title: T-cell depletion of bone marrow transplants: assessment of standard immunological methods of quantification.
abstract: The efficacy of bone marrow transplant (BMT) T-cell depletion for the prevention of acute graft-versus-host disease (GVHD) has been demonstrated in animal models and in clinical studies. The importance of T-cell depletion has to be evaluated with standardized methods suitable for routine purposes. We report herein an in vitro mature T-cell depletion using a cocktail of three monoclonal antibodies (CD2, CD5, and CD7) and baby rabbit complement in 38 histocompatibility leucocyte antigen (HLA)-identical BMT with no more than grade II acute GVHD. The T-cell depletion was quantified using three prestandardized immunological methods: immunofluorescence (IF) analysis, SRBC-rosetting assay, and PHA proliferation assay. A mean of 97.5% IF-assessed T-cell depletion was achieved in the 38 BMT. The immediate IF analysis using three distinct sets of anti-T-cell monoclonal antibodies allowed us to detect a mean of 1.2% residual T cells. The SRBC-rosetting assay was not useful to quantify T-cell depletion because no residual SRBC-rosette-forming cells could be detected in every case. The results of a prestandardized PHA-induced proliferation assay gave a mean 96.7% inhibition of proliferation, and they were correlated with the IF results although the IF threshold of detection was higher. From these data we conclude that our in vitro T-cell-depletion procedure is reproducible and that standardized simple immunological methods such as immediate immunofluorescence analysis and PHA proliferation assay provide good tools to assess a T-cell depletion effective in the prevention of acute GVHD.
authors.affiliation: Laboratoire d'Immunologie Cellulaire, Hôpital Pitié-Salpétrière, Paris, France.
authors.forename: B
authors.initials: B
authors.lastname: Autran
authors.forename: F
authors.initials: F
authors.lastname: Beaujean
authors.forename: C
authors.initials: C
authors.lastname: Pillier
authors.forename: M
authors.initials: M
authors.lastname: Kuentz
authors.forename: V
authors.initials: V
authors.lastname: Leblond
authors.forename: P
authors.initials: P
authors.lastname: Debré
authors.forename: A
authors.initials: A
authors.lastname: Bernard
authors.forename: J L
authors.initials: JL
authors.lastname: Binet
authors.forename: N
authors.initials: N
authors.lastname: Duedari
authors.forename: J P
authors.initials: JP
authors.lastname: Vernant
mesh_terms: D000293:Adolescent; D000328:Adult; D001853:Bone Marrow / Q000276:immunology; D016026:Bone Marrow Transplantation; D002469:Cell Separation; D003602:Cytotoxicity, Immunologic; D005455:Fluorescent Antibody Technique; D006086:Graft vs Host Disease / Q000517:prevention & control; D006801:Humans; D008213:Lymphocyte Activation; D012397:Rosette Formation; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology