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Transgenic Human CD3 Zeta Reconstitutes T Cell Development in CD3 Zeta Knockout Mice
Abstracts AB143
J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 2
Common Variable Immunodeficiency: A Patient With Anaphylaxis To Intravenous And Subcutaneous Immunoglobulin S. Thobani1, E. Hu2, P. Huynh1, L. Scott1; 1University of Southern California+LAC, Los Angeles, CA, 2University of Southern California+LAC, Los angeles, CA. RATIONALE: Patient with Common Variable Immunodeficiency developed anaphylaxis to intravenous immunoglobulin. Literature reports patients with history of anaphylaxis to IVIG demonstrating tolerance to subcutaneous immunoglobulin. METHODS: Case Report. RESULTS: 23 year old male with Common Variable Immunodeficiency, diagnosed age sixteen. Patient’s first episode of anaphylaxis occurred with 10 cc of IVIG, (Gammagard). Patient then treated with Gammagard SD IV at only subtherapeutic doses due to chills, headaches, and rigors. He now developed anaphylaxis to Gammagard SD. Patient was continued on prophylactic antibiotics, Augmentin 500mg bid, and IVIG was discontinued. Initial immune work-up: deficient IG A, low IG M, IG G, with normal levels of IG E. Omalizumab was initiated for severe persistent asthma/ bronchiectesis with secondary potential benefits of down-regulation of FcERI mast cell receptors. Within four months of not receiving IVIG, patient was admitted to Medical ICU with pneumonia and sepsis. Literature reports cases of patients with previous anaphylaxis to IVIG tolerating subcutaneous IG. A trial of SCIG was initiated. Patient skin-prick test negative to SCIG (Vivaglobulin). Small escalation doses of SCIG administered to patient (total dose 0.6cc). Within 30 minutes, patient developed anaphylaxis lasting 72 hours. Patient required intubation, Epinephrine, Nor-epinephrine drip, and steroids. Patient was discharged home on antibiotics; levaquin and clindamycin. IgG and IgE anti-IgA antibodies pending. CONCLUSIONS: To our knowledge, this is the first reported case of a CVID patient with anaphylaxis to intravenous and subcutaneous immunoglobulin. We are currently in discussions with experts world-wide for treatment options for this patient and plan to fully report the outcomes of this case.
561
Transgenic Human CD3 Zeta Reconstitutes T Cell Development in CD3 Zeta Knockout Mice T. Atkinson, Y. Dai, T. R. Schoeb; University of Alabama at Birmingham, Birmingham, AL. RATIONALE: The CD3z protein is critically important for transducing TCR signals during thymic development and peripheral T cell activation. A cryptic splice acceptor site at the 5Õ end of exon 5 of the CD3z gene produces an mRNA isoform bearing a CAG insert resulting in a glutamine (Q) insertion between the first and second Immunoreceptor Tyrosine-based Activation Motifs (ITAMs). The Q insert disrupts a conserved loop with homology to the C-terminal portion of heterotrimeric G-proteins g subunits in a region associated with activation of Phospholipase C b (PLCb). Previous data obtained using transfected CD3z-deficient MA5.8 T cells demonstrated reduced T cell receptor (TCR)-induced phosphatidylinositol hydrolysis in cells transfected with the Q isoform compared to those transfected with the canonical CD3z sequence. The relative proportion of the Q isoforms also appears to increase in human T cells following activation. METHODS: We cloned human cDNA for the two CD3z isoforms into a vector containing a CD2 minigene cassette and developed strains of mice transgenic for the two human CD3z isoforms. These transgenic animals were bred into a CD3z -/- strain, and the resultant mice bearing the human Q isoform have been partially characterized. RESULTS: The human CD3zQ transgenic mice have normal thymic and splenic architecture with normal populations of double positive, and single positive CD4/CD8 thymocytes and CD4/CD8 single positive splenocytes by flow cytometry. CONCLUSIONS: Expression of the human CD3zQ isoform in a CD3z deficient murine strain permits normal thymic and splenic development as well as differentiation of CD4 and CD8 single positive T cell populations.
562
Helios Is A Marker Of Human Thymic-derived Foxp3+ Regulatory T Cells D. Q. Tran; University of Texas Medical School at Houston, Houston, TX. RATIONALE: FOXP3+ regulatory T cells (Tregs) are central to the maintenance of self-tolerance and the prevention of autoimmunity. Since they are generated both in the thymus and the periphery, the ability to distinguish these two populations would be critical to understand their development, particularly in allergic and autoimmune diseases. METHODS: Peripheral blood of healthy adult donors and patients with systemic lupus erythematosus as well as cord blood were analyzed for FOXP3 and Helios expression within the CD4+ T cell population by flow cytometry. Cytokine secretion and suppressive function were studied in the Tregs and correlated with Helios expression. RESULTS: Helios is expressed selectively on the FOXP3+ Tregs and is decreased with age. Helios identifies thymic-derived, cytokine non-producing FOXP3+ Tregs and not peripheral or TGFbeta-induced FOXP3+ T cells. Knockdown of Helios with siRNA does not alter the cytokine profile and suppressor function of Tregs, suggesting that Helios is not critical for their anergic and suppressive properties. CONCLUSIONS: Helios is expressed uniquely in human FOXP3+ Tregs and distinguishes the thymic over the peripheral derived Tregs. While it is expressed predominately in the Tregs, it does not appear to play a critical role in their anergic and suppressor functions.
