Research ArticleImmunology

Blockade of surface-bound TGF-β on regulatory T cells abrogates suppression of effector T cell function in the tumor microenvironment

See allHide authors and affiliations

Science Signaling  29 Aug 2017:
Vol. 10, Issue 494, eaak9702
DOI: 10.1126/scisignal.aak9702
  • Fig. 1 Schematic representation of the experimental setup for the 3D collagen-fibrin gel killing assay.

    (A to D) Illustration and representation of the model and technique used in this study. (A) Melanoma tumors expressing the T cell antigens OVA and Pmel-1 (B16-OVA) are excised from C57BL/6 mice 10 days after implantation and dissociated into single-cell suspensions. Collagen-fibrin gels are prepared in 48-well tissue culture plates containing B16-OVA cells from in vitro culture or B16-OVA cells from the dissociated tumors in the presence or absence of antigen-specific CD8+ T cells. The gels are lysed daily with collagenase and trypsin, and the numbers of remaining viable B16-OVA cells are assessed with a clonogenic assay as previously described (11). (B to D) Illustration of the use of the 3D collagen-fibrin gel killing assay to qualitatively measure the suppression of T cell killing by the tumor microenvironment with hypothetical representation of semilog plots showing the expected numbers of viable B16 cells recovered from collagen-fibrin gels in which T cell–mediated killing (B) or immunosuppression of killing (C) occurred. (D) Equation modeling the T cell–mediated killing of tumor cells in collagen-fibrin gels to calculate the killing efficiency, k, as previously described (11).

  • Fig. 2 Ex vivo collagen-fibrin gel cultures maintain the immunosuppression of in vivo tumor microenvironment.

    (A to E) B16-OVA tumors were excised and digested with collagenase and then disaggregated mechanically into single-cell suspensions. Dissociated tumors were co-embedded in collagen-fibrin gels with in vitro–activated OT-1 cells at a 50:1 effector-to-target (E:T) ratio. (A) The numbers of viable tumor cells and immune cell infiltrates isolated from dissociated 10-day B16-OVA tumors were determined. Data are means ± SEM of eight experiments. (B) The numbers of viable melanoma cells recovered from the gels at the indicated times were measured. Data are means ± SEM of three independent experiments performed in duplicate. (C) The percentages of B16 cells killed were determined. Data are means ± SEM of eight experiments as performed in (A). (D) Calculated value of k ± SEM from the experiments performed in (A) using the equation bt = b0e−kpt+gt, as described in Materials and Methods. (E) The percentages of B16 tumor cells killed were determined. Data are means ± SEM at 24 hours using equivalent numbers (5 × 105 cells per gel) of OT-1 or Pmel CD8+ T cells in collagen-fibrin gel cocultures of B16-OVA cells. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005. ns, not significant.

  • Fig. 3 In vivo depletion of Tregs in Foxp3-DTR mice restores CD8+ T cell–mediated tumor cell killing.

    (A to D) Foxp3-DTR mice were treated with DT to deplete Tregs 2 days before tumor excision was performed, as described in Materials and Methods. The dissociated tumors were co-embedded in collagen-fibrin gels with in vitro–activated OT-1 cells at a 50:1 effector-to-target ratio. (A) At the indicated times, the gels were dissolved, and the numbers of remaining B16 cells were measured using a clonogenic assay. Data are means of the number of viable B16 cells ± SEM from three experiments performed in duplicate. (B) Representative plots (gated on viable CD45+ immune cells) of CD4+Foxp3+ Tregs in B16-OVA tumors with or without DT treatment. (C) The percentages of B16 cells killed under the indicated conditions were determined. Data are means ± SEM from three experiments performed in duplicate as described in (A). (D) Mean values of k ± SEM from the experiments performed in (A). *P ≤ 0.05 and **P ≤ 0.01.

  • Fig. 4 TGF-β blockade reverses the suppression of tumor cell killing ex vivo.

