Flavell Lab Research

While transcriptional programming of immune cell differentiation and function has been a well-studied topic of research, the last decade has witnessed a resurgence of interest in how cell metabolism potentiates the biochemistry of these processes. Our lab is interested both in how cell metabolic processes dictate immune responses on the cellular level as well as how organismal and tissue nutrient environments impact immune function. Our current research focuses on lipid, carbohydrate, and amino acid metabolism of lymphocytes and innate immune cells, as well as the biology of intestinal, mucosal, and adipose tissues.

Integrating our long-term focus on T cell differentiation, recent work by Bailis, Shyer et al. demonstrated separable roles for mitochondrial complex I and complex II driven electron transport systems in CD4+ T cell proliferation and Th1 differentiation/IFNg secretion. We found that during early T cell activation, TCA cycling enables the malate/aspartate shuttle and citrate export to produce acetyl-CoA necessary for histone acetylation and epigenetic remodeling, highlighting the reciprocal regulation of cell metabolism and differentiation through changes in gene expression, an ongoing area of interest for our lab. While much immunometabolism has focused on central nodes such as mTOR, we continue to investigate the complex network of biochemical interactions comprising the metabolic-immune axis in multiple cell types with high resolution via forward genetic and chemical screening approaches including CRISPR/Cas9 screening.

Highlighted Work:

Bailis W, Shyer JA, Zhao J, Canaveras JCG, Al Khazal FJ, Qu R, Steach HR, Bielecki P, Khan O, Jackson R, Kluger Y, Maher LJ 3rd, Rabinowitz J, Craft J, Flavell RA. Distinct modes of mitochondrial metabolism uncouple T cell differentiation and function.

(2019) Jul;571(7765):403-407. doi: 10.1038/s41586-019-1311-3. Epub 2019 Jun 19;573(7773):E2. PMID: 31217581; PMCID: PMC6939459.

The mucosa of higher organisms are the primary interfaces between much of the outside world and our bodies. How does our mucosal immune system interconnect with the rest of the tissue environment, and how does it recognize and respond to pathogens yet tolerate commensals? This environment is a complex tissue composed of all the expected candidates, among others: epithelium, stroma, blood vessels, immune cells and an extensive nervous system. Thus, a major part of our immune system is localized at this barrier. Around 10 years ago, our lab searched for the immune sensors which must initiate the dialog with microbes and found that NOD-like receptors (NLR) plan a key role.

A major focus of research in the Flavell lab since the 1990s has been the biology of the NLR family of pattern recognition receptors and their associated caspases. As cytosolic sensors of bacterial components and signals of cellular stress or infection, NLRs act as a critical nexus of innate immune signaling by activating the assembly of the large multimolecular complex called the inflammasome, and ultimately culminates in the secretion of pro-inflammatory cytokines IL-1beta and IL-18. Because our greatest exposure to microbial signals occurs in the gut, we became interested in the function of NLRs in the intestinal epithelium. Indeed, we discovered that the NLRP6 inflammasome was essential for IL-18 processing in the intestinal epithelium, and that loss of this pathway resulted in the outgrowth of pathogenic microbes that caused gut and systemic inflammation. We further showed that in response to invasive microbes NLRP6-dependent pathways drove mucus secretion and mucosal anti-microbial protein (AMP) expression. Together with Eran Elinav’s lab, we discovered that this was regulated by bacterial metabolites in an IL-18 autocrine loop, thereby facilitating homeostasis. In contrast, we discovered that excessive IL-18 drives pathogenic inflammation unless mitigated by secretion of the IL-18 decoy receptor, IL-18BP, setting up a dichotomy wherein IL-18 can be both pathogenic and protective.

We have become interested in understanding this dichotomy. To this end, we recently discovered that the homeostatic IL-18 which controlled bacterial infection in the intestine was not derived from the intestinal epithelium, but was instead derived from the enteric nervous system (ENS). Remarkably, IL-18 derived from enteric neurons drove an antimicrobial program in the secretory epithelium, while epithelial and immune cell-derived IL-18 dictated distinct immuno-inflammatory programs. A major focus of current studies in this area are identification of the NLRs and cellular pathways which lead to the secretion of ENS-derived IL-18.

