UCSF

T cells constitute an essential component of the adaptive immune response toimmunogenic antigens both endogenous and exogenous such as those from allergens or viral infections. We present here two studies of T cells, one in which we investigate a non-coding RNA circuit in the control of cytotoxic T cells and one in which we characterize allergen reactive T helper cells in human allergic asthmatic subjects. Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses and intracellular bacteria. Through biochemical analysis of RNA:protein interactions, we uncovered a novel non-coding RNA circuit regulating cytotoxic T cells composed of the long non-coding RNA (lncRNA) Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed miRNA family important for cell proliferation and survival. miR-15/16 also play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and T cell memory. Comparative Argonaute-2 high throughput sequencing of crosslinking immunoprecipitation (AHC) combined with gene expression profiling in normal and miR-15/16- deficient T cells revealed a network of several hundred direct miR-15/16 targets, many with functional relevance for T cell activation, survival and memory formation. Among these targets, the long non-coding RNA Malat1 contained the largest absolute magnitude miR-15/16- dependent AHC peak in T cells. This binding site was also among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16 binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of ixother miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of IL-2 and a broader CD28- responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of lncRNAs in the immune system by ascribing cell-intrinsic, sequence- specific in vivo function to Malat1. These findings have implications for T cell-mediated immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed. Beyond cytotoxic T cells, T helper cells can differentiate into distinct states marked by stereotypical cytokine expression. In the context of allergic diseases type 2 helper T cells which produce the type 2 cytokines, IL-5, IL-4, and IL-13 can become pathogenic and contribute to a consistently elevated level of type 2 inflammation. In asthma, the lung is the primary site of aberrant inflammation and understanding how this tissue responds to allergens is key. In this study, human subjects were locally exposed to allergen via bronchoscopy. We then assayed the immunological and cellular state of these bronchoscopy samples by multiple high dimensional ‘omic’ technologies: RNAseq, Cytof, and scRNAseq. We then define heterogeneity across these allergic subjects corresponding to type 2 high and type 2 low individuals. In the type 2 high individuals we find that inflammatory monocyte derived populations enter the lung and induce the expression of inflammatory chemokines CCL3, CCL17, and CCL22 likely contributing to increased inflammation in the lung. Further, through scRNAseq we are able to identify Th2 cells recruited specifically by the allergen challenge. In a subset of individuals we could identify TCR clones which were allergen reactive and expanded in the blood post allergen challenge. Indicating that, while these cells are rare in the lung, dampening their function systemically may lead to decreased local inflammation in the lung as well. Understanding all these facets of allergic inflammation in this detailed way is essential for proper clinical understanding of asthma as well as the potential development of novel therapeutic strategies.