563
Signaling through the PGI2 Receptor Promotes IL-17 Production by CD4 T Cells M. M. Huckabee1, W. Zhou1, D. C. Newcomb1, K. Goleniewska1, G. A. FitzGerald2, R. S. Peebles, Jr,1; 1Vanderbilt University, Nashville, TN, 2 University of Pennsylvania, Philadelphia, PA. RATIONALE: Mounting evidence reveals that IL-17 production of CD4 T cells can be affected not only by cytokine factors, but also by lipid molecules. To investigate the impact of the lipid products in cyclooxygenase (COX) pathway on Th17 differentiation, we determined the effect of PGI2 and PGI2 receptor (IP) signaling on IL-17 production and Th17 differentiation. METHODS: Splenic CD4 T cells of BALB/c mice were activated with anti-CD3 and anti-CD28 in the presence or absences of the PGI2 analogs iloprost or cicaprost for 5 days. CD4+CD62L+ T cells of IP KO/OT II mice and control OT II mice, both on a C57BL/6 background, were activated with ovalbumin (323-339) peptide and anti-CD28 in the presence or absences of iloprost or cicaprost for 5 days. The levels of IL-17 in the culture supernatant were determined by ELISA. RESULTS: Iloprost and cicaprost dose-dependently increased IL-17 production by CD4 T cells activated through either pan-TCR stimulation with anti-CD3 or ovalbumin peptide-specific stimulation. The effect of iloprost and cicaprost on IL-17 production by IP KO/OT II T cells was significantly attenuated compared to the effect on control OT II T cells. Therefore, the augmentation of IL-17 production by iloprost and cicaprost was independent of the mode of TCR stimulation and the mouse strain, but dependent on the IP-mediated signaling. CONCLUSIONS: The stimulatory effect of the PGI2 analogs iloprost and cicaprost suggests that COX pathway may have a regulatory function in IL-17 production and the pathogenesis of Th17-mediated diseases.
MONDAY
560
J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 2
Common Variable Immunodeficiency: A Patient With Anaphylaxis To Intravenous And Subcutaneous Immunoglobulin S. Thobani1, E. Hu2, P. Huynh1, L. Scott1; 1University of Southern California+LAC, Los Angeles, CA, 2University of Southern California+LAC, Los angeles, CA. RATIONALE: Patient with Common Variable Immunodeficiency developed anaphylaxis to intravenous immunoglobulin. Literature reports patients with history of anaphylaxis to IVIG demonstrating tolerance to subcutaneous immunoglobulin. METHODS: Case Report. RESULTS: 23 year old male with Common Variable Immunodeficiency, diagnosed age sixteen. Patient’s first episode of anaphylaxis occurred with 10 cc of IVIG, (Gammagard). Patient then treated with Gammagard SD IV at only subtherapeutic doses due to chills, headaches, and rigors. He now developed anaphylaxis to Gammagard SD. Patient was continued on prophylactic antibiotics, Augmentin 500mg bid, and IVIG was discontinued. Initial immune work-up: deficient IG A, low IG M, IG G, with normal levels of IG E. Omalizumab was initiated for severe persistent asthma/ bronchiectesis with secondary potential benefits of down-regulation of FcERI mast cell receptors. Within four months of not receiving IVIG, patient was admitted to Medical ICU with pneumonia and sepsis. Literature reports cases of patients with previous anaphylaxis to IVIG tolerating subcutaneous IG. A trial of SCIG was initiated. Patient skin-prick test negative to SCIG (Vivaglobulin). Small escalation doses of SCIG administered to patient (total dose 0.6cc). Within 30 minutes, patient developed anaphylaxis lasting 72 hours. Patient required intubation, Epinephrine, Nor-epinephrine drip, and steroids. Patient was discharged home on antibiotics; levaquin and clindamycin. IgG and IgE anti-IgA antibodies pending. CONCLUSIONS: To our knowledge, this is the first reported case of a CVID patient with anaphylaxis to intravenous and subcutaneous immunoglobulin. We are currently in discussions with experts world-wide for treatment options for this patient and plan to fully report the outcomes of this case.
