Catchup: a mouse model for imaging-based tracking and modulation of neutrophil granulocytes.

Neutrophil granulocyte biology is a central issue of immunological research, but the lack of animal models that allow for neutrophil-selective genetic manipulation has delayed progress. By modulating the neutrophil-specific locus Ly6G with a knock-in allele expressing Cre recombinase and the fluorescent protein tdTomato, we generated a mouse model termed Catchup that exhibits strong neutrophil specificity. Transgene activity was found only in very few eosinophils and basophils and was undetectable in bone marrow precursors, including granulomonocytic progenitors (GMPs). Cre-mediated reporter-gene activation allowed for intravital two-photon microscopy of neutrophils without adoptive transfer. Homozygous animals were Ly6G deficient but showed normal leukocyte cellularity in all measured organs. Ly6G-deficient neutrophils were functionally normal in vitro and in multiple models of sterile or infectious inflammation in vivo. However, Cre-mediated deletion of FcγRIV in neutrophils reduced the cells' recruitment to immune-complex-mediated peritonitis, suggesting a cell-intrinsic role for activating Fc receptors in neutrophil trafficking.

A mutation within the SH2 domain of slp-76 regulates the tissue distribution and cytokine production of iNKT cells in mice.

TCR ligation is critical for the selection, activation, and integrin expression of T lymphocytes. Here, we explored the role of the TCR adaptor protein slp-76 on iNKT-cell biology. Compared to B6 controls, slp-76(ace/ace) mice carrying a missense mutation (Thr428Ile) within the SH2-domain of slp-76 showed an increase in iNKT cells in the thymus and lymph nodes, but a decrease in iNKT cells in spleens and livers, along with reduced ADAP expression and cytokine response. A comparable reduction in iNKT cells was observed in the livers and spleens of ADAP-deficient mice. Like ADAP(-/-) iNKT cells, slp-76(ace/ace) iNKT cells were characterized by enhanced CD11b expression, correlating with an impaired induction of the TCR immediate-early gene Nur77 and a decreased adhesion to ICAM-1. Furthermore, CD11b-intrinsic effects inhibited cytokine release, concanavalin A-mediated inflammation, and iNKT-cell accumulation in the liver. Unlike B6 and ADAP(-/-) mice, the expression of the transcription factors Id3 and PLZF was reduced, whereas NP-1-expression was enhanced in slp-76(ace/ace) mice. Blockade of NP-1 decreased the recovery of iNKT cells from peripheral lymph nodes, identifying NP-1 as an iNKT-cell-specific adhesion factor. Thus, slp-76 contributes to the regulation of the tissue distribution, PLZF, and cytokine expression of iNKT cells via ADAP-dependent and -independent mechanisms.

The adhesion- and degranulation-promoting adaptor protein and its role in the modulation of experimental autoimmune encephalomyelitis.

Adaptor proteins mediate protein-protein interactions in signal transduction cascades. These signaling molecules are organized in multimolecular complexes that translate information from cell surface receptors into cellular responses. The cytosolic adhesion- and degranulation-promoting adaptor protein (ADAP) is expressed in T cells, natural killer cells, myeloid cells, and platelets. Here we summarize the data about the function of ADAP in these cells with respect to their contribution to the pathogenesis of experimental autoimmune encephalomyelitis. We discuss possible mechanisms of strongly attenuated experimental autoimmune encephalomyelitis in ADAP-deficient mice.

T cell-independent modulation of experimental autoimmune encephalomyelitis in ADAP-deficient mice.

The adhesion- and degranulation-promoting adaptor protein (ADAP), expressed in T cells, myeloid cells, and platelets, is known to regulate receptor-mediated inside-out signaling leading to integrin activation and adhesion. In this study, we demonstrate that, upon induction of active experimental autoimmune encephalomyelitis (EAE) by immunization with the myelin oligodendrocyte glycoprotein35-55 peptide, ADAP-deficient mice developed a significantly milder clinical course of EAE and showed markedly less inflammatory infiltrates in the CNS than wild-type mice. Moreover, ADAP-deficient recipients failed to induce EAE after adoptive transfer of myelin oligodendrocyte glycoprotein-specific TCR-transgenic T cells (2D2 T cells). In addition, ex vivo fully activated 2D2 T cells induced significantly less severe EAE in ADAP-deficient recipients. The ameliorated disease in the absence of ADAP was not due to expansion or deletion of a particular T cell subset but rather because of a strong reduction of all inflammatory leukocyte populations invading the CNS. Monitoring the adoptively transferred 2D2 T cells over time demonstrated that they accumulated within the lymph nodes of ADAP-deficient hosts. Importantly, transfer of complete wild-type bone marrow or even bone marrow of 2D2 TCR-transgenic mice was unable to reconstitute EAE in the ADAP-deficient animals, indicating that the milder EAE was dependent on (a) radio-resistant nonhematopoietic cell population(s). Two-photon microscopy of lymph node explants revealed that adoptively transferred lymphocytes accumulated at lymphatic vessels in the lymph nodes of ADAP-deficient mice. Thus, our data identify a T cell-independent mechanism of EAE modulation in ADAP-deficient mice.

The adapter protein ADAP is required for selected dendritic cell functions.

BACKGROUND: The cytosolic adaptor protein ADAP (adhesion and degranulation promoting adapter protein) is expressed by T cells, natural killer cells, myeloid cells and platelets. ADAP is involved in T-cell-receptor-mediated inside-out signaling, which leads to integrin activation, adhesion and reorganization of the actin cytoskeleton. However, little is known about the role of ADAP in myeloid cells. In the present study, we analyzed the function of ADAP in bone-marrow-derived dendritic cells (BMDCs) from ADAP-deficient mice. RESULTS: ADAP-deficient BMDCs showed almost normal levels of antigen uptake, adhesion, maturation, migration from the periphery to the draining lymph nodes, antigen-specific T-cell activation, and production of the proinflammatory cytokines IL-6 and TNF-∝. Furthermore, we provide evidence that the activation of signaling pathways after lipopolysaccharide (LPS) stimulation are not affected by the loss of ADAP. In contrast, ADAP-deficient BMDCs showed defects in CD11c-mediated cellular responses, with significantly diminished production of IL-6, TNF-∝ and IL-10. Actin polymerization was enhanced after CD11c integrin stimulation. CONCLUSIONS: In summary, we propose that the adapter molecule ADAP is critical for selected CD11c integrin-mediated functions of dendritic cells.

Protein kinase B/Akt signals impair Th17 differentiation and support natural regulatory T cell function and induced regulatory T cell formation.

Protein kinase B (PKB)/Akt signals control T cell proliferation and differentiation but their effect on the generation and function of regulatory T cells (Treg) and Th17 cells is not well understood. In this study, we show that elevated PKB signals antagonize the immunosuppressive effect of TGF-beta1 on cell size, CD25 and CD98 expression, and proliferation of CD3-stimulated naive CD4(+) T cells from wild-type and CD28-deficient mice. Conventional CD4(+) T cells expressing active PKB are less susceptible to suppression by natural regulatory T cells. Although PKB signals do not affect the development of natural regulatory T cells, they enhance their suppressor capacity. Upon TCR triggering and TGF-beta1 costimulation, wild-type and CD28-deficient CD4(+) T cells transgenic for PKB readily express Foxp3, thereby acquiring suppressor capacity. These effects of elevated PKB signals on T cell function involve a marked and sustained activation of STAT5 and Foxp3 and reduction in nuclear NFATc1 levels. In contrast, PKB signals impair TGF-beta1/IL-6-mediated differentiation of naive CD4(+) T cells into the Th17 lineage. This correlates with an increased signaling of ERK, STAT5, and STAT6. Finally, elevated PKB signals reduced the severity of experimental autoimmune encephalomyelitis in wild-type mice but induced experimental autoimmune encephalomyelitis in mice deficient for CD28. Altogether, these data indicate an important role of PKB signals on control of TGF-beta1-mediated T cell responses and, thereby, on tolerizing and inflammatory immune processes.

NFATc1 affects mouse splenic B cell function by controlling the calcineurin--NFAT signaling network.

By studying mice in which the Nfatc1 gene was inactivated in bone marrow, spleen, or germinal center B cells, we show that NFATc1 supports the proliferation and suppresses the activation-induced cell death of splenic B cells upon B cell receptor (BCR) stimulation. BCR triggering leads to expression of NFATc1/αA, a short isoform of NFATc1, in splenic B cells. NFATc1 ablation impaired Ig class switch to IgG3 induced by T cell-independent type II antigens, as well as IgG3(+) plasmablast formation. Mice bearing NFATc1(-/-) B cells harbor twofold more interleukin 10-producing B cells. NFATc1(-/-) B cells suppress the synthesis of interferon-γ by T cells in vitro, and these mice exhibit a mild clinical course of experimental autoimmune encephalomyelitis. In large part, the defective functions of NFATc1(-/-) B cells are caused by decreased BCR-induced Ca(2+) flux and calcineurin (Cn) activation. By affecting CD22, Rcan1, CnA, and NFATc1/αA expression, NFATc1 controls the Ca(2+)-dependent Cn-NFAT signaling network and, thereby, the fate of splenic B cells upon BCR stimulation.

PKB rescues calcineurin/NFAT-induced arrest of Rag expression and pre-T cell differentiation.

Protein kinase B (PKB), an Ag receptor activated serine-threonine kinase, controls various cellular processes including proliferation and survival. However, PKB function in thymocyte development is still unclear. We report PKB as an important negative regulator of the calcineurin (CN)-regulated transcription factor NFAT in early T cell differentiation. Expression of a hyperactive version of CN induces a profound block at the CD25+CD44- double-negative (DN) 3 stage of T cell development. We correlate this arrest with up-regulation of Bcl-2, CD2, CD5, and CD27 proteins and constitutive activation of NFAT but a severe impairment of Rag1, Rag2, and intracellular TCR-beta as well as intracellular TCR-gammadelta protein expression. Intriguingly, simultaneous expression of active myristoylated PKB inhibits nuclear NFAT activity, restores Rag activity, and enables DN3 cells to undergo normal differentiation and expansion. A correlation between the loss of NFAT activity and Rag1 and Rag2 expression is also found in myristoylated PKB-induced CD4+ lymphoma cells. Furthermore, ectopic expression of NFAT inhibits Rag2 promoter activity in EL4 cells, and in vivo binding of NFATc1 to the Rag1 and Rag2 promoter and cis-acting transcription regulatory elements is verified by chromatin immunoprecipitation analysis. The regulation of CN/NFAT signaling by PKB may thus control receptor regulated changes in Rag expression and constitute a signaling pathway important for differentiation processes in the thymus and periphery.