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Purpose A male infant developed generalized rash, intestinal inflammation and severe infections including persistent cytomegalovirus. Family history was negative, T cell receptor excision circles were normal, and engraftment of maternal cells was absent. No defects were found in multiple genes associated with severe combined immunodeficiency. A 9/10 HLA matched unrelated hematopoietic cell transplant (HCT) led to mixed chimerism with clinical resolution. We sought an underlying cause for this patient’s immune deficiency and dysregulation. Methods Clinical and laboratory features were reviewed. Whole exome sequencing and analysis of genomic DNA from the patient, parents and 2 unaffected siblings was performed, revealing 2 MALT1 variants. With a host-specific HLA-C antibody, we assessed MALT1 expression and function in the patient’s post-HCT autologous and donor lymphocytes. Wild type MALT1 cDNA was added to transformed autologous patient B cells to assess functional correction. Results The patient had compound heterozygous DNA variants affecting exon 10 of MALT1 (isoform a, NM_006785.3), a maternally inherited splice acceptor c.1019-2A > G, and a de novo deletion of c.1059C leading to a frameshift and premature termination. Autologous lymphocytes failed to express MALT1 and lacked NF-κB signaling dependent upon the CARMA1, BCL-10 and MALT1 signalosome. Transduction with wild type MALT1 cDNA corrected the observed defects. Conclusions Our nonconsanguineous patient with early onset profound combined immunodeficiency and immune dysregulation due to compound heterozygous MALT1 mutations extends the clinical and immunologic phenotype reported in 2 prior families. Clinical cure was achieved with mixed chimerism after nonmyeloablative conditioning and HCT.

The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (Pak2) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. Pak2 was required for pre-TCR β-selection and positive selection. Surprisingly, Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, Pak2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2, uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation. T cells are a key element of the immune system. There are many different types of T cells, and they all have their origins in hematopoietic stem cells that are found in the bone marrow. These stem cells leave the bone marrow and circulate in the body until they reach an organ called the thymus, where they become early thymic progenitor cells. These progenitor cells then undergo a process called differentiation to become specific types of T cells, which mature in the thymus before moving to the blood. Although various molecules and mechanisms are known to be involved in the development of T cells, many details of this process are not understood. One group of molecules that has been implicated in the differentiation of T cells is the p21-activated kinases. Kinases are proteins that activate or deactivate other proteins by adding phosphate groups to specific amino acids. Pak2 adds phosphorylate groups to various proteins that are involved in the reorganization of an important structure inside the cell called the cytoskeleton. A kinase called Pak2 has an important role in the reorganization of the cytoskeleton, and since this reorganization is involved in almost all aspects of T cell biology, it seems plausible that Pak2 is also involved in the development of T cells. However, it has not been possible to test this idea because deleting the gene for Pak2 in mice results in their death. Now, Phee et al. have overcome this problem by performing experiments in which the gene for Pak2 was only deleted in T cells. These mice had significantly fewer mature T cells than healthy mice. In particular, the absence of Pak2 in thymocytes (the cells that become T cells) prevented them from maturing into T cells, and also prevented them from producing a receptor protein that is needed for mature T cells to leave the thymus. This work implies that disruption of the Pak2-mediated signaling pathway that regulates the cytoskeleton may weaken the immune system in humans.

Thymic selection requires that thymocytes transit through distinct anatomical compartments. Weiss and colleagues report that the G protein regulator GIT2 integrates T cell antigen receptor and chemokine receptor signaling necessary for proper migratory activity and positive selection. Thymocytes are highly motile cells that migrate under the influence of chemokines in distinct thymic compartments as they mature. The motility of thymocytes is tightly regulated; however, the molecular mechanisms that control thymocyte motility are not well understood. Here we report that G protein–coupled receptor kinase-interactor 2 (GIT2) was required for efficient positive selection. Notably, Git2 −/− double-positive thymocytes showed greater activation of the small GTPase Rac, actin polymerization and migration toward the chemokines CXCL12 (SDF-1) and CCL25 in vitro . By two-photon laser-scanning microscopy, we found that the scanning activity of Git2 −/− thymocytes was compromised in the thymic cortex, which suggests GIT2 has a key role in regulating the chemokine-mediated motility of double-positive thymocytes.

Despite decades of research, mechanisms controlling T cell activation remain only partially understood, which hampers T cell-based immune cancer therapies. Here, we performed a genome-wide CRISPR screen to search for genes that regulate T cell activation. Our screen confirmed many of the known regulators in proximal T cell receptor signaling and, importantly, also uncovered a previously uncharacterized regulator, FAM49B (family with sequence similarity 49 member B). FAM49B deficiency led to hyperactivation of Jurkat T cells following T cell receptor stimulation, as indicated by enhancement of CD69 induction, PAK phosphorylation, and actin assembly. FAM49B directly interacted with the active form of the small GTPase Rac, and genetic disruption of the FAM49B–Rac interaction compromised FAM49B function. Thus, FAM49B inhibits T cell activation by repressing Rac activity and modulating cytoskeleton reorganization.

Systematic characterization of intercellular signaling approximating the physiological conditions of stimulation that involve direct cell–cell contact is challenging. We describe a proteomic strategy to analyze physiological signaling mediated by the T-cell costimulatory receptor CD28. We identified signaling pathways activated by CD28 during direct cell–cell contact by global analysis of protein phosphorylation. To define immediate CD28 targets, we used phosphorylated forms of the CD28 cytoplasmic region to obtain the CD28 interactome. The interaction profiles of selected CD28-interacting proteins were further characterized in vivo for amplifying the CD28 interactome. The combination of the global phosphorylation and interactome analyses revealed broad regulation of CD28 and its interactome by phosphorylation. Among the cellular phosphoproteins influenced by CD28 signaling, CapZ-interacting protein (CapZIP), a regulator of the actin cytoskeleton, was implicated by functional studies. The combinatorial approach applied herein is widely applicable for characterizing signaling networks associated with membrane receptors with short cytoplasmic tails. Significance Intracellular signaling during complex cell–cell interactions, such as between immune cells, provides essential cues leading to cell responses. Global characterization of these signaling events is critical for systematically exploring and understanding how they eventually control cell fate. However, proteome-wide characterization of intercellular signaling under physiologically relevant conditions involving multiple interacting receptors during cell–cell interactions remains challenging. We developed an integrated proteomic strategy for quantitatively profiling intercellular-signaling events mediated by protein phosphorylation and protein–protein interaction. We applied this approach to determine the influence of a single receptor-ligand pair during T-cell stimulation by blocking the interaction of the CD28 costimulatory receptor with its ligand. This approach is generally applicable to other transmembrane receptors involved in signaling during complex cell interactions.