Detection of Wilms' tumor antigen--specific CTL in tumor-draining lymph nodes of patients with early breast cancer.
OBJECTIVE
The Wilms' tumor antigen (WT1) is overexpressed in approximately 90% of breast tumors and, thus, is a potential target antigen for the immunotherapy of breast cancer. We have tested the working hypotheses that WT1 can be immunogenic in patients with breast cancer and can stimulate CTL of sufficient avidity to kill tumor cells.


METHODS
Paired tumor-draining lymph node and peripheral blood samples were analyzed from five HLA-A2-positive patients with stage I/II breast cancer. Fluorescent HLA-A*0201/WT1 tetramers were used to quantify WT1-specific CTL and the functional capacity of the CTL was assessed using cytotoxicity assays and intracellular cytokine staining.


RESULTS
WT1 tetramer-binding T cells expanded from all lymph node samples but none of the corresponding peripheral blood samples. Functional assays were carried out on T cells from the patient who had yielded the highest frequency of HLA-A*0201/WT1 tetramer-positive cells. The cytotoxicity assays showed WT1 peptide--specific killing activity of the CTL, whereas intracellular cytokine staining confirmed that the tetramer--positive T cells produced IFN-gamma after stimulation with WT1 peptide. These WT1-specific T cells killed HLA-A2-positive breast cancer cell lines treated with IFN-gamma but no killing was observed with untreated tumor cells.


CONCLUSIONS
These results show that WT1-specific CTL can be expanded from the tumor-draining lymph nodes of breast cancer patients and that they can display peptide-specific effector function. However, the CTL only killed IFN-gamma-treated tumor targets expressing high levels of HLA-A2 and not tumor cells with low HLA expression. This suggests that induction of autologous WT1-specific CTL may offer only limited tumor protection and that strategies that allow a high level of peptide/MHC complex presentation and/or improve CTL avidity may be required.
Gillmore
Roopinder
R
Department of Immunology and Molecular Pathology, Royal Free Hospital, London, United Knigdom.
Xue
Shao-An
SA
Holler
Angelika
A
Kaeda
Jaspal
J
Hadjiminas
Dimitri
D
Healy
Vourneen
V
Dina
Roberto
R
Parry
Suzanne C
SC
Bellantuono
Ilaria
I
Ghani
Yasmeen
Y
Coombes
R Charles
RC
Waxman
Jonathan
J
Stauss
Hans J
HJ
D000368:Aged; D000951:Antigens, Neoplasm / Q000276:immunology*; D045744:Cell Line, Tumor; D003601:Cytotoxicity Tests, Immunologic; D005260:Female; D005434:Flow Cytometry; D015789:HLA-A2 Antigen / Q000276:immunology; D006801:Humans; D008198:Lymph Nodes / Q000276:immunology*; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D025721:WT1 Proteins / Q000276:immunology*
D000951:Antigens, Neoplasm; D015789:HLA-A2 Antigen; D025721:WT1 Proteins