Healthy individuals have T-cell and antibody responses to the tumor antigen cyclin B1 that when elicited in mice protect from cancer

Abstract

We previously identified the aberrantly expressed cell cycle regulator cyclin B1 as a tumor antigen recognized by antibodies and T cells from patients with breast, lung, and head and neck cancers. Ordinarily expressed only transiently in the G2/M stage of the cell cycle in normal cells, cyclin B1 is constitutively expressed at high levels in the cytoplasm of these and many other tumor types, leading to its recognition by the cancer patient's immune system. We report here an unexpected observation that cyclin B1-specific antibody and memory CD4 and CD8 T cells are also found in many healthy individuals who have no history of cancer. Moreover, young as well as older healthy people have these responses suggesting that events other than cancer, which occur either early in life or throughout life, may lead to aberrant cyclin B1 expression and anti-cyclin B1 immunity. The role, if any, of immunity to this tumor-associated antigen is not known. We wanted to determine specifically whether immunity to cyclin B1 might be important in the immunosurveillance of cyclin B1+ tumors. We therefore tested in mice the effectiveness of vaccine-elicited anti-cyclin B1 immunity against a cyclin B1+ mouse tumor that was chosen based on our published observation that cyclin B1 overexpression is associated with the lack of p53 function. We found that cyclin B1 DNA prime-protein boost vaccine protected mice from a challenge with a tumor cell line that was established from a tumor arising in the p53(-/-) mouse that spontaneously overexpresses cyclin B1.

Fig. 1.

Healthy individuals have anti-cyclin B1 IgG. Plasma samples from healthy donors with no history of cancer (n = 65) were tested for anti-cyclin B1 IgG (dilution of 1:400) and IgG subtypes (1:20). (A) Anti-cyclin B1 IgG from all individuals was normalized to five standard controls. Bar, mean OD. (B) Levels of anti-cyclin B1 IgG are independent of age (Pearson correlation, P = 0.63). (C) Anti-cyclin B1 IgG subtypes were tested in 23 healthy individuals who had above the median levels of anti-cyclin B1 IgG. Each individual is represented by the same symbol in all four assays.

Fig. 2.

Healthy individuals have memory T cells specific for cyclin B1. (A) All T cells: Monocyte-depleted PBMC were cultured with autologous DCs that were loaded with ovalbumin (OVA), cyclin B1 (CB1), or unloaded. Supernatant from the seventh day of culture was tested by ELISA for IFNγ. W6/32: MHC class I blocking antibody. (B) CD4⁺ T cells: PBMC from the same donor as in A were labeled with CFSE and cultured with autologous DCs in the presence or absence of indicated antigen or without DC. Percentage of proliferating CD4⁺ T cells was assessed after seven days. (C) CD8⁺ T cells: CD8⁺ T cells were purified from PBMC (a second donor) and cultured with autologous DCs with and without indicated antigens. Supernatants were tested for IFNy after 10 days. Bars indicate standard deviation. (D–G) Brefeldin A was added for 11 h to one set of a triplicate culture at 6, 30, and 54 h after combination of DCs with PBL. After the incubation periods, CD4⁺ T cells (D and F) and CD8⁺ T cells (E and G) were assessed for intracellular IFNγ. (D and E) Flow cytometric measurement of IFNγ. (Top) PBL stimulated with cyclin B1-loaded DCs. (Center) PBL stimulated with OVA-loaded DCs. (Lower) PBL stimulated with unloaded DCs. (F and G) show a graphical representation of the percentage of IFNγ-positive cells for CD4⁺ (F) and CD8⁺ (G) T cells.

Fig. 3.

Identification of commonly recognized cyclin B1 peptides. PBMC were labeled with CFSE and stimulated with 2 μg/mL recombinant cyclin B1 peptides. After six days of culture, PBMC were stained with cell surface markers and proliferation was assessed by flow cytometry. peptide 61: KFRLLQETMYMTVSI; peptide 62: LQETMYMTVSIIDRF; and peptide 63: MYMTVSIIDRFM.

Fig. 4.

Cyclin B1 DNA prime-protein boost vaccination elicits cyclin B1-specific cellular and humoral responses and delays tumor growth. (A and B) Mice were primed with either pcDNA 3.1 empty vector (group 1), mouse cyclin B1 (mCB1, group 2), or human cyclin B1 (hCB1, group 3) cDNA. All three groups were boosted with human cyclin B1 recombinant protein and the LT/ IS patch two times in three-week intervals. (A) ELISPOT performed on mouse splenocytes. Error bars, SE. (B) ELISA for anti-human (Left) and anti-mouse (Right) cyclin B1 IgG. Bars, geometric mean. (C and D) Mice from groups 1, 2, and 3 with the addition of untreated and pcDNA3.1 empty vector + LT/IS patch controls were challenged with LO2 tumor cells. (C) Tumor growth on day 28 after tumor challenge. Bars, mean tumor size. (D) Survival after tumor challenge (Logrank test, P < 0.0001).