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β+CD45RO+ T Cells and CD4+ T cells are required for induction of human ragweed specific memory IgE responses
Abstracts S257
J ALLERGY CLIN IMMUNOL VOLUME 115, NUMBER 2
CD8+CD60+ TCR /+CD45RO+ T Cells and CD4+ T Cells Are Required for Induction of Human Ragweed Specific Memory IgE Responses T. A. Smith-Norowitz, K. B. Norowitz, M. H. Bluth, S. Chice, R. O. Joks, K. Liao, M. Nowakowski, H. G. Durkin; Center for Allergy and Asthma Research, SUNY Downstate Medical Center, Brooklyn, NY. RATIONALE: CD4+ T cells are required for induction of IgE responses. Although CD8+CD60+ T cell numbers are increased in blood of serum IgE+ ragweed sensitized (IgE+), but not IgE negative nonatopic (IgE-), humans their role in induction of memory IgE responses has not been determined. METHODS: CD8+CD60+ T cell subset (TCR /, TCR /, CD45RA, CD45RO, CD23, IL-4R) distributions in IgE+ and IgE- humans and requirement for CD8+CD60+ and CD4+ T cells for in vitro induction of ragweed specific memory IgE responses by their PBMC were determined (flow cytometry, ELISA). RESULTS: CD8+CD60+ T cells were increased in IgE+, compared with IgE- humans (p=0.001); all were TCR /+. In IgE+, but not IgE-, humans, nearly all were CD45RO+ (80-98%, <3%, respectively). A subset of CD8+CD60+ T cells in IgE+, but not IgE-, humans expressed CD23. When PBMC from IgE+ humans were cultured 0-12 days with ragweed antigen, but not with noncrosssreacting antigen, peak IgE responses occurred on day 10. PBMC of IgE- donors never produced IgE to any stimulant. Depletion and reconstitution studies established that both CD8+CD60+ and CD4+ T cells are required for induction of ragweed specific memory IgE responses. Addition of low numbers of purified CD8+CD60+ T cells to PBMC depleted of these cells reconstituted IgE responses, but higher numbers suppressed them. CD4+ T cells reconstituted IgE responses in dose dependent fashion. CONCLUSIONS: (1) CD8+CD60+TCR /+ (CD45RO+) T cells serve as epsilon memory/regulatory T cells. (2) CD4+ T cells also are required for induction of IgE responses. Funding: NY State Allergy Center Grant
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Heterogeneity of CD4+CD25+ T Cells Among Subjects With Distinct Allergic Phenotypes A. J. Reefer1, S. Satinover1, B. Wilson2, J. A. Woodfolk3; 1Asthma and Allergic Diseases Center, University of Virginia, Charlottesville, VA, 2Department of Dermatology, University of Virginia, Charlottesville, VA, 3Internal Medicine, University of Virginia, Charlottesville, VA. RATIONALE: Cat-sensitized patients with atopic eczema (AE)(IgEhigh) and cat-tolerant subjects (IgEnegIgGhigh) without AE may represent opposite ends of the allergic spectrum. The association of putative markers of effector and regulatory T cells with distinct allergic phenotypes was examined. METHODS: Peripheral blood mononuclear cells were isolated from AE patients (mean total IgE=1,523IU/ml and IgE ab to cat=29IU/ml)(n=7) and cat-tolerant responders (mean total IgE=43IU/ml and IgE ab to cat <0.35IU/ml)(n=7). CD4+ T cells were analyzed by flow cytometry for expression of CD45RO, CLA, CD25, CXCR3 and CCR4. Mean percentages of cells were compared between groups. RESULTS: While expression of CCR4 (Th2 marker) on memory CD4+ T cells was high in both groups (>35%), CXCR3 (Th1) was increased in AE subjects (29% versus 12%, p=0.05). AE patients had markedly increased CD4+ T cells co-expressing CD45RO and CD25 compared with tolerant responders (25% versus 2.8% respectively, p<0.01). Expression of CCR4 on CD4+CD25+ T cells was high in both groups (>50%) and CXCR3 expression in the absence of CCR4 was moderately increased in AE subjects (11% versus 4.8%, p=0.1). Despite a similar frequency of CLA+ (skin-homing) CD4+ T cells between groups (~7%), CD25 expression on these cells was markedly enhanced in AE patients (83% versus 13%, p<0.01). Furthermore, CCR4 expression segregated with CD25 on CLA+CD4+ T cells derived from AE patients but not tolerant responders. CONCLUSIONS: CD4+CD25+ T cells comprise a heterogeneous subset which appear to be expanded in vivo in AE patients with high IgE. Distinct differences in CD4+CD25+ T cells are associated with IgEhigh and IgElow phenotypes. Funding: National Institutes of Health
