The spleen tyrosine kinase family members Syk and Zap-70 are pivotal signal transducers downstream of antigen receptors and exhibit overlapping expression patterns at early lymphocytic developmental stages. To assess their differential kinase fitness in vivo, we generated mice, which carry a Zap-70 cDNA knock-in controlled by intrinsic Syk promoter elements that disrupts wild-type Syk expression. Kinase replacement severely compromised Erk1/2-mediated survival and proper selection of developing B cells at central and peripheral checkpoints, demonstrating critical dependence on BCR signalling quality. Furthermore, ITAM-and hemITAM-mediated activation of platelets and neutrophils was completely blunted, while surprisingly FccR-mediated phagocytosis in macrophages was retained. The alteration in BCR signalling quality resulted in preferential development and survival of marginal zone B cells and prominent autoreactivity, causing the generation of anti-insulin antibodies and agerelated glomerulonephritis. Development of concomitant fasting glucose intolerance in knock-in mice highlights aberrant B cell selection as a potential risk factor for type 1 diabetes, and suggests altered BCR signalling as a mechanism to cause biased cellular and Ig repertoire selection, ultimately contributing to B cell-mediated autoimmune predisposition.

CD1d-restricted invariant natural killer T cells (iNKT) constitute an important immunoregulatory T-cell subset that can be activated by the synthetic glycolipid a-galactosylceramide (a-GalCer) and play a dominant role in antitumor immunity. Clinical trials with a-GalCer-pulsed monocyte-derived dendritic cells (moDC) have shown anecdotal antitumor activity in advanced cancer. It was reported that phosphoantigen (pAg)-activated Vg9Vd2-T cells can acquire characteristics of professional antigen-presenting cells (APC). Considering the clinical immunotherapeutic applications, Vg9Vd2-T APC can offer important advantages over moDC, potentially constituting an attractive novel APC platform. Here, we demonstrate that Vg9Vd2-T APC can present antigens to iNKT. However, this does not result from de novo synthesis of CD1d by Vg9Vd2-T, but critically depends on trogocytosis of CD1d-containing membrane fragments from pAg-expressing cells. CD1d-expressing Vg9Vd2-T cells were able to activate iNKT in a CD1d-restricted and a-GalCer-dependent fashion. Although a-GalCerloaded moDC outperformed Vg9Vd2-T APC on a per cell basis, Vg9Vd2-T APC possess unique features with respect to clinical immunotherapeutic application that make them an interesting platform for consideration in future clinical trials. Cancer Immunol Res; 2(8); 732-40. Ó2014 AACR.

Keywords: BCR r GC r Interclonal competition r Syk r Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe investigation of activation processes triggered by the B-cell antigen receptor has kept the scientific community busy for many years, in particular, understanding the spatial BCR organization Correspondence: Dr. Friedemann Kiefer e-mail: fkiefer@gwdg.de and the orchestration of kinase/phosphatase activity on resting versus activated B-cell membranes. Recent evidence argues that classical multimerization models for activation, postulating n ࣙ 2 BCRs to be cross-linked by antigen, have certain caveats and that receptor preoligomerization as well as protein islands might not only be a feature of T-cell surfaces, but also apply to B cells [1][2][3]. Besides this incomplete picture of BCR organization and autoinhibition, the importance of Syk as the central kinase conferring signaling potential to the B-cell antigen receptor is C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 604Sebastian Königsberger et al. Eur. J. Immunol. 2015. 45: 603-611 well established. Being inducibly recruited to phosphorylated Igα/β ITAMs that serve as allosteric kinase activatory sites [4], Syk phosphorylates a still growing list of signaling substrates, including SPL-65, PLCγ2, Btk, and Vav1. Thereby, Syk is central for the direct translation of receptor-mediated antigenic recognition into cellular responses such as Ca 2+ mobilization from the ER, actin remodeling, and initiation of de novo gene transcription [4][5][6]. The quality of the BCR signal is essential at every selection step during B-cell development, no matter if the signal is acute or tonic in nature [7]. Of note, the integrated signaling output during B-cell activation is a complex function of costimulatory activity and antigen receptor affinity/avidity, of which the latter is the result of RAG-mediated central recombination as well as AID-driven hypermutation in germinal centers (GCs) [8][9][10].We recently described a mouse model of constitutive Syk family kinase switch, in which we inserted a Zap-70 cDNA after the initiating ATG codon (exon 2) of the Syk gene, thereby disrupting endogenous Syk expression and driving Zap-70 from the endogenous Syk promoter (Syk ) [11]. In brief, the analysis of the B-cell compartment showed that developing Syk Zap-70/Zap-70 B cells less efficiently traverse the pre-BCR checkpoint, exhibit a bias toward marginal zone B-cell fate, and decrease BCR signaling potential due to inferior kinase activity. Of note, constitutive loss of Syk was shown to lead to a complete arrest of B-cell development at the immature stage [12,13]. Therefore, we were curious about what impact inducibly lowering Syk kinase levels, modeled here by monoallelic loxP site-flanked Syk in the presence of a knock-in Zap-70 allele that served as a hypoactive rescue allele in case of Syk deletion, would have on the propensity of B cells to participate in GC formation. To t...

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