Establishment of Effective B Lymphocyte Ex Vivo Expansion on Human Cord Blood Using TPO, SCF, FL, IL-4, IL-10, and CD40L.

In respect to B lymphocyte-mediated immunity, characteristics of human cord blood are low counts of mature B lymphocytes, deficient expression of CD40L and cytokine production in CD4+ T lymphocytes, defect in the isotype switch of immunoglobulin and the activation of B lymphocytes, and low IgG production of B lymphocytes. These characteristics of the B lymphocyte from human cord blood lead to a delayed B lymphocyte-mediated immune reconstitution and an increased susceptibility to infections after a cord blood transplantation. The mechanism of immunological recostitution after cord blood transplantation has been examined from a variety of viewpoints in experimental models as well as clinical studies. However, problems of sustained immunodeficiency after cord blood transplantation remain to be resolved.

The aim of the present study is to establish culture conditions that support the effective B lymphocyte expansion of human cord blood using IL-4, IL-10, and CD40L, to which cytokines are defected in B lymphocyte of human cord blood, and established conditions are compared to previously established cytokine combinations, TPO+SCF+FL in our Lab (Br J Haematol 107:176–185, 1999 & Stem Cells 21:228–235, 2003). To elucidate the effective B lymphocyte-mediated immune reconstitution of cord blood after ex vivo expansion, mononuclear cells, separated from density gradient of Ficoll system, and CD34+ purified cells, isolated from immunomicrobead(MiniMACS) system, were cultured with various combinations of cytokines (TPO+FL+SCF and/or IL-4, IL-10 and CD40L) for 2 weeks or 4 weeks. This then allowed for cytometric analysis after immunofluorescence stain with CD34, CD38 (for HSC analysis) and CD19, IgG and IgM (for B lymphocyte-mediated immune reconstitution) and CD4 (for T helper cell) and CD25 (for lymphocyte activation assay) to be performed.

In the B lymphocyte expansion aspect, the immunoglobulin expression, and functional activity, expansion with the TPO+FL+SCF+IL-4+IL-10 combination showed best results in the expression of CD19, CD25, IgG, and IgM. However, the addition of CD40L to those culture condition did not increase expression of CD19, CD25, IgG, and IgM after the expansion of human cord blood. Expansion of CD34+ purified cells was superior to MNCs in the expression of CD19, CD25, IgG, and IgM. In consideration for the duration of cultures, the 2 week culture was superior to the 4 week culture with respect to graft stemness (CD34+CD38- fraction).

Our data suggests most superior results were observed from the ex vivo expansion of CD34+ purified cells cultured for 2 weeks with TPO+FL+SCF+IL-4+IL-10, in the B lymphocyte-mediated immune reconstitution and graft stemness aspect. The results of this study warrant further investigation on effective B lymphocyte-mediated immune reconstitution after cord blood transplantation in vivo using ex vivo expanded cord blood.