1024
Low Thresholds for Activation and Apoptosis of Th1 Cells as a Mechanism of Th2 Predominance in Atopic Diseases T. Akkoc1, J. Zumkehr2, P. de Konig2, I. B. Barlan3, K. Blaser2, M. Akdis2, C. A. Akdis2; 1Marmara University Medical Faculty, Pediatric Allergy and Immunology Department, Istanbul, TURKEY, 2Swiss Institute of Allergy and Asthma Research (SIAF), Davos, SWITZERLAND, 3Marmara University Medical Faculty Pediatric Allergy Immunology Dep, Istanbul, TURKEY. RATIONALE: A dysregulated and Th2-biased immune response appears to be an important pathogenetic factor in atopic diseases. The aim of the study was to compare thresholds for activation and apoptosis of Th1 and Th2 cells in atopic diseases, because preferential activation and subsequent cell death of one subset over the other might be pertinent in Th2 immune response. METHODS: Freshly purified CD45RA+ (naive) and CD45RO+ (memory) T cells and in vitro differentiated Th1 and Th2 cells were investigated in atopic dermatitis patients and healthy controls. Differences in T cell proliferation and several features of apoptosis were determined. In addition, apoptosis and proliferation of spleen CD4+ T cells from high IgE responder BALB/c mice and low IgE responder C56BL6 mice were analyzed for comparison. RESULTS: Naive and memory cells of atopic individuals both showed a low threshold for proliferation with low amounts of T cell receptor triggers compared to healthy individuals. In vitro differentiated Th1 cells of atopic donors were highly susceptible to apoptosis with increased Fas and Fas-ligand expression and caspase degradation compared to healthy individuals. Neutralization of INF- in Th1 cells blocked increased apoptosis in atopic donors. Supporting these findings, T cells of high IgE responder BALB/c mice showed increased apoptosis and secreted increased Th2 cytokines compared to low IgE responder C57B6 mice. CONCLUSIONS: Predominant Th2 profile in atopic diseases might be due to the increased tendency to activation and apoptosis of Th1-like cells in atopic diseases. Funding: Swiss National Foundation grants
1025
TUESDAY
Responses of Fresh and Cryopreserved PBMCs to In Vitro Mitogen and Allergen Stimulation Z. Y. Kucuk1, R. R. Castro1, W. G. Schreffler1, C. Visness2, J. Gern3, H. A. Sampson1; 1Mt. Sinai/NYU Medical Center, New York, NY, 2Rho, Chapel Hill, NC, 3University of Wisconsin, Madison, WI. RATIONALE: Multi-center trials that include in vitro cellular studies must contend with site-to-site variation of assays or utilize cryopreserved cells for central processing. There is little data regarding allergen responsiveness in cryopreserved PBMCs. METHODS: PBMCs were obtained from volunteers with/without positive skin tests to house dust mite (HDM) or cockroach extracts. Cells were divided for cryopreservation and later processing or for immediate culture. Two methods of cryopreservation, constant rate freezer (CRF) and Nalgene®, were compared. PBMCs were stimulated with PHA, LPS, rBla g2, rDer f1, HDM or medium. Supernatant IFN-, TNF-, IL-2, IL-10, IL-4, IL-5 were measured by multiplex cytokine bead assay. RESULTS: PBMC recovery and viability were comparable between preservation groups. Spontaneous and PHA-stimulated levels of IFN-, IL-2, IL-4, IL-5, and IL-10 were similar, although spontaneous TNF- was higher from frozen cells (median 3 pg/ml fresh vs. preserved group: 16 pg/ml Nalgene®, 16.8 pg/ml CRF; p<0.0001). Frozen vs. fresh PBMCs produced similar levels of most cytokines except LPS-induced IL-10, which was significantly lower in the Nalgene® preserved cells only (median 796 pg/ml vs. 98.8 pg/ml). There were no significant differences in cytokine responses to rDerf1 between patients and controls. Fresh and cryopreserved patient PBMCs produced less IFN- than controls in response to HDM. CONCLUSION: While the response of fresh and cryopreserved PBMCs respond to various stimuli in a similar manner, a number of notable differences were observed, e.g. decreased IL-10 production in Nalgene® cryopreserved cells, and these differences must be carefully considered when using cryopreserved cells. Funding: NIH-N01-25496
