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(Multi) virus specific T cells

(Multi) virus specific T cells. Spyridonidis Alexandros SCAT Lab Lab Meeting 03/22/2012. Cellular Immunotherapy for the treatment of Viral infection and reactivation after Bone Marrow Transplantation. major cause of morbidity and mortality CMV, EBV, AdV, BK, HHV-6, HHV-7, RSV

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(Multi) virus specific T cells

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  1. (Multi) virus specific T cells SpyridonidisAlexandros SCAT Lab Lab Meeting 03/22/2012

  2. Cellular Immunotherapy for the treatment of Viral infection and reactivation after Bone Marrow Transplantation • major cause of morbidity and mortality • CMV, EBV, AdV, BK, HHV-6, HHV-7, RSV • Depending on protocol, patient characteristics etc.. • Obligatory for Haplo protocols • drug treatments • Efficacy • Partial effective (CMV, …..Adv?, RSV?) • Not effective (EBV, BK, HHV-6, HHV-7) • Side effects (cytopenias, renal insufficiency) • Expensive • Render viruses resistant • Why cellular immunotherapy • Central role of immune system • inverse relationship between viral antigenemia and circulating virus-reactive T cells • 40% CMV reactivation after autologous BMt but <5% disease • Efficacy • Clinical studies (Seattle, Baylor, Wurzburg, Leiden)

  3. Adoptive cell therapy IFN-g capture

  4. Variables in generation of virus specific T cells • APCs (DCs) • Mo DCsvs Plasmacytoid DCs • TLR • Virus stimuli • peptide mix (CD4, CD8, polyclonal) • Other • Culture • Media • Cytokines, others • IL-7 (10 IU/ml), IL-15 (5 IU/ml), IL-2 (100 IU), IL-12 • Wnt β- catenin inhibitor • T cell Donor • Initial donor • Patient (?) • Third party

  5. Product • Potency markers • Viable • CD4 and CD8 • Polyclonal? (minimize viral escape) • Tcm, IL-21R expression (for long term in vivo repopulation) • Efficacy • IFNγ –producing • Other tests (lysing ability etc) • Number • Probably few are enough. Efficacy has been shown with • IFNγ- capture assay: 1.2 x10e3 cells/ kg (AdV) • If a cellular product of 10x10e6 cells/kg contains 1% virus specific cells = 1x10e5 cells / kg (virus specific) • Reproducible • Lack of alloreactivity • Purity? • GMP criteria / release • GMP materials • Sterility • Identity

  6. Generation of human dendritic cells Immature DCs LPS 30ng/mL IFN-γ 100IU/mL elutriated monocytes GM-CSF 800IU/mL IL-4 1000IU/mL mature DCs • 10% FCS, NABS • X Vivo serum Day 3-5 Day7 Day 0 CD11b HLA-DR CD80 CD86 CD83 FSC Mature DC Immature DC Vivid SSC Vivid SSC

  7. Generation of virus specific CTLs Lymphos Ellutriated Monocytes Immature DCs mature DCs (Re-expansion) Patient 1st stim (x days) 2nd stim (x days) D14? D21? Day 0 D3-5 D5-7 In vitro evaluation In vivo evaluation pulse stimulate Re-stimulate • Yes or no • DC or monos • Pulse? • Days? • competition? Which virus? • Which virus (CMV, EBV, • BK, ADV, … HHV-6?) • Which pep mix? • Peptide dose • Wash vs not wash • Competition? • DC: lympho ratio • Cytokines (IL-7, • IL-15, IL-2, IL-4?) • Days? • methodology • methodology

  8. Important variable: Split the cells!! • Split • No split CD3 CD14-CD19-Vivid

  9. FACS analysis • CD3, CD4, CD8, (CD27 CD45RO) • IFN-g • TNFa • IL-2 • CD154 • CD107 CD4 V500 FCS CD3 efluo 610 SCS FCS CD8 H7-APC FCS CD14-CD19-Vivid

  10. Donor 32102, CMV+, 1st stim CD8+ cells CD4 uDC/SEB uDC/- CD8 EBV/LMP1- EBV/BLFZ1- BK/ST- ADV- MIX- CMV- BK/LT- SS EBV/LMP1+ EBV/BLFZ1+ BK/ST+ ADV+ MIX+ CMV+ BK/LT+ IFN-g(PE)

  11. Donor 32102, CMV+, 1st stim CD4+ cells CD4 uDC/SEB uDC/- CD8 EBV/LMP1- EBV/BLFZ1- BK/ST- ADV- MIX- CMV- BK/LT- SS EBV/LMP1+ EBV/BLFZ1+ BK/ST+ ADV+ MIX+ CMV+ BK/LT+ IFN-g (PE)

  12. Donor 32102, CMV+, 2nd stim CD8+ cells CD4 uDC/SEB uDC/- CD8 EBV/LMP1- EBV/BLFZ1- BK/ST- ADV- MIX- CMV- BK/LT- SS EBV/LMP1+ EBV/BLFZ1+ BK/ST+ ADV+ MIX+ CMV+ BK/LT+ IFNg (PE)

  13. Donor 100108, CMV+, 1st stim CD4+ cells CD4 uDC/- uDC/SEB CD8 MIX- EBV/LMP2- EBV/BLFZ1- BK/VP1- CMV- BK/LT- SS EBV/LMP2+ EBV/BLFZ1+ BK/VP1+ MIX+ CMV+ BK/LT+ IFN-g (PE)

