Naive T cell activation requires both TCR and CD28 signals. The CARMIL2 cytosolic protein enables CD28-dependent activation of the NF-κB transcription factor via its ability to link CD28 to the CARD11 adaptor protein. Here, we developed mice expressing a mutation named Carmil2QE and mimicking a mutation found in human T cell malignancies. Naive T cells from Carmil2QE mice contained preformed CARMIL2QE-CARD11 complexes in numbers comparable to those assembling in wild-type T cells after CD28 engagement. Such ready-made CARMIL2QE-CARD11 complexes also formed in CD28-deficient mice where they unexpectedly induced most of the functions that normally result from CD28 engagement in a manner that remains antigen-dependent. In turn, tumor-specific T cells expressing Carmil2QE do not require CD28 engagement and thereby escape to both PD-1 and CTLA-4 inhibition. In conclusion, we uncovered the overarching role played by CARMIL2-CARD11 signals among those triggered by CD28 and exploited them to induce potent solid tumor-specific T cell responses in the absence of CD28 ligands and immune checkpoint inhibitors.
© 2025 Zhang et al.

Checkpoint blockade revolutionized cancer therapy, but we still lack a quantitative, mechanistic understanding of how inhibitory receptors affect diverse signaling pathways. To address this issue, we developed and applied a fluorescent intracellular live multiplex signal transduction activity reporter (FILMSTAR) system to analyze PD-1-induced suppressive effects. These studies identified pathways triggered solely by TCR or requiring both TCR and CD28 inputs. Using presenting cells differing in PD-L1 and CD80 expression while displaying TCR ligands of distinct potency, we found that PD-1-mediated inhibition primarily targets TCR-linked signals in a manner highly sensitive to peptide ligand quality. These findings help resolve discrepancies in existing data about the site(s) of PD-1 inhibition in T cells while emphasizing the importance of neoantigen potency in controlling the effects of checkpoint therapy.
© 2023 Chan et al.

Mice with a loss-of-function mutation in the LAT adaptor (LatY136F) develop an autoimmune and type 2 inflammatory disorder called defective LAT signalosome pathology (DLSP). We analyzed via single-cell omics the trajectory leading to LatY136F DLSP and the underlying CD4+ T cell diversification. T follicular helper cells, CD4+ cytotoxic T cells, activated B cells, and plasma cells were found in LatY136F spleen and lung. Such cell constellation entailed all the cell types causative of human IgG4-related disease (IgG4-RD), an autoimmune and inflammatory condition with LatY136F DLSP-like histopathological manifestations. Most previously described T cell-mediated autoimmune manifestations require persistent TCR input. In contrast, following their first engagement by self-antigens, the autoreactive TCR expressed by LatY136F CD4+ T cells hand over their central role in T cell activation to CD28 costimulatory molecules. As a result, all subsequent LatY136F DLSP manifestations, including the production of autoantibodies, solely rely on CD28 engagement. Our findings elucidate the etiology of the LatY136F DLSP and qualify it as a model of IgG4-RD.
© 2023 Joachim et al.

The CARD11 scaffold controls antigen receptor signaling to NF-κB, JNK, and mTOR. Three classes of germline mutations in CARD11 cause Primary Immunodeficiency, including homozygous loss-of-function (LOF) mutations in CARD11 deficiency, heterozygous gain-of-function (GOF) mutations in BENTA disease, and heterozygous dominant-negative LOF mutations in CADINS. Here, we characterize LOF CARD11 mutants with a range of dominant-negative activities to identify the mechanistic properties that cause these variants to exert dominant effects when heterozygous. We find that strong dominant negatives can poison signaling from mixed wild-type:mutant oligomers at two steps in the CARD11 signaling cycle, at the Opening Step and at the Cofactor Association Step. Our findings provide evidence that CARD11 oligomer subunits cooperate in at least two steps during antigen receptor signaling and reveal how different LOF mutations in the same oligomeric signaling hub may cause disease with different inheritance patterns.
© 2022 The Author(s).

T cell activation initiates protective adaptive immunity, but counterbalancing mechanisms are critical to prevent overshooting responses and to maintain immune homeostasis. The CARD11-BCL10-MALT1 (CBM) complex bridges T cell receptor engagement to NF-κB signaling and MALT1 protease activation. Here, we show that ABIN-1 is modulating the suppressive function of A20 in T cells. Using quantitative mass spectrometry, we identified ABIN-1 as an interactor of the CBM signalosome in activated T cells. A20 and ABIN-1 counteract inducible activation of human primary CD4 and Jurkat T cells. While A20 overexpression is able to silence CBM complex-triggered NF-κB and MALT1 protease activation independent of ABIN-1, the negative regulatory function of ABIN-1 depends on A20. The suppressive function of A20 in T cells relies on ubiquitin binding through the C-terminal zinc finger (ZnF)4/7 motifs, but does not involve the deubiquitinating activity of the OTU domain. Our mechanistic studies reveal that the A20/ABIN-1 module is recruited to the CBM complex via A20 ZnF4/7 and that proteasomal degradation of A20 and ABIN-1 releases the CBM complex from the negative impact of both regulators. Ubiquitin binding to A20 ZnF4/7 promotes destructive K48-polyubiquitination to itself and to ABIN-1. Further, after prolonged T cell stimulation, ABIN-1 antagonizes MALT1-catalyzed cleavage of re-synthesized A20 and thereby diminishes sustained CBM complex signaling. Taken together, interdependent post-translational mechanisms are tightly controlling expression and activity of the A20/ABIN-1 silencing module and the cooperative action of both negative regulators is critical to balance CBM complex signaling and T cell activation.
© 2022. The Author(s).