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J ALLERGY CLIN IMMUNOL VOLUME 115, NUMBER 2
CD8+CD60+ TCR /+CD45RO+ T Cells and CD4+ T Cells Are Required for Induction of Human Ragweed Specific Memory IgE Responses T. A. Smith-Norowitz, K. B. Norowitz, M. H. Bluth, S. Chice, R. O. Joks, K. Liao, M. Nowakowski, H. G. Durkin; Center for Allergy and Asthma Research, SUNY Downstate Medical Center, Brooklyn, NY. RATIONALE: CD4+ T cells are required for induction of IgE responses. Although CD8+CD60+ T cell numbers are increased in blood of serum IgE+ ragweed sensitized (IgE+), but not IgE negative nonatopic (IgE-), humans their role in induction of memory IgE responses has not been determined. METHODS: CD8+CD60+ T cell subset (TCR /, TCR /, CD45RA, CD45RO, CD23, IL-4R) distributions in IgE+ and IgE- humans and requirement for CD8+CD60+ and CD4+ T cells for in vitro induction of ragweed specific memory IgE responses by their PBMC were determined (flow cytometry, ELISA). RESULTS: CD8+CD60+ T cells were increased in IgE+, compared with IgE- humans (p=0.001); all were TCR /+. In IgE+, but not IgE-, humans, nearly all were CD45RO+ (80-98%, <3%, respectively). A subset of CD8+CD60+ T cells in IgE+, but not IgE-, humans expressed CD23. When PBMC from IgE+ humans were cultured 0-12 days with ragweed antigen, but not with noncrosssreacting antigen, peak IgE responses occurred on day 10. PBMC of IgE- donors never produced IgE to any stimulant. Depletion and reconstitution studies established that both CD8+CD60+ and CD4+ T cells are required for induction of ragweed specific memory IgE responses. Addition of low numbers of purified CD8+CD60+ T cells to PBMC depleted of these cells reconstituted IgE responses, but higher numbers suppressed them. CD4+ T cells reconstituted IgE responses in dose dependent fashion. CONCLUSIONS: (1) CD8+CD60+TCR /+ (CD45RO+) T cells serve as epsilon memory/regulatory T cells. (2) CD4+ T cells also are required for induction of IgE responses. Funding: NY State Allergy Center Grant
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Heterogeneity of CD4+CD25+ T Cells Among Subjects With Distinct Allergic Phenotypes A. J. Reefer1, S. Satinover1, B. Wilson2, J. A. Woodfolk3; 1Asthma and Allergic Diseases Center, University of Virginia, Charlottesville, VA, 2Department of Dermatology, University of Virginia, Charlottesville, VA, 3Internal Medicine, University of Virginia, Charlottesville, VA. RATIONALE: Cat-sensitized patients with atopic eczema (AE)(IgEhigh) and cat-tolerant subjects (IgEnegIgGhigh) without AE may represent opposite ends of the allergic spectrum. The association of putative markers of effector and regulatory T cells with distinct allergic phenotypes was examined. METHODS: Peripheral blood mononuclear cells were isolated from AE patients (mean total IgE=1,523IU/ml and IgE ab to cat=29IU/ml)(n=7) and cat-tolerant responders (mean total IgE=43IU/ml and IgE ab to cat <0.35IU/ml)(n=7). CD4+ T cells were analyzed by flow cytometry for expression of CD45RO, CLA, CD25, CXCR3 and CCR4. Mean percentages of cells were compared between groups. RESULTS: While expression of CCR4 (Th2 marker) on memory CD4+ T cells was high in both groups (>35%), CXCR3 (Th1) was increased in AE subjects (29% versus 12%, p=0.05). AE patients had markedly increased CD4+ T cells co-expressing CD45RO and CD25 compared with tolerant responders (25% versus 2.8% respectively, p<0.01). Expression of CCR4 on CD4+CD25+ T cells was high in both groups (>50%) and CXCR3 expression in the absence of CCR4 was moderately increased in AE subjects (11% versus 4.8%, p=0.1). Despite a similar frequency of CLA+ (skin-homing) CD4+ T cells between groups (~7%), CD25 expression on these cells was markedly enhanced in AE patients (83% versus 13%, p<0.01). Furthermore, CCR4 expression segregated with CD25 on CLA+CD4+ T cells derived from AE patients but not tolerant responders. CONCLUSIONS: CD4+CD25+ T cells comprise a heterogeneous subset which appear to be expanded in vivo in AE patients with high IgE. Distinct differences in CD4+CD25+ T cells are associated with IgEhigh and IgElow phenotypes. Funding: National Institutes of Health
