In Vitro CD4+ T Cells Expansion: Induction of Regulatory T Cells with Sirolimus and CD3/CD28 Dynabeads

Reviewer: Eric Shinohara MD, MSCI
Abramson Cancer Center of the University of Pennsylvania
Last Modified: March 23, 2007

Presenter: Borelli, G
Presenter's Affiliation: Department of Cellular Therapy, Rikshospitalet-Radiumhospitalet HF
Type of Session: Scientific


  • T regulatory cells (Treg) are a subset of T lymphocytes defined by CD4+CD25+ markers and high FOXP3 expression. They play a key role in self-reactivity and alloreactivity control.
  • Treg cells have many functions which are beneficial including: T-cell homeostasis, preventing autoimmune disease, preventing the allergic response, and preventing graft versus host disease (GVHD).
  • Treg cells have functions which may be detrimental in tumor treatment as well, including: downregulation of tumor suppression and increased risk of infection.
  • In allogenic hematopoietic stem cell transplantation (HSCT), Treg cells have the potential to be useful in reducing GVHD without impairing graft versus tumor effect.
  • The population of Treg cells is small, making up only 1-2% of the CD4+ helper T-cell population, and a means to expand this population of cells is needed to provide sufficient numbers of cells for experimental/clinical use.
  • The purpose of this study was to create a protocol which optimized the in vitro T- cell expansion conditions for clinical grade production of Treg cells.

Materials and Methods

  • CD4+ cells were obtained by positive and negative immunomagnetic selection from lymphocytes derived from peripheral blood.
  • CD4+ cells were stimulated with anti CD3/CD28-coated Dynabeads at a bead/cell ratio of 3:1. Dynabeads functioned as antigen presenting cells (APC). The cells were cultured with X-VIVO20 media, autologous plasma, IL2, IL4 and with and without Sirolimus (rapamycin).
  • At day 12 cells were washed to remove the CD3/CD28 Dynabeads. After 24 hours, cells were reincubation with CD3/CD28 Dynabeads and cytokine secretion was analyzed in the supernatant by BioPlex. Cell phenotype and FOXP3 expression was evaluated by flow cytometry and suppressive capacitive was measured using standard proliferation assay.


  • 80% of CD4+ cells cultured with Sirolimus expressed CD25 and secreted low levels of both Th1 and Th2 cytokines. In contrast, only 20% of CD4+ cells cultured without Sirolimus expressed CD25 and secreted high level of Th1 cytokines. Culture conditions either with or without Sirolimus resulted in the same number of FOXP3+ cells.
  • Cells cultured with Sirolimus showed strong suppressive capacity and could suppress CD4+CD25- T cell proliferation by 80% even at 1:32 ratio of CD4+CD25-:CD4+CD25+ cells.

Author's Conclusions

  • Sirolimus induces generation of Treg cells with a strong inhibitory power over CD4+ CD25- cells proliferation.

Clinical/Scientific Implications

This study presented a novel protocol for the production of Treg cells with the capacity to strongly inhibit CD4+CD25- cell proliferation. They were able to obtain a fairly pure population of CD4+CD25+, however it is not clear why the FOXP3+ cell expression was the same between the two groups. Generally, FOXP3+ expression is high in Treg cells. Hence, it remains to be seen how Treg cells produced in this manner function in vivo. In particular, do these cells truly suppress the immune system selectively with no effect on the antitumor effects of the immune system, but with adequate suppression of the autoimmune component in vivo. However, if effective in vivo, this protocol would allow for the large scale production of Treg cells which could be used not only to suppress graft versus host disease, but also in other autoimmune diseases to help selectively suppress the autoimmunity.