Presence of low dose of fludarabine in cultures blocks regulatory T cell expansion and maintains tumor-specific cytotoxic T lymphocyte activity generated with peripheral blood lymphocytes.
BACKGROUND
For tumor vaccine-based immunotherapy of cancer, the expansion of tumor antigen-specific cytotoxic T lymphocytes (CTL) in the patients by blocking induced regulatory T (Treg) cells is the most important objective now. Fludarabine (FLU), a known anticancer drug, has been shown to downregulate Treg cells in vivo in chronic leukemia patients. Melanoma tumor antigen Mart-1(27-35)-specific CD8+ CTLs generated in vitrowith total peripheral blood lymphocytes (PBL) lose their activity within 14-21 days with concomitant expansion of Treg cells. When CD4+ cells are removed from PBL and CTL are generated with purified CD8+ cells, the CTL survive and maintain their activity for a significantly longer period.


METHODS
We used a low dose of FLU in the cultures in Mart-1-specific CTL generation assays with total PBL. Blood samples were taken from HLA-A2-positive melanoma patients and normal donors. Autologous matured dendritic cells pulsed with Mart-1(27-35) peptide were used to generate CTL responses using purified CD8+ cells or total PBL.


RESULTS
The presence of FLU in the cultures with PBL helped to generate a significantly higher number of antigen-specific CTLs as detected by Mart-1 HLA-A2 tetramer staining. Specificity of such CTLs was determined by IFN-gamma secretion or by cytotoxicity against the target cells bearing the specific antigen. The presence of FLU stopped the expansion of IL-10-producing CD4+ Treg cells in the cultures with PBL. Analyses of expanded CD4+ cells isolated from PBL in vitro cocultures with FLU showed a Th1 type of function. Those cells secreted higher amounts of IFN-gamma and very low levels of IL-10, or no IL-10 at all, upon restimulation.


CONCLUSIONS
The observations of the study are as important for adaptive immunotherapy of cancer as they are for vaccine-based approaches.
Department of Medicine, Carole and Ray Neag Comprehensive Cancer Center, Farmington, Conn., USA.
Upendra
U
Hegde
Arvind
A
Chhabra
Subhasis
S
Chattopadhyay
Raja
R
Das
Swagatam
S
Ray
Nitya G
NG
Chakraborty
D000970:Antineoplastic Agents / Q000494:pharmacology*; D002455:Cell Division; D002478:Cells, Cultured; D018920:Coculture Techniques; D016207:Cytokines / Q000032:analysis / Q000096:biosynthesis; D003601:Cytotoxicity Tests, Immunologic; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007963:Leukocytes, Mononuclear; D008213:Lymphocyte Activation / Q000187:drug effects; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology
D000970:Antineoplastic Agents; D016207:Cytokines; D014740:Vidarabine; C024352:fludarabine