MK-571, an inhibitor of Multidrug Resistant Protein 1 (MRP1) selectively blocks T cell activation and may reveal novel d

1. MK-571, an inhibitor of Multidrug Resistant Protein 1 (MRP1) selectively blocks T cell activation and may reveal novel drug targets for immunomodulation Ronald L. Rabin, M.D. Laboratory of Immunobiochemistry rabin@cber.fda.gov

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

3. 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: • Doxorubicin • Ritonavir • Calcein-AM • Verapamil • MDR-associated Resistant Protein-1 (MRP1) described in 1992; 15% homologous to MDR, more closely related to CFTR: • organic anions • glutathione, glucuronide, & sulfate conjugates • LTC4 • Calcein

4. MDR Family members Percent identity between MDR members

5. MDR1 MRP1 MRP2 MRP3 MRP5 MRP6 MXR MDR family gene expression in T cells35 cycle RT-PCR specific products CD4 CD8 Cord CD4 naive memory naive memory B cells NK cells 3 days 0 day Monocytes ACTIN MRP4

6. MK-571 is an inhibitor of MRP1 TSST-1 10 ng/ ml Control MK-571 (100µM) 40x

7. The MRP1 inhibitor MK-571 decreases expression of CD69 in response to superantigens

8. The MRP1 inhibitor MK-571 decreases IFN-g but not IL-4 TSST-1 stimulated human CD4 T cells

9. IFN-g % of max IFN-g+ (Percent of CD4 T cell) Relative Induction IFN-g (Log2) The MRP1 inhibitor MK-571 blocks IFN-g synthesis

10. Log10 cytokine concentration (pg/ml) The MRP1 inhibitor MK-571 blocks cytokine secretion

11. The MRP1 inhibitor MK-571 blocks TNF-a secretion by human macrophages TNF-a (pg/ml x 103) in response to LPS TNF-a (pg/ml x 103) in response to SAC MK-571 (µM)

12. IL-6 (pg/ml) in response to LPS or SAC The MRP1 inhibitor MK-571 does not block IL-6 secretion by human macrophages MK-571 (µM)

13. The MRP1 inhibitor MK-571 blocks calcium flux in response to T cell receptor cross-linking Ratio fluorescence (violet/blue) Relative number of cells

14. unst cntrl 0 MK-571 25 MK-571 50 MK-571 Relative number of cells Ratio fluorescence violet/blue) The MRP1 inhibitor MK-571 blocks calcium flux in response to the chemokine SDF-1a (CXCL12)

15. MRP1 blockade is reversible Percent IFN-g+ CD4 T cells Percent Inhibition

16. The MRP1 inhibitor MK-571 is ineffective if added two hours after T cell stimulation Percent IFN-g+ CD4 T cells

17. Is MRP4 the Th2 counterpart to MRP1? •MRP4 transports “the other” arachidonic acid products, PGA2, PGE1, PGE2, and PGF1a and PGF2a, and PGD2. •PGE1 down-modulates Th1 activity and up-modulates Th2 activity •The PGD2 receptor, CRTH2 is used to define Th2 cells •Do Th2 cell lines express more MRP4 than Th1 lines? Reid et al. PNAS 100:9244; 2003

18. Th1 Th2 MRP1 MRP4 MRP1/MRP4 Relative level of expression Ratio expression MRP1/MRP4 Increased expression of MRP4 in Th2 cell lines

19. MRP1 MRP4 Normalized expression Relative level of expression RSV may increase expression of MRP4 in Human Monocyte Derived Dendritic Cells

20. Conclusions MRP1 blockade abrogates: type 1 (e.g. IFN-g) T cell function calcium flux after TcR stimulation calcium flux after chemokine receptor stimulation MRP4 may regulate type 2 T cell responses analogous to MRP1/Type 1. Defining the mechanisms by which MRP1 and MRP4 regulate T cell function may provide novel drug targets for immunomodulation.

21. 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 Laboratory of Biophysics CBER, FDA Steve Perfetto Mario Roederer Rich Pastor Acknowledgments