Highlighted Work:

Elinav E, Strowig T, Kau AL, Henao-Mejia J, Thaiss CA, Booth CJ, Peaper DR, Bertin J, Eisenbarth SC, Gordon JI, Flavell RA. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell.(2011) May 27;145(5):745-57. doi: 10.1016/j.cell.2011.04.022. Epub 2011 May 12. PMID: 21565393; PMCID: PMC3140910.

Jarret A, Jackson R, Duizer C, Healy ME, Zhao J, Rone JM, Bielecki P, Sefik E, Roulis M, Rice T, Sivanathan KN, Zhou T, Solis AG, Honcharova-Biletska H, Vélez K, Hartner S, Low JS, Qu R, de Zoete MR, Palm NW, Ring AM, Weber A, Moor AE, Kluger Y, Nowarski R, Flavell RA. Enteric Nervous System-Derived IL-18 Orchestrates Mucosal Barrier Immunity. Cell. (2020) Jan 9;180(1):50-63.e12. doi: 10.1016/j.cell.2019.12.016. PMID: 31923399; PMCID: PMC7339937.

Nowarski R, Jackson R, Gagliani N, de Zoete MR, Palm NW, Bailis W, Low JS, Harman CC, Graham M, Elinav E, Flavell RA. Epithelial IL-18 Equilibrium Controls Barrier Function in Colitis. Cell. (2015) Dec 3;163(6):1444-56. doi: 10.1016/j.cell.2015.10.072. PMID: 26638073; PMCID: PMC4943028.

Wlodarska M, Thaiss CA, Nowarski R, Henao-Mejia J, Zhang JP, Brown EM, Frankel G, Levy M, Katz MN, Philbrick WM, Elinav E, Finlay BB, Flavell RA. NLRP6 inflammasome orchestrates the colonic host-microbial interface by regulating goblet cell mucus secretion. Cell. (2014) Feb 27;156(5):1045-59. doi: 10.1016/j.cell.2014.01.026. PMID: 24581500; PMCID: PMC4017640.

NLRs in the intestine sense bacteria, but do they recognize viruses? We identified Nlrp6 and the novel NLR, Nlrp9b protect against major enteric viruses, rotavirus and norovirus respectively. Nlrp6 recognizes long dsRNA via RNA helicase Dhx15, driving the interferon response through Mavs/IFN/ISGs. Nlrp9b identifies short dsRNA through helicase Dhx9, driving GasderminD-pyroptosis to eliminate infected cells. As Nlrp6 is highly expressed in the proximal SI while Nlrp9b is highly expressed in distal SI, it seems that NLRs form a differential network of protection in the intestine against distinct viral threats.

Highlighted Work:

Wang P, Zhu S, Yang L, Cui S, Pan W, Jackson R, Zheng Y, Rongvaux A, Sun Q, Yang G, Gao S, Lin R, You F, Flavell R, Fikrig E. Nlrp6 regulates intestinal antiviral innate immunity. Science. (2015) Nov 13;350(6262):826-30. doi: 10.1126/science.aab3145. Epub 2015 Oct 22. PMID: 26494172; PMCID: PMC4927078.

Zhu S, Ding S, Wang P, Wei Z, Pan W, Palm NW, Yang Y, Yu H, Li HB, Wang G, Lei X, de Zoete MR, Zhao J, Zheng Y, Chen H, Zhao Y, Jurado KA, Feng N, Shan L, Kluger Y, Lu J, Abraham C, Fikrig E, Greenberg HB, Flavell RA. Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells. Nature. (2017) Jun 29;546(7660):667-670. doi: 10.1038/nature22967. Epub 2017 Jun 21. PMID: 28636595; PMCID: PMC5787375.

