IL-2 treatment enhanced OX40 expression on CD8 T cells in tumor-bearing hosts.

<p><b>A, B</b>) C57BL/6 OX40-cre x ROSA-YFP reporter mice received 1×10⁶ MCA-205 sarcoma tumor cells (day 0) and two weeks later, the tumor-bearing mice were treated with IL-2 cytokine/mAb complexes (day 14, 15; i.p.). Twenty four hours later (day 16 post-tumor inoculation) the extent of CD25, YFP (OX40 reporter), and OX40 expression on CD8 T cells isolated from the <b>A</b>) tumor and <b>B</b>) spleen were assessed. Graphs depict the results obtained from 3–4 individual animals from 1 out of 2 independent experiments.</p

Common gc cytokines regulate OX40 via JAK/STAT signaling.

<p><b>A</b>) Naïve WT OT-I T cells were stimulated with peptide-pulsed APCs (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034467#pone-0034467-g003" target="_blank">Fig. 3A</a>). <b>A</b>) Two days later, OT-I T cells were harvested and re-cultured (5×10⁵ cells/ml) with media or rmIL-2 (100 ng/ml) and the expression of the indicated proteins was assessed by Western blot. <b>B</b>) WT OT-I T cells were stimulated (as in (<b>A</b>)) +/− a JAK3 inhibitor (PF-956980; 100 ng/ml). Twenty-four hours later, cells were harvested and the extent of CD25 and OX40 expression was determined. <b>C, D</b>) WT or OX40^−/− OT-I cells were stimulated for 2 days, harvested, and then re-stimulated with media alone, rmIL-2, rmIL-4, rmIL-7, rmIL-9, rmIL-15, or rmIL-21 (100 ng/ml). Twenty-four hours later, cells were harvested and the extent of <b>C</b>) CD25 and <b>D</b>) OX40 expression (% positive and MFI) were determined. <b>E</b>) WT OT-I T cells were activated and then re-stimulated with the indicated common gc cytokines and protein expression was assessed by Western blot. <b>B–D</b>) Bar graphs depict the mean+/−SD from <b>B</b>) n = 2–3/group or <b>C, D</b>) n = 3–8/group. Data are representative of one out of two to ten independent experiments. *P<0.05; ** P<0.01; *** P<0.001.</p

OX40 is regulated by TCR stimulation and IL-2Ralpha (CD25) expression.

<p>(<b>A, B</b>) Naïve wild-type or OX40^−/− OT-I T cells (2×10⁵/ml) were stimulated with peptide-pulsed APCs (2×10³/ml). <b>A</b>) Three days later, OT-I T cells were harvested and the extent of CD25 and OX40 expression were determined. <b>B</b>) Kinetics of CD25 and OX40 expression following TCR stimulation were determined at the indicated time points by flow cytometry. (<b>C</b>) Naïve polyclonal wild-type or CD25^−/− CD8 T cells (3×10⁵/well) were CFSE-labeled and then stimulated with anti-CD3 and anti-CD28 (1 and 5 mcg/ml, respectively). One to three days later, CD8 T cells were harvested and the extent of CD25 and OX40 expression were determined. <b>B, C</b>) Bar graphs depict the mean+/−SD (n = 2–3/group). Data are representative of one out of two to three independent experiments. *P<0.05.</p

Combined anti-OX40/IL-2c therapy boosts anti-tumor immunity through a T cell-dependent mechanism.

<p><b>A, B</b>) Wild-type mice received 1×10⁶ MCA-205 sarcoma tumor cells (n = 8/group). Tumor-bearing mice were treated with anti-OX40 or rat IgG Ab (days 10, 14; i.p.) along with IL-2 cytokine/mAb complexes (days 10–13; i.p.) and the extent of <b>A</b>) tumor growth and <b>B</b>) survival of tumor-bearing mice were assessed. Data are representative of one out of 2 independent experiments. <b>C</b>) MCA-205 tumor-bearing mice (as in (<b>A</b>)) received no treatment (n = 9), anti-CD4 (n = 6), anti-CD8 (n = 6), or anti-CD4+anti-CD8 (n = 3) (200 mcg/dose; i.p.) 9, 17, and 24 days post-tumor implantation. Mice were then treated with anti-OX40 (days 10, 14) and IL-2c (days 10–13) and the extent of survival of tumor-bearing mice was assessed.</p

Dual anti-OX40/IL-2c therapy reverses CD8 T cell anergy and increases the survival of mice with long-term well-established tumors.

