An autoimmune-mediated strategy for prophylactic breast cancer vaccination

Journal name:
Nature Medicine
Volume:
16,
Pages:
799–803
Year published:
DOI:
doi:10.1038/nm.2161
Received
Accepted
Published online

Although vaccination is most effective when used to prevent disease, cancer vaccine development has focused predominantly on providing therapy against established growing tumors1. The difficulty in developing prophylactic cancer vaccines is primarily due to the fact that tumor antigens are variations of self proteins and would probably mediate profound autoimmune complications if used in a preventive vaccine setting2. Here we use several mouse breast cancer models to define a prototypic strategy for prophylactic cancer vaccination. We selected α-lactalbumin as our target vaccine autoantigen because it is a breast-specific differentiation protein expressed in high amounts in the majority of human breast carcinomas3, 4, 5 and in mammary epithelial cells only during lactation6, 7, 8, 9. We found that immunoreactivity against α-lactalbumin provides substantial protection and therapy against growth of autochthonous tumors in transgenic mouse models of breast cancer and against 4T1 transplantable breast tumors in BALB/c mice. Because α-lactalbumin is conditionally expressed only during lactation, vaccination-induced prophylaxis occurs without any detectable inflammation in normal nonlactating breast tissue. Thus, α-lactalbumin vaccination may provide safe and effective protection against the development of breast cancer for women in their post–child-bearing, premenopausal years, when lactation is readily avoidable and risk for developing breast cancer is high10.

At a glance

Figures

  1. Immunogenicity of recombinant mouse a-lactalbumin.
    Figure 1: Immunogenicity of recombinant mouse a-lactalbumin.

    (a) Western blot showing purified recombinant mouse α-lactalbumin detected with His tag–specific antibody. (b) Recall responses to recombinant proteins elicited by whole LNCs taken 10 d after immunization of SWXJ female mice with α-lactalbumin. (c) Recall responses to recombinant proteins elicited by CD4+ and CD8+ T cells purified from LNCs taken 10 d after immunization of SWXJ female mice with α-lactalbumin. (d) Cytokine production in response to recombinant α-lactalbumin by LNCs taken 10 d after immunization of SWXJ mice with α-lactalbumin. (eg) Immunocytochemical staining for CD8. In e, arrows show CD3+ surveillance T cells in mammary parenchyma of nonlactating SWXJ female mice 6 weeks after immunization with α-lactalbumin. In f, arrows show extensive inflammatory infiltrates of CD3+ T cells in mammary parenchyma of lactating SWXJ female mice 6 weeks after immunization with α-lactalbumin. In g, CD3+ infiltrates were never observed in mammary tissue from lactating control mice immunized with CFA alone. Scale bar for eg, 50 μm. (h) Flow cytometry analysis of breast infiltrates showing high frequencies of CD3+CD4+ (left) and CD3+CD8+ T cells (right) expressing the CD44high activated phenotype. (i) Real-time RT-PCR analysis of lactating mammary tissue showing significantly elevated expression of IFN-γ (*P = 0.001) but not IL-10 (P > 0.10). All error bars show means ± s.e.m.

  2. [alpha]-lactalbumin vaccination delays and treats breast tumor growth.
    Figure 2: α-lactalbumin vaccination delays and treats breast tumor growth.

    (a) Growth of autochthonous breast tumors in 10-month-old MMTV-neu mice immunized with α-lactalbumin in CFA or CFA alone at 8 weeks of age. (b) Growth of transplanted 4T1 tumors after prophylactic immunization with α-lactalbumin in CFA or CFA alone 13 d before tumor inoculation. (c) H&E staining of tissue extracted from the flank of a representative mouse 5 d after s.c. inoculation of 2 × 104 4T1 tumor cells. Arrows outline a 4T1 tumor. Scale bar, 100 μm. (df) 4T1 tumor size after α-lactalbumin immunization 5 d after tumor inoculation (P < 0.01) (d), 13 d after tumor inoculation (P < 0.01) (e) and 21 d after tumor inoculation (P > 0.10) (f). (g) Tumor size of very aggressive autochthonous tumors after α-lactalbumin immunization of MMTV-PyVT transgenic mice at 6 weeks of age (P < 0.0006). Tumors in MMTV-PyVT mice were amenable to measurement in only one direction. All error bars show ± s.e.m. Each * indicates significance.