An integral part of all tissues is the mesenchyme, which is primarily composed of extracellular matrix and heterogeneous resident mesenchymal cell types. Our recent work provided a detailed characterization of mesenchymal cell diversity in the mouse intestine at the single cell level and uncovered hitherto unknown fibroblast subsets and specific niches occupied by them within the tissue. Based on this work and by employing mouse models and organoids, we discovered that a rare population of Ptgs2 (Cox-2)-expressing pericryptal fibroblasts constitutively secretes prostaglandin E2, a labile lipid metabolite, which signals to the neighboring mutant intestinal stem cells and orchestrates colorectal cancer initiation. This work demonstrated for the first time that intestinal tumorigenesis is controlled by the mesenchymal microenvironment of mutant stem cells and may explain why Cox-2 antagonists, such as aspirin, reduce the risk of colorectal cancer in humans.

Highlighted Work

Roulis M, Kaklamanos A, Schernthanner M, Bielecki P, Zhao J, Kaffe E, Frommelt LS, Qu R, Knapp MS, Henriques A, Chalkidi N, Koliaraki V, Jiao J, Brewer JR, Bacher M, Blackburn HN, Zhao X, Breyer RM, Aidinis V, Jain D, Su B, Herschman HR, Kluger Y, Kollias G, Flavell RA. Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche. Nature. (2020) Apr;580(7804):524-529. doi: 10.1038/s41586-020-2166-3. Epub 2020 Apr 1. PMID: 32322056

We continue to examine the critical role of the mesenchyme in tissue biology and pathology Fibrosis involves a complex communication network between immune and mesenchymal cell types. Multiple innate immune cells have been identified to be essential for driving the expression of extracellular matrix genes by mesenchymal cells, and the degree of response may be modulated by regulatory T cells. However, the particular immune signals that mediate these effects are not known. In the Flavell lab, we are studying immune regulation of fibrosis in patients with scleroderma, the prototypic autoimmune disease that causes fibrosis of multiple organs, including skin, lungs, kidneys, and GI tract. Intra-abdominal fibrosis may also occur in a variety of inflammatory states; we are interested in how this occurs and how to prevent or treat it.

Inflammatory cells run the gauntlet from blood to infected/compromised organs. Differential force is exerted upon these cells during this process. We found myeloid cells expressing the mechanosensory receptor Piezo1 detects cyclical pressure which triggers pulmonary inflammation through an Endothelin1/HIF1a autocrine loop that is not only critical for anti-bacterial defense, but also exacerbates lung damage in fibrotic disease.

Highlighted Work

Solis AG, Bielecki P, Steach HR, Sharma L, Harman CCD, Yun S, de Zoete MR, Warnock JN, To SDF, York AG, Mack M, Schwartz MA, Dela Cruz CS, Palm NW, Jackson R, Flavell RA. Mechanosensation of cyclical force by PIEZO1 is essential for innate immunity. Nature. (2019) Sep;573(7772):69-74. doi: 10.1038/s41586-019-1485-8. Epub 2019 Aug 21. PMID: 31435009; PMCID: PMC6939392.

Our lab has been working on T cell biology for 25 years. Effector T cells originate from naïve precursors which differentiate according to environment. Naïve T cells remain quiescent to constrain activation, but are also poised to respond rapidly. This explosive awakening involves rewiring of much of the cells’ molecular machinery. A major component of this is in RNA metabolism.

Dynamic chemical modifications post-transcriptionally decorate coding and non-coding RNAs, and affect RNA structure and functions like degradation and translation. At least 140 different RNA modifications are identified with N6-methyladenosine (m6A) perhaps the most abundant on mRNAs. We hope to identify the underlying principles whereby the RNA epitranscriptomic system enables immune cells to rapidly adapt to their microenvironment. We have already identified that the levels of m6A and its ‘writer.’ METTL3 increases upon T cell activation and serves to guide cell differentiation. We are now investigating if these modifications serve to regulate other aspects of T cell biology including T cell subtype differentiation and cytokine production.

