Exacerbated anti–tumor response in CD69−/−RAG-deficient mice. Mice were injected intraperitoneally with 10⁶ RMA-S tumor cells. Total unfractionated peritoneal cells from CD69+/+ and CD69−/−–RAG2−/− mice were examined 72 h after tumor inoculation. Forward and scattered FACS^® analysis. Marked gates correspond to RMA-S tumor cells. One representative mouse out of 10 per group is shown. Results shown are representative of two independent experiments (n = 8).

Peritoneal cell accumulation in CD69−/− mice in response to MHC class I⁻ tumor cells. Mice were injected intraperitoneally with PBS or with 10⁵ RM-1 cells (n = 7, mice/group). Cells collected from the peritoneal lavage were analyzed by FACS^® and a Multisizer cell counter, and total leukocyte numbers (×10⁻⁶) determined at the indicated times.

Attenuation of spontaneous cell death of CD69−/− lymphocytes. (A) Unfractionated peritoneal cells of untreated mice were seeded in culture medium and cell survival was measured by PI staining. Values show the percentage of PI⁺ cells (mean ± SD; n = 3 for each group). Results are representative of three independent experiments. (B) Analysis of intracellular caspase-3 activity of spleen cells from WT (left) and CD69−/− (right) challenge mice (3 d with 10⁵ RM-1). Caspase-3 activation was detected with fluorogenic substrate PhiPhiLux-G1D2. PhiPhiLux staining on FL-1 versus forward scatter channels was displayed. (C) After gating on DX5⁺ cells from spleen, caspase-3 activity was assayed. At least 100,000 events were collected per sample. Experiments were done twice with four and six mice per group; and similar results were obtained.

Enhanced anti-tumor activity by blocking anti–TGF-β in WT mice. Survival plot of WT and CD69−/− mice intraperitoneally injected on day 0 with 10⁴ RMA-S cells and treated with either 1D11 anti–TGF-β mAb (500 μg/injection) or PBS on days −3 and −1 (before tumor challenge), and at day +1 and every week after tumor inoculation. (n = 4). Results shown are representative of two independent experiments.

Therapeutic anti-tumor activity of anti-CD69 mAbs. 8-wk-old C57BL/6 mice were treated intraperitoneally with 500 µg anti-CD69.2.2 or with the isotype control (mouse IgG1). (A) Thymocytes were prepared from the treated mice, and were subjected to flow cytometric analysis. Representative profiles of CD4/CD8, CD2/CD69 (10 d after mAb injection), and CD3/CD69 (15 d after mAb injection) are shown with the percentage of cells in each quadrant. (B) Peritoneal cells of WT mice were examined at day 3 after intraperitoneal injection of 10⁶ RMA-S tumor cells. Mice were treated with mAb 1 d before tumor inoculation. Cells collected from the peritoneal lavage were analyzed and forward and scattered FACS^® analysis is shown. Marked gates correspond to RMA-S tumor cells. (C) Mice were injected intravenously with 10⁴ RM-1 and were treated with mAb on day −1 (before tumor challenge). Number of lung metastases was counted 14 d after tumor inoculation. (C) Open bar, control mice; closed bar, anti-CD69 mAb treated WT mice (bars ± SD). Results are representative of two independent experiments (n = 3).

