Large ex vivo expansion of human umbilical cord blood CD4+ and CD8+ T cells.

The use of human umbilical cord (UC) blood as a source of transplantable hematopoietic stem cells and progenitor cells may present some advantages over the use of BM. For example, it has been suggested that the degree of HLA matching may be less stringent, and the risk of GvHD may be lower. We have been studying the ex vivo expansion of UC blood T lymphocytes with a view to their use in the adoptive immunotherapy of cancer, autoimmunity, and infectious disease. We have developed a new method involving the use of a conditioned medium (XLCM) that consistently results in levels of UC blood T cell expansion not hitherto possible. Primary cultures of unfractionated low-density MNC (LDMNC) derived from UC blood treated with 5% XLCM routinely show expansions greater than 10,000-fold within 4 weeks. By contrast, similar FBS-free cultures treated with IL-2 expand less than 10-fold and not after 1 week, and cultures treated with IL-2 and concanavalin A (ConA) expand to a maximum of only 300-500-fold over 2 weeks and fail to continue to proliferate thereafter. The MAb, OKT3, which, when combined with IL-2 and FBS, is known to stimulate proliferation of adult peripheral blood lymphocytes, permitted only a 17-fold expansion of UC blood lymphocytes under the same conditions. Thus, XLCM, which can also stimulate adult peripheral blood lymphocyte expansion to levels exceeding 100,000-fold in 3-4 weeks, is uniquely able to stimulate proliferation of UC blood lymphocytes to high levels. From initiation of the UC blood or adult peripheral blood LDMNC/XLCM cultures up to approximately 2 weeks, the cultures are dominated by CD4+ T lymphocytes. By 4 weeks, >80% of the cultured cells bear the CD8+ phenotype, whereas UC blood T lymphocytes cultured in the presence of IL-2 are predominantly CD8+. Thus, XLCM not only allows high levels of expansion of UC blood T lymphocytes not heretofore possible but also permits the selective expansion of different T lymphocyte subsets from a single source.

The selective expansion of functional T cell subsets.

Adoptive cellular immunotherapy is considered a potential treatment for a wide range of diseases, including cancer, infectious diseases, and autoimmune disorders. We have developed a method using a conditioned medium, XLCM, that selectively expands several different T cell subsets with a view to their use in cell therapy. Primary FBS-free suspension cultures of human peripheral blood low-density mononuclear cells treated with XLCM reproducibly expand over 100,000-fold within a period of 4 weeks. CD4+ T cells and CD8+ T cells expand sequentially in the unfractionated cultures, and relatively pure populations of CD4+ and CD8+ T cells may be expanded from populations first enriched in the respective T cell subset. CD4+ T cells cultured in XLCM produce cytokines consistent with the expansion of Th1, Th2, and Th0 subsets, whereas CD8+ T cells cultured in XLCM are cytolytically competent. An interesting feature of T cells cultured in XLCM is the persistence of 5%-10% CD4+CD8+ double-positive T cells in spite of substantial single-positive T cell expansion, suggesting that these cells also proliferate in XLCM. In addition to subsets of TCRalphabeta+ T cells, TCRgammadelta+ T cells are also significantly expanded by XLCM. These results demonstrate that XLCM efficiently expands several functional T cell subsets and provides a means of obtaining selected populations suitable for use in cellular immunotherapy.

Development of an enzyme-linked immunosorbent assay for measurement of serum-associated ALX40-4C.

ALX40-4C is an antiretrovirus agent that has been found to have some inhibitory properties against human immunodeficiency virus (HIV) replication in vitro. The compound was designed as a competitor of the HIV Tat protein for TAR binding. In addition to its anti-HIV properties, it has demonstrated the ability to inhibit in vitro replication of herpes simplex virus types 1 and 2 as well as human cytomegalovirus. Subsequently, in vivo pharmacokinetic evaluation of ALX40-4C necessitated the establishment of a detection system for the measurement of ALX40-4C in subject serum. For this purpose, an indirect-competition enzyme-linked immunosorbent assay with generated rabbit anti-ALX40-4C antiserum was developed. The original assay took 12 h to complete and required many manipulations. Herein, we describe alterations to the system that resulted in the overall reduction in assay time and manipulation. We demonstrate that our alterations do not affect the specificity or sensitivity of the assay compared to that of the original system. ALX40-4C levels in spiked serum samples as well as drug levels from patient samples were used to validate the assay.