T follicular helper (T _FH ) cells are essential for effective antibody responses, but deciphering the intrinsic wiring of mouse T _FH cells has long been hampered by the lack of a reliable protocol for their generation in vitro. We report that transforming growth factor–β (TGF-β) induces robust expression of T _FH hallmark molecules CXCR5 and Bcl6 in activated mouse CD4 ⁺ T cells in vitro. TGF-β–induced mouse CXCR5 ⁺ T _FH cells are phenotypically, transcriptionally, and functionally similar to in vivo–generated T _FH cells and provide critical help to B cells. The study further reveals that TGF-β–induced CXCR5 expression is independent of Bcl6 but requires the transcription factor c-Maf. Classical TGF-β–containing T helper 17 (T _H 17)–inducing conditions also yield separate CXCR5 ⁺ and IL-17A–producing cells, highlighting shared and distinct cell fate trajectories of T _FH and T _H 17 cells. We demonstrate that excess IL-2 in high-density T cell cultures interferes with the TGF-β–induced T _FH cell program, that T _FH and T _H 17 cells share a common developmental stage, and that c-Maf acts as a switch factor for T _FH versus T _H 17 cell fates in TGF-β–rich environments in vitro and in vivo.

The RNA-binding proteins Roquin-1/2 and Regnase-1 exert essential regulation by controlling proinflammatory mRNA expression to prevent autoimmune disease. More recently, inhibition of this post-transcriptional gene regulatory program has been demonstrated to enable enhanced anti-tumor responses by tumor antigen-specific CD8 ⁺ T cells. In this review we describe the functions of these RNA-binding proteins and the phenotypes that arise in association with genetic inhibition or inactivation. We discuss how inducible inactivation of the system reprograms CD4 ⁺ and CD8 ⁺ T cell fates by changing cell metabolism, activation, differentiation or effector/memory decisions. We furthermore outline what we need to know to precisely modulate this system in order to dampen autoimmune reactions or boost the efficacy of adoptively transferred T cells or chimeric antigen receptor (CAR) T cells in cancer immunotherapies.

Constitutive activation of the MALT1 paracaspase in conventional T cells of Malt1 ^TBM/TBM (TRAF6 Binding Mutant = TBM) mice causes fatal inflammation and autoimmunity, but the involved targets and underlying molecular mechanisms are unknown. We genetically rendered a single MALT1 substrate, the RNA-binding protein (RBP) Roquin-1, insensitive to MALT1 cleavage. These Rc3h1 ^Mins/Mins mice showed normal immune homeostasis. Combining Rc3h1 ^Mins/Mins alleles with those encoding for constitutively active MALT1 (TBM) prevented spontaneous T cell activation and restored viability of Malt1 ^TBM/TBM mice. Mechanistically, we show how antigen/MHC recognition is translated by MALT1 into Roquin cleavage and derepression of Roquin targets. Increasing T cell receptor (TCR) signals inactivated Roquin more effectively, and only high TCR strength enabled derepression of high-affinity targets to promote Th17 differentiation. Induction of experimental autoimmune encephalomyelitis (EAE) revealed increased cleavage of Roquin-1 in disease-associated Th17 compared to Th1 cells in the CNS. T cells from Rc3h1 ^Mins/Mins mice did not efficiently induce the high-affinity Roquin-1 target IκB _NS in response to TCR stimulation, showed reduced Th17 differentiation, and Rc3h1 ^Mins/Mins mice were protected from EAE. These data demonstrate how TCR signaling and MALT1 activation utilize graded cleavage of Roquin to differentially regulate target mRNAs that control T cell activation and differentiation as well as the development of autoimmunity.

The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca²⁺ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21–bound transcriptome reveals strong interactions with the Rag1 3′-UTR. Arpp21–deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3′-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.

T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to a persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3, and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct, with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.