561
Transgenic Human CD3 Zeta Reconstitutes T Cell Development in CD3 Zeta Knockout Mice T. Atkinson, Y. Dai, T. R. Schoeb; University of Alabama at Birmingham, Birmingham, AL. RATIONALE: The CD3z protein is critically important for transducing TCR signals during thymic development and peripheral T cell activation. A cryptic splice acceptor site at the 5Õ end of exon 5 of the CD3z gene produces an mRNA isoform bearing a CAG insert resulting in a glutamine (Q) insertion between the first and second Immunoreceptor Tyrosine-based Activation Motifs (ITAMs). The Q insert disrupts a conserved loop with homology to the C-terminal portion of heterotrimeric G-proteins g subunits in a region associated with activation of Phospholipase C b (PLCb). Previous data obtained using transfected CD3z-deficient MA5.8 T cells demonstrated reduced T cell receptor (TCR)-induced phosphatidylinositol hydrolysis in cells transfected with the Q isoform compared to those transfected with the canonical CD3z sequence. The relative proportion of the Q isoforms also appears to increase in human T cells following activation. METHODS: We cloned human cDNA for the two CD3z isoforms into a vector containing a CD2 minigene cassette and developed strains of mice transgenic for the two human CD3z isoforms. These transgenic animals were bred into a CD3z -/- strain, and the resultant mice bearing the human Q isoform have been partially characterized. RESULTS: The human CD3zQ transgenic mice have normal thymic and splenic architecture with normal populations of double positive, and single positive CD4/CD8 thymocytes and CD4/CD8 single positive splenocytes by flow cytometry. CONCLUSIONS: Expression of the human CD3zQ isoform in a CD3z deficient murine strain permits normal thymic and splenic development as well as differentiation of CD4 and CD8 single positive T cell populations.
562
Helios Is A Marker Of Human Thymic-derived Foxp3+ Regulatory T Cells D. Q. Tran; University of Texas Medical School at Houston, Houston, TX. RATIONALE: FOXP3+ regulatory T cells (Tregs) are central to the maintenance of self-tolerance and the prevention of autoimmunity. Since they are generated both in the thymus and the periphery, the ability to distinguish these two populations would be critical to understand their development, particularly in allergic and autoimmune diseases. METHODS: Peripheral blood of healthy adult donors and patients with systemic lupus erythematosus as well as cord blood were analyzed for FOXP3 and Helios expression within the CD4+ T cell population by flow cytometry. Cytokine secretion and suppressive function were studied in the Tregs and correlated with Helios expression. RESULTS: Helios is expressed selectively on the FOXP3+ Tregs and is decreased with age. Helios identifies thymic-derived, cytokine non-producing FOXP3+ Tregs and not peripheral or TGFbeta-induced FOXP3+ T cells. Knockdown of Helios with siRNA does not alter the cytokine profile and suppressor function of Tregs, suggesting that Helios is not critical for their anergic and suppressive properties. CONCLUSIONS: Helios is expressed uniquely in human FOXP3+ Tregs and distinguishes the thymic over the peripheral derived Tregs. While it is expressed predominately in the Tregs, it does not appear to play a critical role in their anergic and suppressor functions.
563
Signaling through the PGI2 Receptor Promotes IL-17 Production by CD4 T Cells M. M. Huckabee1, W. Zhou1, D. C. Newcomb1, K. Goleniewska1, G. A. FitzGerald2, R. S. Peebles, Jr,1; 1Vanderbilt University, Nashville, TN, 2 University of Pennsylvania, Philadelphia, PA. RATIONALE: Mounting evidence reveals that IL-17 production of CD4 T cells can be affected not only by cytokine factors, but also by lipid molecules. To investigate the impact of the lipid products in cyclooxygenase (COX) pathway on Th17 differentiation, we determined the effect of PGI2 and PGI2 receptor (IP) signaling on IL-17 production and Th17 differentiation. METHODS: Splenic CD4 T cells of BALB/c mice were activated with anti-CD3 and anti-CD28 in the presence or absences of the PGI2 analogs iloprost or cicaprost for 5 days. CD4+CD62L+ T cells of IP KO/OT II mice and control OT II mice, both on a C57BL/6 background, were activated with ovalbumin (323-339) peptide and anti-CD28 in the presence or absences of iloprost or cicaprost for 5 days. The levels of IL-17 in the culture supernatant were determined by ELISA. RESULTS: Iloprost and cicaprost dose-dependently increased IL-17 production by CD4 T cells activated through either pan-TCR stimulation with anti-CD3 or ovalbumin peptide-specific stimulation. The effect of iloprost and cicaprost on IL-17 production by IP KO/OT II T cells was significantly attenuated compared to the effect on control OT II T cells. Therefore, the augmentation of IL-17 production by iloprost and cicaprost was independent of the mode of TCR stimulation and the mouse strain, but dependent on the IP-mediated signaling. CONCLUSIONS: The stimulatory effect of the PGI2 analogs iloprost and cicaprost suggests that COX pathway may have a regulatory function in IL-17 production and the pathogenesis of Th17-mediated diseases.
MONDAY
560