Adaptive immune responses are tightly controlled by proteins including CARD11 that regulate signaling events downstream of the T- and B-cell receptors. Germline mutations in CARD11 cause several distinct monogenic inborn errors of immunity with early childhood onset and potentially fatal prognoses. Somatic CARD11 gain-of-function mutations are associated with B-cell malignancies. Precisely how various CARD11 mutations culminate in unique clinical entities, and the mechanisms of CARD11-driven B-cell proliferation, is not fully understood. We sought to identify the genetic basis of disease and characterize immune-cell phenotypes and functions in a patient with apparent BENTA (B-cell expansion with nuclear factor-κB [NF-κB] and T-cell anergy) disease and a history of sibling death in early childhood. We used whole-exome sequencing, flow and mass cytometry, whole blood mRNA analyses, in vitro T-cell proliferation and B-cell differentiation assays, and single-cell RNA sequencing of patient-derived samples to identify the genetic basis of disease and define its molecular and cellular mechanisms. We also ectopically expressed wild-type and mutant forms of CARD11 in T and B cells and assessed their functional impacts on NF-κB-dependent responses. We report a surprising new genetic basis of BENTA caused by homozygosity for the novel CARD11 mutation R331P and characterized by massive expansion of B cells with a naive surface phenotype and aberrant transcriptional program. This autosomal-recessive form of BENTA features exaggerated B-cell lymphocytosis relative to monoallelic BENTA. Furthermore, we have identified patterns of gene expression that distinguish B cells of patients with the autosomal-recessive form of BENTA from those of healthy controls and from monoallelic BENTA. We found that ectopic CARD11 R331P expression induced constitutive NF-κB activity in T and B cells. These data suggest that R331P is a gain-of-function mutation and causes BENTA in homozygosity. These results define a novel autosomal-recessive form of BENTA disease. Additional analysis of mutation-driven changes in B-cell function may shed light on the mechanisms of B lymphomagenesis in patients with germline or somatic CARD11 variants.

The CARD11 (Caspase Recruitment Domain Family Member 11) gene encodes a scaffold protein critical for NF-κB signaling, regulating B-cell differentiation and T-cell effector functions. Gain-of-function (GOF) mutations in CARD11 cause BENTA disease (B cell Expansion with NF-κB and T cell Anergy), an autosomal dominant disorder typically presenting with early-onset polyclonal B-cell lymphocytosis, splenomegaly, lymphadenopathy, and recurrent infections. We describe three related patients harboring a novel CARD11-GOF mutation (D357E), presenting with a BENTA phenotype with atypical features, including high IgM levels and a normal B-cell count, with life-threatening HLH in one case. Additionally, we conducted a systematic literature review using PubMed and EMBASE to identify previously reported cases of CARD11 GOF mutations. In vitro functional analysis demonstrated that the D357E variant activates the NF-κB signaling pathway in primary lymphocytes and in HEK293T cells transfected with mutant CARD11. Our literature review identified 13 studies describing 29 patients. Notably, HLH emerged as a common complication of CARD11 GOF mutations (18.8 %), while B-lymphocytosis -though frequent- was not universally present. We identified a novel pathogenic CARD11 variant and described its atypical phenotype, further expanding the clinical spectrum of CARD11 GOF disorders. These findings underscore the need for increased awareness of HLH risk in patients with CARD11 GOF mutations.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare primary cutaneous non-Hodgkin lymphoma involving CD8+ T cells, the genetic underpinnings of which remain incompletely understood. Here we report two unrelated patients with B cell Expansion with NF-κB and T cell Anergy (BENTA) disease and a novel presentation of SPTCL. Patient 1 presented early in life with recurrent infections and B cell lymphocytosis, linked to a novel gain-of-function (GOF) CARD11 mutation (p.Lys238del). He developed SPTCL-like lesions and membranoproliferative glomerulonephritis by age 2, treated successfully with cyclosporine. Patient 2 presented at 13 months with splenomegaly, lymphadenopathy, and SPTCL with evidence of hemophagocytic lymphohistiocytosis. Genetic analysis revealed two in cis germline GOF CARD11 variants (p.Glu121Asp/p.Gly126Ser). Autologous bone marrow transplant resulted in SPTCL remission despite persistent B cell lymphocytosis. These cases illuminate an unusual pathological manifestation for BENTA disease, suggesting that CARD11 GOF mutations can manifest in cutaneous CD4+and CD8+ T cell malignancies.

Germline CARD11 gain-of-function (GOF) mutations cause B cell Expansion with NF-κB and T cell Anergy (BENTA) disease, whilst somatic GOF CARD11 mutations recur in diffuse large B cell lymphoma (DLBCL) and in up to 30% of the peripheral T cell lymphomas (PTCL) adult T cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL) and Sezary Syndrome. Despite their frequent acquisition by PTCL, the T cell-intrinsic effects of CARD11 GOF mutations are poorly understood. Here, we studied B and T lymphocytes in mice with a germline Nethyl-N-nitrosourea (ENU)-induced Card11M365K mutation identical to a mutation identified in DLBCL and modifying a conserved region of the CARD11 coiled-coil domain recurrently mutated in DLBCL and PTCL. Our results demonstrate that CARD11.M365K is a GOF protein that increases B and T lymphocyte activation and proliferation following antigen receptor stimulation. Germline Card11M365K mutation was insufficient alone to cause B or T-lymphoma, but increased accumulation of germinal center (GC) B cells in unimmunized and immunized mice. Card11M365K mutation caused cell-intrinsic over-accumulation of activated T cells, T regulatory (TREG), T follicular (TFH) and T follicular regulatory (TFR) cells expressing increased levels of ICOS, CTLA-4 and PD-1 checkpoint molecules. Our results reveal CARD11 as an important, cell-autonomous positive regulator of TFH, TREG and TFR cells. They highlight T cell-intrinsic effects of a GOF mutation in the CARD11 gene, which is recurrently mutated in T cell malignancies that are often aggressive and associated with variable clinical outcomes.