Laboratory of Immunobiochemistry

1
Laboratory of Immunobiochemistry

• Research update

2
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
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)4363-9.
• 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
Publications

• Review
• Slater JE. Recombinant allergens in the US.
  Methods 2004 32(3)209-11.
• Slater JE. Latex allergens. Clin Allergy Immunol
  200418369-86.
• Slater JE. Standardized allergen extracts in the
  United States. Clin Allergy Immunol
  200418421-32.
• 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

5
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 115S226
• 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 115S163
• 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
  115S164
• 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 115S255
• C Valerio, LG Arlian, JE Slater. Bacterial 16S
  ribosomal DNA sequences isolated from house dust
  mites. J Allergy Clin Immunol 2005 115S163

6
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
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
Outside collaborations

• 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

• 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

9
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
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
MK-571, an inhibitor of Multidrug Resistant
Protein 1 (MRP1) blocks T cell activation
12
observation
13
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
• LTC4

14
MDR family gene expression in T cells35 cycle
RT-PCR specific products
CD4
CD8
Cord CD4
memory
naive
memory
naive
B cells
NK cells
0 day
3 days
Monocytes
ACTIN
15
MK-571 blocks morphologic changes associated with
PBMC activation
TSST-1 10 ng/ ml
Control
MK-571 (100µM)
40x
16
MK-571 decreases expression of CD69 in response
to superantigens
17
The MRP1 inhibitor MK-571 decreases IFN-g and
IL-4 by superantigen stimulated CD4 T cells
18
The MRP1 inhibitor MK-571 blocks cytokine
secretion
19
Increased PPARg activation in Jurkat T cells
treated with MK-571
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 50uM CE Ciglitazone
20uM PGJ2 Prostaglandin J2 10uM
20
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 PPARg
• Hypothesis endogenous ligands for PPARg are
  retained in MRP1 blocked cells

21
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
Regulation of T cell responses by the Respiratory
Syncytial Virus
23
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-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).

24
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
Inhibition of SEB-induced T cell proliferation by
RSV
DC monocyte-derived dendritic cells SEB
staphylococcal enterotoxin B
26
RSV significantly inhibits CMV specific and
SEB-activated T cell proliferation
27
Do CD8 T cells, NK cells, or B cells mediate
immunosuppression?
28
CD4 T cells and DC are sufficient for suppression
of proliferation by RSV
Pgt0.05
Plt 0.05
P0.003
Plt0.05
SEB SEB
SEB SEB
RSV FLU paraflu
SEB SEB
SEB SEB
RSV FLU paraflu
29
Dendritic cells are productively infected by
GFP-RSV
30
Brightfield view of dendritic cells
31
Can suppressive activity be transferred with DC
supernatants?
32
Inhibition of CD4 T cell proliferation by DC
supernatant
P 0.001
Pgt0.05
DC sup UV RSV control RSV
33
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
Acknowledgments