  14. Mutlivirus CTLs from different donors CD8+ 1 stim CD4+ CD8+ 2 stim CD4+ IFN-g (PE)

  15. The role of CMV in CMV seropos patients

  16. Donor 100108, CMV+, 2nd stim 2nd stim Irradiated monos +/- CMV pulsed CD4+ cells SEB unpulsed CMV- EBV/LMP2- EBV/BLFZ1- BK/LT- BK/VP1- MIX- MIX+ (CMV CTLs) CMV+ EBV/LMP2+ EBV/BLFZ1+ BK/LT+ BK/VP1+ MIX+

  17. Donor 100108, CMV+, 2nd stim 2nd stim Irradiated monos +/- CMV pulsed CD8+ cells SEB unpulsed CMV- EBV/LMP2- EBV/BLFZ1- BK/LT- BK/VP1- MIX- MIX+ (CMV CTLs) CMV+ EBV/LMP2+ EBV/BLFZ1+ BK/LT+ BK/VP1+ MIX+

  18. Donor 60607, CMV+, 2nd stim CD8+ cells CMVpp65 - CMV/IE1 - CMVpp65 + CMV/IE1 + SS IFN-g (PE)

  19. Which peptides?

  20. 1 or 2 stimulations?

  21. Variability

  22. The tough case CMV seronegative donors One stimulation

  23. Exp #13 Donor 11310, CMV- 1st stim

  24. Donor 11310, CMV-, 1st stim CD4+ cells uDC/SEB uDC/- CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ IFN-g (PE)

  25. Donor 11310, CMV-, 1st stim CD4+ cells uDC/SEB uDC/- CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ IL-2 (PE-efluo710)

  26. Donor 11310, CMV-, 1st stim CD8+ cells uDC/- uDC/SEB CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ IFN-g (PE)

  27. Donor 11310, CMV-, 1st stim CD8+ cells uDC/- uDC/SEB CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ TNFa (PE-Cy7)

  28. Donor 11310, CMV-, 1st stim CD8+ cells uDC/- uDC/SEB CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ IL-2 (PE-efluo710)

  29. Donor 11310, CMV-, 1st stim CD4+ CD8+ CMVpp65+ EBV/ BLFZ1+ ADV + MIX+ CD45 RO (APC) CD45 RO (APC) SS SS IFNg(PE) CD27 (FITC) IFNg(PE) CD27 (FITC)

  30. Exp. #14, Stim #2, Donor 101310 EBV EBNA1 EBV EBNA1 BKV ST-1 BKV ST-1 Adv. Hex5 Adv. Hex5 CMV IE-1 CMV IE-1 EBV BLZF1 EBV BLZF1 CMV pp65 CMV pp65 Mix-CMV Mix-CMV Mix +CMV Mix +CMV CD4+ SSC-A IFN-γ CD8+ SSC-A IFN-γ

  31. Exp. #14, Stim #2, Donor 101310 EBV EBNA1 EBV EBNA1 BKV ST-1 BKV ST-1 Adv. Hex5 Adv. Hex5 CMV IE-1 CMV IE-1 EBV BLZF1 EBV BLZF1 CMV pp65 CMV pp65 Mix-CMV Mix-CMV Mix +CMV Mix +CMV CD4+ SSC-A TNF-α CD8+ SSC-A TNF-α

  32. Exp #13 Donor 012180, CMV- 1st stim

  33. Donor 012180, CMV-, 1st stim CD4+ cells uDC/SEB uDC/- CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ IFN-g (PE)

  34. Donor 012180, CMV-, 1st stim CD4+ cells uDC/SEB uDC/- CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ IL-2 (PE-efluo710)

  35. Donor 012180, CMV-, 1st stim CD8+ cells uDC/- uDC/SEB CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ IFN-g (PE)

  36. Donor 012180, CMV-, 1st stim CD8+ cells uDC/- uDC/SEB CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ TNFa (PE-Cy7)

  37. Donor 012180, CMV-, 1st stim CD8+ cells uDC/- uDC/SEB CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ IL-2 (PE-efluo710)

  38. Exp #13 Donor 062311, CMV- 1st stim

  39. Donor 062311, CMV-, 1st stim CD4+ cells uDC/SEB uDC/- CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ IFN-g (PE)

  40. Donor 062311, CMV-, 1st stim CD4+ cells uDC/SEB uDC/- CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ TNF-a (PE-Cy7)

  41. Donor 062311, CMV-, 1st stim CD8+ cells uDC/- uDC/SEB CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ IFN-g (PE)

  42. Donor 062311, CMV-, 1st stim CD8+ cells uDC/- uDC/SEB CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ TNFa (PE-Cy7)

  43. Exp #13 Donor 090110, CMV- 1st stim

  44. Donor 090110, CMV-, 1st stim CD4+ cells uDC/SEB uDC/- CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ IFN-g (PE)

  45. Donor 090110, CMV-, 1st stim CD8+ cells uDC/- uDC/SEB CMVpp65- CMV-IE1- EBV-EBNA1- EBV/BLFZ1- BK-LT- ADV - MIX- SS CMVpp65+ CMV-IE1+ EBV-EBNA1+ EBV/BLFZ1+ BK-LT+ ADV + MIX+ IFN-g (PE)

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