Molecular mechanisms controlling age-associated B cells in autoimmunity.

Age-associated B cells (ABCs) have emerged as critical components of immune responses. Their inappropriate expansion and differentiation have increasingly been linked to the pathogenesis of autoimmune disorders, aging-associated diseases, and infections. ABCs exhibit a distinctive phenotype and, in addition to classical B cell markers, often express the transcription factor T-bet and myeloid markers like CD11c; hence, these cells are also commonly known as CD11c+ T-bet+ B cells. Formation of ABCs is promoted by distinctive combinations of innate and adaptive signals. In addition to producing antibodies, these cells display antigen-presenting and proinflammatory capabilities. It is becoming increasingly appreciated that the ABC compartment exhibits a high degree of heterogeneity, plasticity, and sex-specific regulation and that ABCs can differentiate into effector progeny via several routes particularly in autoimmune settings. In this review, we will discuss the initial insights that have been obtained on the molecular machinery that controls ABCs and we will highlight some of the unique aspects of this control system that may enable ABCs to fulfill their distinctive role in immune responses. Given the expanding array of autoimmune disorders and pathophysiological settings in which ABCs are being implicated, a deeper understanding of this machinery could have important and broad therapeutic implications for the successful, albeit daunting, task of targeting these cells.

SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1.

SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca(2+) signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4(+) T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4(+) T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6(-/-)) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling.

Regulation of systemic autoimmunity and CD11c+ Tbet+ B cells by SWEF proteins.

Recent studies have revealed the existence of a T-bet dependent subset of B cells, which expresses unique phenotypic and functional characteristics including high levels of CD11c and CD11b. In the murine system this B cell subset has been termed Age/autoimmune-associated B cells (ABCs) since it expands with age in non-autoimmune mice and it prematurely accumulates in autoimmune-prone strains. The molecular mechanisms that promote the expansion and function of ABCs are largely unknown. This review will focus on the SWEF proteins, a small family of Rho GEFs comprised of SWAP-70 and its homolog DEF6, a newly identified risk variant for human SLE. We will first provide an overview of the SWEF proteins and then discuss the complex array of biological processes that they control and the autoimmune phenotypes that spontaneously develop in their absence, highlighting the emerging involvement of these proteins in regulating ABCs. A better understanding of the pathways controlled by the SWEF proteins could help provide new insights into the mechanisms responsible for the expansion of ABCs in autoimmunity and potentially guide the design of novel therapeutic approaches.

Swap70b is required for convergent and extension cell movement during zebrafish gastrulation linking Wnt11 signalling and RhoA effector function.

Swap70 functions as a guanine nucleotide exchange factor for Rac and RhoA regulating F-actin cytoskeletal rearrangements and playing a crucial role in mammalian cell activation, migration, adhesion and invasion. Here we show that the zebrafish orthologue, Swap70b, is required for convergent and extension cell movement during gastrulation. Swap70b morphants exhibited broader and shorter body axis but cell fate specification appeared normal. While ectopic Swap70b expression robustly rescued Wnt11 morphants, RhoA overexpression was sufficient to rescue Swap70b morphants, establishing Swap70b as a novel member of the non-canonical Wnt/PCP pathway downstream of Wnt11 and upstream of RhoA. This is distinct from the related Def6a protein that acts downstream of Wnt5b. Def6a/Swap70b morphants resemble Ppt/Slb double mutant embryos suggesting that Swap70b and Def6a delineate Wnt11 and Wnt5b signalling pathways.

DEF6 has potential to be a biomarker for cancer prognosis: A pan-cancer analysis.

