The emerging and diverse roles of the SLy/SASH1-protein family in health and disease-Overview of three multifunctional proteins.

Intracellular adaptor proteins are indispensable for the transduction of receptor-derived signals, as they recruit and connect essential downstream effectors. The SLy/SASH1-adaptor family comprises three highly homologous proteins, all of them sharing conserved structural motifs. The initial characterization of the first member SLy1/SASH3 (SH3 protein expressed in lymphocytes 1) in 2001 was rapidly followed by identification of SLy2/HACS1 (hematopoietic adaptor containing SH3 and SAM domains 1) and SASH1/SLy3 (SAM and SH3 domain containing 1). Based on their pronounced sequence similarity, they were subsequently classified as one family of intracellular scaffold proteins. Despite their obvious homology, the three SLy/SASH1-members fundamentally differ with regard to their expression and function in intracellular signaling. On the contrary, growing evidence clearly demonstrates an important role of all three proteins in human health and disease. In this review, we systematically summarize what is known about the SLy/SASH1-adaptors in the field of molecular cell biology and immunology. To this end, we recapitulate current research about SLy1/SASH3, SLy2/HACS1, and SASH1/SLy3, with an emphasis on their similarities and differences.

SLy2-overexpression impairs B-cell development in the bone marrow and the IgG response towards pneumococcal conjugate-vaccine.

BACKGROUND: Infections with Streptococcus pneumoniae can cause severe diseases in humans including pneumonia. Although guidelines for vaccination have been established, S. pneumoniae is still responsible for a serious burden of disease around the globe. Currently, two pneumococcal immunizations are available, namely the pure polysaccharide vaccine Pneumovax23 (P23) and the conjugate-vaccine Prevenar13 (PCV13). We recently reported impaired thymus-independent antibody responses towards P23 in mice overexpressing the immunoinhibitory adapter SLy2. The purpose of this study was to evaluate adaptive B-cell responses towards the thymus-dependent vaccine PCV13 in SLy2-overexpressing mice and to study their survival rate during pneumococcal lung infection. Moreover, we investigated B-cell developmental stages within the bone marrow (BM) in the context of excessive SLy2-expression. METHODS: B-cell subsets and their surface immune globulins were investigated by flow cytometry. For class-switch assays, isolated splenic B cells were stimulated in vitro with lipopolysaccharide and interleukin-4 and antibody secretion was quantified via LEGENDplex. To study PCV13-specific responses, mice were immunized and serum antibody titers (immunoglobulin M, immunoglobulins IgG1 , IgG2 , and IgG3 ) were examined by enzyme-linked immunosorbent assay. Survival rates of mice were assessed within 7 days upon intranasal challenge with S. pneumoniae. RESULTS: Our data demonstrate impaired IgG1 and IgG3 antibody responses towards the pneumococcal conjugate-vaccine PCV13 in SLy2-overexpressing mice. This was accompanied by reduced frequencies and numbers of BM-resident plasmablasts. In addition, we found drastically reduced counts of B-cell precursors in the BM of SLy2-Tg mice. The survival rate upon intranasal challenge with S. pneumoniae was mostly comparable between the genotypes. CONCLUSION: Our findings demonstrate an important role of the adapter protein SLy2 in the context of adaptive antibody responses against pneumococcal conjugate-vaccine. Interestingly, deficits in humoral immunity seemed to be compensated by cellular immune effectors upon bacterial challenge. Our study further shows a novel relevance of SLy2 for plasmablasts and B-cell progenitors in the BM.

Enhanced IgG1 -mediated antibody response towards thymus-dependent immunization in CXCR1-deficient mice.

BACKGROUND: Chemokine receptors and their corresponding ligands are key players of immunity by regulation of immune cell differentiation and migration. CXCR1 is a high-affinity receptor for CXCL8. Differential expression of CXCR1 is associated with a variety of human pathologies including cancer and inflammatory diseases. While various studies have highlighted the importance of CXCR1-mediated CXCL8-sensing for neutrophil trafficking and function, its role in B-cell responses remains unsolved. Therefore, our aim was to investigate innate and adaptive antibody responses in CXCR1-deficient mice. METHODS: Cell populations of the spleen and the peritoneal cavity were identified and quantified via flow cytometry. To investigate thymus-independent (TI) and thymus-dependent (TD) antibody responses, mice were immunized intraperitoneally with TNP-Ficoll, Pneumovax23, and TNP-Chicken Gamma Globulin. Mice were bled before as well as 7 and 14 days after vaccination to collect serum. Serum antibody levels overtime were analyzed according to their specificity by enzyme-linked immunosorbent assay. B-1 cell functionality was examined by IL-5/IL-5Rα-dependent stimulation of peritoneal and splenic cells in vitro. To analyze CXCR1/2-expression, CD19+ splenocytes were enriched by magnetic-activated cell sorting before isolation of total RNA contents, followed by reverse transcription and real-time polymerase chain reaction. RESULTS: The distribution of natural B-1 cell populations was disturbed in the absence of CXCR1, while their responsiveness towards TI antigens and in vitro stimulation remained functional. Besides, CXCR1-deficiency was accompanied by increased frequencies of follicular B-2 cells in the spleen. Interestingly, these mice produced elevated levels of antigen-specific IgG1 upon TD immunization and harbored a significantly enlarged proportion of CXCR5-expressing T helper (H) cells. CXCR1-expression was detectable in CD19+ splenocytes derived from wild-type, but not CXCR1-deficient mice. CONCLUSION: Our data demonstrate a previously unknown relevance of CXCR1 for the production of specific IgG1 in response to vaccination. These findings identify CXCR1 as a promising candidate for future studies on the regulation of adaptive antibody responses.