Increased NK anti-tumor activity in CD69−/− mice. Mice were injected intraperitoneally with 10⁴ RMA-S (A) or RMA (C) tumor cells. Mice were observed daily for tumor growth up to 12 wk by monitoring body weight and ascites development. Similar results were observed in mice injected subcutaneously with 10⁵ RMA-S (B). (A, n = 9; B, n = 6; C, n = 9.) (D) NK or T lymphocytes were depleted from CD69−/− mice before intraperitoneal inoculation with 10⁴ RMA-S tumor cells. Mice were treated for NK cell depletion on day −1 (before tumor challenge), and at days +2 and +4 after tumor inoculation with either control diluent or anti–asialo-GM1 antiserum (100 μl/injection). Mice were treated for lymphocyte T CD4⁺ depletion on day −1 (before tumor challenge), and at days +2 and +4 after tumor inoculation with either GK1.5 anti-CD4 or isotype control mAbs (100 μl/injection). (D, n = 7) Results shown are representative of two independent experiments. (E) Photographs are of lungs from CD69−/− and WT mice 2 wk after the challenge with 10⁴ RM-1 cells intravenous inoculation (top). One representative mouse out of 10 per group is shown. The number of lung metastases were counted (bottom) and each symbol represents one mouse. Open symbols, WT mice; closed symbols, CD69−/− mice. Results are representative of two independent experiments.

Increased ex vivo NK cytotoxicity in CD69−/− mice. Mice received 10⁴ RMA-S (A) or RM-1 cells (B) intraperitoneally 72 h after inoculation, total unfractionated peritoneal cells (A and B) from CD69−/− and WT mice were analyzed for cytotoxicity against YAC target cells. Two animals were used per experimental group (mean ± SE). (C and D) Purified NK cells were used, untreated or treated with IL-2, respectively. (D) Two animals were used per experimental group (mean ± SE). Open symbols, WT; closed symbols, CD69−/−. The results are representative of three similar experiments.

Similar trafficking of activated CD69−/− and WT CD4⁺ T cells. Short-term (A) and 24-h (B) trafficking patterns of activated CD69−/− and WT CD4⁺ T cells do not differ. Distribution of radioactivity in various organs at 1 or 24 h, respectively, after intravenous injection of activated ⁵¹Cr-labeled CD4⁺ T cells. Note the numerical scales for organs shown on the right. Four animals were used per experimental group (bars ± SD).

Altered chemokine and cytokine pattern expression in CD69−/− mice. (A) Relative levels of cytokine and chemokine mRNA in peritoneal cells from CD69−/− (▪) and WT (□) mice were analyzed 3 d after intraperitoneally inoculation of 10⁵ RM-1 cells. Results are expressed in arbitrary densitometric units normalized for the expression of GAPDH or L32 in each sample. Five animals were used per experimental group, and results are representative of four independent experiments. (B) MCP-1 levels in LPS-activated peritoneal cells from thioglycollated-treated CD69−/− and WT mice. Data represent the mean ± SE (n = 4 for each group) of one experiment representative of three independent experiments. *, P < 0.004. (C) Quantitative real-time RT-PCR analysis of mRNA from CD69−/− and WT purified peritoneal macrophages (left) and NK cells (right) obtained from mice after 6 d of 10⁵ i.p. RM-1 challenge. Three animals were used per experimental group, and results are representative of one experiment.

CD69 engagement induces TGF-β1 secretion in NK and CD3⁺ T lymphocytes. Antibody engagement of CD69 induces TGF-β1 secretion in mouse-purified NK and T lymphocytes. Anti-CD69 (CD69.2.2) or the isotypic control (murine IgG1) was added followed by cross-linking with goat anti–mouse IgG antibody. (A) Active (left) and total (right) TGF-β1 were determined in NK cells after 24 h in culture supernatants. (B) Purified resting CD3⁺ T cells were added to 96-well plates coated with anti-CD3. Expression of CD69 after activation with anti-CD3 were analyzed by staining with FITC–anti-murine CD69 mAb (shaded line) after 24 h of culture. Dotted line represents negative control. (C) Antibody engagement of CD69 induces TGF-β1 secretion in mouse T lymphocytes. Cells were incubated in serum-free medium for 72 h and TGF-β1 was determined by ELISA. Data are expressed as mean ± SD of replicate wells and are representative of three independent experiments. (D) Cultured cells were removed and trypan blue was added. Dead cells (trypan blue positive) was determined after 48 h of culture. Data represent means of triplicate wells (bars ± SD).