Treatment with anti-CD137 mAbs causes intense accumulations of liver T cells without selective antitumor immunotherapeutic effects in this organ

Abstract

Background/aims: Cancer therapy with agonist anti-CD137 mAbs has been shown to induce immune-mediated tumor rejections in mice, and equivalent agents of this kind are currently being tested in cancer patients. Previous reports indicated that CD137 stimulation induced polyclonal infiltrates of T lymphocytes in the liver. This study characterizes the liver infiltrates and the target dependency of the phenomena and addresses the question of whether tumors nested in the liver are a more favorable target for CD137-based immunotherapy.

Methods: Liver infiltrates were studied with conventional histology and multiple color flow cytometry of total liver leukocytes. CD137(-/-) mice, mice with a single rearrangement of the TCR (OT-1 mice) and Rag(-/-) mice were used to clarify molecular requirements. Mice implanted with MC38 colon carcinomas either subcutaneously or inside the liver were used for comparative studies under treatment with agonist anti-CD137 mAbs.

Results: CD137 treatment caused mononuclear inflammation in the portal spaces of the liver, which gave rise to moderate increases in transaminases without signs of cholestasis. Marked increases in the numbers of CD8+ T cells were observed, including CD8+ T lymphocytes co-expressing CD11c. Infiltrates were absent in CD137(-/-) mice and mitigated in mice harboring a single transgenic TCR on their CD8 T cells. Despite the tumor-independent accumulation of T cells in the liver, immunotherapeutic effects were not more prominent against tumors located in this organ.

Conclusions: Target-dependent effects of CD137 stimulation lead to liver infiltration with T cells, but lymphocyte enrichment in this organ does not privilege this site for immunotherapeutic effects against transplanted tumors.

Fig. 1

Treatment of mice with anti-CD137 mAb gives rise to liver inflammation in a CD137-dependent fashion. Representative microphotographs of H&E-stained paraffin-embedded sections from the liver of C57BL/6 mice at different magnifications from the indicated mice treated with three weekly doses of 150 μg of 2A anti-CD137 mAb or rat IgG as indicated in each figure (5 mice per group were analyzed). The right column of the microphotographs shows CD3 immunohistochemistry stainings with anti-CD3 mAb of the same samples at ×200 magnification. Livers were collected 1 week following the last dose of antibody

Fig. 2

Treatment of mice with anti-CD137 mAb causes increases in transaminases and moderate hepatocyte apoptosis. a Sequential serum samples were collected from mice treated as in Fig. 1 and the concentration of SGOT and SGPT are given on day 14 when these parameters peaked. This experiment is representative of two similarly performed experiments. b Tunel staining of liver sections from the indicated mice as in a are shown. Arrows indicate fluorescence in the apoptotic nuclei

Fig. 3

Liver infiltrates dependent on CD137 stimulation are composed mainly of CD8+ T lymphocytes, while the intensity of the infiltrates is mitigated in transgenic animals with a single TCR (OT-1 mice). The indicated groups of mice (6 per group) were treated with three weekly doses of 150 μg of anti-CD137 mAb 2A and the liver leukocytes were purified with percoll gradients. a The total numbers of recovered liver leukocytes. b, c Individual absolute numbers (left) and percentage (right) of CD3+CD8+ (b) and T cells CD4+ (c). A representative dot plot of CD3/CD8 and CD3/CD4 double stainings of samples from WT mice treated with anti-CD137 mAb is presented next to the graphs. The set of experiments is representative of two similarly performed experiments. Similar results were obtained on repeated treatment with 1D8 anti-CD137 mAb instead of 2A

Fig. 4

Liver infiltrates contain CD8 T cells coexpressing CD11c. Multiple color flow cytometry analyses quantify in individual mice from Fig. 3 the absolute numbers and percentages of a CD8+CD11c+ T cells, b CD3-NK1.1 NK cells and c CD3+NK1.1+ NKT cells in the indicated groups of mice

Fig. 5

Transplanted tumors in the liver and in subcutaneous tissue respond similarly to CD137-mediated immunotherapy with agonist monoclonal antibodies. Independent groups of mice were implanted subcutaneously in the right flank or in the left liver lobe under laparotomy. Mice were treated with 2A anti-CD137 mAb (a) or 1D8 anti-CD137 mAb (b) at 100 μg/mL doses given on days 8, 11 and 14 following tumor cell inoculation in separate experiments. Polyclonal rat IgG-treated mice at the same doses are included in each of the experiments as a control. Subcutaneous tumors are represented as individual follow-up of the tumor sizes and the fraction of mice that actually rejected the tumor is provided in each graph. Liver tumors were inspected and measured by laparotomy on day 19 and individual sizes on the surface of the liver are given. The fraction of mice that completely rejected the tumor is given in each case