Citations

Abstract HIV-1 infection causes the depletion of host CD4 T cells through direct and indirect (bystander) mechanisms. Although HIV Env has been implicated in apoptosis of uninfected CD4 T cells via gp120 binding to either CD4 and/or the chemokine receptor 4 (CXCR4), conflicting data exist concerning the molecular mechanisms involved. Using primary human CD4 T cells, we demonstrate that gp120 binding to CD4 T cells activates proapoptotic p38, but does not activate antiapoptotic Akt. Because ligation of the CD4 receptor alone or the CXCR4 receptor alone causes p38 activation and apoptosis, we used the soluble inhibitors, soluble CD4 (sCD4) or AMD3100, to delineate the role of CD4 and CXCR4 receptors, respectively, in gp120-induced p38 activation and death. sCD4 alone augments gp120-induced death, suggesting that CXCR4 signaling is principally responsible. Supporting that model, AMD3100 reduces death caused by gp120 or by gp120/sCD4. Finally, prevention of gp120-CXCR4 interaction with 12G5 Abs blocks p38 activation and apoptosis, whereas inhibition of CD4-gp120 interaction with Leu-3a has no effect. Consequently, we conclude that gp120 interaction with CXCR4 is required for gp120 apoptotic effects in primary human T cells.

Abstract Studies in cancer patients have suggested that breast tumors recruit regulatory T cells (Tregs) into the tumor microenvironment. The extent to which local Tregs suppress antitumor immunity in breast cancer is unknown. We questioned whether inhibiting systemic Tregs with an IL-2 immunotoxin in a model of neu-mediated breast cancer, the neu-transgenic mouse, could impact disease progression and survival. As in human breast cancer, cancers that develop in these mice attract Tregs into the tumor microenvironment to levels of ∼10–25% of the total CD4+ T cells. To examine the role of Tregs in blocking immune-mediated rejection of tumor, we depleted CD4+CD25+ T cells with an IL-2 immunotoxin. The treatment depleted Tregs without concomitant lymphopenia and markedly inhibited tumor growth. Depletion of Tregs resulted in a persistent antitumor response that was maintained over a month after the last treatment. The clinical response was immune-mediated because adoptive transfer of Tregs led to a complete abrogation of the therapeutic effects of immunotoxin treatment. Further, Treg down-modulation was accompanied by increased Ag-specific immunity against the neu protein, a self Ag. These results suggest that Tregs play a major role in preventing an effective endogenous immune response against breast cancer and that depletion of Tregs, without any additional immunotherapy, may mediate a significant antitumor response.

Abstract Respiratory failure during Pneumocystis pneumonia is mainly a consequence of exaggerated inflammatory responses to the organism. Dendritic cells (DCs) are the most potent APCs in the lung and are key to the regulation of innate and adaptive immune responses. However, their participation in the inflammatory response directed against Pneumocystis infection has not been fully elucidated. Therefore, we studied the role of Pneumocystis carinii, as well as Saccharomyces cerevisiae, cell wall-derived β-glucans, in DC costimulatory molecule expression. We further studied the impact of β-glucans on subsequent T cell activation. Because cytokine secretion by DCs has recently been shown to be regulated by Fas ligand (FasL), its role in β-glucan activation of DCs was also investigated. β-Glucan-induced DC activation occurred in part through dectin-1 receptors. We demonstrated that DC activation by β-glucans elicits T cell activation and polarization into a Th1 patterned response, but with the conspicuous absence of IL-12. These observations differed from LPS-driven T cell polarization, suggesting that β-glucans and LPS signal DC activation through different mechanisms. We additionally determined that IL-1β and TNF-α secretion by β-glucan-stimulated DCs was partially regulated by Fas-FasL. This suggests that dysregulation of FasL could further enhance exuberant and prolonged cytokine production by DCs following DC-T cell interactions, further promoting lung inflammation typical of Pneumocystis pneumonia.

