Laboratory of Immunobiochemistry Research update

2/34 Active research projects PI Rabin MDR proteins in T cell activation Regulation of T cell responses by the Respiratory Syncytial Virus PI Slater Cockroach allergen standardization Determination of optimal surrogate test Depletion analysis of CR extracts Endotoxin in allergen vaccines

3/34 Publications Research  Spann KM, Tran KC, Chi B, Rabin RL, Collins PL. Suppression of the induction of alpha, beta, and lambda interferons by the NS1 and NS2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages. J Virol 2004; 78(8):  Song K, Rabin RL, Douek D, Roederer M, Farber JM. Novel Subsets of CD4+ 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 (MRP1) is induced upon activation of human T cells, and its inhibition blocks T cell function, in preparation

4/34 Publications Review  Slater JE. Recombinant allergens in the US. Methods 2004; 32(3):  Slater JE. Latex allergens. Clin Allergy Immunol 2004;18:  Slater JE. Standardized allergen extracts in the United States. Clin Allergy Immunol 2004;18:  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

5/34 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:S226 NC deVore, WJJ Finlay, EN Dobrovolskaia, A Gam, JE Slater. Cloning and analysis of mono-specific single chain fragment variable scFv 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:S163 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:S164 J Zhang, MA Alston, H Huang, RA Houghtling, RW Pastor, RL Rabin. Human type 1 CD4 T cell cytokine responses are selectively dependent on Multidrug Resistance Protein1. J Allergy Clin Immunol 2005; 115:S255 C Valerio, LG Arlian, JE Slater. Bacterial 16S ribosomal DNA sequences isolated from house dust mites. J Allergy Clin Immunol 2005; 115:S163

6/34 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

7/34 Invited presentations - Slater  American Contact Dermatitis Society, October 2004 meeting:  Natural rubber latex allergy  ACAAI Annual Meeting, November 2004, Immunotherapy Collegium:  Allergen immunotherapy in the era of uncertainty  AAAAI Annual Meeting, March 2005:  Allergen identification

8/34 Outside collaborations Larry G. Arlian, PhD Director, Microbiology and Immunology, Department of Biological Sciences, Wright State University, Dayton, Ohio Patrick R. Murray, PhD Chief, Microbiology Service, NIH Clinical Center, Bethesda, Maryland Immunotherapy Committee, AAAAI Harold Nelson, MD Peter L. Collins, PhD Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland Mario Roederer, PhD Vaccine Research Center, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland

9/34 Allergy and asthma are T cell dependent 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 Understanding T cell responses to respiratory viruses will provide insight into the mechanisms of allergy and asthma

10/34 Allergy and asthma are T cell dependent 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 Understanding T cell responses to respiratory viruses will provide insight into the mechanisms of allergy and asthma

11/34 MK-571, an inhibitor of Multidrug Resistant Protein 1 (MRP1) blocks T cell activation

12/34 observation Activation affects [probe] [probe] reflects gene expression of probe transporter Does probe transporter modulate activation?

13/34 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 MDR1 (P-glycoprotein, P-gp); best substrates are large hydrophobic cations. MDR-associated Resistant Protein-1 (MRP1) described in 1992; substrates are organic anions, and also: glutathione, glucuronide, & sulfate conjugates LTC 4

14/34 MDR family gene expression in T cells 35 cycle RT-PCR specific products ACTIN MDR1 MRP1MRP3 B cellsNK cellsMonocytes 0 day 3 daysmemorynaivememorynaive CD4CD8Cord CD4

15/34 MK-571 blocks morphologic changes associated with PBMC activation TSST-1 10 ng/ ml Control 40x MK-571 (100µM)

16/34 MK-571 decreases expression of CD69 in response to superantigens

17/34 The MRP1 inhibitor MK-571 decreases IFN-  and IL-4 by superantigen stimulated CD4 T cells

18/34 The MRP1 inhibitor MK-571 blocks cytokine secretion

19/34 MK: MK uM CE: Ciglitazone 20uM PGJ 2 :Prostaglandin J 2 10uM Lane Med PHA PHA+MK PHA+CE PHA+PGJ 2 Blank Increased PPAR  activation in Jurkat T cells treated with MK-571 Jurkat T cells treated with PHA for 24 hours, and then PPAR  agonists or MK-571 for 1 hour prior to harvest.

20/34 Conclusions Inhibition of MRP1 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 PPAR  Hypothesis: endogenous ligands for PPAR  are retained in MRP1 blocked cells

21/34 Allergy and asthma are T cell dependent 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 Understanding T cell responses to respiratory viruses will provide insight into the mechanisms of allergy and asthma

22/34 Regulation of T cell responses by the Respiratory Syncytial Virus

23/34 RSV inhibition of T cell proliferation 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-  (Preston et al, 1995). Direct contact with RSV F (fusion protein) is necessary and sufficient to inhibit proliferation of lymphocytes (Schlender et al, 2002).

24/34 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.

25/34 Inhibition of SEB-induced T cell proliferation by RSV DC 4 hr RSV + SEB 4 days H 3 thymidine uptake assay L + DC = monocyte-derived dendritic cells SEB = staphylococcal enterotoxin B

26/34 RSV significantly inhibits CMV specific and SEB-activated T cell proliferation

27/34 Do CD8 T cells, NK cells, or B cells mediate immunosuppression? DC L + 4 hr Virus + SEB 4 days H 3 thymidine uptake assay CD4 + 4 hr Virus + SEB 4 days DC H 3 thymidine uptake assay

28/34 CD4 T cells and DC are sufficient for suppression of proliferation by RSV SEB SEB SEB SEB RSV FLU paraflu SEB SEB SEB SEB RSV FLU paraflu P< 0.05 P=0.003 P<0.05 P>0.05

29/34 Dendritic cells are productively infected by GFP-RSV

30/34 Brightfield view of dendritic cells

31/34 Can suppressive activity be transferred with DC supernatants? DC + ON Supernatant Add to CD4 + SEB4 days H 3 thymidine uptake assay RSV

32/34 Inhibition of CD4 + T cell proliferation by DC supernatant DC sup: UV RSV control RSV P>0.05 P = 0.001

33/34 Conclusions We have simplified the experimental system of RSV- mediated immunosuppression to monocyte-derived DC and CD4 T cells Immunosuppressive activity can be transferred with supernatants from infected DC, and is not due to carry over of virus within the supernatants

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