Two patients with chronic mucocutaneous candidiasis caused by TRAF3IP2 deficiency.

BACKGROUND: TRAF3 interacting protein 2 (TRAF3IP2) (Act1) is an adapter protein that interacts with IL-17R via its similar expression to fibroblast growth factor genes and IL-17R domain and coordinates 2 separate proinflammatory pathways following IL-17 cytokine stimulation. OBJECTIVE: We sought to elucidate the immunologic consequences of TRAF3IP2 homozygous mutations to improve treatments for immunodeficiency patients with chronic mucocutaneous candidiasis. METHODS: We describe 2 patients presenting with chronic mucocutaneous candidiasis who harbor biallelic nonsense mutations in TRAF3IP2. The cellular and molecular features of this genetic defect were assessed using in vitro cytokine assays and protein analysis. RESULTS: We show that the homozygous mutation causes complete loss of protein expression. We also show that the absence of TRAF3IP2 was associated with a defective response to combined IL-2/IL-25 (IL-17E) stimulation. CONCLUSIONS: Failure to initiate normal signaling downstream of IL-17R engagement likely contributes to the patients' recurrent fungal infections. These findings add to our molecular understanding of genetic defects affecting this critical pathway of antifungal immunity.

CD55 Deficiency, Early-Onset Protein-Losing Enteropathy, and Thrombosis.

BACKGROUND: Studies of monogenic gastrointestinal diseases have revealed molecular pathways critical to gut homeostasis and enabled the development of targeted therapies. METHODS: We studied 11 patients with abdominal pain and diarrhea caused by early-onset protein-losing enteropathy with primary intestinal lymphangiectasia, edema due to hypoproteinemia, malabsorption, and less frequently, bowel inflammation, recurrent infections, and angiopathic thromboembolic disease; the disorder followed an autosomal recessive pattern of inheritance. Whole-exome sequencing was performed to identify gene variants. We evaluated the function of CD55 in patients' cells, which we confirmed by means of exogenous induction of expression of CD55. RESULTS: We identified homozygous loss-of-function mutations in the gene encoding CD55 (decay-accelerating factor), which lead to loss of protein expression. Patients' T lymphocytes showed increased complement activation causing surface deposition of complement and the generation of soluble C5a. Costimulatory function and cytokine modulation by CD55 were defective. Genetic reconstitution of CD55 or treatment with a complement-inhibitory therapeutic antibody reversed abnormal complement activation. CONCLUSIONS: CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein-losing enteropathy (the CHAPLE syndrome) is caused by abnormal complement activation due to biallelic loss-of-function mutations in CD55. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Opposing roles for RhoH GTPase during T-cell migration and activation.

T cells spend the majority of their time perusing lymphoid organs in search of cognate antigen presented by antigen presenting cells (APCs) and then quickly recirculate through the bloodstream to another lymph node. Therefore, regulation of a T-cell response is dependent upon the ability of cells to arrive in the correct location following chemokine gradients ("go" signal) as well as to receive appropriate T-cell receptor (TCR) activation signals upon cognate antigen recognition ("stop" signal). However, the mechanisms by which T cells regulate these go and stop signals remain unclear. We found that overexpression of the hematopoietic-specific RhoH protein in the presence of chemokine signals resulted in decreased Rap1-GTP and LFA-1 adhesiveness to ICAM-1, thus impairing T-cell chemotaxis; while in the presence of TCR signals, there were enhanced and sustained Rap1-GTP and LFA-1 activation as well as prolonged T:APC conjugates. RT-PCR analyses of activated CD4(+) T cells and live images of T-cell migration and immunological synapse (IS) formation revealed that functions of RhoH took place primarily at the levels of transcription and intracellular distribution. Thus, we conclude that RhoH expression provides a key molecular determinant that allows T cells to switch between sensing chemokine-mediated go signals and TCR-dependent stop signals.

HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease.

Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)-dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses.

Human PI3Kγ deficiency and its microbiota-dependent mouse model reveal immunodeficiency and tissue immunopathology.

Phosphatidylinositol 3-kinase-gamma (PI3Kγ) is highly expressed in leukocytes and is an attractive drug target for immune modulation. Different experimental systems have led to conflicting conclusions regarding inflammatory and anti-inflammatory functions of PI3Kγ. Here, we report a human patient with bi-allelic, loss-of-function mutations in PIK3CG resulting in absence of the p110γ catalytic subunit of PI3Kγ. She has a history of childhood-onset antibody defects, cytopenias, and T lymphocytic pneumonitis and colitis, with reduced peripheral blood memory B, memory CD8+ T, and regulatory T cells and increased CXCR3+ tissue-homing CD4 T cells. PI3Kγ-deficient macrophages and monocytes produce elevated inflammatory IL-12 and IL-23 in a GSK3α/ß-dependent manner upon TLR stimulation. Pik3cg-deficient mice recapitulate major features of human disease after exposure to natural microbiota through co-housing with pet-store mice. Together, our results emphasize the physiological importance of PI3Kγ in restraining inflammation and promoting appropriate adaptive immune responses in both humans and mice.

