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IVIg and IVIg Mimetic: HLA-reactivity and Immunosuppression

IVIg and IVIg Mimetic: HLA-reactivity and Immunosuppression. Mepur H. Ravindranath, Paul I. Terasaki, Tho Pham, Vadim Jucaud, Satoru Kawakita, Dong Zhu, Michiko Taniguchi, Judy Hopefield. Terasaki Foundation Laboratory. Los Angeles, CA 90064. Background. Investigations of

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IVIg and IVIg Mimetic: HLA-reactivity and Immunosuppression

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  1. IVIg and IVIg Mimetic: HLA-reactivity and Immunosuppression Mepur H. Ravindranath, Paul I. Terasaki, Tho Pham, Vadim Jucaud, Satoru Kawakita, Dong Zhu, Michiko Taniguchi, Judy Hopefield Terasaki Foundation Laboratory. Los Angeles, CA 90064

  2. Background Investigations of Dr. Stanley Jordan, Dr. Denis Glotz, Dr. Srini Kaveri on suppression of antibody production by IVIg & Dr. Steve Woodle’spaper (1994, J Immunol. 153(3):1054-67) on suppression of T cell activation by a mAb binding to the a3 of HLA-I prompted us to have a closer look into the properties of IVIg & the potential of mAbs that mimic IVIg HLA-reactivity.

  3. IVIg reacts with HLA class I Blood 2013, 21(11):2013- 28 While this is so, how come IVIg is capable of suppressing Anti-HLA DSA and NDSA?

  4. All IVIgs have high level of anti-HLA-E GamaSTANTM Blood 2013, 21(11):2013- 28

  5. Adsorption of anti-HLA-E Abs from IVIg abolishes its HLA-Ia reactivity Serial dilution of HLA-E Absorbed and Unabsorbed IVIg Loss of HLA-Ia reactivity after adsorption of anti-HLA-E. Loss of anti-HLA-E Abs after adsorption. Blood 2013, 21(11):2013- 28

  6. IVIg may recognize HLA- class I shared-peptides like some anti-HLA-E mAbs, Mol. Immunol. 47(5): 1121-1131, 2010 Number of HLA-Alleles Examined Sharedversus HLA-E specific Peptides A B C F G

  7. HLA-E b2m-free heavy chain generated mAbs HLA-E restricted (Group 1) versus Class-I-shared (Group 8) peptides

  8. HLA-I shared peptide sequences inhibited the binding of Group 8 to both HLE-E & HLA-I Mol. Immunol. 48(4): 423-430, 2011

  9. Binding site explains the behavior of Group 8 mAbs

  10. PHA activates CD4+ T-cells Proliferation Blastogenesis 93 No PHA No PHA 684 with PHA with PHA

  11. PHA-Activation = CD3-phosphorylation PHA TCR TCR CD4 CD4 CD3 PHA ACTIVATION OF T-Cells CD3 RESTING T-LYMPHOCYTES CD3 CD3 P P P SH2 P P SH2 SH3 LCK P SH2 P P P • (Time sequence 0 -12 Hrs) Signal Transduction Cell Tissue Res. 201(1):101-27. Biochem. J. (2013) 454, 169–179

  12. IVIg (GlobEx) suppresses Activated CD4+ T lymphocytes Blastogenesis Proliferation With PHA Note the decrease PHA + IVIg (1/10) X

  13. Anti-HLA-E mAbs (TFL-006/TFL-007) mimic IVIg Blood 2013 21(11):2013- 28 • Are they capable of suppressing Activated T cells?

  14. TFL-007s suppresses Activated CD4+ & CD8+ T lymphocytes Blastogenesis Proliferation with PHA Note the decrease in the numbers PHA + TFL-007s (1/10) PHA + TFL-007s (1/100) X

  15. Dosimetric Inhibition of Blastogenesis of CD4+ & CD8+ T cells by TFL-006 CD4+ CD8+

  16. IVIg-mimeticsTFL-006s/TFL-007s suppress blastogenesis of activated CD4+ T cells but not the non-mimetic of IVIg (TFL-037s) Blastogenesis

  17. IVIg-mimetic TFL-006s suppresses proliferation of activated CD4+ T cells but not the non-mimetic of IVIg (TFL-037s) Proliferation Proliferation No PHA with PHA PHA + TFL-006s (1/10) + TFL-037s (1/10) + TFL-006s (1/100)

