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Article History

Received: 10 September 2018

Accepted: 26 March 2019

First Online: 8 June 2019

Compliance with ethical standards

:

: The authors declare that they have no conflict of interest.

: All animal procedures were conducted in accordance with accredited institution guidelines and the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. The animal protocol for this study IACUC-17-10-0356 was approved by the Wayne State University Institutional Animal Care and Use Committee (IACUC).

: Wild-type (wt) C57BL/6 and BALB/c mice were purchased from Charles River Laboratory (Frederick, MD, US). Heterozygous B6 HER-2 Tg mice (B6 HER-2 Tg) which express full-length wt HER-2 under the whey acidic protein (WAP) promoter were generated in our laboratory and maintained by mating with wt B6 mice []. BALB/c HER-2 Tg (BALB HER-2 Tg) mice were generated by back-crossing B6 HER-2 Tg mice to wild-type BALB/c mice [] and are maintained by mating with BALB/c mice. B6 HER2 Tg mice have been deposited at Jackson lab repository (B6.Cg-Tg(Wap-ERBB2)229Wzw/J). Transgene positive mice were identified by PCR as described [].

: Antigen-presenting cells (APC) used in ELISPOT assay including 3T3/KB, 3T3/EKB and TC-1/E2 were generated as previously described []. BALB/c NIH 3T3 fibroblasts were transfected with both Kd and B7.1 (CD80) to generate 3T3/KB, or with the addition of HER2 for 3T3/EKB. 3T3/NKB similarly generated to express neu was used for measuring anti-neu Ab levels in the immune serum. The expression of the transgenes is validated by flow cytometry using mAb to Kd (SF1-1.1, Biolegend), B7.1 (CD80, BD), HER2 (TA-1/Ab5, Calbiochem) and neu (Ab4, EMD Millipore). C57BL/6 lung epithelial cell line TC-1 expressing Kb and B7.1 was a gift from Dr. T.C. Wu (The Johns Hopkins University, Baltimore, MD). TC-1/E2 cells were transfected with wt HER-2 as previously described []. TC-1 and TC-1/E2 cells are validated by tumor growth in B6 mice and by their expression of Kb as detected by mAb Af6-88.5.5.3 (eBioscience, Thermo Fisher). D2F2 is a mouse mammary tumor line that arose originally in a BALB/c hyperplastic alveolar nodule line, D2, [–]. D2F2/E2 cells were generated by co-transfection with a HinDIII WAP-HER-2 expression cassette (6.9-kb) and linearized pRSV/neo as reported []. D2F2/E2t cells were selected from D2F2/E2 cells by serial passage in BALB/c mice. D2F2 cells and derivatives are validated by tumor growth in BALB/c mice and by their expression of Kd as detected by mAb SF1-1.1. Expression of HER2 in D2F2/E2 and D2F2/E2t is verified by mAb Ab5, using flow cytometry. SK-BR-3 and SKOV3 cells were purchased from the American Type Culture Collection. Authentication of SKBR-3 and SKOV3 cells by short tandem repeat (STR) profiling was carried out with Promega’s Cell ID System.