IN-VITRO T-LYMPHOPOIESIS OF HUMAN AND RHESUS CD34(+) PROGENITOR CELLS

Differentiation of hematopoietic progenitor cells into T lymphocytes g enerally occurs in the unique environment of the thymus, a feature tha t has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34(+) progenitor cells. Cu lture of rhesus or human CD34(+) bone marrow-derived cells depleted of CD3(+) lymphocytes on rhesus thymic stromal monolayers yielded CD3(+) CD4(+)CD8(+), CD3(+)CD4(+)CD8(-), and CD3(+)CD4(-)CD8(+) cells after 1 0 to 14 days. In addition to classical T lymphocytes, a discrete popul ation of CD3(+)CD8(lo)CD16(+)CD56(+) cells was detected after 14 days in cultures inoculated with rhesus CD34(+) cells. CD3(+) T cells arisi ng from these cultures were not derived from contaminating T cells pre sent in the CD34(+) cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activatio n gene RAG-2, which is selectively expressed in developing lymphocytes , was detectable in thymic cultures inoculated with CD34(+) cells but not in CD34(+) cells before thymic culture or in thymic stromal monola yers alone, Reverse transcriptase-polymerase chain reaction analysis o f T cells derived from thymic stromal cultures of rhesus and human CD3 4(+) cells showed a polyclonal T-cell receptor repertoire. T-cell prog eny derived from rhesus CD34(+) cells cultured on thymic stroma suppor ted vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome. (C) 1996 by The American Society of Hematology.