Comparison of polyclonal expansion methods to improve the recovery of cervical cytobrush-derived T cells from the female genital tract of HIV-infected women.
Cervical cytobrushing is a useful and non-invasive method for obtaining mucosal mononuclear cells from the female genital tract, but yields few cells. The aim of this study was to compare in vitro expansion protocols (anti-CD3, anti-CD3/CD28 or Dynal anti-CD3/CD28 beads) and cytokine combinations (IL-2, IL-7 and IL-15) to improve cervical T cell yields and viability. Eighteen HIV-infected women were included in this study to compare methods for polyclonal expansion of T cells from the female genital tract and blood. Comparison of T cell yields, viability and maturational status (by differential staining with CD45RO, CCR7 and CD27) was determined following 7 days of in vitro expansion. Anti-CD3 and IL-2 resulted in a 4.5-fold (range 3.7-5.3) expansion of cervical CD3+ T cells in 7 days compared to day 0. Inclusion of anti-CD28 or addition of IL-7 and IL-15 to this combination did not improve expansion. Culturing cells with Dynal beads (1:1) and IL-2, IL-7 and IL-15 gave rise to the highest yields after 7 days in both blood (7.1-fold) and cervix (5.6-fold). While expansion with anti-CD3 led to the accumulation of effector memory T cells (CD45RO+CCR7-CD27-), expansion with Dynabeads selected for accumulation of central memory T cells (CD45RO+CCR7+CD27+). We conclude that in vitro expansion with Dynabeads (1:1) in the presence of IL-2, IL-7 and IL-15 resulted in the greatest increase in viable T cells from both blood and cytobrush. Irrespective of the expansion method used, the T cell memory profile was altered following expansion.
Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, South Africa.
Alfred
A
Bere
Lynette
L
Denny
Willem
W
Hanekom
Wendy A
WA
Burgers
Jo-Ann S
JA
Passmore
D000328:Adult; D015703:Antigens, CD / Q000378:metabolism*; D018106:CD28 Antigens / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation; D002470:Cell Survival; D002478:Cells, Cultured; D002584:Cervix Uteri / Q000276:immunology* / Q000821:virology; D005260:Female; D005434:Flow Cytometry; D015658:HIV Infections / Q000097:blood / Q000276:immunology* / Q000821:virology; D006801:Humans; D007156:Immunologic Memory; D016130:Immunophenotyping; D019409:Interleukin-15 / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D015851:Interleukin-7 / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism*; D017493:Leukocyte Common Antigens / Q000378:metabolism; D009092:Mucous Membrane / Q000276:immunology / Q000821:virology; D010641:Phenotype; D054400:Receptors, CCR7 / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013048:Specimen Handling; D013601:T-Lymphocytes / Q000276:immunology* / Q000821:virology; D013997:Time Factors; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7 / Q000378:metabolism
D015703:Antigens, CD; C515370:CCR7 protein, human; D018106:CD28 Antigens; D017252:CD3 Complex; D019409:Interleukin-15; D007376:Interleukin-2; D015851:Interleukin-7; D007378:Interleukins; D054400:Receptors, CCR7; D011994:Recombinant Proteins; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7; D017493:Leukocyte Common Antigens