Determination of the immune system that suppresses tumor formation of the tumor cybrids containing allogenic mtDNA. (A–F and H) The tumor cybrids were inoculated under the skin of B6 β2m^−/− and B6 Tap1^−/− mice (A), B6 Abβ^−/− mice (B), B6 Rag2^−/− mice (C), B6 Rag2^−/−/γc^−/− mice (D), B6 NK^Red mice (E), B6mtNZB NK^Red mice (F), and B6 Myd88^−/− and B6 Myd88^−/−/NK^Red mice (H) to study the involvement of the immune system and immune cells in the suppression of tumor formation (n = 6). Similar results were obtained in two independent experiments. Error bars represent SD. (G) Contribution of perforin but not FasL in preferential killing of P29mtNZB by NK cells derived from B6 Rag2^−/− mice. Cytotoxic assay of B6 Rag2^−/− splenocytes that had been stimulated with poly I:C in vivo for 20 h was tested against P29mtB6 or P29mtNZB at the indicated E/T ratios. An 8-h ⁵¹Cr releasing assay was performed in the presence or absence of 20 nM concanamycin A or 10 µg/ml anti-FasL antibody. In all experiments, spontaneous ⁵¹Cr release from the P29 cybrids was <15%. Data represent the mean ± SD of triplicate samples. Similar results were obtained in two independent experiments (*, P < 0.0005; **, P < 0.0001).

Rejection of the transplanted ES cybrids containing allogenic mtDNA. (A) Identification of the mtDNA genotypes of the ES cybrids. B6 and NZB represent mtDNAs from B6 and NZB mice, respectively. (B–E) Examination of tumor formation of the ES cybrids by their inoculation into B6 mice (B), B6mtNZB mice (C), B6 β2m^−/− mice (D), and B6 NK^Red mice (E). Similar results were obtained in two independent experiments. Error bars represent SD.

Effects of allogenic mtDNA on the expression of tumor phenotypes. (A) Experimental (left) and spontaneous (right) lung metastasis phenotypes of the tumor cybrids. Metastatic phenotypes were examined by inoculation of the tumor cybrids containing allogenic mtDNA derived from different strain mice into the tail veins or under the skin of B6 mice. (B) Tumor formation phenotype of the tumor cybrids. Total numbers of 5 × 10⁶ tumor cybrids were inoculated under the skin of B6 mice (left). A mixture of P29mtB6 (2.5 × 10⁶) and P29mtNZB (2.5 × 10⁶) was also inoculated (left) to confirm the selective tumor formation of P29mtB6 (n = 6 for each group). Data are representative of two independent experiments (*, P < 0.005; **, P < 0.0005). Restriction enzyme (SphI) digestions of PCR products were performed to identify the mtDNA genotypes (right). After the digestion, NZB mtDNA produced 143- and 31-bp fragments, whereas B6 mtDNA remained 174-bp PCR products. Mixed cybrids represent a simple mixture of P29mtB6 and P29mtNZB. Tumors represent primary tumors formed by inoculation of the mixed cybrids. (A and B) Error bars represent SD. (C) Tumor formation phenotype of the tumor cybrids inoculated under the skin of the B6mtNZB mice. Data represent the mean ± SD (n = 6). Similar results were obtained in two independent experiments.

Infiltration of NK cells and DCs into cell clusters formed by inoculation of the tumor cybrids into the B6 mice. (A) Identification by histochemical analysis of the mononuclear cells that infiltrated into cell clusters. P29mtB6 and P29mtNZB formed small cell clusters on day 4 after inoculation into B6 mice. P29mtB6 showed an increase in cluster size from day 4–12, but P29mtNZB showed a decrease in cluster size. H&E staining showed preferential infiltration of the mononuclear cells into cell clusters of P29mtNZB. Photographs of higher magnification of boxed areas are shown below. Immunohistochemical analyses showed preferential infiltration of CD11c⁺ DCs into cell clusters of P29mtNZB at day 4. Bars: (gray) 5 mm; (black) 20 µm. (B) Quantitative estimation of the NK cells and DCs that infiltrated into cell clusters by flow cytometric analysis using CD45⁺ lymphocytes isolated from the cell clusters. CD11c, B220, CD3, and NK1.1 are lineage-specific markers for DCs, B cells, T cells, and NK cells, respectively. The red boxes in the top and bottom panels indicate NK cells (CD3⁻ and NK1.1⁺) and DCs (CD11c⁺ and B220⁻), respectively. (C) Transcription of cytokines in cell clusters. Total RNA was prepared from cell clusters, and RT-PCR analyses were performed. Similar results were obtained in two independent experiments.