  18. Negative Control Abs & anti-HLA-E IVIg-non-mimitics (TFL-033s/TFL-037s) do not to inhibit blastogenesis of CD4+ T cells in contrast to IVIg-mimetics (TFL-006/TFL-007)

  19. Suppression of Blastogenesis and Proliferation of Activated CD4+ T cells by IVIg-mimetics may involve CD3-dephosphorylation PHA TCR CD4 IL2Rα CD3 CD3 Expression of open conformer with exposed shared peptide on the cell surface after Activation Step 1 TFL-006/007 & IVIg binding to the shared peptides on the Open conformer P P P SH2 P P Step 2 SH2 SH3 TFL-006/007 X LCK P P P P SH2 Signals Dephosphorylation of CD3 Step 3 Signal Transduction X P Reversible Phosphorylation after mAb binding to the shared peptides on the Open conformer P

  20. How does TFL-IVIg mimetic compare with IVIg in suppressing Allo-HLA IgG production by lymphokine-stimulated B lymphocytes ?

  21. First case Activated Bmem cells from PBL of a woman alloimmunized postpartum 23 years ago, and grown in a well-defined culture system generated by Dr.Toru Miyazaki.

  22. JH developed postpartum alloantibodies against DRB allele of her husband >20 yrs ago. The long-lived B cells still secrete the Abs for >20 years. Dilutions

  23. Bmem cells (CD19+/CD20-/CD27+/CD38+) were isolated, activated, cultured & examined for secretion of anti-HLA IgG Day 0 Day 7 CD19+/CD 20- CD38 CD27 Before Activation No Activation Post-activation from fresh Blood from culture from culture

  24. Primary Alloantibody DRB1*0101 Medium Control Paired sample (12-72 hrs) IVIg-GamaSTAN p2=0.0005 p2=0.01 mAb TFL-007 4500 43% 3000 28% 46% 40% 72% MFI of secreted IgG 1500 62% 62% 0 0 12 24 72 48 IgG production by B cells in vitro (hours)

  25. Secondary Alloantibody DRB1*1402 Medium Control Paired sample (12-72 hrs) IVIg-GamaSTAN p2=0.0005 p2=0.01 mAb TFL-007 4500 3000 MFI of secreted IgG 1500 58% 40% 63% 75% 0 0 12 24 72 48 IgG production by B cells in vitro (hours)

  26. Second Case an hybridoma cell line (HML16) that secretes IgG that reacts with B*0702 & B*0801, from another postpartum alloimmunized womengenerated by Dr. Toru Miyazaki

  27. Anti-HLA B*0801 IgG production by the hybridoma cell line(HML16) mAb TFL-007 IVIg 0.1 0.05 0.025 0.0125 Concentration of mAb (mg/ml) 15 10 7.5 5 3.8 2.5 Concentration of IVIg (mg/ml) mAb TFL-007 suppresses the Production of anti-B*0801 IgG Significantly IVIg fails to suppress the Production of anti-B*0801 IgG

  28. Anti-HLA IgG production by the hybridoma cell line (HML16) B*0702 IVIg mAb TFL-007 15 10 7.5 5 3.8 2.5 Concentration of IVIg (mg/ml) 0.1 0.05 0.025 0.0125 Concentration of mAb (mg/ml) IVIg fails to suppress the Production of anti-B*0702 IgG mAb TFL-007 suppresses the Production of anti-B*0702 IgG Significantly

  29. Conclusions Anti-HLA-E mAbs TFL-006/007 produced at TFL mimics HLA class-I reactivity of IVIg. These, but not those that do not mimic IVIg, suppressed blastogenesis & proliferation of activated CD4+ T-cells. mAb TFL-007 suppressed significantly the allo-HLA Abs produced by stimulated Bmem cells and by the immortalized B cells, much better than IVIg. IVIg-mimetics are well defined & purified monoclonal IgG2a capable of better Immunosuppression than IVIg. Suppression mechanism involves binding of F(ab’)2 of the mAb to shared epitopes on the HLA open conformers. Concentration of TFL-mAbs required for suppression of activated CD4+ T cells and IgG production of B cells is 50 to 100-fold less than that of IVIg.

  30. We, the TFL Family, Pray and Wish Professor Terasaki the Best of Health, Happiness and Continuous Guidance

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