<p><b>A</b>) Tumor model. 2.5×10⁶ TRAMP-C1-mOVA tumor cells were injected into POET-1 mice. Twenty days later, mice (∼50 mm² tumors) received 5×10⁵ naïve OT-I T cells. Seventeen days after T cell adoptive transfer (37 days post-tumor inoculation), the anergic donor OT-I T cells were re-stimulated with anti-OX40 or control (rat IgG) Ab (d37-38), 500 mcg OVA (d37), 10 mcg LPS (d37), +/− IL-2 cytokine/mAb complexes (d37-44). <b>B</b>) Seven days after the initial dose of Ag/anti-OX40 the extent of donor CD8 T cell expansion (% OT-I of total CD8 T cells; pre- vs. post-treatment), Ki-67 (proliferation), granzyme B, and KLRG-1 expression on the donor OT-I T cells were determined. <b>C</b>) Tumor-bearing mice were treated as in (<b>B</b>) and then seven days later OVA peptide-pulsed (CFSE^high) and control HA peptide-pulsed (CFSE^low) target cells were mixed at a 1∶1 ratio and injected into recipient mice. Four hours later, spleens were harvested and the ratio of % CFSE^low/% CFSE^high target cells from individual mice (n = 5/group) was determined. <b>D, E</b>) The extent of tumor growth (mean+/−SD; n = 5/group) and <b>D</b>) survival (n = 11/group) of tumor-bearing mice were assessed. Data are representative of one out of 2 to 3 independent experiments or <b>E</b>) the cumulative survival from 2 independent experiments. *P<0.05, **P<0.01.</p

STAT3 and STAT5 are required for optimal up-regulation of OX40 following stimulation with common gc cytokines.

<p><b>A</b>) WT or STAT3^−/− OT-I T cells were stimulated for 2 days, harvested, and then re-cultured with media alone, rmIL-2, rmIL-4, or rmIL-21 (100 ng/ml); 24 hours later cells were harvested and the extent of CD25 and OX40 expression (% positive and MFI) were measured. <b>B</b>) Polyclonal endogenous WT or STAT5^−/− CD8 T cells were stimulated for 2 days with 2 mcg/ml anti-CD3 mAb, harvested, and then re-cultured with media alone, rmIL-2, rmIL-4, or rmIL-21 (100 ng/ml) and then 24 hours later, cells were harvested and the extent of CD25 and OX40 expression (% positive and MFI) were determined. <b>A, B</b>) Bar graphs depict the mean+/−SD (n = 2–3/group). Data are representative of one out of two independent experiments. *P<0.05; ** P<0.01; *** P<0.001; NS = no statistically significant difference.</p

OX40 is regulated on murine and human T cells by TCR stimulation and IL-2.

<p><b>A</b>) Purified naïve wild-type or OX40^−/− OT-I T cells (1×10⁶/ml) were stimulated with peptide-pulsed APCs (6×10⁶/ml). Two days later, OT-I T cells were harvested and re-cultured (5×10⁵ cells/ml) +/− rmIL-2 (100 ng/ml). Twenty-four hours later, cells were harvested and the extent of CD25 and OX40 expression were determined. Bar graphs depict the mean+/−SEM (n = 6/group). <b>B, C</b>) Human CD8 or <b>C</b>) CD4 T cells collected from PBMC were stimulated with media, rhIL-2 (5,000 IU/ml, equivalent to 300 ng/ml), and/or 1 mcg/ml anti-CD3 mAb (OKT-3). Forty-eight hours later, cells were harvested, washed, and stimulated with media or rhIL-2 (5,000 IU/ml). Twenty-four hours later, the extent of CD25 and OX40 expression were measured. <b>C</b>) Bar graphs depict the mean+/−SD (n = 3–5/group). Data are pooled from five independent experiments. *P<0.05; **P<0.01; ***P<0.001.</p