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Laboratory of Immunobiochemistry Research update Laboratory of Immunobiochemistry Research update

Active research projects n PI Rabin n MDR proteins in T cell activation Regulation Active research projects n PI Rabin n MDR proteins in T cell activation Regulation of T cell responses by the Respiratory Syncytial Virus PI Slater n Cockroach allergen standardization n Determination of optimal surrogate test Depletion analysis of CR extracts Endotoxin in allergen vaccines 2

Publications Research § Spann KM, Tran KC, Chi B, Rabin RL, Collins PL. Suppression Publications Research § Spann KM, Tran KC, Chi B, Rabin RL, Collins PL. Suppression of the induction of alpha, beta, and lambda interferons by the NS 1 and NS 2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages. J Virol 2004; 78(8): 4363 -9. § § Song K, Rabin RL, Douek D, Roederer M, Farber JM. Novel Subsets of CD 4+ Memory T Cells Reveal Early Branched Pathways of T Cell Differentiation in Humans. Proc Natl Acad Sci, in press Zhang J, Alston MA, Huang H, Rabin RL. Multidrug Resistant Protein 1 (MRP 1) is induced upon activation of human T cells, and its inhibition blocks T cell function, in preparation 3

Publications Review § § § Slater JE. Recombinant allergens in the US. Methods 2004; Publications Review § § § Slater JE. Recombinant allergens in the US. Methods 2004; 32(3): 209 -11. Slater JE. Latex allergens. Clin Allergy Immunol 2004; 18: 369 -86. Slater JE. Standardized allergen extracts in the United States. Clin Allergy Immunol 2004; 18: 421 -32. Rabin RL. Respiratory Syncytial Virus exploits genetic and environmental risk factors for asthma; Business Briefing, US Pediatric Care 2005, in press Rabin RL, Levinson AI, Apter AJ. Coincidence of autoimmune and allergic diseases: epidemiologic and mechanistic analyses, in preparation 4

Abstracts B Chi, M Alston, KM Spann, PL Collins, RL Rabin. A critical role Abstracts B Chi, M Alston, KM Spann, PL Collins, RL Rabin. A critical role for dendritic cells in immunosuppression caused by the respiratory syncytial virus (RSV). J Allergy Clin Immunol 2005; 115: S 226 NC de. Vore, WJJ Finlay, EN Dobrovolskaia, A Gam, JE Slater. Cloning and analysis of mono-specific single chain fragment variable sc. Fv fragments that recognize German cockroach allergens Bla g 1, Bla g 2, Bla g 4, and Bla g 5. J Allergy Clin Immunol 2005; 115: S 163 E Dobrovolskaia, A Gam, JE Slater. Competition ELISA can be a sensitive method for the specific detection of small quantities of allergen in a complex mixture. J Allergy Clin Immunol 2005; 115: S 164 J Zhang, MA Alston, H Huang, RA Houghtling, RW Pastor, RL Rabin. Human type 1 CD 4 T cell cytokine responses are selectively dependent on Multidrug Resistance Protein 1. J Allergy Clin Immunol 2005; 115: S 255 C Valerio, LG Arlian, JE Slater. Bacterial 16 S ribosomal DNA sequences isolated from house dust mites. J Allergy Clin Immunol 2005; 115: S 163 5

Invited presentations - Rabin § AAAAI Annual Meeting, March 2004: § § FDA Food Invited presentations - Rabin § AAAAI Annual Meeting, March 2004: § § FDA Food and Cosmetics Act as it Applies to Research Studies Paul-Ehrlich-Seminar, October 2005: § Recombinant and modified allergens: The US perspective 6

Invited presentations - Slater § American Contact Dermatitis Society, October 2004 meeting: § § Invited presentations - Slater § American Contact Dermatitis Society, October 2004 meeting: § § ACAAI Annual Meeting, November 2004, Immunotherapy Collegium: § § Natural rubber latex allergy Allergen immunotherapy in the era of uncertainty AAAAI Annual Meeting, March 2005: § Allergen identification 7

Outside collaborations n Larry G. Arlian, Ph. D n n n Director, Microbiology and Outside collaborations n Larry G. Arlian, Ph. D n n n Director, Microbiology and Immunology, Department of Biological Sciences, Wright State University, Dayton, Ohio Patrick R. Murray, Ph. D n Chief, Microbiology Service, NIH Clinical Center, Bethesda, Maryland Immunotherapy Committee, AAAAI n n Harold Nelson, MD Peter L. Collins, Ph. D n n Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland Mario Roederer, Ph. D n Vaccine Research Center, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland 8

