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Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications
- Source :
- Clinical and experimental immunology. 156(2)
- Publication Year :
- 2009
-
Abstract
- Although CD4(+)/CD25(+) T regulatory cells (T(regs)) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell-separation strategy complying with good manufacturing practice guidelines. We isolated T(regs) from standard leukapheresis products using double-negative selection (anti-CD8 and anti-CD19 monoclonal antibodies) followed by positive selection (anti-CD25 monoclonal antibody). The final cell fraction (CD4(+)/CD25(+)) showed a mean purity of 93.6% +/- 1.1. Recovery efficiency was 81.52% +/- 7.4. The CD4(+)/CD25(+bright) cells were 28.4% +/- 6.8. The CD4(+)/CD25(+) fraction contained a mean of 51.9% +/- 15.1 FoxP3 cells and a mean of 18.9% +/- 11.5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4(+)CD25(+)FoxP3(+) cells were in line with flow cytometric results. In Vbeta spectratyping the complexity scores of CD4(+)/CD25(+) cells and CD4(+)/CD25(-) cells were not significantly different, indicating that T(regs) had a broad T cell receptor repertoire. The inhibition assay showed that CD4(+)/CD25(+) cells inhibited CD4(+)/CD25(-) cells in a dose-dependent manner (mean inhibition percentages: 72.4 +/- 8.9 [ratio of T responder (T(resp)) to T(regs), 1:2]; 60.8% +/- 20.5 (ratio of T(resp) to T(regs), 1:1); 25.6 +/- 19.6 (ratio of T(resp) to T(regs), 1:0.1)). Our study shows that negative/positive T(reg) selection, performed using the CliniMACS device and reagents, enriches significantly CD4(+)CD25(+)FoxP3(+) cells endowed with immunosuppressive capacities. The CD4(+)CD25(+)FoxP3(+) population is a source of natural T(reg) cells that are depleted of CD8(+) and CD4(+)/CD25(-) reacting clones which are potentially responsible for triggering graft-versus-host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft-versus-tumour effect.
- Subjects :
- Adult
CD4-Positive T-Lymphocytes
Translational Studies
medicine.drug_class
Immunology
Population
chemical and pharmacologic phenomena
Biology
Monoclonal antibody
Immunophenotyping
Interleukin-7 Receptor alpha Subunit
Interleukin-5 Receptor alpha Subunit
medicine
Immunology and Allergy
Humans
IL-2 receptor
Leukapheresis
education
Interleukin-7 receptor
education.field_of_study
Immunomagnetic Separation
Reverse Transcriptase Polymerase Chain Reaction
Spectral Karyotyping
Interleukin-2 Receptor alpha Subunit
FOXP3
hemic and immune systems
Forkhead Transcription Factors
Middle Aged
medicine.disease
Graft-versus-host disease
Phenotype
CD8
Subjects
Details
- ISSN :
- 13652249
- Volume :
- 156
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- Clinical and experimental immunology
- Accession number :
- edsair.doi.dedup.....e5153991b2f6c12c6c09477c116e1133