Optimized clinical-scale culture conditions for ex vivo selective depletion of host-reactive donor lymphocytes: a strategy for GvHD prophylaxis in allogeneic PBSC transplantation.
BACKGROUND
Ex vivo selective depletion (SD) is a strategy to prevent GvHD, in which host-reactive donor lymphocytes are selectively eliminated from a PBSC allograft while conserving useful donor immune function. Prior to testing this strategy in patients, our goal was to develop a clinical-scale SD process, which involves co-culture of donor lymphocytes and irradiated recipient cells, followed by the addition of an immunotoxin (IT) directed against the alpha-chain of the IL-2 receptor (CD25), expressed on activated donor T cells.


METHODS
Stimulator cells were generated from immunomagnetically selected and expanded recipient T lymphocytes. Donor PBMCs from G-CSF-mobilized peripheral blood were co-cultured for 72 h with irradiated stimulator cells. Alloreactive T cells were targeted for elimination by the addition of the anti-CD25 IT, RFT5-SMPT-dgA, and the IT enhancer, NH(4)Cl.


RESULTS
Stimulator-cell selection/expansion yielded > 2 x 10(10) highly enriched CD3(+) cells (98.9 +/- 2.2%). After SD, cell recovery was 68.5 +/- 23.3% and viability was 84.6 +/- 6.4%. This permitted a potential T-cell dose >/= 1 x 10(8) CD3(+) cells kg(-1) to transplant recipients. Although SD donor lymphocytes retained little proliferative capacity against the original stimulator cells (2.6 +/- 0.6%), responses were conserved against third party cells (107.6 +/- 18.6%), the bacterial superantigen staphylococcus enterotoxin B (108.2 +/- 4.2%), and CMV Ag (72.1 +/- 3.8%).


CONCLUSIONS
We have demonstrated that ex vivo SD is feasible in clinical-scale culture conditions. The ability of this strategy to prevent GvHD is the subject of an ongoing clinical trial, in which the SD lymphocyte product is transplanted in conjunction with a T cell-depleted PBSC allograft.
Solomon
S R
SR
Stem Cell Allotransplantation Section, Hematology Branch, NHLBI, National Institutes of Health, Bethesda, MD 20892-1652, USA.
Tran
T
T
Carter
C S
CS
Donnelly
S
S
Hensel
N
N
Schindler
J
J
Bahceci
E
E
Ghetie
V
V
Michálek
J
J
Mavroudis
D
D
Read
E J
EJ
Vitetta
E S
ES
Barrett
A J
AJ
D017252:CD3 Complex / Q000096:biosynthesis; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D003470:Culture Media; D016207:Cytokines / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005615:Freezing; D006086:Graft vs Host Disease / Q000517:prevention & control*; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D016130:Immunophenotyping; D007963:Leukocytes, Mononuclear; D008213:Lymphocyte Activation; D008212:Lymphocyte Depletion; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000502:physiology*; D013997:Time Factors; D014184:Transplantation, Homologous / Q000379:methods*
D017252:CD3 Complex; D003470:Culture Media; D016207:Cytokines; D015375:Receptors, Interleukin-2