Figure 1.

Accumulation of CD11b⁺Gr-1⁺ cells in 4T1 tumour-bearing mice. 4T1 mammary tumour cells (5 × 10⁴/mouse) were injected orthotopically in five individual female CB6F1/J (H-2^b/d) mice. Control (denoted as ‘C’) mice did not receive a tumour injection. Upon reaching the ethical limit of 2 cm³ tumour volume (at day 26), all mice were euthanized, and splenocytes recovered for enumeration. (A, left panel) Unfractionated splenocytes were evaluated for the percentage of CD11b⁺Gr-1⁺ cells using two-colour flow cytometry. (A, right panel) Absolute numbers of CD11b⁺Gr-1⁺ cells were calculated by multiplying the percentages determined in (A) by total splenocyte counts. For both panels, splenocytes from the control group were pooled and an average result determined, whereas splenocytes from the 4T1 tumour-bearing mice were analysed separately. Comparable results were observed when BALB/c mice were similarly challenged with 4T1 cells. In a separate experiment, the percentages (B, left panel) and absolute numbers (B, right panel) of CD11b⁺Gr-1⁺ cells were determined, as described above from unfractionated bone marrow cells of control and 4T1 tumour-bearing mice. Each data point represents the results of an individual mouse. (C) Splenic CD4⁺ or CD8⁺ T cells were purified from non-tumour-bearing wild-type (WT) CB6F1/J mice. T cells were mixed with CD11b⁺Gr-1⁺ cells purified from the spleens of either WT or 4T1 tumour-bearing (TB) CB6F1/J mice (1:1 ratio). In the case of the control group, both T cells and CD11b⁺Gr-1⁺ cells were obtained from the same mouse. In the case of tumour-induced CD11b⁺Gr-1⁺ cells, T cells from a wild-type mouse were used to avoid any in vivo tumour-induced effect on T-cell function. Cultures were then incubated in the absence or presence of immobilized anti-CD3 mAb for 48 hrs. Proliferation was measured by ³H-thymidine uptake after an additional 24 hrs of incubation. Results represent the mean ± S.E.M. of triplicate wells.