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. 2014 Dec 21;3(11):e963395.
doi: 10.4161/21624011.2014.963395. eCollection 2014 Nov.

Differential potency of regulatory T cell-mediated immunosuppression in kidney tumors compared to subcutaneous tumors

Christel Devaud  1 , Jennifer A Westwood  1 , Michele Wl Teng  2 , Liza B John  1 , Carmen Sm Yong  1 , Connie Pm Duong  1 , Mark J Smyth  3 , Phillip K Darcy  4 , Michael H Kershaw  4
Affiliations

Differential potency of regulatory T cell-mediated immunosuppression in kidney tumors compared to subcutaneous tumors

Christel Devaud et al. Oncoimmunology. .

Abstract

In many cancers, regulatory T cells (Treg) play a crucial role in suppressing the effector immune response thereby permitting tumor development. Indeed, in mouse models, their depletion can promote the regression of tumors of various origins, including renal cell carcinoma when located subcutaneous (SC). In the present study, we aimed to assess the importance of Treg immunosuppression in the physiologic context of metastatic renal carcinoma (Renca) disease. To that purpose we inoculated renal tumors orthotopically, intra-kidney (IK), in mice. Treg depletions were performed using anti-CD4 antibody in wild type mice or diphtheria toxin (DT) in Foxp3DTR transgenic mice. Our main observation was that Treg were not the key immunosuppressive component of the IK tumoral microenvironment, compared to the same tumors located SC. We demonstrated that the CD8+ effector immune response was still suppressed in IK tumors when compared to SC tumors, following Treg depletion. Furthermore, the level of program cell death protein (PD)-1 was increased on the surface of CD4+ T cells infiltrating IK tumors compared to SC tumors. Finally, the Treg-independent immunosuppression, occurring in IK tumors, was potent enough to inhibit regression of concomitant SC tumors, normally responsive to Treg depletion. Our findings provide further insight into the immunosuppressive nature of the immune response generated in the kidney microenvironment, suggesting that it can have additional mechanisms in addition to Treg. These observations might help to identify better targets from the kidney tumor microenvironment for future cancer therapies.

Keywords: CD, cluster of differentiation; CTLA-4, cytotoxic T lymphocyte antigen 4; DEREG, Depletion of Regulatory T cells; DT, diphtheria toxin; DTR, diphtheria toxin receptor; FCS, fetal calf serum; FR, folate receptor; Foxp3, Forkhead box protein P3; IFN, interferon; IK, intra-kidney; IL, interleukin; IP, intra-peritoneal; IV, intravenously; M2, type-2 immunosuppressive macrophages; PBS, phosphate-buffered saline; PD-1, program cell death protein 1; PD-L1, PD ligand 1; Renca Ch+ L+, Renca Cherry Luciferase; SC, subcutaneous; T regulatory cells; TCR, T cell receptor; Th, T helper cells; Treg, regulatory T cells; depletion; immunosuppression; kidney tumors; mAb, monoclonal antibody; tumor microenvironment.

