Functional assessment and specific depletion of alloreactive human T cells using flow cytometry.
Human T-cell alloreactivity plays an important role in many disease processes, including the rejection of solid organ grafts and graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. To develop a better understanding of the T cells involved in alloreactivity in humans, we developed a cytokine flow cytometry (CFC) assay that enabled us to characterize the phenotypic and functional characteristic of T cells responding to allogeneic stimuli. Using this approach, we determined that most T-cell alloreactivity resided within the CD4(+) T-cell subset, as assessed by activation marker expression and the production of effector cytokines (eg, tumor necrosis factor alpha [TNF]alpha) implicated in human GVHD. Following prolonged stimulation in vitro using either allogeneic stimulator cells or viral antigens, we found that coexpression of activation markers within the CD4(+) T-cell subset occurred exclusively within a subpopulation of T cells that significantly increased their surface expression of CD4. We then developed a simple sorting strategy that exploited these phenotypic characteristics to specifically deplete alloreactive T cells while retaining broad specificity for other stimuli, including viral antigens and third-party alloantigens. This approach also was applied to specifically enrich or deplete human virus-specific T cells.
Martins
Sergio L R
SL
Transplant Immunology Section, Department of Blood and Marrow Transplantation, MD Anderson Cancer Center, SCRB 3.3019, Unit 900, 7455 Fannin St, Houston, TX 77030, USA.
St John
Lisa S
LS
Champlin
Richard E
RE
Wieder
Eric D
ED
McMannis
John
J
Molldrem
Jeffrey J
JJ
Komanduri
Krishna V
KV
D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis; D001781:Blood Component Removal; D015704:CD4 Antigens / Q000032:analysis; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D016207:Cytokines / Q000096:biosynthesis; D005434:Flow Cytometry; D006801:Humans; D007111:Immunity, Cellular; D007700:Kinetics; D008213:Lymphocyte Activation / Q000276:immunology; D008212:Lymphocyte Depletion / Q000379:methods*; D013601:T-Lymphocytes / Q000276:immunology*; D015854:Up-Regulation
D000945:Antigens, Differentiation, T-Lymphocyte; D015704:CD4 Antigens; D016207:Cytokines