Regulation of T Cell Receptor Signaling by DENND1B in TH2 Cells and Allergic Disease.

The DENN domain is an evolutionary conserved protein module found in all eukaryotes and serves as an exchange factor for Rab-GTPases to regulate diverse cellular functions. Variants in DENND1B are associated with development of childhood asthma and other immune disorders. To understand how DENND1B may contribute to human disease, Dennd1b(-/-) mice were generated and exhibit hyper-allergic responses following antigen challenge. Dennd1b(-/-) TH2, but not other TH cells, exhibit delayed receptor-induced T cell receptor (TCR) downmodulation, enhanced TCR signaling, and increased production of effector cytokines. As DENND1B interacts with AP-2 and Rab35, TH2 cells deficient in AP-2 or Rab35 also exhibit enhanced TCR-mediated effector functions. Moreover, human TH2 cells carrying asthma-associated DENND1B variants express less DENND1B and phenocopy Dennd1b(-/-) TH2 cells. These results provide a molecular basis for how DENND1B, a previously unrecognized regulator of TCR downmodulation in TH2 cells, contributes to asthma pathogenesis and how DENN-domain-containing proteins may contribute to other human disorders.

Neutrophils influence the level of antigen presentation during the immune response to protein antigens in adjuvants.

Neutrophils modulated Ag presentation following immunization with Ags in CFA or IFA or alum. The neutrophils had an important negative role in the CD4 T cell and B cell responses to three protein Ags: hen egg white lysozyme, OVA, and listeriolysin O. In their absence (by depleting with Abs for only the first 24 h, or using genetically neutropenic mice), the cellular responses increased several-fold. The CD8 response was not affected or slightly decreased. Competition for Ag between the presenting cells and the neutrophils, as well as an effect on the response to Ag-bearing dendritic cells (DCs), was documented. Neutrophils entered the draining lymph nodes rapidly and for a brief period of several hours, localizing mainly to the marginal sinus and superficial cortex. There they established brief contact with DCs and macrophages. Moreover, neutrophils imprinted on the quality of the subsequent DC-T cell interactions, despite no physical contact with them; by intravital microscopy, the clustering of Ag-specific T cells and DCs was improved in neutropenic mice. Thus, neutrophils are obligate cells that briefly enter sites of immunization and set the level of Ag presentation. A brief depletion may have a considerably positive impact on vaccination.

Beneficial effects of treatment with transglutaminase inhibitor cystamine on macrophage response in NZB/W F1 mice.

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disorder of unknown etiology. However, the definitive mechanisms remain obscure. Recently, transglutaminase 2 (TG2) was implicated in the pathogenesis of SLE. Cystamine, which inactivates TG2 activity by forming a mixed disulfide, may interfere with and inhibit other thiol-dependent enzymes such as caspases. To investigate the effects of cystamine in SLE pathogenesis, this in vivo study assessed the serum and macrophage response after administration of cystamine to NZB/W F(1) mice. The experimental results demonstrated for the first time a significant reduction in TG2 and matrix metalloproteinase (MMP)-9 activity; tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, TG2, tumor necrosis factor alpha, and tumor growth factor beta mRNA expression; and anticardiolipin autoantibodies (aCL) in NZB/W F(1) mice following cystamine administration. It strongly suggests the therapeutic potential of cystamine in SLE.

Neutrophils control the magnitude and spread of the immune response in a thromboxane A2-mediated process.

Neutrophils are obligate cells entering lymph nodes shortly after immunization with protein antigens in adjuvants, starting during the first hour and continuing for several days in two distinct waves. Previously, we demonstrated the strong suppressive effects of neutrophils on CD4 T cell and B cell responses, using either neutrophil-depleting antibodies or genetically neutropenic mice. In this study, we find that neutrophils are the major cells controlling the spread of T cell responses to distal lymph nodes. Although in the presence of neutrophils, ∼75% of the response was restricted to the draining node, in their absence, most of the response was found in distal nodes. Prostanoids were responsible for the rapid entry of neutrophils into the draining nodes, as well as for the two distinct neutrophil effects: the modulation of the magnitude of the cellular response, and in its spread outside the draining nodes. Neutrophil-produced thromboxane A(2) was the key eicosanoid controlling both effects. Adoptive transfer of neutrophils into mice genetically deficient in neutrophils indicated their role in both. These functions of neutrophils are important in infections and vaccinations with adjuvants where neutrophils are abundant in the initial stages.

Gamma-herpesvirus kinase actively initiates a DNA damage response by inducing phosphorylation of H2AX to foster viral replication.

DNA virus infection can elicit the DNA damage response in host cells, including ATM kinase activation and H2AX phosphorylation. This is considered to be the host cell response to replicating viral DNA. In contrast, we show that during infection of macrophages murine gamma-herpesvirus 68 (gammaHV68) actively induces H2AX phosphorylation by expressing a viral kinase (orf36). GammaHV68-encoded orf36 kinase and its EBV homolog, BGLF4, induce H2AX phosphorylation independently of other viral genes. The process requires the kinase domain of Orf36 and is enhanced by ATM. Orf36 is important for gammaHV68 replication in infected animals, and orf36, H2AX, and ATM are all critical for efficient gammaHV68 replication in primary macrophages. Thus, activation of proximal components of the DNA damage signaling response is an active viral kinase-driven strategy required for efficient gamma-herpesvirus replication.

Dendritic cells cross-present exogenous fungal antigens to stimulate a protective CD8 T cell response in infection by Histoplasma capsulatum.

The contribution of CD8 T cells in host defense against histoplasmosis is minor in the CD4 T cell-intact mouse, as it has been shown that depleting CD8 T cells only marginally affects fungal clearance. However, it remains to be determined whether the CD8 T cells are protective in a host lacking functional CD4 T cells. In this study, MHC class II-deficient mice infected with Histoplasma capsulatum (Histoplasma) kept the fungus in check for up to 16 wk, indicating that CD8 T cells are able to limit fungal replication. Ex vivo studies showed that CD8 T cells from Histoplasma-infected mice expressed both intracytoplasmic IFN-gamma and granzyme B. Furthermore, CD8 T cells exhibited cytotoxic activity against macrophage targets containing Histoplasma. We demonstrated that the macrophage, being the primary host cell as well as the effector cell, can also serve as Ag donor to dendritic cells. Histoplasma-specific CD8 T cells are stimulated by dendritic cells that present exogenous Histoplasma Ags, either through direct ingestion of yeasts or through uptake of apoptotic macrophage-associated fungal Ags, a process known as "cross-presentation." Based on these results, we present a model detailing the possible sequence of events leading to a cell-mediated immune response and fungal clearance in Histoplasma-infected hosts.