Introduction: DEF6 is a gene associated with the immune system and is thought to play a crucial role in autoimmunity. There are few DEF6-related studies in cancer, and it is assumed that DEF6 is a proto-oncogene. There is currently no pan-cancer analysis of DEF6, and we performed a systematic and comprehensive pan-cancer analysis of DEF6 in an attempt to reveal its role and function in cancer. Methods: The data were analyzed by mining databases available to the public and by using R software. Moreover, immunohistochemistry was used to validate the results. Results: Our results revealed that DEF6 is commonly aberrantly expressed in cancer and its expression is strongly correlated with survival prognosis in a variety of cancer types. Through correlation analysis we found that DEF6 was associated with multiple immune genes and was closely related to immune infiltration. In the enrichment analysis, DEF6 may have cross-talk with multiple cancer pathways and exert oncogenic or pro-cancer functions. In addition, we collected pathological samples from colorectal cancer patients for immunohistochemical analysis and found that patients with higher immunohistochemical scores had more lymph node metastases, higher CA199, and bigger tumor size. Discussion: Overall, DEF6 expression is closely related to cancers and has the potential to act as a cancer biomarker.

Different Apples, Same Tree: Visualizing Current Biological and Clinical Insights into CTLA-4 Insufficiency and LRBA and DEF6 Deficiencies.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a crucial immune checkpoint that is constitutively expressed in regulatory T (Treg) cells. Following T-cell activation, CTLA-4 is rapidly mobilized from its intracellular vesicle pool to the cell surface to control the availability of co-stimulatory B7 molecules, thereby maintaining immune homeostasis. Heterozygous mutations in CTLA-4 lead to defects in (i) CTLA-4 ligand binding, (ii) homo-dimerization, (iii) B7-transendocytosis, and (iv) CTLA-4 vesicle trafficking, resulting in an inborn error of immunity with predominant autoimmunity. CTLA-4 vesicle trafficking impairment is also observed in patients with lipopolysaccharide-responsive beige-like anchor protein (LRBA) deficiency or the differentially expressed in FDCP6 homolog (DEF6) deficiency, caused by biallelic mutations in LRBA and DEF6, respectively. Therefore, patients with CTLA-4 insufficiency, LRBA deficiency, and-most recently reported-DEF6 deficiency present an overlapping clinical phenotype mainly attributed to a defective suppressive activity of Tregs, as all three diseases reduce overall surface expression of CTLA-4. In this paper, we describe the clinical phenotypes of these immune checkpoint defects, their patho-mechanisms, and visually compare them to other immune regulatory disorders (IPEX syndrome, CD27, and CD70 deficiencies) by using the immune deficiency and dysregulation (IDDA version 2.1) "kaleidoscope" score. This illustrates the variability of the degrees and manifestations of immune deficiency and dysregulation. Patients characteristically present with an increased risk of infections, autoimmune cytopenias, multi-organ autoimmunity, and inflammation, which are often severe and life-threatening. Furthermore, these patients suffer an increased risk of developing malignancies, especially Non-Hodgkin's lymphoma. Successful treatment options include regular administration of soluble CTLA-4-Ig fusion protein, Treg cell-sparing immune suppressants like sirolimus or mycophenolate mofetil, and hematopoietic stem cell transplantation. This mini-review highlights the most relevant biological and clinical features as well as treatment options for CTLA-4 insufficiency and LRBA and DEF6 deficiencies.

Intrinsic Restriction of TNF-Mediated Inflammatory Osteoclastogenesis and Bone Resorption.

TNF (Tumor necrosis factor) is a pleiotropic cytokine that plays an important role in immunity and inflammatory bone destruction. Homeostatic osteoclastogenesis is effectively induced by RANKL (Receptor activator of nuclear factor kappa-B ligand). In contrast, TNF often acts on cell types other than osteoclasts, or synergically with RANKL to indirectly promote osteoclastogenesis and bone resorption. TNF and RANKL are members of the TNF superfamily. However, the direct osteoclastogenic capacity of TNF is much weaker than that of RANKL. Recent studies have uncovered key intrinsic mechanisms by which TNF acts on osteoclast precursors to restrain osteoclastogenesis, including the mechanisms mediated by RBP-J signaling, RBP-J and ITAM (Immunoreceptor tyrosine-based activation motif) crosstalk, RBP-J mediated regulatory network, NF-κB p100, IRF8, and Def6. Some of these mechanisms, such as RBP-J and its mediated regulatory network, uniquely and predominantly limit osteoclastogenesis mediated by TNF but not by RANKL. As a consequence, targeting RBP-J activities suppresses inflammatory bone destruction but does not significantly impact normal bone remodeling or inflammation. Hence, discovery of these intrinsic inhibitory mechanisms addresses why TNF has a weak osteoclastogenic potential, explains a significant difference between RANKL and TNF signaling, and provides potentially new or complementary therapeutic strategies to selectively treat inflammatory bone resorption, without undesirable effects on normal bone remodeling or immune response in disease settings.