Abstract TCR engagement by peptide-MHC class I (pMHC) ligands induces a conformational change (Δc) in CD3 (CD3Δc) that contributes to T cell signaling. We found that when this interaction took place between primary T lineage cells and APCs, the CD8 coreceptor was required to generate CD3Δc. Interestingly, neither enhancement of Ag binding strength nor Src kinase signaling explained this coreceptor activity. Furthermore, Ag-induced CD3Δc was developmentally attenuated by the increase in sialylation that accompanies T cell maturation and limits CD8 activity. Thus, both weak and strong ligands induced CD3Δc in preselection thymocytes, but only strong ligands were effective in mature T cells. We propose that CD8 participation in the TCR/pMHC interaction can physically regulate CD3Δc induction by “translating” productive Ag encounter from the TCR to the CD3 complex. This suggests one mechanism by which the developmentally regulated variation in CD8 sialylation may contribute to the developmental tuning of T cell sensitivity.

Abstract In activated CD4+ T cells, TCR restimulation triggers apoptosis that depends on interactions between the death receptor Fas and its ligand, FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but what pathways mediate this process are not known. In this study we identify the Rho GTPases Rac1 and Rac2 as essential components in restimulation-induced cell death. RNA interference-mediated knockdown of Rac GTPases greatly reduced Fas-dependent, TCR-induced apoptosis. The ability of Rac1 to sensitize T cells to Fas-induced apoptosis correlated with Rac-mediated cytoskeletal reorganization, dephosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal linker proteins, and the translocation of Fas to lipid raft microdomains. In primary activated CD4+ T cells, Rac1 and Rac2 were independently required for maximal TCR-induced apoptosis. Activating Rac signaling may be a novel way to sensitize chronically stimulated lymphocytes to Fas-induced apoptosis, an important goal in the treatment of autoimmune diseases.

Abstract Human eosinophil granule major basic protein (MBP1) is an exceedingly basic (isoelectric point >11) 14-kDa protein, comprising the core of the secondary eosinophil granule. Recently, a less cationic homolog of MBP, termed MBPH or simply, MBP2, has been discovered. We prepared a panel of mAbs to MBP2 and used these Abs to localize and quantitate this molecule in leukocytes and biological fluids. Specific mAbs for MBP2 were selected using slot-blot analyses and used in a two-site immunoassay, Western blotting, and immunofluorescence microscopy. The sensitivity of the immunoassay was markedly improved by reduction and alkylation of MBP2. MBP1 is more abundant than MBP2 in lysates of eosinophils and their granules, as judged by immunoassay and Western blotting. By immunofluorescence, MBP1 is present in eosinophils, basophils, and a human mast cell line (HMC1), whereas MBP2 is only detected in eosinophils. Neither MBP1 nor MBP2 could be detected in any other peripheral blood leukocyte. MBP2 levels measured in plasma and serum were essentially identical. In contrast to past measurements for MBP1, MBP2 was not detected above normal levels in sera from pregnant donors. However, measurement of serum MBP2 discriminated patients with elevated eosinophils from normal subjects, and MBP2 was also detectable in other biological specimens, such as bronchoalveolar lavage, sputum, and stool. These results indicate that MBP2 is present only in eosinophils and that it may be a useful biomarker for eosinophil-associated diseases.

Abstract T cell activation is characterized by a vast expansion of Ag-specific T cells followed by an equally extensive reduction in T cell numbers. This decline is due, in part, to activation-induced apoptosis of the responding T cells during repeated encounter with Ag. In the current study, we used solid-phase MHC class I/peptide monomers to cause activation-induced cell death (AICD) of previously activated CD8 T cells in an Ag-specific manner. AICD occurred rapidly and was mediated primarily by Fas–FasL interactions. Most interestingly, we observed that Th cells could provide survival signals to CTL significantly reducing the level of AICD. Both Th1 and Th2 subsets were capable of protecting CTL from AICD, and a major role for soluble factors in this protection was ruled out, as cell-to-cell contact was an essential component of this Th-mediated protection. Upon encounter with Ag-expressing tumor cells, CTL underwent significant apoptosis. However, in the presence of Th cells, the CTL not only were protected against death, but also had significantly greater lytic ability. In vivo tumor protection studies using peptide immunization showed that the activation of Ag-specific Th cells was crucial for optimal protection, but did not affect the magnitude of the CTL response in the lymphoid tissues. In this study, we examine the type of help that CD4 T cells may provide and propose a model of Th cell–CTL interaction that reduces CTL death. Our results show a novel role for Th cells in the maintenance of CTL responses.

