In vivo CRISPR screens reveal a HIF-1α-mTOR-network regulates T follicular helper versus Th1 cells

Abstract

T follicular helper (Tfh) cells provide signals to initiate and maintain the germinal center (GC) reaction and are crucial for the generation of robust, long-lived antibody responses, but how the GC microenvironment affects Tfh cells is not well understood. Here we develop an in vivo T cell-intrinsic CRISPR-knockout screen to evaluate Tfh and Th1 cells in an acute viral infection model to identify regulators of Tfh cells in their physiological setting. Using a screen of druggable-targets, alongside genetic, transcriptomic and cellular analyses, we identify a function of HIF-1α in suppressing mTORC1-mediated and Myc-related pathways, and provide evidence that VHL-mediated degradation of HIF-1α is required for Tfh development; an expanded in vivo CRISPR screen reveals multiple components of these pathways that regulate Tfh versus Th1 cells, including signaling molecules, cell-cycle regulators, nutrient transporters, metabolic enzymes and autophagy mediators. Collectively, our data serve as a resource for studying Tfh versus Th1 decisions, and implicate the VHL-HIF-1α axis in fine-tuning Tfh generation.

A CRISPR screen targeting PI3K effectors identifies RASA3 as a negative regulator of LFA-1–mediated adhesion in T cells

“…In addition, 162 proteins without PH domains, but which have been suggested to have affinity for PIP 3 ( 30 , 31 ), were included. From this list, we designed a retroviral library of 2298 sgRNAs targeting 570 genes, together with sgRNAs targeting genes encoding the phosphoinositol-modifying enzymes PTEN, PI3K p110α, p110γ, and p110δ, as well as Rap1 and CD11a; nontargeting (NT) sgRNAs were included as controls (data file S1) ( 28 , 32 ). OT-I × Cas9 CD8 + T cells were transduced with the library, isolated by fluorescence-activated cell sorting (FACS) on the basis of TCR-induced ICAM-1 binding, and the abundance of sgRNAs was quantified by next-generation sequencing (Fig.…”

“…sgRNAs were subcloned into an optimized retroviral backbone, MSCV sgRNA IRES GFP (MRIG) ( 28 , 32 ), containing a mU6-sgRNA fragment and an SV40-spCas9 fragment from pQCiG2 subcloned into MSCV-IRES-GFP-retroviral 1 (MIGR1) upstream of internal ribosomal entry site (IRES)–green fluorescent protein (GFP). All cloning was confirmed by Sanger sequencing.…”

“…Individually cloned oligos were designed with a partial mU6 and Trans activating crRNA (TracrRNA) sequence at 5′ and 3′ ends, respectively [5′-ggagaaaagccttgtttg-N20-gttttagagctaggatcctagc-3′ (where N20 is the sgRNA sequence)] and ordered as individual oligos (Integrated DNA Technologies) ( 28 , 32 ). For the PIP 3 -binding protein library, oligos were ordered as individual oligos and combined at equimolar ratios.…”

T cell help in the autoreactive germinal center

“…ApoE‐deficient bone marrow chimeras reconstituted with BXD2 bone marrow develop increased TFH cells, GCs, and autoantibodies, 176 suggesting that dyslipidemia may contribute to TFH associated loss of GC tolerance. Bcl6 177 and PD‐1 178 suppress glycolysis, but mTOR signaling is necessary for TFH activation 179–181 . CD4 T cells from SLE patients have increased mTORC1 signaling and glycolysis 182–190 and mTORC1 promotes Bcl6 expression in autoreactive TFH cells 191,192 .…”

“…Bcl6 177 and PD-1 178 suppress glycolysis, but mTOR signaling is necessary for TFH activation. [179][180][181] CD4 T cells from SLE patients have increased mTORC1 signaling and glycolysis [182][183][184][185][186][187][188][189][190] and mTORC1 promotes Bcl6 expression in autoreactive TFH cells. 191,192 Inhibition of glycolysis in B6.Sle1.Sle2.Sle3, NZB/W F1, BXSB.yaa, or B6.lpr mice with 2-deoxyglucose and metformin reduced autoantibody and TFH levels but preserved the ability to mount humoral responses to foreign antigen.…”