  3. [alpha]-lactalbumin-specific T cells induce tumor inflammation and cytotoxicity.
    Figure 3: α-lactalbumin–specific T cells induce tumor inflammation and cytotoxicity.

    (a,b) Immunocytochemical staining for CD3. In a, arrows show extensive infiltration of tumors with CD3+ T cells in BALB/c mice 32 d after vaccination with α-lactalbumin and inoculation with 4T1 cells. In b, T cell infiltrates were never observed in tumor-inoculated mice immunized with CFA alone. Scale bar, 50 μm. (c) Flow cytometry analysis of TILs showing a predominance of CD4+ (64.3%) over CD8+ (14.4%) T cells. (d) Cytokine production in response to 50 μg ml−1 α-lactalbumin by TILs taken from 4T1 tumors. (e) ELISPOT analysis of TILs showing production of IFN-γ in response to 50 μg ml−1 α-lactalbumin in the presence of MHC class I– and class II–specific antibodies (anti–class I and anti–class II). (f) Cytotoxicity of 4T1 tumor cells induced by LNCs taken from BALB/c female 10 d after immunization with α-lactalbumin. All data shown are representative of three experiments providing similar results. All error bars show means ± s.e.m.

  4. Inhibition of tumor growth by [alpha]-lactalbumin vaccination is mediated by T cells.
    Figure 4: Inhibition of tumor growth by α-lactalbumin vaccination is mediated by T cells.

    (ac) Growth of 4T1 tumors after transfer of α-lactalbumin–primed or OVA-primed LNCs into naive recipient BALB/c mice on the same day as inoculation with 4T1 tumor cells (a), numbers of tumor-bearing mice after transfer of α-lactalbumin–primed or OVA primed LNCs into naive recipient BALB/c mice on the same day as inoculation with 4T1 tumor cells (b) and final tumor weights after transfer of α-lactalbumin–primed or OVA primed LNCs into naive recipient BALB/c mice on the same day as inoculation with 4T1 tumor cells (c). (d) Growth of 4T1 tumors after transfer of α-lactalbumin–primed or OVA primed CD4+ T cells (left) and CD8+ T cells (right) into naive recipient BALB/c mice on the same day as inoculation with 4T1 tumor cells. All error bars show means ± s.e.m. Each * indicates significance.

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Author information

  1. These authors contributed equally to this work.

    • Ritika Jaini &
    • Pavani Kesaraju

Affiliations

  1. Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.

    • Ritika Jaini,
    • Pavani Kesaraju,
    • Justin M Johnson,
    • Cengiz Z Altuntas,
    • Daniel Jane-wit &
    • Vincent K Tuohy
  2. Department of Biology, Cleveland State University, Cleveland, Ohio, USA.

    • Pavani Kesaraju &
    • Vincent K Tuohy
  3. Current addresses: Department of Urology, Case Western Reserve University, School of Medicine, Cleveland, Ohio, USA (C.Z.A.) and Yale University School of Medicine, Section of Cardiovascular Medicine, New Haven, Connecticut, USA (D.J.-w.).

    • Cengiz Z Altuntas &
    • Daniel Jane-wit

Contributions

R.J. performed the adoptive tumor immunotherapy studies and the TIL analysis and evaluated the effects of α-lactalbumin vaccination in MMTV-PyVT mice. P.K. evaluated the phenotype of autoimmune-mediated breast failure and performed the initial tumor immunotherapy studies. J.M.J. participated in the cloning and generation of α-lactalbumin. C.Z.A. and D.J.-w. provided technical assistance with molecular and immunocytochemical assays, and V.K.T. designed the experiments, supervised and obtained funding for the project, analyzed the data and wrote the manuscript. All authors discussed the results and implications and commented on the manuscript at all stages.

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The authors declare no competing financial interests.

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