BENTA (B cell Expansion with NF-κB and T cell Anergy) disease is a rare, selective B cell lymphoproliferative disorder caused by gain-of-function (GOF) mutations in the lymphocyte scaffolding molecule CARD11. CARD11 is required for downstream activation of NF-κB following antigen receptor engagement, which normally triggers recruitment of BCL10 and MALT1. MALT1 serves both scaffolding and proteolytic functions that initiate and amplify canonical NF-κB signaling. It still remains unclear how continuous CARD11 signaling ultimately drives massive B cell accumulation in BENTA patients. Here we show that in contrast to normal human B cells, BENTA patient B cells exhibited a striking resistance to apoptosis in cell culture, particularly after stimulation. Enhanced survival of patient cells was dependent on MALT1 protease, which is activated constitutively by CARD11 GOF mutants. Treatment with a novel, specific low molecular weight inhibitor of MALT1 protease completely restored apoptosis sensitivity in BENTA patient B cells. RNA-Seq analyses revealed that MALT1 protease inhibition induced a specific subset of pro-apoptotic genes in activated patient B cells, and downregulated multiple genes associated with cell cycle progression and metabolism, in addition to several NF-κB target genes. Although not strictly required for CARD11-dependent NF-κB activation, our results imply that MALT1 protease function governs a pro-survival signaling program in BENTA B cells that likely contributes to the profound B cell lymphocytosis that distinguishes this disease. MALT1 protease thus represents an attractive therapeutic target for reducing B cell burden in these patients, thereby lowering the risk of B cell malignancy later in life.

The proliferation and contraction of activated effector T cells must be carefully coordinated to maintain immune homeostasis. This is achieved in part through restimulation-induced cell death (RICD), a pre-programmed apoptosis program triggered by antigen restimulation through the T cell receptor (TCR). Forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) also constrain conventional T cell (Tcon) responses, but can resist RICD themselves despite frequent TCR stimulation. We previously showed that FOXP3 protects Tregs from RICD by suppressing SLAM-associated protein (SAP), a key adaptor protein that amplifies TCR signal strength. Mysteriously, FOXP3 expression is also transiently induced in human Tcons after activation, with a kinetic expression profile that correlates inversely with acquired RICD sensitivity. Hence we asked whether FOXP3 protects expanding human effector T cells from premature RICD by modulating SAP expression. Our results show that although siRNA-mediated FOXP3 knockdown sensitizes early effector CD4 and CD8 T cells to RICD, SAP expression remains unaffected. Unlike late stage effector T cells, a low level of RICD in expanding Tcons was entirely dependent on de novo transcription, and knockdown of SAP failed to reduce death. Subsequent RNA-Seq analyses revealed that CD48, a SLAM family receptor, was markedly reduced upon FOXP3 knockdown. Blockade or knockdown of CD48 also increased RICD in CD4 and CD8 T cells. We now show that CD48 protects early effector T cells both by promoting autophagy and upregulation of pro-survival genes such as BATF. These findings implicate FOXP3 as the central governor of a distinct transcriptional program that promotes RICD resistance early in the effector T cell response.

The adaptive immune response relies on specific apoptosis programs to regulate effector T cell expansion and maintain homeostasis. Restimulation-induced cell death (RICD) is an apoptotic pathway triggered by repeated stimulation through the T cell receptor (TCR), which ensures that effector T cell proliferation remains in check. Constitutive fork head box P3 (FOXP3) expression renders regulatory T cells (Tregs) resistant to RICD. Although FOXP3 is also expressed transiently in human conventional T cells (Tcons) during initial rounds of activation-induced proliferation, its function in this context remains unclear. Here we describe a novel role for FOXP3 in protecting both CD4+ and CD8+ human Tcons from premature RICD during the expansion phase. SiRNA-mediated silencing of FOXP3 sensitized early effector Tcons to RICD, and was dependent on de novo transcription. RNA-Seq analysis revealed FOXP3-dependent effects on genes involved in metabolic pathways, autophagy, and cell surface receptors. FOXP3 silencing increased glycolysis and reduced autophagy in Tcons, which has been linked to changes in apoptosis sensitivity. Indeed, blocking autophagy increased RICD in control Tcons without augmenting death of FOXP3-silenced cells, suggesting FOXP3 reduces RICD sensitivity through an autophagy-dependent mechanism. Additionally, FOXP3 knockdown markedly reduced expression of CD48, a SLAM-family receptor. We now show that CD48 protects expanding Tcons from RICD. Surprisingly, CD48 also promoted protective autophagy, illuminating a novel connection between SLAM receptor signaling, autophagy regulation, and RICD sensitivity that is governed by FOXP3 expression in early Tcons.