    (A to C) B16-OVA tumors were excised and dissociated as described in Fig. 2. The dissociated tumors were co-embedded in collagen-fibrin gels with in vitro–activated OT-1 cells at a 50:1 effector-to-target ratio in the presence or absence of blocking antibodies (Ab) against TGF-β, IL-10R, MHC II, or IL-35 (all at 10 μg/ml). (A) The percentages of B16 cells killed at 48 hours were determined. Data are means ± SEM of three experiments performed in duplicate. (B) The mean values of k ± SEM from the experiments performed in (A) were determined. (C) The mean numbers of clonogenic B16 remaining at the indicated times were determined. Data are means ± SEM of three experiments performed in duplicate. ***P ≤ 0.005.

  • Fig. 5 Pmel-1 T cells are located near to and interact with Tregs in YFP-B16 tumors.

    (A and B) YFP-B16 tumor cells were suspended in Matrigel and inoculated subcutaneously into Foxp3-GFP mice. Three days later, 3 × 105 naïve CFP-Pmel CD8+ T cells were transferred by tail vein injection. The mice were then imaged as described in Materials and Methods. Time-lapse mages were analyzed for the interactions of CD8+ CFP-Pmel T cells (cyan) with Tregs (red) as described in Materials and Methods. (A) Top: Representative frames from a single region of a six-region time-lapse image. Bottom: Magnified images from the region surrounded by the yellow box. Foxp3-GFP Tregs are depicted in red, CFP-Pmel cells are depicted in cyan, and YFP-B16-OVA tumor cells are depicted in green. Frames are separated in time by 6 min 12 s. (B) Points on plots represent individual cells scored for proximity (within 10 μm) or contact with a Treg during imaging (see Materials and Methods). Top: Data are means ± SEM of the entire population of cells. Bottom: Data are means ± SEM of only those cells that had interactions with Tregs. All cells tracked for over five time points from four mice are represented.

  • Fig. 6 Adding back Tregs to depleted tumors partially restored the suppression.

    (A) B16-OVA tumors from nondepleted mice were excised and dissociated as described in Materials and Methods. Left: The cell surface expression of TGF-β on CD4+ effectors, CD8+ T cells, and CD4+Foxp3+ (GFP+) Tregs was assessed by flow cytometry. Representative histograms for the isotype control and anti–TGF-β antibody are shown. Right: Data represent the mean fluorescence intensity (MFI) ± SEM for TGF-β relative to that or an isotype control antibody for four mice per group. Teff, effector T cell. (B and C) GFP+ Tregs from tumors in Foxp3-GFP mice were sorted by FACS from nondepleted tumors and preincubated with blocking antibody against TGF-β. Tregs (2 × 104 cells) were then co-embedded with dissociated Treg-depleted B16-OVA tumors (B) or cultured B16-OVA cells (C). At the indicated times, the gels were dissolved, and the numbers of the remaining B16 cells were measured using a clonogenic assay. Data are means ± SEM of the numbers of B16 cells from three experiments performed in duplicate. (D) OT-1 CD8+ T cells were recovered from collagen-fibrin gels at 48 hours after culture under the indicated conditions and were analyzed by flow cytometry to determine the relative abundances of granzyme B (left) and PD-1 (right). Data are means ± SEM of the MFIs from triplicate analyses. *P ≤ 0.05 and **P ≤ 0.01.

  • Table 1 Calculation of k values for Treg-depleted tumors.

    The value of k (min) for each condition was calculated from the equation bt = b0ekpt+gt and the values obtained in Fig. 6B. The percentage decreases compared to Treg-depleted tumors at each time point are listed in parentheses.

    Time
    (hours)
    Treg-depleted
    tumor
    Treg-depleted
    tumor + Tregs
    Treg-depleted tumor +
    Tregs + anti–TGF-β
    240.77 × 10−90.66 × 10−90.66 × 10−9
    481.44 × 10−90.84 × 10−9 (42%)1.41 × 10−9 (2%)
    721.79 × 10−90.88 × 10−9 (51%)1.40 × 10−9 (21%)
  • Table 2 Calculation of k values for cultured B16-OVA cells.

