Allergen specific Th2 cytokine expression is independent of CD28 costimulation

J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2

immune response to allergens present in natural rubber latex. Since successful immunotherapy induces IL-10 producing CD4+CD25+T regulatory cells (Tr) that can inhibit proliferative responses of Th2 cells, we investigated the immune responses of latex sensitization in mice depleted of Tr cells. METHODS: Mice (3 groups) were sensitized with latex protein MNA (100 microgram/animal) by 3 SC injections at weekly intervals. In order to deplete Tr cells at different time points of sensitization, group 2 mice were treated with anti CD25 prior to each latex sensitization and group 3 received anti CD25 treatment after the last SC immunization. Animals were sacrificed 24 h after MNA challenges (intranasal) for 3 consecutive days. RESULTS: Depletion of Tr cells before latex sensitization (group 2) exhibited higher levels of allergen specific IgG and total lgE in BAL and serum samples as compared with latex sensitized animals (group i ). Th2 cytokines 1L-4, IL-5 and IL-13 in culture supernatants of PBMCs from CD4+CD25+ Tr cell depleted mice (n=5) exhibited higher levels in comparison to latex sensitized animals (P<0.05). The effect is more pronounced in animals receiving anti CD25 treatment before allergen exposure (group 2) than the group receiving treatment after allergen sensitization (group 3). C O N C L U S I O N S : Depletion of Tr cells resulted in exaggerated cell mediated and humoral immune responses against latex allergens, hence, indicating a major role for Tr cells in the development and regulation of peripheral immune responses and tolerance against invading allergens. Funding: American Lung Association of Wisconsin

166 Allergic Effect of TGF-beta on Cow's Milk-Specific T Cells Derived from and Non-Allergic Children M. M. Tiemessen ] , S. Kunzmann2, C. B. Schmidt-Weber2, M. A. Hoijer 3, C. A. E Bruijnzeel-Koomenl E. Van Hoffen I, E. E Knoll ; ]Department Dermatology/Allergology, University Medical Center Utrecht, Utrecht, NETHERLANDS, 2Swiss Institute of Allergy and Asthma Research, Davos, SWITZERLAND, 3Numico Research, Wageningen, NETHERLANDS. A natural state of oral tolerance prevents harmful immune responses against ingested food in healthy individuals. The mechanisms underlying oral tolerance are abrogated in patients with food allergy, resulting in a Th2-mediated allergic reaction. The immunosuppressive cytokines TGF[3 and IL-10 are hypothesized to have an important role in oral tolerance to food antigens. We examined whether cow's milk-specific T-cell clones derived from cow's milk allergic and non-allergic children differed in their response towards TGF-13. TGF-~ (1 ng/ml) inhibited antigen-specific Tcell proliferation of T-cells from both allergic and non-allergic patients. IL-10 (25 ng/ml), previously shown to modulate the effect of TGF-13 via increased TGF-[3RII expression, was not able to enhance the suppressive effect of TGF-[3 nor the mRNA-expression of TGF-I]RII. The sensitivity of individual T-cell clones for inhibition by TGF-I~ was not related to the intracellular expression of SMAD7 or SMAD3, signaling molecules involved in TGF-I3 signal-transduction. To examine the cellular mechanism, via which TGF-[3 could exert its suppressive function, apoptosis (Annexin-V) and cell cycle progression (CFSE) were analyzed with flowcytometry. The percentage of apoptotic T-cells in the presence of TGF-[3 was not increased. However, the cell cycle progression of T-cells was clearly inhibited by TGF-13. in conclusion, cow's milk-specific T-cells derived from allergic and non-allergic donors are equally inhibited by TGF-t3. This suppression is not mediated by induction of apoptosis but seems the result of interference with the cell cycle. These results suggest that oral intolerance in food allergy is not explained by differences in sensitivity of allergen-specific T-cells towards TGF-~. Funding: Numico Research

Abstracts

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67 bleExpression of SNARE Proteins by Human Lymphocytes:Possi Role in Regulated Release of Mediators of Immune Response S. O. O d e m u y i w a I, K. Lo J, P. Lacy j, R. C. Bleackley 2, R. MoqbelZ; IMedicine, University of Alberta, Edmonton, AB, CANADA, 2Biochemistry, University of Alberta, Edmonton, AB, CANADA. RATIONALE: Lymphocytes play a central role in the maintenance and regulation of immune response, including allergic and asthmatic inflammation, through the release of soluble mediators. There is a paucity of information on the intracellular mechanisms that lead to mediator release from lymphocytes. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) on vesicular membranes (vSNAREs) and target membranes (tSNAREs) are critical for the docking of intracytoplasmic vesicles to plasma membrane. SNARE proteins are important in mediator release from hemopoietic cells, including eosinophils, mast cells, macrophages, and neutrophils. We have investigated the expression of SNARE proteins in human lymphocytes to understand the possible mechanisms of exocytosis in these cells. HYPOTHESIS: Lymphocytes express SNARE fusion complex important in mediator release. METHODS: Specific primer pairs and RT-PCR were used to probe lor syntaxin 4, SNAP-23, and VAMP-2 mRNA in a YT-Indy cell line (NK phenotype), a CTL clone, and Jurkat cells. Western blotting and confocal microscopic examination of stained cells with monoclonal antibodies were used to determine expression of SNARE proteins. These findings were confirmed on CD4 + cells from human peripheral blood lymphocytes (PBLs) by FACS. RESULTS: YT-Indy, CTL clone, CD4+ PBLs, and Jurkat cells expressed both mRNA and protein for SNAP-23, syntaxin-4, and VAMP-2. Confocal microscopy revealed colocalization of VAMP-2 protein with granzyme B-containing vesicles in YT-Indy and CTL clones. VAMP-2 staining was associated with small vesicular structures in the cytoplasm of Triton Xpermeabilized Jurkat cells. CONCLUSIONS: Human lymphocytes express SNARE proteins that may play a role in regulated release of immune mediators. Funding: CIHR, AHFMR

168 AllergencD28 CostimulationSpeci Th2 fic Cytokine Expression Is Independent of H. N. Thai, B. Foster, C. Prussin; Laboratory of Allergic Diseases, National Institutes of Health, Bethesda, MD. RATIONALE: CD28 is the major costimulatory receptor for T cell cytokine responses. However, its role in costimulating allergen specific Th2 responses has not been determined. METHODS: Allergen (D. pteronyssinus, D. farinae) specific human T cell cytokine responses were studied using intracellular cytokine staining. Costimulation was blocked by the addition of either CTLA-4 Ig or a cocktail of mAbs to B7-1 and B7-2. Conversely, to determine whether maximal CD28 costimulation could augment allergen specific cytokine production, we examined dust mite specific Th2 responses with and without the addition of an anti-CD28 mAb. RESULTS: The addition of either CTLA-4 Ig (n=2) or anti-B7 mAbs (n=2) had no effect on either IL-2 or 1L-4 production. Augmenting costimulation by the addition of anti-CD28 mAb had no significant effect on IL-2 or IL-4 production (n=7: p=0.gt25; p=0.3910 respectively). CONCLUSIONS: Using mAbs to both block and augment costimulation, we found that allergen specific Th2 cytokine production is independent of CD28 costimulation. These data suggest that other costimulatory receptors may play a role in regulating human Th2 responses. Funding: NIAID Division of lntrumural Research