Allergy and asthma are T cell dependent n n T cells are allergen responsive Allergy and asthma are T cell dependent n n T cells are allergen responsive and secrete cytokines involved in the allergic response (IL-4, IL-5, and IL-13) Allergen immunotherapy works by modifying T cell responses Novel approaches towards modifying T cell responses may provide novel therapeutics for treatment of allergic diseases and asthma The link between respiratory viral infections and wheezing is mediated by T cells n Understanding T cell responses to respiratory viruses will provide insight into the mechanisms of allergy and asthma 9

Allergy and asthma are T cell dependent n n T cells are allergen responsive Allergy and asthma are T cell dependent n n T cells are allergen responsive and secrete cytokines involved in the allergic response (IL-4, IL-5, and IL-13) Allergen immunotherapy works by modifying T cell responses Novel approaches towards modifying T cell responses may provide novel therapeutics for treatment of allergic diseases and asthma The link between respiratory viral infections and wheezing is mediated by T cells n Understanding T cell responses to respiratory viruses will provide insight into the mechanisms of allergy and asthma 10

MK-571, an inhibitor of Multidrug Resistant Protein 1 (MRP 1) blocks T cell activation MK-571, an inhibitor of Multidrug Resistant Protein 1 (MRP 1) blocks T cell activation 11

observation Activation affects [probe] transporter [probe] reflects gene expression of probe Does probe transporter observation Activation affects [probe] transporter [probe] reflects gene expression of probe Does probe transporter modulate activation? 12

MDR Family • Proteins that transport substances across cellular membranes, against a concentration gradient, MDR Family • Proteins that transport substances across cellular membranes, against a concentration gradient, in an energy dependent manner. • ABC proteins (ATP Binding Cassette) that contain distinctive nucleotide binding domains (NBD). • Genes are highly conserved across species. • First member is MDR 1 (P-glycoprotein, P-gp); best substrates are large hydrophobic cations. • MDR-associated Resistant Protein-1 (MRP 1) described in 1992; substrates are organic anions, and also: glutathione, glucuronide, & sulfate conjugates LTC 4 13

MDR family gene expression in T cells 35 cycle RT-PCR specific products CD 4 MDR family gene expression in T cells 35 cycle RT-PCR specific products CD 4 CD 8 Cord CD 4 3 days 0 day Monocytes NK cells B cells memory naive ACTIN MDR 1 MRP 3 14

MK-571 blocks morphologic changes associated with PBMC activation TSST-1 10 ng/ ml Control MK-571 MK-571 blocks morphologic changes associated with PBMC activation TSST-1 10 ng/ ml Control MK-571 (100µM) 40 x 15

MK-571 decreases expression of CD 69 in response to superantigens 16 MK-571 decreases expression of CD 69 in response to superantigens 16

The MRP 1 inhibitor MK-571 decreases IFN-g and IL-4 by superantigen stimulated CD 4 The MRP 1 inhibitor MK-571 decreases IFN-g and IL-4 by superantigen stimulated CD 4 T cells 17

The MRP 1 inhibitor MK-571 blocks cytokine secretion 18 The MRP 1 inhibitor MK-571 blocks cytokine secretion 18

Increased PPARg activation in Jurkat T cells treated with MK-571 PHA+PGJ 2 Med PHA+MK Increased PPARg activation in Jurkat T cells treated with MK-571 PHA+PGJ 2 Med PHA+MK PHA+CE Blank Jurkat T cells treated with PHA for 24 hours, and then PPARg agonists or MK-571 for 1 hour prior to harvest. MK: MK-571 50 u. M CE: Ciglitazone 20 u. M PGJ 2: Prostaglandin J 2 10 u. M Lane 1 2 3 4 5 6 19

Conclusions n n n Inhibition of MRP 1 with MK-571 blocks T cell activation Conclusions n n n Inhibition of MRP 1 with MK-571 blocks T cell activation MK-571 does not decrease the viability of the activated cells (not shown) “Washout” of MK-571 reverses the inhibition of activation (not shown) Treatment of cells with MK-571 activates the transcriptional repressor PPARg Hypothesis: endogenous ligands for PPARg are retained in MRP 1 blocked cells 20