Highlighted Work

Li HB, Tong J, Zhu S, Batista PJ, Duffy EE, Zhao J, Bailis W, Cao G, Kroehling L, Chen Y, Wang G, Broughton JP, Chen YG, Kluger Y, Simon MD, Chang HY, Yin Z, Flavell RA. m⁶ A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways. Nature. (2017) Aug 17;548(7667):338-342. doi: 10.1038/nature23450. Epub 2017 Aug 9. PMID: 28792938; PMCID: PMC5729908

A second level of the maintenance of T cell quiescence is in the stability of mRNA. By compiling super-enhancer datasets with RNA-seq/GWAS, we recently identified Btg1/2 as novel regulators of the naïve T cell state. Btg1/2-deficient naïve T cells exhibited increased proliferation and spontaneous activation. Btg1/2 is part of the CNOT-deadenylase complex and its loss increased global poly(A)-tail length, extending mRNA half-life, global increase in mRNA abundance and increases in key positive regulatory proteins delivering a reduced activation threshold. Thus, BTG1/2 promotes deadenylation and degradation of mRNAs to secure T cell quiescence, a previously unidentified mechanism underlying this process. Upon activation, these brakes on gene expression are released, RNA stabilized and protein synthesis enabled.

Highlighted Work:

Hwang SS, Lim J, Yu Z, Kong P, Sefik E, Xu H, Harman CCD, Kim LK, Lee GR, Li HB, Flavell RA. mRNA destabilization by BTG1 and BTG2 maintains T cell quiescence. Science. (2020) Mar 13;367(6483):1255-1260. doi: 10.1126/science.aax0194. PMID: 32165587.

The Flavell Lab, along with others, have shown that activated CD4 T cells are functionally plastic, and T_H17 cells are uniquely able to acquire regulatory properties by expressing anti-inflammatory IL-10 with or without the Treg marker Foxp3. Our fate mapping system suggested that effector T_H17 derived IL-10-expressing cells (T_R1exT_H17), also contribute to the control of intestinal immune homeostasis and inflammation. Recently, this fate mapping system also showed that tissue resident memory CD4 T cells can be derived from effector T_H17 cells, which are important for host immune defense against secondary infection.

In addition to T cell plasticity, we seek to understand and manipulate the capacity of ILCs to change fate and control disease. Although psoriasis is driven by type 3 cytokines like IL-17 and IL-22, little is known about dynamics as well as the source of these cytokines. By using type 2 cytokine reporter mice, together with single cell ATACseq and scRNAseq techniques (in collaboration with Dr. Aviv Regev), we recently find that resident skin ILC are epigenetically poised to transdifferentiate into ILC3, which produce pathogenic IL-17/22 and further drive psoriatic phenotypes. Uncovering the signals/mechanisms controlling trans-differentiation may reveal novel therapies for patients refractory to today’s treatments.

Highlighted Work:

Gagliani N, Amezcua Vesely MC, Iseppon A, et al. Th17 cells transdifferentiate into regulatory T cells during resolution of inflammation. Nature. (2015); 523(7559):221-225. doi:10.1038/ nature14452. PMCID: PMC4498984.

Amezcua Vesely MC, Pallis P, Bielecki P, et al. Effector TH17 Cells Give Rise to Long-Lived TRM Cells that Are Essential for an Immediate Response against Bacterial Infection. Cell. (2019);178(5):1176-1188.e15. doi:10.1016/j.cell.2019.07.032. PubMed PMID: 31442406