1024
Low Thresholds for Activation and Apoptosis of Th1 Cells as a Mechanism of Th2 Predominance in Atopic Diseases T. Akkoc1, J. Zumkehr2, P. de Konig2, I. B. Barlan3, K. Blaser2, M. Akdis2, C. A. Akdis2; 1Marmara University Medical Faculty, Pediatric Allergy and Immunology Department, Istanbul, TURKEY, 2Swiss Institute of Allergy and Asthma Research (SIAF), Davos, SWITZERLAND, 3Marmara University Medical Faculty Pediatric Allergy Immunology Dep, Istanbul, TURKEY. RATIONALE: A dysregulated and Th2-biased immune response appears to be an important pathogenetic factor in atopic diseases. The aim of the study was to compare thresholds for activation and apoptosis of Th1 and Th2 cells in atopic diseases, because preferential activation and subsequent cell death of one subset over the other might be pertinent in Th2 immune response. METHODS: Freshly purified CD45RA+ (naive) and CD45RO+ (memory) T cells and in vitro differentiated Th1 and Th2 cells were investigated in atopic dermatitis patients and healthy controls. Differences in T cell proliferation and several features of apoptosis were determined. In addition, apoptosis and proliferation of spleen CD4+ T cells from high IgE responder BALB/c mice and low IgE responder C56BL6 mice were analyzed for comparison. RESULTS: Naive and memory cells of atopic individuals both showed a low threshold for proliferation with low amounts of T cell receptor triggers compared to healthy individuals. In vitro differentiated Th1 cells of atopic donors were highly susceptible to apoptosis with increased Fas and Fas-ligand expression and caspase degradation compared to healthy individuals. Neutralization of INF- in Th1 cells blocked increased apoptosis in atopic donors. Supporting these findings, T cells of high IgE responder BALB/c mice showed increased apoptosis and secreted increased Th2 cytokines compared to low IgE responder C57B6 mice. CONCLUSIONS: Predominant Th2 profile in atopic diseases might be due to the increased tendency to activation and apoptosis of Th1-like cells in atopic diseases. Funding: Swiss National Foundation grants
1025
TUESDAY
Responses of Fresh and Cryopreserved PBMCs to In Vitro Mitogen and Allergen Stimulation Z. Y. Kucuk1, R. R. Castro1, W. G. Schreffler1, C. Visness2, J. Gern3, H. A. Sampson1; 1Mt. Sinai/NYU Medical Center, New York, NY, 2Rho, Chapel Hill, NC, 3University of Wisconsin, Madison, WI. RATIONALE: Multi-center trials that include in vitro cellular studies must contend with site-to-site variation of assays or utilize cryopreserved cells for central processing. There is little data regarding allergen responsiveness in cryopreserved PBMCs. METHODS: PBMCs were obtained from volunteers with/without positive skin tests to house dust mite (HDM) or cockroach extracts. Cells were divided for cryopreservation and later processing or for immediate culture. Two methods of cryopreservation, constant rate freezer (CRF) and Nalgene®, were compared. PBMCs were stimulated with PHA, LPS, rBla g2, rDer f1, HDM or medium. Supernatant IFN-, TNF-, IL-2, IL-10, IL-4, IL-5 were measured by multiplex cytokine bead assay. RESULTS: PBMC recovery and viability were comparable between preservation groups. Spontaneous and PHA-stimulated levels of IFN-, IL-2, IL-4, IL-5, and IL-10 were similar, although spontaneous TNF- was higher from frozen cells (median 3 pg/ml fresh vs. preserved group: 16 pg/ml Nalgene®, 16.8 pg/ml CRF; p<0.0001). Frozen vs. fresh PBMCs produced similar levels of most cytokines except LPS-induced IL-10, which was significantly lower in the Nalgene® preserved cells only (median 796 pg/ml vs. 98.8 pg/ml). There were no significant differences in cytokine responses to rDerf1 between patients and controls. Fresh and cryopreserved patient PBMCs produced less IFN- than controls in response to HDM. CONCLUSION: While the response of fresh and cryopreserved PBMCs respond to various stimuli in a similar manner, a number of notable differences were observed, e.g. decreased IL-10 production in Nalgene® cryopreserved cells, and these differences must be carefully considered when using cryopreserved cells. Funding: NIH-N01-25496
1022