Scott A. Wetzel

In vitro exposure to the herbicide atrazine inhibits T cell activation, proliferation, and cytokine production and significantly increases the frequency of Foxp3+ regulatory T cells.

The herbicide atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) is the most common water contaminant in the United States. Atrazine is a phosphodiesterase inhibitor and is classified as an estrogen disrupting compound because it elevates estrogen levels via induction of the enzyme aromatase. Previous studies have shown that atrazine exposure alters the function of innate immune cells such as NK cells, DC, mast cells, and macrophages. In this study we have examined the impact of in vitro atrazine exposure on the activation, proliferation, and effector cytokine production by primary murine CD4(+) T lymphocytes. We found that atrazine exposure significantly inhibited CD4(+) T cell proliferation and accumulation as well as the expression of the activation markers CD25 and CD69 in a dose-dependent manner. Interestingly, the effects were more pronounced in cells from male animals. These effects were partially mimicked by pharmacological reagents that elevate intracellular cAMP levels and addition of exogenous rmIL-2 further inhibited proliferation and CD25 expression. Consistent with these findings, atrazine exposure during T cell activation resulted in a 2- to 5-fold increase in the frequency of Foxp3(+) CD4(+) T cells.

Live-Cell Dynamics and the Role of Costimulation in Immunological Synapse Formation

Using transfected fibroblasts expressing both wild-type I-Ek and green fluorescent protein-tagged I-Ek with covalently attached antigenic peptide, we have monitored movement of specific MHC:peptide complexes during CD4+ T cell-APC interactions by live-cell video microscopy. Ag recognition occurs within 30 s of T cell-APC contact, as shown by a sharp increase in cytoplasmic calcium ion concentration. Within 1 min, small MHC:peptide clusters form in the contact zone that coalesce into an immunological synapse over 3–20 min. When T cells conjugated to APC move across the APC surface, they appear to drag the synapse with them. This system was used to examine the role of costimulation in the formation of the immunological synapse. Blocking CD80/CD28 or ICAM-1/LFA-1 interactions alters synapse morphology and reduces the area and density of accumulated complexes. These reductions correlate with reduced T cell proliferation, while CD69 and CD25 expression and TCR down-modulation remain unaffected. Thus, costimulation is essential for normal mature immunological synapse formation.

Peptide-Specific Intercellular Transfer of MHC Class II to CD4+ T Cells Directly from the Immunological Synapse upon Cellular Dissociation

The transfer of membrane proteins from APC to T cells was initially described in the 1970s, and subsequent work has described two mechanisms of transfer: APC-derived exosomes and direct transfer of small packets, while cells remain conjugated. Using fibroblast APC expressing a GFP-tagged I-Ek molecule with covalently attached antigenic peptide, we observed a third mechanism in live cell imaging: T cells spontaneously dissociating from APC often capture MHC:peptide complexes directly from the immunological synapse. Using two I-Ek-restricted murine TCR transgenic T cells with different peptide specificity, we show in this study that the MHC transfer is peptide specific. Using blocking Abs, we found that MHC:peptide transfer in this system requires direct TCR-MHC:peptide interactions and is augmented by costimulation through CD28-CD80 interactions. Capture of the GFP-tagged MHC:peptide complexes correlates with an activated phenotype of the T cell, elevated CD69 with down-modulated TCR. The transferred MHC:peptide molecules transferred to the T cell are associated with molecules that imply continued TCR signaling; p56lck, phosphotyrosine, and polarization of the actin cytoskeleton.

Th1 and Th2 cells form morphologically distinct immunological synapses.

The arrangement of molecules at the interface between T cells and APCs is known as the immunological synapse (IS). We conducted experiments with supported planar bilayers and transfected fibroblast APC to examine the IS formed by polarized Th1 and Th2 cells. Th1 cells formed typical “bull’s-eye” IS with a ring of adhesion molecules surrounding MHC/TCR interactions at all Ag concentrations tested, while Th2 cells formed multifocal IS at high concentrations of Ag. At low Ag concentrations, the majority of Th2 cells formed IS with a compact, central accumulation of MHC/TCR, but ICAM-1 was not excluded from the center of the IS. Additionally, CD45 was excluded from the center of the interface between Th1 cells and APC, while CD45 was found at the center of the multifocal IS formed by Th2 cells. Finally, phosphorylated signaling molecules colocalized with MHC/TCR to a greater extent in Th2 IS. Together, our results indicate that the IS formed by Th1 and Th2 cells are distinct in structure, with Th2 cells failing to form bull’s-eye IS.

Regulation of the Small GTPase Rap1 and Extracellular Signal-Regulated Kinases by the Costimulatory Molecule CTLA-4

ABSTRACT The mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) is activated following engagement of the T-cell receptor and is required for interleukin 2 (IL-2) production and T-cell proliferation. This activation is enhanced by stimulation of the coreceptor CD28 and inhibited by the coreceptor CTLA-4. We show that the small G protein Rap1 is regulated in the opposite manner; it is inhibited by CD28 and activated by CTLA-4. Together, CD3 and CTLA-4 activate Rap1 in a sustained manner. To delineate T-cell function in the absence of Rap1 activity, we generated transgenic mice expressing Rap1GAP1, a Rap1-specific GTPase-activating protein. Transgenic mice showed lymphadenopathy, and transgenic T cells displayed increased ERK activation, proliferation, and IL-2 production. More significantly, the inhibitory effect of CTLA-4 on T-cell function in Rap1GAP1-transgenic T cells was reduced. We demonstrate that CTLA-4 activates Rap1, and we propose that intracellular signals from CTLA-4 antagonize CD28, at least in part, at the level of Rap1.

Trogocytosis Results in Sustained Intracellular Signaling in CD4+ T Cells

CD4+ T cells capture membrane and membrane-bound molecules from APCs directly from the immunological synapse in a process termed trogocytosis. The function and biological consequences of trogocytosis are largely unknown. In this study, we examine the biological significance of this phenomenon on the trogocytosis-positive T cell. We used murine fibroblasts expressing GFP-tagged I-Ek molecules loaded with a covalently attached antigenic peptide (moth cytochrome c 88–103) to present Ag to primary TCR transgenic T cells. Using a combination of high-resolution light microscopy and flow cytometry, we show that the trogocytosed molecules are retained on the surface of the T cell in association with the TCR and elevated phosphorylated ZAP-70, phosphorylated tyrosine, and phosphorylated ERK 1/2. Through the use of the Src inhibitor PP2, we demonstrate that trogocytosed molecules directly sustain TCR signaling. In addition, after removal of APC, trogocytosis-positive cells preferentially survive in culture over several days. These novel findings suggest that trogocytosed molecules continue to engage their receptors on the T cell surface and sustain intracellular signaling leading to selective survival of these cells.

Antimicrobial activity of AmBisome and non-liposomal amphotericin B following uptake of Candida glabrata by murine epidermal Langerhans cells

The antifungal efficacy and cellular toxicity of AmBisome(R) and non-liposomal amphotericin B were compared in cultured epidermal Langerhans cells infected with Candida glabrata. Uptake of the yeast was determined by light and electron microscopy, and viability was assessed by plating dilutions of lysates from yeast-infected Langerhans cells and counting colony forming units. The Candida-infected Langerhans cells were incubated for 6, 24 or 48 h with 12.5 micro ml-1 of AmBisome or non-liposomal amphotericin B, non-drug-containing liposomes or media. Intracellular C. glabrata incubated with media or non-drug-containing liposomes showed a 2 log increase in cfu, and microscopic examination revealed budding yeast within the Langerhans cells. Both liposomal and non-liposomal amphotericin B treatment reduced intracellular growth of C. glabrata by 5 logs over 48 h of incubation. A morphometric analysis of cell ultrastructure demonstrated that AmBisome-treated Langerhans cells retained their cell architecture, but Langerhans cells treated with non-liposomal amphotericin B were characterized by the absence of intact organelles, disrupted non-granular cytoplasm and the presence of many large vacuoles. In conclusion, AmBisome was significantly less toxic for epidermal Langerhans cells than amphotericin B, but demonstrated comparable antifungal efficacy. After 48 h of drug exposure, both forms of amphotericin B effectively inhibited intracellular growth of C. glabrata, but only AmBisome did not damage the Langerhans cells.

Autoantibodies from mice exposed to Libby amphibole asbestos bind SSA/Ro52-enriched apoptotic blebs of murine macrophages.

Asbestos exposure is associated with increased autoimmune responses in humans. For example, in Libby, MT where significant asbestos exposure has occurred due to an asbestos-contaminated vermiculite mine near the community, residents have developed increased autoimmune responses compared to an unexposed population. However, the exact mechanism by which Libby amphibole asbestos generates autoimmune responses is unclear. A murine model of amphibole asbestos-induced autoimmunity was recently established, and one of the targets of the autoantibodies (AAs) was the SSA/Ro52 autoantigen. The purpose of this study was to determine whether the SSA/Ro52 autoantigen is exposed at the surface of cells as a result of asbestos exposure as a possible mechanism leading to antigenicity. Our results indicate that Libby asbestos induces apoptosis in murine macrophages as determined by phosphatidylserine exposure, cleavage of poly(ADP-ribose) polymerase and morphological changes such as nuclear condensation. Moreover, asbestos-induced apoptosis results in the formation of apoptotic cell surface blebs enriched in SSA/Ro52 as determined by confocal microscopy. Most importantly, apoptotic cell surface blebs are recognized by AAs from mice exposed to amphibole asbestos suggesting that these cell surface structures may be antigenic when presented in a pro-inflammatory context. This study supports the hypothesis that the induction of apoptosis plays a key role in environmentally induced autoimmunity through cell surface exposure of a known autoantigen.

Anergic CD4+ T cells form mature immunological synapses with enhanced accumulation of c-Cbl and Cbl-b

CD4+ T cell recognition of MHC:peptide complexes in the context of a costimulatory signal results in the large-scale redistribution of molecules at the T cell–APC interface to form the immunological synapse. The immunological synapse is the location of sustained TCR signaling and delivery of a subset of effector functions. T cells activated in the absence of costimulation are rendered anergic and are hyporesponsive when presented with Ag in the presence of optimal costimulation. Several previous studies have looked at aspects of immunological synapses formed by anergic T cells, but it remains unclear whether there are differences in the formation or composition of anergic immunological synapses. In this study, we anergized primary murine CD4+ T cells by incubation of costimulation-deficient, transfected fibroblast APCs. Using a combination of TCR, MHC:peptide, and ICAM-1 staining, we found that anergic T cells make mature immunological synapses with characteristic central and peripheral supramolecular activation cluster domains that were indistinguishable from control synapses. There were small increases in total phosphotyrosine at the anergic synapse along with significant decreases in phosphorylated ERK 1/2 accumulation. Most striking, there was specific accumulation of c-Cbl and Cbl-b to the anergic synapses. Cbl-b, previously shown to be essential in anergy induction, was found in both the central and the peripheral supramolecular activation clusters of the anergic synapse. This Cbl-b (and c-Cbl) accumulation at the anergic synapse may play an important role in anergy maintenance, induction, or both.

CD4 + T Cell Differentiation and Activation

The activation and differentiation of CD4+ T cells play a critical role in establishing and subsequently controlling protective adaptive immune responses. Flow cytometry is a powerful technique with which to assess the potential of xenobiotics to influence CD4+ T cell activation and differentiation. With flow cytometry, cells are stained with fluorochrome-conjugated antibodies and/or specific fluorescent probes to assess T cell activation, proliferation, effector cytokine production, and transcription factor expression. This technique allows for complex phenotypic analysis of tens to hundreds of thousands of individual cells very rapidly to assess the potential impact of a xenobiotic on CD4 effector differentiation and activation state.