Figure 3. Tumor infiltrating CD4⁺ T cells stimulate pulmonary metastasis.

a–b, Freshly-isolated MMTV-ErbB2 PCaM cells (1×10⁶) were transplanted into the #2 mammary glands of indicated mice. a, Lung metastasis incidence and multiplicity were determined 56 days later. Means +/− s.e.m. (n=4–6). Each point represents a value from a single mouse. b, Pulmonary metastasis in indicated mice inoculated with PCaM cells was quantified by qRT-PCR analyses of lung RNA with MMTV-ErbB2 specific primers. Means +/− s.e.m. (n=4). c, MT2 cells were transplanted as above. After 56 days, pulmonary metastasis incidence and multiplicity were quantified. Means +/− s.e.m. (n=6). d, MT2 cells were transplanted into Rag1^−/− mice reconstituted with indicated cell types, 3 days after reconstitution. After 35 days, lung metastasis incidence and multiplicity were quantified. Means +/− s.e.m. (n=3). e, MT2-inoculated Rag1^−/− mice were treated with BSA or RANKL (80 μg/kg) twice weekly, starting one week post-inoculation. After 35 days, lung metastasis incidence and multiplicity were determined. Means +/− s.e.m. (n=6–7). f, MT2 cells were transplanted into Rag1^−/− mice reconstituted with indicated cell types as in d. Lung metastases were measured by Q-RT-PCR of ErbB2 mRNA on day 56 post-inoculation. Each plot represents ErbB2 mRNA expression from an independent littermate group. Means +/− s.e.m. (n=3). g, MT2 cells were transplanted into Rag1^−/− littermate females reconstituted with PBS, CD4⁺CD25⁻ or CD4⁺CD25⁺ cell, as above. After one week, the CD4⁺CD25⁺ reconstituted mice were treated with control Fc or RANK-Fc (2.5 mg/kg) twice weekly. Pulmonary metastasis multiplicity was calculated on day 35 post-inoculation. Means +/− s.e.m. (n=4–5). *: P<0.05; **: P<0.01.