Abstract Little is known about the regulatory roles of specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in cytotoxic lymphocytes. Recent information suggests that mutations in the SNARE protein syntaxin 11 result in a form of familial hemophagocytic lymphohistiocytosis (FHL). Because genetic abnormalities in key granule components (e.g., perforin) or in regulators of secretion (e.g., Munc13–4) underlie the other identified forms of FHL, we assessed whether syntaxin 11 might also serve a related regulatory role. We determined that syntaxin 11 is expressed in NK cells and activated CTLs and is located in discrete membrane-associated structures in the cytoplasm. Enhanced expression of syntaxin 11 augments the secretion and killing of tumor targets, and suppression of syntaxin 11 expression inhibits these functions. Our data identify and characterize a role for syntaxin 11 in granule exocytosis and in the generation of cell-mediated killing. These results also provide new insights on the mechanisms of hemopoietic dysregulation in FHL.

Abstract Lymphocyte differentiation from naive CD4+ T cells into mature Th1, Th2, Th17, or T regulatory cell (Treg) phenotypes has been considered end stage in character. In this study, we demonstrate that dendritic cells (DCs) activated with a novel immune modulator B7-DC XAb (DCXAb) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DCXAb interaction is Ag-specific, IL-6-dependent, and results in the functional reprogramming of the mature T cell phenotype. The reprogrammed Tregs cease to express IL-10 and TGFβ, fail to suppress T cell responses, and gain the ability to produce IFN-γ, IL-17, and TNF-α. The ability of IL-6+ DCXAb and the inability of IL-6−/− DCXAb vaccines to protect animals from lethal melanoma suggest that exogenously modulated DC can reprogram host Tregs. In support of this hypothesis and as a test for Ag specificity, transfer of DCXAb into RIP-OVA mice causes a break in immune tolerance, inducing diabetes. Conversely, adoptive transfer of reprogrammed Tregs but not similarly treated CD25− T cells into naive RIP-OVA mice is also sufficient to cause autoimmune diabetes. Yet, treatment of normal mice with B7-DC XAb fails to elicit generalized autoimmunity. The finding that mature Tregs can be reprogrammed into competent effector cells provides new insights into the plasticity of T cell lineage, underscores the importance of DC-T cell interaction in balancing immunity with tolerance, points to Tregs as a reservoir of autoimmune effectors, and defines a new approach for breaking tolerance to self Ags as a strategy for cancer immunotherapy.

Abstract MUC1, a membrane tethered mucin glycoprotein, is overexpressed and aberrantly glycosylated in >80% of human ductal pancreatic adenocarcinoma. However, the role of MUC1 in pancreatic cancer has been elusive, partly due to the lack of an appropriate model. We report the characterization of a novel mouse model that expresses human MUC1 as a self molecule (PDA.MUC1 mice). Pancreatic tumors arise in an appropriate MUC1-tolerant background within an immune-competent host. Significant enhancement in the development of pancreatic intraepithelial preneoplastic lesions and progression to adenocarcinoma is observed in PDA.MUC1 mice, possibly due to increased proliferation. Tumors from PDA.MUC1 mice express higher levels of cyclooxygenase-2 and IDO compared with PDA mice lacking MUC1, especially during early stages of tumor development. The increased proinflammatory milieu correlates with an increased percentage of regulatory T cells and myeloid suppressor cells in the pancreatic tumor and tumor draining lymph nodes. Data shows that during pancreatic cancer progression, MUC1-mediated mechanisms enhance the onset and progression of the disease, which in turn regulate the immune responses. Thus, the mouse model is ideally suited for testing novel chemopreventive and therapeutic strategies against pancreatic cancer.