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Figures

Figure 1.
Figure 1.
Treg depletion eradicates some Renca tumors established subcutaneously but not intra-kidney (IK). Mice were inoculated at day 0 with 2 × 105 Renca Ch+ L+ tumor cells subcutaneous (SC) or intra-kidney (IK). All mice were then injected intra-peritoneal (IP), at days 11, 12, 13, with anti-CD4 antibody (αCD4) or isotype control antibody (Ctl) when tumors were established (∼20–30 mm2). (A) Tumor growth, following SC injection, was monitored using a caliper and size was calculated by multiplying the longest diameter by its perpendicular diameter (n = 11–13, representative experiment of 3). (B) Bioluminescence emission imaging was used to monitor Renca Ch+ Luc + IK tumors development following IP injection of D-luciferin. Bioluminescent counts were analyzed from a region of interest applied around mice IK tumors. (n = 9–10, representative experiment of 2). (C) Survival of mice inoculated SC or IK (n = 29–37 per group, 3 experiments pooled). (D and E) Survival of Foxp3DTR mice injected with 2 × 105 Renca cells (D) SC and (E) IK and treated IP with one dose of diphtheria toxin (DT) and PBS as control (Ctl) at (D) day 13 or (E) day 9. (D) (n = 4–6 per group). (E) One experiment (n = 5–7 per group). Statistical analyses of survivals were performed using Log-rank (Mantel–Cox) test ** P < 0.005. *** P < 0.0005.
Figure 2.
Figure 2.
Treg are depleted in the blood and in tumors following anti-CD4 antibody or diphtheria toxin (DT) injections. Mice were inoculated at day 0 with 2 × 105 Renca Ch+ L+ tumor cells subcutaneous (SC) or intra-kidney (IK) (A–D) Representative FACS analysis of the gating strategy for Treg, and (E and F) quantitative data of percentage of Treg of CD4+ cells analyzed in blood and tumors from (A and C) SC and (B and D) IK tumor-bearing mice. (A, B and E) Wild-type mice were injected with anti-CD4 depleting antibody (CD4 d.) or not depleted as control (Ctl) at days 11, 12, 13 after tumor cell inoculation n = 3 mice per group, from one representative experiment of three. (C and F, SC left panel) Foxp3DTR tumor-bearing mice were injected with DT at day 12 after tumor cells inoculation, n = 4 mice per group, from one experiment. (D and F, IK right panel) Tumor-bearing mice were injected with DT at day 9 after tumor cells inoculation, n = 3 mice per group, from one representative experiment of two. Leukocytes from blood and tumors were analyzed 7 d after last anti-CD4 mAb injection and 2 d (for SC tumors) or 5 d (for IK tumors) after DT injection, by flow cytometry using anti-CD45.2, anti-CD4, anti-T cell receptor (TCR) β, anti-CD25 and anti-Foxp3 antibodies or Foxp3-GFP expression in Foxp3DTR mice. Numbers in the dot plots represent the percentage of cells of total CD4+ cells. Statistical analyses analysis were performed using Mann–Whitney test ** P < 0.005, **** P < 0.0001.
Figure 3.
Figure 3.
Treg depletion increased the CD8+ T cell effector immune response in subcutaneous (SC) tumors. Mice were injected with 2 × 105 Renca Ch+ L+ SC or intra-kidney (IK) at day 0 and depleted using anti-CD4 antibody (mAb) at days 11, 12, 13 (CD4 d.) or PBS as control (Ctl). CD8+ T cells infiltrating tumors were analyzed, 7 d after the last injection of anti-CD4 mAb, by flow cytometry using anti-CD45.2, anti-T cell receptor (TCR) β, anti-CD8 and anti-interferon (IFN)γ antibodies. Numbers in the dot plots represent the percentage of cells of total CD45.2+ cells. (A) Representative FACS analysis of the gating strategy for IFNγ production in CD8+ T cells infiltrating SC tumors (left panels as depicted) and IK tumors (right panels as depicted). (B–E) Quantitative data of (B) the percentage of TCRβ+/CD8+ T cells of CD45.2+ cells, (c) the percentage of IFNγ+ cells of CD8+ T cells, (D) mean of fluorescence (MFI) for IFNγ on CD8+ T cells, (e) MFI for CD8 surface marker on CD8+ T cells. (n = 3 mice per group, one experiment). Statistical analyses were performed using Mann–Whitney test * P < 0.05, ** P < 0.005, **** P < 0.0001.
Figure 4.
Figure 4.
CD4+ T cells infiltrating intra-kidney (IK) tumors express more program cell death protein 1 (PD1) then in subcutaneous (SC) tumors. Mice were inoculated at day 0 with 2 × 105 Renca Ch+ L+ SC or IK. CD4+ T cells and regulatory T cells (Treg) infiltrating tumors were analyzed at day 14, by flow cytometry, using anti-CD45.2, anti- TCRβ, anti-CD4, anti-CD25 and anti-Foxp3 antibodies (A and C) Representative FACS analysis and (B and D) quantitative data of percentage of (A and B) CD4+ T cells and (C and D) Treg infiltrating SC and IK tumors and expressing high level of PD1 (determined by the dashed gate-line). (A and C) The gray line histograms represent isotype control antibody staining, the black line histogram represents anti-PD-1 antibody staining. Numbers in the histograms represent the percentage of cells of total CD45.2+ cells. (B and D) (n = 3 mice per group, one experiment). Statistical analyses were performed using Mann–Whitney test *, P < 0.05, **, P < 0.005. (E and F) Day 14 Renca Ch+ L+ SC and IK in vivo tumors were analyzed, by flow cytometry, using the Cherry expression of the tumor cells and anti-CD45.2, anti-PD-L1 antibodies. (e) Representative histograms from FACS analysis and (F) quantitative data of the mean fluorescence intensity (MFI) of PD-L1 on the surface of the tumor cells. (E) The gray curve represents the isotype control and the black curve represents the anti-PD-L1 staining. (n = 2–3 mice per group, one experiment).
Figure 5.
Figure 5.
Splenocytes from Treg-depleted mice do not exert a strong antitumor immune response in Treg depleted intra-kidney (IK) tumor-bearing recipient mice. Donor mice were injected subcutaneous (SC) with 2 × 105 Renca Ch+ L+ and depleted by anti-CD4 antibody injection at days 11, 12, 13. At day 20, donor mice-spleens were harvested and prepared splenocytes were transferred intravenously (IV), into IK tumor-bearing recipient mice at day 9. After receiving the splenocytes, the IK-tumor-bearing recipient mice were treated with three doses of an anti-CD4 antibody (αCD4) at days 11, 12, 13 or isotype control (Ctl). (A and C) Survival of IK tumor-bearing mice and (B and D) bioluminescence emission imaging of IK tumors from mice transferred with (A and B) 2 × 107 or (C and D) 1.1 × 108 splenocytes. Bioluminescent counts were analyzed from a region of interest applied around mice IK tumors. (A and B) n = 8 mice per group, one experiment. (C) n = 14–15 mice per group, two experiments pooled. (D) n = 8 mice per group, one experiment.
Figure 6.
Figure 6.
Concomitant intra-kidney (IK) tumors inhibit the response of subcutaneous (SC) tumors to Treg depletion. Mice were injected at day 0 with 2 × 105 Renca Ch+ Luc+ cells in one location: SC or IK, in both locations simultaneously (SC+IK) or two tumors SC (SC+SC). Anti-CD4 antibody (αCD4) or isotype control (Ctl) was administrated at days 11, 12, 13 after tumor inoculation. (A–C) Individual SC tumor growth followed using a caliper and size was calculated by multiplying the longest diameter by its perpendicular diameter. (D) Survival of mice bearing SC and IK tumors in one or two concomitant sites. (A–C) n = 4–9 mice per group, one representative experiment of 2. (D), n = 10–24, three experiments pooled. Statistical analyses of survivals were performed using Log-rank (Mantel–Cox) test **** P < 0.0001.
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