High expression levels of DEF6 predicts a poor prognosis for patients with clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is one of the most common types of malignant tumors and early detection contributes to a better prognosis. Finding new biomarkers for the diagnosis or treatment remains meaningful. DEF6 guanine nucleotide exchange factor (DEF6) is upregulated in ccRCC compared to normal controls, but the relationship between DEF6 expression and prognosis in ccRCC is unclear. Moreover, the potential biological functions of DEF6 in ccRCC remains unclear. In the present study, the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), TISIDB and the clinical database of the Peking University First Hospital were used to analyze DEF6 expression in ccRCC. Immunohistochemistry (IHC), western blotting and reverse transcription­quantitative PCR were used to examine the DEF6 protein and mRNA expression levels in cell lines and clinical samples. Subsequently, the Kaplan­Meier method and Cox regression analyses were used to determine the impact of DEF6 expression on the overall survival of patients alongside other clinical variables in both the TCGA database and the present clinical database. The results showed that both DEF6 mRNA and protein expression levels were upregulated in ccRCC compared to normal controls. The Kaplan­Meier survival analysis showed that patients with high DEF6 expression had poor prognoses from both the TCGA database and the present clinical database. Univariate survival analysis and multivariate survival analysis revealed that DEF6 could be an independent prognostic factor for ccRCC. Additionally, bioinformatics analysis indicated that differentially expressed genes related to DEF6 expression influenced ccRCC by regulating the tumor immune microenvironment. In conclusion, overexpression of DEF6 is significantly correlated with a poor prognosis for patients with ccRCC and DEF6 may influence the biological processes involved with ccRCC by regulating the immune microenvironment.

Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells.

Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4+CD25- T cells (Tcons) independently of IP3 levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP3 receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer.

DEF6 expression in ovarian carcinoma correlates with poor patient survival.

BACKGROUND: Increased expression of DEF6 is correlated with the malignant behavior of various cancers. Both DEF6 and p16 contribute to the regulation of cell cycle progression, and p53 plays important role in the cell cycle checkpoints. This study was designed to elucidate the prognostic significance of DEF6, p53 and p16 immunoexpressions in different histology subtypes of ovarian carcinoma. METHODS: Immunohistochemistry results of DEF6, p53 and p16 on ovarian carcinoma were compared with histology subtypes, clinical data, overall survival (OS) and disease-free survival (DFS) by Cox regression and Kaplan-Meier analysis. RESULTS: We studied 180 cases of ovarian carcinomas (75 high-grade serous, 41 clear cell, 36 mucinous and 28 endometrioid), including 109 FIGO stage I-II cases and 71 FIGO stage III-IV cases. Ovarian carcinomas positive for both DEF6 and p16 expression were associated with the worst OS (P = 0.027) and DFS (P = 0.023), whereas those negative for both DEF6 and p16 had the best OS and DFS. Aberrant p53 expression combined with positive DEF6 was associated with worst OS (P = 0.031) and DFS (P = 0.028). Kaplan-Meier analysis showed that significantly shorter survival rates were seen in patients with high expressions of DEF6 (P = 0.008) and p16 (P = 0.022). Patients with aberrant p53 expression in high-grade serous carcinoma (P = 0.012) and patients with high DEF6 expression in clear cell carcinoma (P = 0.001) were also associated with shorter overall survival. In univariate analysis, FIGO stage, DEF6 and p16 were associated with poor prognosis. DEF6 expression was the only independent prognostic factor correlated with shorted OS (HR 2.115; P = 0.025) and DFS (HR 2.248; P = 0.016) upon multivariate analysis. CONCLUSIONS: DEF6 expression may serve as an independent prognostic factor, and interacted positively with p16 toward high tumor stage and shorter survival.