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Blood, 1 December 2004, Vol. 104, No. 12, pp. 3429-3436.
Prepublished online as a Blood First Edition Paper on July 29, 2004; DOI 10.1182/blood-2004-05-1918.
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PLENARY PAPERS
Functional assessment and specific depletion of alloreactive human T cells using flow cytometry
Sergio L. R. Martins,
Lisa S. St. John,
Richard E. Champlin,
Eric D. Wieder,
John McMannis,
Jeffrey J. Molldrem, and
Krishna V. Komanduri
From the Transplant Immunology Section, Department of Blood and Marrow Transplantation, MD Anderson Cancer Center, Houston, TX; and the Department of Hematology, Fleury-Diagnostic Medical Center, Jabaquara, Sao Paulo, Brazil.
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  [TNF]) 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.
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