    The value of k (min) for each condition was calculated from the equation bt = b0ekpt+gt and the values obtained in Fig. 6C. The percentage changes compared to B16-OVA cells alone at each time point are listed in parentheses.

    Time (hours)B16-OVA cellsB16-OVA + TregsB16-OVA + Tregs +
    anti–TGF-β
    240.69 × 10−90.74 × 10−91.07 × 10−9
    480.94 × 10−90.19 × 10−9
    (−80%)
    0.74 × 10−9
    (−21%)
    721.60 × 10−90.93 × 10−9
    (−42%)
    1.68 × 10−9
    (+5%)

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/494/eaak9702/DC1

    Summary of statistical analyses

    Fig. S1. Analysis of immune cell infiltrates in tumors from DT-treated Foxp3-DTR mice.

    Fig. S2. In vivo depletion of CCR2+ cells in CCR2-DTR mice has no effect on the suppression of CD8+ T cell–mediated killing by the tumor microenvironment.

    Fig. S3. Depleting Tregs ex vivo with anti-CD25 MicroBeads has no effect on the immunosuppression of CD8+ T cells.

    Fig. S4. Tregs cause minor alterations to the mobility of CD8+ T cells in the tumor.

    Fig. S5. Suppression of CD8+ T cells by Tregs is contact- or proximity-dependent.

    Fig. S6. Expression of TGF-β and CD51 (αV integrin) in immune cell subsets from the tumors and spleens of B16 tumor–bearing mice.

    Fig. S7. Effect of DT on the expression of PD-1 and granzyme B on the surface of endogenous CD8+ T cells.

    Movie S1. CFP-Pmel T cells are found in regions highly infiltrated by Tregs.

    Movie S2. CFP-Pmel T cells are found within proximity to or make contact with Tregs.

  • Supplementary Materials for:

    Blockade of surface-bound TGF-β on regulatory T cells abrogates suppression of effector T cell function in the tumor microenvironment

    Sadna Budhu, David A. Schaer, Yongbiao Li, Ricardo Toledo-Crow, Katherine Panageas, Xia Yang, Hong Zhong, Alan N. Houghton, Samuel C. Silverstein, Taha Merghoub,* Jedd D. Wolchok*

    *Corresponding author. Email: merghout{at}mskcc.org (T.M.); wolchokj{at}mskcc.org (J.D.W.)

    This PDF file includes:

    • Summary of statistical analyses
    • Fig. S1. Analysis of immune cell infiltrates in tumors from DT-treated Foxp3-DTR mice.
    • Fig. S2. In vivo depletion of CCR2+ cells in CCR2-DTR mice has no effect on the suppression of CD8+ T cell–mediated killing by the tumor microenvironment.
    • Fig. S3. Depleting Tregs ex vivo with anti-CD25 MicroBeads has no effect on the immunosuppression of CD8+ T cells.
    • Fig. S4. Tregs cause minor alterations to the mobility of CD8+ T cells in the tumor.
    • Fig. S5. Suppression of CD8+ T cells by Tregs is contact- or proximity-dependent.
    • Fig. S6. Expression of TGF-β and CD51 (αV integrin) in immune cell subsets from the tumors and spleens of B16 tumor–bearing mice.
    • Fig. S7. Effect of DT on the expression of PD-1 and granzyme B on the surface of endogenous CD8+ T cells.
    • Legends for movies S1 and S2

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 789 KB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). CFP-Pmel T cells are found in regions highly infiltrated by Tregs.
    • Movie S2 (.mov format). CFP-Pmel T cells are found within proximity to or make contact with Tregs.

    Citation: S. Budhu, D. A. Schaer, Y. Li, R. Toledo-Crow, K. Panageas, X. Yang, H. Zhong, A. N. Houghton, S. C. Silverstein, T. Merghoub, J. D. Wolchok, Blockade of surface-bound TGF-β on regulatory T cells abrogates suppression of effector T cell function in the tumor microenvironment. Sci. Signal. 10, eaak9702 (2017).

    © 2017 American Association for the Advancement of Science

Navigate This Article