal epithelial cells prevented CD4-IEL differentiation. However, TCR ablation on developed CD4-IELs did not affect their accumulation. Overall, our results reveal an inter- and intra-tissue specialization of anti-inflammatory CD4+ T cells shaped by discrete niches of the intestine. We uncovered the stepwise molecular mechanisms and TCR-signaling requirements for T cells to adapt to the intestinal epithelium. We found that the coordinated replacement of the circulating lymphocyte program with site–specific transcriptional and chromatin changes is necessary for tissue imprinting. Furthermore, our results indicate that local recognition of possibly a limited set of antigens is an essential signal for the differentiation and adaptation of T cells to the epithelium. Taken together, the work presented in this thesis demonstrates that a combination of genetic, TCR, and environmental triggers is crucial in driving T cell plasticity and adaptation to the tissues within the intestine

Retinoic Acid is essential for Th1 cell lineage stability and prevents transition to a th17 cell program

SummaryCD4+ T cells differentiate into phenotypically distinct T helper cells upon antigenic stimulation. Regulation of plasticity between these CD4+ T-cell lineages is critical for immune homeostasis and prevention of autoimmune disease. However, the factors that regulate lineage stability are largely unknown. Here we investigate a role for retinoic acid (RA) in the regulation of lineage stability using T helper 1 (Th1) cells, traditionally considered the most phenotypically stable Th subset. We found that RA, through its receptor RARα, sustains stable expression of Th1 lineage specifying genes, as well as repressing genes that instruct Th17-cell fate. RA signaling is essential for limiting Th1-cell conversion into Th17 effectors and for preventing pathogenic Th17 responses in vivo. Our study identifies RA-RARα as a key component of the regulatory network governing maintenance and plasticity of Th1-cell fate and defines an additional pathway for the development of Th17 cells

Downregulation of chemokine receptor 9 facilitates CD4+CD8αα+ intraepithelial lymphocyte development

Abstract Intestinal intraepithelial lymphocytes (IELs) reside in the gut epithelial layer, where they help in maintaining intestinal homeostasis. Peripheral CD4+ T cells can develop into CD4+CD8αα+ IELs upon arrival at the gut epithelium via the lamina propria (LP). Although this specific differentiation of T cells is well established, the mechanisms preventing it from occurring in the LP remain unclear. Here, we show that chemokine receptor 9 (CCR9) expression is low in epithelial CD4+CD8αα+ IELs, but CCR9 deficiency results in CD4+CD8αα+ over-differentiation in both the epithelium and the LP. Single-cell RNA sequencing shows an enriched precursor cell cluster for CD4+CD8αα+ IELs in Ccr9 −/− mice. CD4+ T cells isolated from the epithelium of Ccr9 −/− mice also display increased expression of Cbfβ2, and the genomic occupancy modification of Cbfβ2 expression reveals its important function in CD4+CD8αα+ differentiation. These results implicate a link between CCR9 downregulation and Cbfb2 splicing upregulation to enhance CD4+CD8αα+ IEL differentiation

A conserved Bacteroidetes antigen induces anti-inflammatory intestinal T lymphocytes

The microbiome contributes to the development and maturation of the immune system. In response to commensal bacteria, intestinal CD4 + T lymphocytes differentiate into functional subtypes with regulatory or effector functions. The development of small intestine intraepithelial lymphocytes that coexpress CD4 and CD8αα homodimers (CD4IELs) depends on the microbiota. However, the identity of the microbial antigens recognized by CD4 + T cells that can differentiate into CD4IELs remains unknown. We identified β-hexosaminidase, a conserved enzyme across commensals of the Bacteroidetes phylum, as a driver of CD4IEL differentiation. In a mouse model of colitis, β-hexosaminidase–specific lymphocytes protected against intestinal inflammation. Thus, T cells of a single specificity can recognize a variety of abundant commensals and elicit a regulatory immune response at the intestinal mucosa. </jats:p

Total Synthesis of a Functional Designer Eukaryotic Chromosome

Designer Chromosome One of the ultimate aims of synthetic biology is to build designer organisms from the ground up. Rapid advances in DNA synthesis has allowed the assembly of complete bacterial genomes. Eukaryotic organisms, with their generally much larger and more complex genomes, present an additional challenge to synthetic biologists. Annaluru et al. (p. 55 , published online 27 March) designed a synthetic eukaryotic chromosome based on yeast chromosome III. The designer chromosome, shorn of destabilizing transfer RNA genes and transposons, is ∼14% smaller than its wild-type template and is fully functional with every gene tagged for easy removal. </jats:p