Down Regulation of Stem Cell Colony Formation by Purified CD8 Lymphocytes and CD8 Conditioned Medium: Potential Importance for Bone Marrow Transplantation in Leukemia

Abstract

A methodology for selection of the CD8 cell subset from the peripheral blood and bone marrow mononuclear cells was developed using anti-T₈ (CD8) antibody and magnetic microspheres coated with anti-mouse IgG. Following optimization of antibody: cell binding ratio and microsphere : cell ratios, CD8⁺-cells in the peripheral blood and bone marrow were effectively removed, with an overall final recovery of 34.9% ± 8.6%, and 56% ± 8.5% respectively with complete recovery of stem cells and very little contamination with effector cells.

CD8⁺-depleted cell preparations demonstrated a 3-4 fold increase in CFU-C and CFU-E colony formation over non-depleted preparations when stimulated with G-CSF, GM-CSF or IL-3 and erythropoietin. The largest increase in colony formation was evident when IL-3 was used to stimulate colony formation. Purified autologous CD8⁺ T-cells or culture supernatant from in vitro cultures of purified autologous CD8⁺ T-cells added back to CD8 depleted preparations, induced 20%-90% suppression of CFU-C and CFU-E colony formation in a dose dependent manner.

Colony formation by CD34⁺ cells, purified by anti CD34 antibodies and magnetic microspheres, were also inhibited by either pure CD8⁺-cell populations or CD8-culture supernatant. Preliminary fractionation studies indicate that the inhibitory factor is a protein of >50kd. In contrast, when purified autologous CD4⁺ cells were added to purified CD34⁺ stem cells, an increase (<50%) in colony formation was observed. These results, taken together, suggest that CD8⁺ T-cells are negative effectors of normal hematopoiesis whereas CD4+ T-cells function as positive effectors.

Key Words:

• Immunomagnetic selection
• CD8
• down regulation
• bone marrow
• colony formation