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Abstract 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.

Abstract 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.

Abstract 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.

Oncogenic signalling via Ca2+‐activated K+ channels of intermediate conductance (SK4, also known as KCa3.1 or IK) has been implicated in different cancer entities including breast cancer. Yet, the role of endogenous SK4 channels for tumorigenesis is unclear. Herein, we generated SK4‐negative tumours by crossing SK4‐deficient (SK4 KO) mice to the polyoma middle T‐antigen (PyMT) and epidermal growth factor receptor 2 (cNeu) breast cancer models in which oncogene expression is driven by the retroviral promoter MMTV. Survival parameters and tumour progression were studied in cancer‐prone SK4 KO in comparison with wild‐type (WT) mice and in a syngeneic orthotopic mouse model following transplantation of SK4‐negative or WT tumour cells. SK4 activity was modulated by genetic or pharmacological means using the SK4 inhibitor TRAM‐34 in order to establish the role of breast tumour SK4 for cell growth, electrophysiological signalling, and [Ca2+]i oscillations. Ablation of SK4 and TRAM‐34 treatment reduced the SK4‐generated current fraction, growth factor‐dependent Ca2+ entry, cell cycle progression and the proliferation rate of MMTV‐PyMT tumour cells. In vivo, PyMT oncogene‐driven tumorigenesis was only marginally affected by the global lack of SK4, whereas tumour progression was significantly delayed after orthotopic implantation of MMTV‐PyMT SK4 KO breast tumour cells. However, overall survival and progression‐free survival time in the MMTV‐cNeu mouse model were significantly extended in the absence of SK4. Collectively, our data from murine breast cancer models indicate that SK4 activity is crucial for cell cycle control. Thus, the modulation of this channel should be further investigated towards a potential improvement of existing antitumour strategies in human breast cancer.