Influence of cell isolation method on the optimization of CD4+T cell expansion using anti-CD3/CD28 coated beads

Backgrounds: Activation of CD4+ T lymphocytes with anti-CD3/CD28 coated magnetic beads promotes intrinsic resistance to HIV as well as cell expansion. The propose of this study is to define the optimal cell isolation protocol for expansion of CD4+ T lymphocytes by using anti-CD3/CD28 coated bead stimulation with an ultimate goal of using these cells for adoptive immunotherapy. Methods: CD4+ T cells were isolated from healthy donor blood samples using three different methods including immunorosette formation, negative selection and CD8 depletion using immunomagnetic beads. These cells were activated with anti-CD3/CD28 coated beads at a bead to cell ratio of 1:1 and cell expansion was carried for 3 weeks. Cell numbers, cell viability and phenotypic characterization were determined by trypan blue exclusion and flow cytometry. Results: Purified CD4+ T lymphocytes which were isolated via immunorosette formation can be expanded up to 1000-fold within 3 weeks with high viability (90%) and high purity of CD4+ T lymphocytes (>95%). However, cell expansion from purified CD4+ T lymphocytes which were isolated by negative selection and CD8-depletion provided approximately 300-fold expansion. Conclusions: The results demonstrate that purified CD4+ T lymphocytes from immunorosette formation provided the highest CD4+ T lymphocyte expansion when stimulated with anti-CD3/CD28 coated beads. This method can be used to obtain a large number of expanded CD4+ T cells for adoptive immunotherapy.