A long-standing focus of the Flavell lab has been the development and utilization of cutting-edge humanized mouse models, systems in which human tissues are engrafted into immunodeficient mice to study human cells in vivo without directly involving human patients. 15 years ago, our lab established a collaboration with Regeneron scientists and the Gates Foundation to establish a robust system to study human immunity and its role in disease. This initiated a longitudinal effort to replace the mouse loci of critical cytokines with human homologs in order to provide the necessary signals for proper human cell development in the mouse context. These modifications were achieved through knock-in replacement of the mouse coding sequence with the human gene counterpart, resulting in the human protein product’s expression being driven by the endogenous mouse promoter and subsequently physiologic expression of the cytokine. Using this method, we initially generated mice with human thrombopoietin knock-in to enhance human hematopoietic stem cell (HSC) maintenance and myeloid differentiation (Rongvaux 2011), hIL-3/GM-CSF resulting in engraftment of human alveolar macrophages and lung innate immune responses to intranasal influenza infection (Willinger 2011), M-CSF yielding robust tissue macrophages and circulating monocytes (Rathinam 2011) and Sirpa (Strowig 2011) protecting against innate destruction of human cells. This effort continued within the Flavell lab with the development of the MISTRG6 mouse, incorporating human knock-ins of M-CSF, IL-3-GM-CSF, SIRPa, TPO, IL-6on a Rag2^-/-IL-2R-Gamma-chain^-/-background (Rongvaux 2014, Das 2016). This strain exhibits enhanced engraftment of human HSCs compared with Nod-SCID-Gamma (NSG) mice and supports an expanded human myeloid compartment in a manner more closely mimicking human blood. Multiple subsets of functional myeloid cells, as well as T, B and NK cells, are generated in this model, indicating this mouse is unique in supporting studies of the innate immune system. The advent of the MISTRG6 strain represents a quantum leap in the field of humanized mouse modeling. In further unpublished work, we are investigating the effects of knock-ins for other factors for human hematopoietic development and function.

In addition to development of these novel mouse strains, our goal is for their use in studying human diseases in a pre-clinical context. This has led to important studies modeling patient responses to HIV (Shan 2015), antibody-mediated cancer immunosurveillance (Herndler-Branstetter 2017), and Ig class switching in humanized mice (Yu 2017). In addition, a number of ongoing projects utilize MISTRG6 or other derived strains to tackle important questions.

Our lab uses MISTRG6 to generate autologously engrafted tumor models, i.e. humanized mice which have been engrafted with a patient’s autologous hematopoietic stem cells and tumor cells. With these models we are studying tumor infiltration and the effects of cancer immunotherapies (anti-PD1 and anti-VEGF), with a focus on the myeloid immune cells which our MISTRG6 model uniquely generates. We have an IRB-approved human investigational study consenting lung cancer patients who are scheduled for surgery to collect bone marrow aspirate and tumor tissue. This allows us to obtain paired samples with autologous human hematopoietic stem cells (HSCs) for engraftment of humanized mice. We have obtained single-cell gene expression profiles from these autologously-engrafted tumors which reveals intriguing differences between tumor-resident and blood-derived T and myeloid cell populations. We have established that immunotherapies such as anti-PD1 drugs are active in this model and are piloting hTIL transfer studies using HSCs, TILs and tumor PDX obtained autologously from patients.

Highlighted Work:

Das R, Strowig T, Verma R, et al. Microenvironment-dependent growth of preneoplastic and malignant plasma cells in humanized mice. Nat Med (2016);22(11):1351-1357. doi:10.1038/nm.4202

Deng K, Pertea M, Rongvaux A, et al. Broad CTL response is required to clear latent HIV-1 due to dominance of escape mutations. Nature. (2015);517(7534):381-385. doi:10.1038/nature14053

Herndler-Brandstetter D, Shan L, Yao Y, Stecher C, Plajer V, Lietzenmayer M, Strowig T, de Zoete MR, Palm NW, Chen J, Blish CA, Frleta D, Gurer C, Macdonald LE, Murphy AJ, Yancopoulos GD, Montgomery RR, Flavell RA. Humanized mouse model supports development, function, and tissue residency of human natural killer cells. Proc Natl Acad Sci USA (2017) Oct 25. PMID: 29078283

Rathinam C, Poueymirou WT, Rojas J, Murphy AJ, Valenzuela DM, Yancopoulos GD, Eynon EE, Manz MG, Flavell RA. Efficient differentiation and function of human macrophages in humanized CSF-1 mice. Blood. (2011);118:3119-3128 PMID: 21791433

