title: Levels of circulating regulatory CD4+CD25+ T cells are decreased in breast cancer patients after vaccination with a HER2/neu peptide (E75) and GM-CSF vaccine.
abstract: OBJECTIVE
  We are conducting clinical trials in breast cancer (BrCa) patients to test the HER2/neu peptide vaccine (E75). We have investigated the impact of this vaccine on circulating levels of regulatory T cells (Treg) and the resulting effects on antitumor responses.
  
  
  METHODS
  Twenty-two blood samples from healthy individuals and from 22 BrCa patients including pre- and post-vaccination samples from seven vaccinated HLA-A2+ patients were stained for CD4, CD25, and CD69 as well as CD8 and E75:HLA-A2 Ig dimer and quantified by flow cytometry. Cytotoxic activity against HER2/neu+ tumors was measured by 51Cr-release. Serum from BrCa patients and normal subjects were analyzed for TGF-beta levels.
  
  
  RESULTS
  BrCa patients have a greater percentage of circulating Treg (CD4+CD25+, 4.45% versus 2.96%; p=0.007) than normal subjects. HLA-A2+ BrCa patients had more Treg compared to the HLA-A2- BrCa patients (CD4+CD25+, 5.63% versus 3.28%; p=0.001). E75 vaccination increased circulating activated CD4+ T cells post-vaccination (CD4+CD69+, 1.23 versus 3.81%; p=0.03). However, T(reg) were significantly reduced after vaccination (CD4+CD25+, 5.31-1.81%; p<0.0001). Furthermore, activated Treg also decreased (CD4+CD25+CD69+, 0.23% versus 0.08%; p=0.06). Importantly, post-vaccination decreases in Treg were temporally associated with increased E75 vaccine-specific CD8+ T cells and corresponding HER2/neu+ tumor cytotoxicity. Serum TGF-beta levels were significantly elevated in BrCa patients compared to normals (3548 pg/ml versus 1007 pg/ml; p=0.007). Four of seven vaccinated patients showed decreased serum TGF-beta levels post-vaccination.
  
  
  CONCLUSIONS
  Treg, are increased in BrCa patients along with serum levels of TGF-beta. E75 vaccination resulted in CD4+ recruitment but was associated with a significant decrease in circulating Treg and TGF-beta levels in the majority of the vaccinated patients. Successful cancer vaccination strategies may require the alteration of complex immune interactions.
authors.affiliation: Clinical Breast Care Project, Department of Surgery, Walter Reed Army Medical Center, Washington, DC, USA.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Hueman
authors.forename: Alexander
authors.initials: A
authors.lastname: Stojadinovic
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Storrer
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Foley
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Gurney
authors.forename: Craig D
authors.initials: CD
authors.lastname: Shriver
authors.forename: Sathibalan
authors.initials: S
authors.lastname: Ponniah
authors.forename: George E
authors.initials: GE
authors.lastname: Peoples
mesh_terms: D015703:Antigens, CD / Q000097:blood; D000945:Antigens, Differentiation, T-Lymphocyte / Q000097:blood; D001943:Breast Neoplasms / Q000097:blood*; D015704:CD4 Antigens / Q000097:blood*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D019496:Cancer Vaccines / Q000627:therapeutic use*; D016022:Case-Control Studies; D005434:Flow Cytometry; D018734:Genes, erbB-2; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D015789:HLA-A2 Antigen / Q000097:blood; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000097:blood*; D037181:Lectins, C-Type; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D010455:Peptides / Q000737:chemistry; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D015704:CD4 Antigens; D019496:Cancer Vaccines; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D015789:HLA-A2 Antigen; D053645:Interleukin-2 Receptor alpha Subunit; D037181:Lectins, C-Type; D010446:Peptide Fragments; D010455:Peptides; D000099192:CD69 Antigens; C474513:HER-2 peptide E75 (369-377), human