title: Rapamycin enriches for CD4(+) CD25(+) CD27(+) Foxp3(+) regulatory T cells in ex vivo-expanded CD25-enriched products from healthy donors and patients with multiple sclerosis.
abstract: BACKGROUND
  CD4(+) CD25(bright+) regulatory T cells (Treg) can be expanded to clinical doses using CD3/CD28 Ab-coated beads plus IL-2. However, this method requires high purity of the starting population to prevent overgrowth by non-regulatory T cells. Rapamycin, an agent that inhibits T-cell proliferation but selectively spares Treg, may be a means to expand Treg from less pure CD25-enriched cells.
  
  
  METHODS
  CD25-enriched cells were prepared by a single-step immunomagnetic-selection using anti-CD25 microbeads. The cells were activated with a single addition of anti-CD3/CD28 beads and expanded in ex vivo 15-5% HS and autologous CD4(+) CD25(-) feeder cells,+/-rapamycin (0.01-20 ng/mL). IL-2 was added on day 3. Cells were rested for 2 days in ex vivo 15-5% HS and tested for phenotype, intracellular Foxp3 protein and suppressor activity.
  
  
  RESULTS
  In the absence of rapamycin, CD25-enriched fractions expanded >17 000-fold by 21 days. Although suppressor activity was detected to day 14, it declined significantly by 21 days as non-regulatory cells expanded. The addition of rapamycin inhibited expansion of non-regulatory T cells at doses > or =1 ng/mL while increasing suppressor activity and the percentage of CD4(+) CD25(+) CD27(+) Foxp3(+) cells. Rapamycin did not enrich for Foxp3(+) cells in expanded cultures of CD4(+) CD25(-) cells. Treg were also readily expanded in cultures of CD25-enriched cells obtained from patients with multiple sclerosis in the presence of rapamycin.
  
  
  CONCLUSIONS
  The addition of 1-20 ng/mL rapamycin to CD25-enriched cultures increased the purity of cells with the phenotype and function of Treg. This approach may alleviate the need for rigorous enrichment of Treg prior to activation and expansion for potential clinical use.
authors: Department of Medicine/Division of Neoplastic Diseases and Related Disorders, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. ckeever@mcw.edu
authors: C A
authors: CA
authors: Keever-Taylor
authors: M B
authors: MB
authors: Browning
authors: B D
authors: BD
authors: Johnson
authors: R L
authors: RL
authors: Truitt
authors: C N
authors: CN
authors: Bredeson
authors: B
authors: B
authors: Behn
authors: A
authors: A
authors: Tsao
mesh_terms: D015704:CD4 Antigens / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D006801:Humans; D016130:Immunophenotyping; D007166:Immunosuppressive Agents / Q000494:pharmacology; D017493:Leukocyte Common Antigens / Q000378:metabolism*; D009103:Multiple Sclerosis / Q000097:blood / Q000276:immunology; D020123:Sirolimus / Q000494:pharmacology*; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7 / Q000378:metabolism*
chemical_list: D015704:CD4 Antigens; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D007166:Immunosuppressive Agents; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7; D017493:Leukocyte Common Antigens; D020123:Sirolimus