James A. Listman

Fas Modulation of Apoptosis during Negative Selection of Thymocytes

A major mechanism maintaining immune tolerance is the deletion of potentially autoreactive thymocytes by apoptosis during development in the thymus. Previous reports suggest that apoptosis is induced by high avidity signals transduced via the T cell receptor; however, the role of signals transduced by other cell surface receptors during thymic selection remains poorly understood. Fas, a member of the TNF receptor family, has been shown to induce apoptosis in mature peripheral T cells; however, the effects of Fas on negative selection of thymocytes have not been previously detected. Using a sensitive terminal deoxynucleotidyl transferase method to detect apoptotic cells, we found that mutant Fas molecules in lpr mice decrease the sensitivity of thymocytes to T cell receptor-mediated apoptosis and that blockade of Fas-Fas ligand interactions in vivo can inhibit antigen-induced apoptosis of thymocytes in non-lpr mice. Thus, we have shown that Fas, in conjunction with antigen-specific signals, can modulate apoptosis during negative selection of thymocytes.

Inhibition of T Cell Costimulation Abrogates Airway Hyperresponsiveness in a Murine Model

Activation of naive T cells requires at least two signals. In addition to the well characterized interaction of the T cell antigen receptor with the antigen/MHC expressed on an antigen-presenting cell, T cell activation also requires costimulation by a second set of signals. The best characterized costimulatory receptor is CD28, which binds to a family of B7 ligands expressed on antigen-presenting cells. In asthma, although activated T cells play a role in the initiation and maintenance of airway inflammation, the importance of T cell costimulation in bronchial hyperresponsiveness had not been characterized. Therefore, we tested the hypothesis that inhibition of the CD28:B7 costimulatory pathway would abrogate airway hyperresponsiveness. Our results show that blockade of costimulation with CTLA4-Ig, a fusion protein known to prevent costimulation by blocking CD28:B7 interactions, inhibits airway hyperresponsiveness, inflammatory infiltration, expansion of thoracic lymphocytes, and allergen-specific responsiveness of thoracic T cells in this murine model of allergic asthma.

Analysis of T-cell activation after bronchial allergen challenge in patients with atopic asthma.

BACKGROUND T helper cells are a heterogeneous group of cells that have phenotypic and functional differences. Activated T helper cells have been found in peripheral blood after allergen challenge of subjects with atopic asthma, but the phenotypes of specific T helper subpopulation involved remains to be identified. OBJECTIVE To characterize the T cell activation markers that may be regulated by allergens, we analyzed peripheral blood lymphocytes obtained before and after allergen challenge from subjects with atopic asthma. METHODS We analyzed the distribution of the cell surface activation markers, interleukin 2 receptor (IL-2R) and major histocompatibility complex class II antigens (MHC II) among T helper subpopulations classified as naive (CD45RA) or memory (CD45RO) phenotypes. Nine adult subjects with atopic asthma underwent bronchoprovacative allergen inhalation and isocapnic cold air hyperventilation (ISH) challenge followed by serial spirometry. Peripheral blood mononuclear cells (PBMC) were isolated at baseline and 2 and 24 hours after challenge. Four-color flow cytometry was used to analyze the expression and distribution in vivo of IL-2R and MHC II activation markers on naive and memory T cell subsets after challenge. RESULTS At 2 and 24 hours after allergen challenge, there was a significant increase in the CD45RO+IL-2R+ T helper cells compared with baseline (mean +/- SE, baseline, 12.5% +/- 1% versus 2 hours, 18.1% +/- 1% and 24 hours, 17.8% +/- 2%, p < 0.025). MHC II expression was not significantly increased after challenge on naive and memory T helper cells and coexpression of IL-2R and MHC II was only found in a small proportion of CD45RO+ T helper cells (2.7% +/- 1%). No changes of IL-2R or MHC II expression on T helper subsets were observed after ISH challenge in the same patients. We also found that 31% to 46% of T helper cells coexpress CD45RA and CD45RO simultaneously, and upregulation of IL-2-R and MHC II expression occurs only on those T helper cells that express CD45RO. CONCLUSIONS We have found that T helper cells express both CD45RA and CD45RO isoforms, which suggests the existence of a transitional phenotype among naive and memory T helper cells in peripheral blood. In subjects with atopic asthma, our in vivo analysis characterizes two populations of activated memory T helper cells based on the expression of IL-2R or MHC II surface molecules after allergen challenge.

Detection of rare apoptotic T cells in vivo.

The flow cytometric analysis of apoptosis in lymphocytes from in vivo samples has been difficult because of the low frequency of apoptotic events. To overcome this obstacle, many investigators have relied on in vitro incubations to increase the number of apoptotic cells before analysis. In this report, we show that an adaptation of the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay for use in flow cytometry can be used to detect rare apoptotic lymphocytes from freshly harvested LN suspensions. This approach is both specific and extremely sensitive. This method also is amenable to multiparameter analyses and allows a phenotypic analysis of these rare apoptotic cells. However, we observed that some monoclonal antibodies can stain apoptotic-but not viable-cells nonspecifically. Therefore, the specificity of all antibodies to stain apoptotic cells was confirmed in competition assays.