Soluble CD80 Protein Delays Tumor Growth and Promotes Tumor-Infiltrating Lymphocytes

Abstract

Tumor cells use various immune-suppressive strategies to overcome antitumor immunity. One such method is tumor expression of programmed death ligand-1 (PD-L1), which triggers apoptotic death or anergy upon binding programmed death-1 (PD-1) on T cells. Our previous in vitro cellular studies with human and mouse PD-L1⁺ tumor cells demonstrated that a soluble form of the costimulatory molecule CD80 prevented PD-L1-mediated immune suppression and restored T-cell activation by binding PD-L1 and blocking interaction with PD-1. We now report that in vivo treatment of established syngeneic PD-L1⁺ CT26 colon carcinoma and B16F10 melanoma tumors with CD80-Fc delays tumor growth and promotes tumor-infiltrating T cells. Studies with PD-1^-/- and CD28^-/- mice demonstrate that soluble CD80 acts in vivo by simultaneously neutralizing PD-1 suppression and activating through CD28. We also report that soluble CD80 mediates its effects by activating transcription factors EGR1-4, NF-κB, and MAPK, downstream signaling components of the CD28 and T-cell receptor pathways. Soluble CD80 binds to CTLA-4 on activated human peripheral blood mononuclear cells. However, increasing quantities of CTLA-4 antagonist antibodies do not increase T-cell activation. These results indicate that soluble CD80 does not suppress T-cell function through CTLA-4 and suggest that CTLA-4 acts as a decoy receptor for CD80, rather than functioning as a suppressive signaling receptor. Collectively, these studies demonstrate that soluble CD80 has therapeutic efficacy in vivo in mouse tumor systems and that its effects are due to its ability to inhibit PD-1-mediated suppression while concurrently activating T cells through CD28. Cancer Immunol Res; 6(1); 59-68. ©2017 AACR.

Figure 1.. Monotherapy with CD80-Fc delays tumor growth in mice with CT26 tumors.

A, BALB/c or B, C57BL/6 mice were inoculated s.c. on day 0 with 1×105 CT26 colon carcinoma or B16F10 melanoma cells. When the average tumor diameter was 4.5mm, mice were started on twice weekly i.t. injections of 20μg IgG or CD80-Fc/mouse. I.t. injections were continued for three weeks. Graphs show average tumor volumes ± SD, survival time, and tumor growth in individual mice. Day 0 is the first day of therapy. n= 14 (CD80-Fc) or 15 (IgG) mice/group and 8 (CD80-Fc and IgG) mice/group for A and B, respectively, and are pooled from multiple experiments. Statistical analyses for A and B were performed using the compare Growth Curves function of the Statmod software package. C, BALB/c mice were inoculated s.c. on day 0 with 5×105 CT26 colon carcinoma cells. I.p. injections of 200μg of mouse CD80-Fc, rat IgG2a, or anti-mouse PDL1 were administered on days 3, 6, 9, and 22 following tumor inoculation. Surviving mice were sacrificed on day 42. Tumors were cryopreserved and analyzed by immunohistochemistry. Images are representative of 5 mice/group.

Figure 2.. Combination i.t. therapy with CD80-Fc plus CpG is more effective than CpG monotherapy.

A, BALB/c mice were inoculated s.c. on day 0 with 1×105 CT26 colon carcinoma cells. On day 14 when the average tumor diameters were 6.4 – 6.7 mm, the mice were started on twice weekly i.t. injections of 20μg IgG, 200 μg CpG/mouse, or 200 μg CpG + 20μg CD80-Fc/mouse. I.t. injections were continued for three weeks. Graphs show average tumor volumes ± SD, percent surviving mice, and tumor growth in individual mice. Day 0 is the day of tumor inoculation. n= 5 mice/group. Statistical analysis was performed using pairwise comparison test.

Figure 3.. CD80-Fc activates transcription factors downstream of CD28 and the TcR.

A, CD80-Fc induces transcription of EGR1/2/3/4. CD3⁺ cells were isolated by negative selection from the blood of healthy human donors, activated with PHA for 72 hours, and then either untreated or incubated for two hours with isotype control mAb, agonist CD28 mAb, TROY-Fc, or CD80-Fc. RNA was then isolated, converted into cDNA, and the DNA analyzed by qRT-PCR for EGR1/2/3/4. Values were normalized to β-actin expression. Results are shown as fold change in expression level compared to untreated T cells. Statistical analysis was performed using Student’s t test. Data are from one of three independent experiments. B, CD3⁺CD28+ Jurkat cells were treated for 30 min at 37OC with isotype mAb, agonist CD28 mAb, TROY-Fc, or CD80-Fc. Cells were then fixed and permeabilized, and stained with mAbs to pMAPK or pNF-κB followed by anti-rabbit IgG-FITC. C, Jurkat cells were untreated or treated for 30 min with agonist anti-CD28 mAb and then lysed and analyzed by western blot for phosphorylated MAPK and NFκB. Data for B and C are representative of one of two and one of three independent experiments, respectively.

Figure 4.. CD80-Fc does not affect T cell activation by interacting with CTLA4.

A, PBMC from a healthy human donor were activated with PHA for 72 hours and subsequently stained with antagonist anti-CTLA4 mAb L3D10 or Ipilimumab. Human melanoma C8161 cells were stained for PDL1 expression (29E.2A3 mAb). B, PHA-treated CTLA4+ T cells were co-cultured with PDL1⁺ C8161 human melanoma cells in the presence or absence of CD80-Fc, Ipilimumab, control recombinant protein TROY-Fc, antagonist CTLA4 mAb L3D10, or irrelevant isotype antibody. After three days of culture T cell activation was assessed by measuring IFNγ in the supernatants. Top panel shows one of three independent experiments; bottom panel shows the average of three independent experiments. Error bars indicate SD. Bars with * are statistically different from all other values and are not statistically different from each other at p < 0.05.

Figure 5.. CD80-Fc prevents CTLA4-mediated suppression by saturating CTLA4.

PHA-activated human PBMC, PDL1⁺ C8161 cells, and CD80-Fc (2 μg/ml) were incubated with increasing amounts of soluble CTLA4-Fc. or control irrelevant protein TROY-Fc. T cell activation was assessed by measuring IFNγ in the supernatants. Statistical analyses were performed using Student’s t test. Data are from one of two independent experiments.

Figure 6.. The in vivo therapeutic effect of CD80-Fc involves neutralization of PD1 suppression and activation through CD28.

C57BL/6 wild type, CD28-deficient, and PD1-deficient mice were inoculated with B16F10 cells in both flanks. When tumors were ~5 mm in diameter (~day 7), tumors on one flank were treated with CD80-Fc (wild type, CD28^−/−, and LPD1^−/− mice) or PBS (wild type mice). Mice were followed for tumor progression. A, Progression of treated tumors. B, Comparison of progression of treated and contralateral untreated tumors.