SLy2-deficiency promotes B-1 cell immunity and triggers enhanced production of IgM and IgG2 antibodies against pneumococcal vaccine.

BACKGROUND: Despite the benefits of existing vaccines, Streptococcus pneumoniae is still responsible for the greatest proportion of respiratory tract infections around the globe, thereby substantially contributing to morbidity and mortality in humans. B-1 cells are key players of bacterial clearance during pneumococcal infection and even provide long-lasting immunity towards S. pneumoniae. Previous reports strongly suggest an essential role of the immunoinhibitory adapter Src homology domain 3 lymphocyte protein 2 (SLy2) for B-1 cell-mediated antibody production. The objective of this study is to evaluate S. pneumoniae-directed B cell responses in the context of SLy2 deficiency. METHODS: B-1 cell populations were analyzed via flow cytometry before and after pneumococcal immunization of SLy2-deficient and wild-type control mice. Global and vaccine-specific immunoglobulin M (IgM) and IgG antibody titers were assessed by enzyme-linked immunosorbent assay. To investigate survival rates during acute pneumococcal lung infection, mice were intranasally challenged with S. pneumoniae (serotype 3). Complementary isolated splenic B cells were stimulated in vitro and their proliferative response was assessed by fluorescent staining. In vitro antibody secretion was quantified by LEGENDplex. RESULTS: We demonstrate increased frequencies of B-1 cells and elevated titers of preantigenic IgM in SLy2-deficient mice. In addition, these mice produce significantly more amounts of IgM and IgG2 upon pneumococcal vaccination. Knocking out SLy2 did not induce survival advantages in our murine model of acute pneumonia, indicating the presence of compensatory mechanisms. CONCLUSION: Our results reveal reinforced specific antibody responses towards pneumococcal polysaccharides and enhanced IgG2 secretion as a consequence of SLy2 deficiency, which could be relevant to the development of more efficient vaccines.

Human monocytic myeloid-derived suppressor cells impair B-cell phenotype and function in vitro.

Myeloid-derived suppressor cells (MDSCs) are key regulators of immunity that initially have been defined by their ability to potently suppress T-cell responses. Recent studies collectively demonstrate that the suppressive activity of MDSCs is not limited to T cells, but rather affects a broad range of immune cell subsets. However, relatively few studies have assessed the impact of MDSCs on B cells, particularly in the human context. Here, we report that human monocytic MDSCs (M-MDSCs) significantly interfere with human B-cell proliferation and function in vitro. We further show that the inhibition occurs independent of direct cell-contact and involves the expression of suppressive mediators such as indoleamine 2, 3-dioxygenase (IDO), arginase-1 (Arg1), and nitric oxide (NO). In addition, our studies demonstrate that the suppression of B cells by M-MDSCs is paralleled by a skewing in B-cell phenotype and gene expression signatures. M-MDSCs induced the downregulation of key surface markers on activated B cells, including IgM, HLA-DR, CD80, CD86, TACI, and CD95. Concurrently, M-MDSCs but not conventional monocytes elicited alterations in the transcription of genes involved in apoptosis induction, class-switch regulation, and B-cell differentiation and function. In summary, this study expands our understanding of the regulatory role of M-MDSCs for human B-cell responses.

Myeloid-derived suppressor cells modulate B-cell responses.

Myeloid-derived suppressor cells (MDSCs) are key regulators of adaptive immunity by suppressing T-cell functions. However, their potential action on or interaction with B cells remained poorly understood. Here we demonstrate that human polymorphonuclear MDSCs differentially modulate B-cell function by suppressing B-cell proliferation and antibody production. We further demonstrate that this MDSC-mediated effect is cell contact dependent and involves established mediators such as arginase-1, nitric oxide (NO), reactive oxygen species (ROS) as well as B-cell death. Collectively, our studies provide novel evidence that human MDSCs modulate B cells, which could have future implications for immunotherapy approaches.