Complexes of trophoblastic peptides and heat shock protein 70 as a novel contraceptive vaccine in a mouse model  Mei Han, Yuan Yao, Wangjiang Zeng, Yanfang Wang, Lin Feng, Jie Zhao  Reproductive BioMedicine Online  Volume 32, Issue 4, Pages 457-465 (April 2016) DOI: 10.1016/j.rbmo.2015.12.009 Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Mouse trophoblast-derived peptides bound to heat-inducible 70-kDa HSP (HSP70) induce trophoblastspecific T cell. (A) HSP70-trophoblastic peptide complexes induced T cell proliferation. Approximately 2 × 104 spleen T cells were co-cultured with 5 × 103 irradiated splenocytes in the presence or absence of HSP70-trophoblastic peptide complexes for 6 days. The anti-CD3 and CD28 antibodies were used to stimulate T cells as a positive control. T-cell proliferation was determined by [3H]-thymidine incorporation assay. Stimulation index was calculated by dividing the proliferation count in the presence of peptide complexes or antibodies by that in the absence of peptide complexes or antibodies; (B) production of IFN-γ and IL-2. The culture supernatants were collected for the determination of cytokine concentrations by enzyme-linked immunosorbent assay. The data in this figure represent the results of six independent experiments. *P < 0.05 compared with the medium group. Reproductive BioMedicine Online 2016 32, 457-465DOI: (10.1016/j.rbmo.2015.12.009) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Cytotoxic effects of T cells on trophoblasts in vitro. (A) Stimulation of T cells by trophoblast-derived peptides and different tissue-derived peptides. Different peptides were bound to heat-inducible 70-kDa HSP (HSP70) for T-cell stimulation and evaluated by [3H]-thymidine incorporation assay. *P < 0.05 compared with the medium group; (B) cytotoxicity of T cells on trophoblasts in vitro. The naïve spleen T cells were stimulated by HSP70-trophoblastic peptide complexes for 7 days as effector cells. The anti-CD3 and CD28 antibodies (1 µg/ml each) that stimulated T cells or naive T cells served as controls. The trophoblasts were isolated from the murine placenta for use as target cells. The cytotoxicity of T cells on trophoblasts was measured at different ratios (T cells: trophoblasts). In certain cases, T cells and trophoblasts were incubated in the supernatant from cultured placental tissue for the cytotoxicity assay. The data in this figure represent the results of six independent experiments. PS, placenta supernatant; TP, trophoblastic peptide. Reproductive BioMedicine Online 2016 32, 457-465DOI: (10.1016/j.rbmo.2015.12.009) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Various components of trophoblasts are capable of inducing T cell activation. (A) Syngeneic male and female trophoblastic peptides stimulated cytotoxic T cells against trophoblasts. BALB/c mice were used for mating. After identifying the genotypic sex of the trophoblasts, the male and female trophoblastic peptides from the placenta were prepared and complexed with heat-inducible 70-kDa HSP (HSP70) to activate T cells. The cytotoxicity assay was carried out through the incubation of different activated T cells with isolated male or female trophoblasts at a ratio of 25:1, *P < 0.01 and **P < 0.05; (B–D) allogeneic trophoblastic peptides induced cytotoxic T cells against trophoblasts. C57BL/6 or BALB/c male mice were mated with BALB/c female mice. The female trophoblastic peptides were prepared and complexed with HSP70 to activate BALB/c splenic T cells. The hybrid peptide increased T-cell proliferation and promoted the production of IFN-γ and IL-2 relative to the inbred peptide (B and C), *P < 0.05. The hybrid T cells also exhibited greater cytotoxicity against hybrid trophoblasts than inbred T cells at different ratios (T cells: trophoblasts) (D); the data in this figure represent the results of six independent experiments. Reproductive BioMedicine Online 2016 32, 457-465DOI: (10.1016/j.rbmo.2015.12.009) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Heat-inducible 70-kDa HSP (HSP70)-trophoblastic peptide complexes induced T cell activation in vivo. (A) Expression of IL-2, IFN-γ, IL-4, and IL-10 by splenocytes in vaccinated mice. Mice (n = 6) were treated with HSP70-peptide complexes (50 µl) or phophate buffered saline control by subcutaneous injection once a day for 5 days. Seven days later, the mice T splenocytes were isolated and the transcripts of genes were measured by conventional reverse transcription polymerase chain reaction (bottom) and real-time polymerase chain reaction (top); (B) induction of cytotoxic T cells in vaccinated mice. The above splenocytes were further stimulated by HSP70-peptide complexes for 3 days and used in the cytotoxicity assay. The cytotoxicity of T cells on trophoblasts/uterine was measured at different ratios (T cells: trophoblasts/uterine). GADPH, glyceraldehyde-3-phosphate dehydrogenase. Reproductive BioMedicine Online 2016 32, 457-465DOI: (10.1016/j.rbmo.2015.12.009) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Heat-inducible 70-kDa HSP (HSP70)-trophoblastic peptide complexes resulted in contraception in vivo. (A) Immunization by HSP70-trophoblastic peptide complexes induced contraception in female mice. Adult female BALB/c mice (six mice per group) were immunized subcutaneously with HSP70-trophoblastic peptide complexes or controls and paired with fertile male mice seven days later. Fertility was monitored over a 90-day period by the number of litters; (B) trophoblast-specific T cell-induced contraception. Fertilization in mice was shown by the presence of vaginal plug. On days 7 and 9 of gestation, the mice were adoptively transferred intravenously with 2 × 106 T cells activated by HSP70-peptide complexes (peptide T) or control spleen T cells (native T). This experiment was repeated by adoptive transfer on days 1 and 3. The number of pups was monitored over a 30-day interval. The data represent the results of two independent experiments; (C) serum levels of glutamate-pyruvate transaminase and creatinine in mice (n = 8) were detected 2 months after vaccination with HSP70-peptide complexes once a day for 5 days. TP, trophoblastic peptide; UP, uterine peptide. GPT, glutamate-pyruvate transaminase. Reproductive BioMedicine Online 2016 32, 457-465DOI: (10.1016/j.rbmo.2015.12.009) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions