Biology subjects

Gagnon, J. D.

Publications and source records attributed to Gagnon, J. D..

5 recordsLinked to original sources

bioRxiv2026

Dual-inactivation of Regnase-1 and SOCS1 rewires exhausted CD8+ T cell fate to enhance anti-tumor functionality

The solid tumor microenvironment inhibits the functionality of tumor infiltrating T cells recognizing cognate tumor antigen, driving their differentiation towards terminal exhaustion. Interventions are sought to enhance the anti-tumor functionality of tumor-reactive T cells for clinical benefit. The functional genome regulating CD8+ T cell function against solid tumors was mapped by performing genome-wide, focused, and combination in vivo CRISPR/Cas9 screens using OT1 and PMEL TCR transgenic T cells in B16-OVA, MC38-gp100 and EG7-OVA syngeneic tumor models. The ability of the top single hits and combinations, which include Regnase-1 and SOCS1, to enhance CD8+ T cell anti-tumor function was evaluated in the OT1/B16-OVA model with large and established tumors, the disseminated PMEL/B16F10 tumor model, and in a novel murine TIL syngeneic model. The impact of Regnase-1 and SOCS1 single and dual-inactivation on the differentiation of exhausted CD8+ T cell subsets and on long-term persistent memory following tumor clearance was evaluated in OT1 CD8+ T cells in the B16-OVA model. The impact of single and dual-inactivation of Regnase-1 and SOCS1 on the anti-tumor function of experimental human T cell therapeutics was characterized in CRISPR/Cas9-engineered human TIL derived in vitro and in mesothelin-targeting CAR-Ts in vivo. NF-{kappa}B and cytokine signaling were identified as the top pathways regulating CD8+ T cell anti-tumor function, with Regnase-1 and Suppressor of Cytokine Signaling 1 (SOCS1) the top single and combination edits regulating the accumulation of tumor-specific TCR transgenic CD8+ T cells in syngeneic tumor models. Dual-inactivation of Regnase-1 and SOCS1 cooperated through non-redundant mechanisms to strongly expand intermediate (Texint) and effector (Texeff) exhausted CD8+ T cells within lymphoid tissues and tumor, with CD8+ T cells rewired to display an enhanced effector state and suppressed expression of TOX. Dual-edited persistent T effector memory cells (Tem) were formed following tumor clearance. Lastly, Regnase-1 and SOCS1 inactivation enhanced human Tumor Infiltrating Lymphocyte (TIL) and chimeric antigen receptor T cells (CAR-T) therapy functionality. Collectively, this study systematically mapped pathways regulating CD8+ T cell anti-tumor functionality, with Regnase-1 and SOCS1 dual-inactivation found to maximize anti-tumor function through non-redundant mechanisms.

immunology↗

bioRxiv2023

The lncRNA Malat1 Inhibits miR-15/16 to Enhance Cytotoxic T Cell Activation and Memory Cell Formation

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, many intracellular bacteria and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor 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 (Ago2 HITS-CLIP, or AHC) combined with gene expression profiling in normal and miR-15/16-deficient T cells revealed a large network of several hundred direct miR-15/16 target mRNAs, 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 other 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 long noncoding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/536843v2_figu1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1ebb1bborg.highwire.dtl.DTLVardef@e19c29org.highwire.dtl.DTLVardef@1d3568forg.highwire.dtl.DTLVardef@1d7d69b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

An essential role for miR-15/16 in Treg suppression and restriction of proliferation

The miR-15/16 family is a highly expressed group of tumor suppressor miRNAs that target a large network of genes in T cells to restrict their cell cycle, memory formation and survival. Upon T cell activation, miR-15/16 are downregulated, allowing rapid expansion of differentiated effector T cells to mediate a sustained immune response. Here, using conditional deletion of miR-15/16 in immunosuppressive regulatory T cells (Tregs) that express FOXP3, we identify new functions of the miR-15/16 family in T cell immunity. miR-15/16 are indispensable to maintain peripheral tolerance by securing efficient suppression by a limited number of Tregs. miR-15/16-deficiency alters Treg expression of critical functional proteins including FOXP3, IL2R/CD25, CTLA4, PD-1 and IL7R/CD127, and results in accumulation of functionally impaired FOXP3loCD25loCD127hi Tregs. Excessive proliferation in the absence of miR-15/16 inhibition of cell cycle programs shifts Treg diversity and produces an effector Treg phenotype characterized by low expression of TCF1, CD25 and CD62L, and high expression of CD44. These Tregs fail to control immune activation of CD4+ effector T cells, leading to spontaneous multi-organ inflammation and increased allergic airway inflammation in a mouse model of asthma. Together, our results demonstrate that miR-15/16 expression in Tregs is essential to maintain immune tolerance. HighlightsO_LITreg-specific miR-15/16 expression is essential to prevent systemic tissue inflammation C_LIO_LImiR-15/16 restrict Treg proliferation and regulate expression of the key functional Treg molecules FOXP3, IL2R, CTLA4, PD-1 and IL7R C_LIO_LImiR-15/16 limit formation of effector Tregs and is necessary for high suppressive capacity C_LI

molecular biology↗

bioRxiv2020

The non-coding RNA miR-17~92 is a central mediator of T cell activation

T cell activation is paramount for productive adaptive immune responses. CD28 is a key clinically targeted immunoregulatory receptor because it provides the prototypical costimulatory signal required for T cell activation. Therefore, a precise understanding of the molecular consequences of CD28 costimulation has direct therapeutic relevance. Here, we uncover that the microRNA cluster miR-17~92 is part of the molecular program triggered by CD28 costimulation and hence T cell activation. Combining genetics, transcriptomics, bioinformatics and biochemical miRNA:mRNA interaction maps we demonstrate that transgenic miR-17~92 can cell-intrinsically largely overcome defects caused by CD28-deficiency. miR-17~92 promotes transcription of a proinflammatory gene signature by enhancing the calcineurin/NFAT pathway. miR-17~92 binds to and represses a network of genes including several negative regulators of T cell activation. Finally, CD28-deficient T cells exhibit derepressed miR-17~92 target genes during activation, demonstrating that this non-coding RNA is required to shape the transcriptome. Thus, we propose that miR-17~92 constitutes a central mediator for T cell activation, integrating signals by the TCR and CD28 costimulation. In this model miR-17~92 facilitates T cell activation by dampening the breaks that prevent T cell activation.

immunology↗

bioRxiv2018

Tumor cell-adipocyte gap junctions activate lipolysis and are essential for breast tumorigenesis

A pro-tumorigenic role for adipocytes has been identified in breast cancer, and reliance on fatty acid catabolism found in aggressive tumors. The molecular mechanisms by which tumor cells coopt neighboring adipocytes, however, remain elusive. Here, we describe a direct interaction linking tumorigenesis to adjacent adipocytes. We examine breast tumors and their normal adjacent tissue from several patient cohorts, patient-derived xenografts and mouse models, and find that lipolysis and lipolytic signaling are activated in neighboring adipose tissue. We find that functional gap junctions form between breast cancer cells and adipocytes. As a result, cAMP is transferred from breast cancer cells to adipocytes and activates lipolysis in a gap junction-dependent manner. We identify connexin 31 (GJB3), which promotes receptor triple negative breast cancer growth and activation of lipolysis in vivo. Thus, direct tumor cell-adipocyte interaction contributes to tumorigenesis and may serve as a new therapeutic target in breast cancer. One sentence summaryGap junctions between breast cancer cells and adipocytes transfer cAMP and activate lipolysis in the breast tumor microenvironment to support growth.

cancer biology↗