Allergy and asthma are T cell dependent n n T cells are allergen responsive Allergy and asthma are T cell dependent n n T cells are allergen responsive and secrete cytokines involved in the allergic response (IL-4, IL-5, and IL-13) Allergen immunotherapy works by modifying T cell responses Novel approaches towards modifying T cell responses may provide novel therapeutics for treatment of allergic diseases and asthma The link between respiratory viral infections and wheezing is mediated by T cells n Understanding T cell responses to respiratory viruses will provide insight into the mechanisms of allergy and asthma 21

Regulation of T cell responses by the Respiratory Syncytial Virus 22 Regulation of T cell responses by the Respiratory Syncytial Virus 22

RSV inhibition of T cell proliferation n RSV depresses proliferation of PBMC to PHA, RSV inhibition of T cell proliferation n RSV depresses proliferation of PBMC to PHA, EBV, or to RSV antigens in vitro (Roberts, 1982; Preston et al, 1992). RSV stimulates inhibitors of proliferation such as prostaglandins (Panuska et al, 1990), “IL-1 inhibitors” (Roberts et al, 1986 and interferon-a (Preston et al, 1995). Direct contact with RSV F (fusion protein) is necessary and sufficient to inhibit proliferation of lymphocytes (Schlender et al, 2002). 23

Experimental approach § § Published data have implicated various cytokines and inhibitors, or contact Experimental approach § § Published data have implicated various cytokines and inhibitors, or contact dependency, most unconfirmed or refuted Goal: Develop a simplified model to determine which cells are necessary and/or sufficient for inhibition of proliferation by RSV. 24

Inhibition of SEB-induced T cell proliferation by RSV DC + RSV 4 hr 4 Inhibition of SEB-induced T cell proliferation by RSV DC + RSV 4 hr 4 days L + SEB H 3 thymidine uptake assay DC = monocyte-derived dendritic cells SEB = staphylococcal enterotoxin B 25

RSV significantly inhibits CMV specific and SEB-activated T cell proliferation 26 RSV significantly inhibits CMV specific and SEB-activated T cell proliferation 26

Do CD 8 T cells, NK cells, or B cells mediate immunosuppression? DC + Do CD 8 T cells, NK cells, or B cells mediate immunosuppression? DC + Virus DC L 4 hr 4 days + Virus CD 4 4 hr + SEB H 3 thymidine uptake assay 4 days + SEB H 3 thymidine uptake assay 27

CD 4 T cells and DC are sufficient for suppression of proliferation by RSV CD 4 T cells and DC are sufficient for suppression of proliferation by RSV P>0. 05 P< 0. 05 P=0. 003 P<0. 05 SEB SEB RSV SEB FLU SEB paraflu 28

Dendritic cells are productively infected by GFP-RSV 29 Dendritic cells are productively infected by GFP-RSV 29

Brightfield view of dendritic cells 30 Brightfield view of dendritic cells 30

Can suppressive activity be transferred with DC supernatants? + DC RSV ON Supernatant Add Can suppressive activity be transferred with DC supernatants? + DC RSV ON Supernatant Add to CD 4 4 days + SEB H 3 thymidine uptake assay 31

Inhibition of CD 4+ T cell proliferation by DC supernatant P = 0. 001 Inhibition of CD 4+ T cell proliferation by DC supernatant P = 0. 001 P>0. 05 DC sup: UV RSV control RSV 32

Conclusions n n We have simplified the experimental system of RSVmediated immunosuppression to monocyte-derived Conclusions n n We have simplified the experimental system of RSVmediated immunosuppression to monocyte-derived DC and CD 4 T cells Immunosuppressive activity can be transferred with supernatants from infected DC, and is not due to carry over of virus within the supernatants 33

Acknowledgments Laboratory of Immunobiochemistry CBER, FDA Marc Alston Jinsong Zhang Hui Huang Bo Chi Acknowledgments Laboratory of Immunobiochemistry CBER, FDA Marc Alston Jinsong Zhang Hui Huang Bo Chi Jay Slater Laboratory of Infectious Diseases NIAID, NIH Peter L. Collins Immunotechnology Section VRC, NIH Steve Perfetto Mario Roederer Laboratory of Biophysics CBER, FDA Rich Pastor 34