Rongvaux A, Willinger T, Takizawa H, Rathinam C, Auerbach W, Murphy A, Valenzuela D, Yancopoulos G, Eynon EE, Stevens S, Manz MG, Flavell RA. Human thrombopoietin knockin mice efficiently support human hematopoiesis in vivo. Proc. Natl. Acad. Sci. USA (2011) 108:2378-2383 PMC3038726

Rongvaux A, Willinger T, Martinek J, Strowig T, Gearty SV, Teichmann LL, Saito Y, Marches F, Halene S, Palucka AK, Manz MG and Flavell RA. Development and function of human innate immune cells in a humanized mouse model. Nature Biotechnology (2014). PMC4017589

Strowig T, Rongvaux A, Rathinam C, Takizawa H, Borsotti C, Philbrick W, Eynon E, Manz M, Flavell RA.Transgenic expression of human SIRPa in Rag2^-/- ?_c^-/- mice improves engraftment of human hematopoietic cells in humanized mice. Proc. Natl. Acad. Sci. USA 108:13218-13223 (2011) PMC3156175

Willinger T, Rongvaux A, Takizawa H, Yancopoulos GD, Valenzuela DM, Murphy A, Auerbach W, Eynon EE, Stevens S, Manz M, Flavell RA. Human IL-3/GM-CSF knock-in mice support human alveolar macrophage development and human immune responses in the lung. Proc. Natl. Acad. Sci. USA (2011) 108:2390-2395 PMC3038773

Yu H, Borsotti C, Schickel JN, Zhu S, Strowig T, Eynon EE, Frleta D, Gurer C, Murphy AJ, Yancopoulos GD, Meffre E, Manz MG, Flavell RA. A novel humanized mouse model with significant improvement of class- switched, antigen-specific antibody production. Blood. (2017). Jan 11 PMID: 28077418

Only a small portion of our transcriptome encodes protein. Research has identified that a species of RNA, long-noncoding RNAs (lncRNAs), regulate cellular homeostasis through interactions with DNA, RNA, and proteins. However, the significance of lncRNAs in maintaining a functional and regulated immune system is not well understood. In collaboration with Dr. John Rinn, we identified immunoregulatory candidate lncRNAs by high-throughput RNA sequencing and established a pipeline based on CRISPR/Cas9 genomic editing in murine models to determine their function. This system proved to be extremely successful and has led to the discovery of multiple lncRNAs which are central to immune cell development and function. One such lncRNA is termed Morrbid. Morrbid is expressed in Eosinophils/Neutrophils/Monocytes and controls cell-lifespan by regulating its neighboring gene, the cell death driver, Bim. Survival cytokines drive Morrbid transcription, repressing Bim and consequently increasing cell survival during infection. Morrbid expression wanes during the resolution phase of inflammation, and cells are removed by Bim-mediated cell death. In this way, Morrbid acts as a rheostat modulating cell death during inflammatory states. We also identified a lncRNA which regulates ILC1 function via a cis element, as well as a Th17-specific lncRNA that regulates expression of the lineage defining cytokines Rorg and Il17a by forming a ribonuclribonucleic-protein complex that regulates gene transcription. In preliminary studies we identified a lncRNA that regulates Treg associated aging related insulin resistance, as well as an IBD-associated lncRNA which regulates expression of central anti-microbial factors. These studies have underlined the central role of lncRNAs in immunity and we are now interested in applying our lncRNA discovery pipeline to a broader range of immune cell populations from healthy and diseased states.

Highlighted Work:

Kotzin JJ, Spencer SP, McCright SJ, Kumar DBU, Collet MA, Mowel WK, Elliott EN, Uyar A, Makiya MA, Dunagin MC, Harman CCD, Virtue AT, Zhu S, Bailis W, Stein J, Hughes C, Raj A, Wherry EJ, Goff LA, Klion AD, Rinn JL, Williams A, Flavell RA, Henao-Mejia J. The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan. Nature. (2016) Sep 8;537(7619):239-243. doi: 10.1038/nature19346. Epub 2016 Aug 15. PMID: 27525555; PMCID: PMC5161578.