Molecular Classification of Primary Immunodeficiencies of T Lymphocytes.

Proper regulation of the immune system is required for protection against pathogens and preventing autoimmune disorders. Inborn errors of the immune system due to inherited or de novo germline mutations can lead to the loss of protective immunity, aberrant immune homeostasis, and the development of autoimmune disease, or combinations of these. Forward genetic screens involving clinical material from patients with primary immunodeficiencies (PIDs) can vary in severity from life-threatening disease affecting multiple cell types and organs to relatively mild disease with susceptibility to a limited range of pathogens or mild autoimmune conditions. As central mediators of innate and adaptive immune responses, T cells are critical orchestrators and effectors of the immune response. As such, several PIDs result from loss of or altered T cell function. PID-associated functional defects range from complete absence of T cell development to uncontrolled effector cell activation. Furthermore, the gene products of known PID causal genes are involved in diverse molecular pathways ranging from T cell receptor signaling to regulators of protein glycosylation. Identification of the molecular and biochemical cause of PIDs can not only guide the course of treatment for patients, but also inform our understanding of the basic biology behind T cell function. In this chapter, we review PIDs with known genetic causes that intrinsically affect T cell function with particular focus on perturbations of biochemical pathways.

Action and Traction: Cytoskeletal Control of Receptor Triggering at the Immunological Synapse.

It is well known that F-actin dynamics drive the micron-scale cell shape changes required for migration and immunological synapse (IS) formation. In addition, recent evidence points to a more intimate role for the actin cytoskeleton in promoting T cell activation. Mechanotransduction, the conversion of mechanical input into intracellular biochemical changes, is thought to play a critical role in several aspects of immunoreceptor triggering and downstream signal transduction. Multiple molecules associated with signaling events at the IS have been shown to respond to physical force, including the TCR, costimulatory molecules, adhesion molecules, and several downstream adapters. In at least some cases, it is clear that the relevant forces are exerted by dynamics of the T cell actomyosin cytoskeleton. Interestingly, there is evidence that the cytoskeleton of the antigen-presenting cell also plays an active role in T cell activation, by countering the molecular forces exerted by the T cell at the IS. Since actin polymerization is itself driven by TCR and costimulatory signaling pathways, a complex relationship exists between actin dynamics and receptor activation. This review will focus on recent advances in our understanding of the mechanosensitive aspects of T cell activation, paying specific attention to how F-actin-directed forces applied from both sides of the IS fit into current models of receptor triggering and activation.

F-actin flow drives affinity maturation and spatial organization of LFA-1 at the immunological synapse.

Integrin-dependent interactions between T cells and antigen-presenting cells are vital for proper T cell activation, effector function, and memory. Regulation of integrin function occurs via conformational change, which modulates ligand affinity, and receptor clustering, which modulates valency. Here, we show that conformational intermediates of leukocyte functional antigen 1 (LFA-1) form a concentric array at the immunological synapse. Using an inhibitor cocktail to arrest F-actin dynamics, we show that organization of this array depends on F-actin flow and ligand mobility. Furthermore, F-actin flow is critical for maintaining the high affinity conformation of LFA-1, for increasing valency by recruiting LFA-1 to the immunological synapse, and ultimately for promoting intracellular cell adhesion molecule 1 (ICAM-1) binding. Finally, we show that F-actin forces are opposed by immobilized ICAM-1, which triggers LFA-1 activation through a combination of induced fit and tension-based mechanisms. Our data provide direct support for a model in which the T cell actin network generates mechanical forces that regulate LFA-1 activity at the immunological synapse.

The dendritic cell cytoskeleton promotes T cell adhesion and activation by constraining ICAM-1 mobility.

Integrity of the dendritic cell (DC) actin cytoskeleton is essential for T cell priming, but the underlying mechanisms are poorly understood. We show that the DC F-actin network regulates the lateral mobility of intracellular cell adhesion molecule 1 (ICAM-1), but not MHCII. ICAM-1 mobility and clustering are regulated by maturation-induced changes in the expression and activation of moesin and α-actinin-1, which associate with actin filaments and the ICAM-1 cytoplasmic domain. Constrained ICAM-1 mobility is important for DC function, as DCs expressing a high-mobility ICAM-1 mutant lacking the cytoplasmic domain exhibit diminished antigen-dependent conjugate formation and T cell priming. These defects are associated with inefficient induction of leukocyte functional antigen 1 (LFA-1) affinity maturation, which is consistent with a model in which constrained ICAM-1 mobility opposes forces on LFA-1 exerted by the T cell cytoskeleton, whereas ICAM-1 clustering enhances valency and further promotes ligand-dependent LFA-1 activation. Our results reveal an important new mechanism through which the DC cytoskeleton regulates receptor activation at the immunological synapse.