Enhanced generation of cytotoxic T lymphocytes by IL-21 mediated FoxP3 suppression
A method of carrying out adoptive immunotherapy by administering a subject an antigen-specific cytotoxic T lymphocytes (CTL) preparation in a treatment-effective amount is described. In the method, the CTL preparation is preferably administered as a preparation of an in vitro antigen-stimulated and expanded primate CTL population, the CTL population: (i) depleted of FoxP3+ T lymphocytes prior to antigen stimulation; (ii) antigen-stimulated in vitro in the presence of interleukin-21; or (iii) both depleted of FoxP3+ T lymphocytes prior to antigen stimulation and then antigen-stimulated in vitro in the presence of interleukin-21. Methods of preparing such compositions, and compositions useful for carrying out the adoptive immunotherapy, are also described.
1. A method of making a cytotoxic T lymphocyte (CTL) preparation useful for adoptive immunotherapy, comprising:
(a) sorting a lymphocyte subpopulation depleted of CD25 + cells from a first lymphocyte population of peripheral blood mononuclear cells (PBMC) to produce a CD8 + CD25 − subpopulation;
(b) promoting the production of antigen-specific CTL cells in said subpopulation by in vitro culturing the sorted CD8 + CD25 − subpopulation with antigen in a medium containing interleukin-21; and
(c) formulating antigen-specific CTL cells produced therefrom into a pharmaceutical formulation.
2. The method of claim 1 , wherein said interleukin-21 is included in said culture in an amount of from 1 to 1000 nanograms per milliliter.
3. The method of claim 1 , wherein the CTL cells are specific for a tumor antigen.
4. The method of claim 1 , wherein (a) the sorting a lymphocyte subpopulation depleted of CD25 + cells from a first lymphocyte population comprises treating the first lymphocyte population with denileukin diftitox.
5. The method of claim 1 , wherein (a) the sorting a lymphocyte subpopulation depleted of CD25 + cells from a first lymphocyte population comprises contacting the first lymphocyte population with anti-CD25 antibodies.
6. The method of claim 1 , wherein (b) the promoting the production of antigen-specific CTL cells in said subpopulation by in vitro culturing comprises culturing the CTLs with antigen presenting cells.
7. The method of claim 1 , wherein the antigen presenting cells comprise dendritic cells (DCs).
8. The method of claim 1 , wherein the antigen-specific CTL cells comprise a transgene.
9. The method of claim 1 , wherein the antigen-specific CTL cells of the formulation consist essentially of CD8 + CD25− antigen-specific CTL cells.
10. The method of claim 1 , wherein the number of antigen specific CTL are enriched in said CD8 + CD25 − subpopulation by at least 100-fold, as compared to that seen in the same lymphocyte population not subjected to either the sorting step (a) or the promoting step (b).
11. The method of claim 1 , wherein the pharmaceutical formulation comprises at least 10 9 CTL cells.