Mowel WK, McCright SJ, Kotzin JJ, Collet MA, Uyar A, Chen X, DeLaney A, Spencer SP, Virtue AT, Yang E, Villarino A, Kurachi M, Dunagin MC, Pritchard GH, Stein J, Hughes C, Fonseca-Pereira D, Veiga-Fernandes H, Raj A, Kambayashi T, Brodsky IE, O'Shea JJ, Wherry EJ, Goff LA, Rinn JL, Williams A, Flavell RA, Henao-Mejia J. Group 1 Innate Lymphoid Cell Lineage Identity Is Determined by a cis-Regulatory Element Marked by a Long Non-coding RNA. Immunity. (2017) Sep 19;47(3):435-449.e8. doi: 10.1016/j.immuni.2017.08.012. PMID: 28930659; PMCID: PMC5761663.

Much work has defined the number of our genes and transcripts. Genome-wide annotation of open reading frames (ORFs) and their proteins, however, occurred through bioinformatic inference, not biological investigation. The definition of a coding gene was inferred from RNA hallmarks; an ATG initiation codon, an ORF greater than 300nts. Expressed RNAs lacking these are classified as non-coding RNAs. However, whether such RNAs generate protein has been overlooked. Using unbiased ribosome association and profiling techniques, we demonstrated many “non-coding” classified RNA encode protein. We investigated several in detail and published one non-canonical ORF of lncRNA Aw112010. Using an epitope tag KI mouse, we revealed its endogenous protein in challenged macrophages. Stop codon inactivation of the ORF rendered mice susceptible to infection and prevented pro-inflammatory cytokine upregulation. From our first analysis we found ~300 candidate ORF translated in just one cell type under a single condition. There are likely thousands yet to be revealed in the whole organism. A genome wide re-evaluation of open reading frames and “non-coding” RNA is required.

Highlighted Work:

Jackson R, Kroehling L, Khitun A, Bailis W, Jarret A, York AG, Khan OM, Brewer JR, Skadow MH, Duizer C, Harman CCD, Chang L, Bielecki P, Solis AG, Steach HR, Slavoff S, Flavell RA. The translation of non-canonical open reading frames controls mucosal immunity. Nature. (2018) Dec;564(7736):434-438. doi: 10.1038/s41586-018-0794-7. Epub 2018 Dec 12. PMID: 30542152; PMCID: PMC6939389.

Bioinformatics is a new and rapidly developing area in the Flavell lab in which we collaborate with Yuval Kluger and colleagues. The main focus of bioinformatics research is on single cell RNA sequencing (scRNA-seq) datasets from various biological systems and tissues (intestinal cells, T cells, tumor infiltrating immune cells, etc.) that provide important insights. The close interaction between biologists and bioinformaticians enables the development of novel tools to better analyze scRNA-seq data. Currently, we are working on the denoising and differential abundance of scRNA-seq data, as well as incorporating other modalities, for instance, scATAC-seq data and TCR/BCR sequencing data into analysis.

To be specific, differential abundance analysis aims at identifying cell populations that have significant differences in local density distribution across two samples, which for example, may indicate the corresponding cell-types are undergoing selection during treatment.

By leveraging TCR/BCR sequencing, we can look at the landscape of clonotypes before and after treatment and then perform differential analysis to identify the clonotypes with distinctive enrichment ratios. We then identify the genes that are enriched in corresponding up/down-regulated clonotypes to speculate the biological pathways involved in the whole process.

Additionally, we have been working on a variety of Next Generation Sequencing data, such as bulk RNA-seq, CRISPR Genome-wide Screen, CHIP-seq, etc. In addition, the advent of spatial transcriptomics sheds light on illustrating the spatial organization of cells in original tissue, from which the proximity among cells can be accurately quantified. Some traditional analysis using standard scRNA-seq including cell-cell interaction analysis and trajectory inference fails to take spatial information into account, which makes it less reliable to construct cell-cell networks. This new field provides more opportunities for us to uncover biological mechanisms, for example, to study the tumor microenvironment, in which we would like to develop new methods and tools in the future.