Tumor-infiltrating regulatory dendritic cells inhibit CD8+ T cell function via L-arginine metabolism

Abstract

Dendritic cells (DC) have a critical effect on the outcome of adaptive immune responses against growing tumors. Whereas it is generally assumed that the presence of phenotypically mature DCs should promote protective antitumor immunity, evidence to the contrary does exist. We describe here a novel mechanism by which tumor-infiltrating dendritic cells (TIDC) actively contribute to the suppression of protective CD8(+) T-cell-based antitumor immunity. Using the BALB/NeuT model of spontaneously arising mammary carcinoma, we found that canonical MHC II(+)/CD11b(+)/CD11c(high) TIDCs act as regulatory DCs to suppress CD8(+) T-cell function, resulting in diminished T-cell-based antitumor immunity in vivo. Stimulation of naive T cells with regulatory TIDCs resulted in an altered cell fate program characterized by minimal T-cell expansion, impaired IFNgamma production, and anergy. Suppression by regulatory TIDCs overcame stimulatory signals provided by standard DCs, occurred in the absence of cognate interactions with T cells, and was mediated primarily by arginase metabolism of l-arginine. Immunosuppressive TIDCs were found in every murine tumor type examined and were phenotypically distinct from tumor-infiltrating CD11c(int-low)/CD11b(+)/Gr-1(+) myeloid-derived suppressor cells. Thus, within the tumor microenvironment, MHC II(+) TIDCs can function as potent suppressors of CD8(+) T-cell immunity.

Figure 1

Canonical CD11c⁺/ MHC II⁺ DCs infiltrate NeuT tumors. A, NeuT tumors were stained for CD11c-Cy3 (red) and CD31 FITC or I-Ad FITC (green). Cells expressing both appear in yellow. One tumor, representative of 5 is shown. B, Spleens from tumor-free BALB/c mice and mammary tumors from NeuT mice were harvested and stained. The percentage of live, gated CD11c^high cells is shown, along with maturation marker expression on these cells. LPS-treated BALB/c mice received 70µg of Salmonella abortus LPS one day prior to harvest and staining of splenocytes. C, Gates for CD11c^high TIDC and CD11c^int-low MDSC are shown, along with surface expression of Gr 1. Specific antibodies = blue lines, red lines = isotype controls. The mean fold increase of Gr1 MFI over isotype MFI is shown at right for 6 individual mice from 3 experiments.

Figure 2

TIDC from NeuT mammary tumors promote tumor outgrowth in vivo. A, CMS5 fibrosarcoma cells were were injected s.c. alone, or together with NeuT TIDC on day 0 into BALB/c mice. Tumor outgrowth through day 16 is shown as means +/− SEM; n = 11 mice for CMS5 alone and n = 10 for CMS5+ TIDC. Open circles = CMS5 alone, closed diamonds = CMS5+TIDC. Data are cumulative from 3 independent experiments. Center panel: Individual tumor regression data for 4 mice receiving CMS5+ TIDC on day −4 with naive DUC18 T cells transferred i.v. on day 0. Right panel: Individual tumor regression data for 4 mice receiving CMS5+ TIDC on day −4, with naive DUC18 T cells transferred i.v. on day 0. B, Cumulative scatter plots of tumor sizes at day 40, from all individual mice used in multiple experiments. C, Progressive tumor outgrowth in NeuT recipient versus BALB/c recipient mice after DUC18 T cell transfer on day 0. For BALB CMS5 vs BALB CMS5+TIDC, p = .0202; for BALB CMS5 vs. NeuT CMS5, p = .1208; for BALB CMS5 vs. NeuT CMS5+TIDC, p= .0001; for NeuT CMS5 vs NeuT CMS5+TIDC, p= .0014 (all, two-tailed t test with Welch’s correction). For NeuT recipients only, the relative % of gated tumor-infiltrating DUC18 T cells making IFNγ at day 6 post-transfer is shown as means from 4 –5 individual mice.

Figure 3

TIDC stimulation of DUC18 T cells leads to impaired proliferation and anergy. A, Proliferation of naive DUC18 T cells cultured with tERK peptide-pulsed spDC or NeuT TIDC for 96 hours, at the indicated T cell: DC ratios. Data from 1 experiment, representative of more than 15 are shown. * = p < .001, versus spDC at each ratio. B, Annexin V staining on gated Thy1.1⁺ T cells after 96 hours of culture. C, Naive DUC18 T cells were CFSE labeled, then cultured at a 10:1 ratio with spDC or NeuT TIDC. Gated on live, CD8⁺ DUC18 T cells at 96 hours, with percentages of divided cells in specific quadrants indicated. Data represent more than 5 experiments. D, Following primary stimulation with TIDC, DUC18 T cells become unresponsive to secondary stimulation with spDCs as evidenced by a lack of IFNγ production at 48 hours.

Figure 4

NeuT TIDC act as regDCs to dominantly suppress naive CD8⁺ T cell proliferation. A, The gate used to sort-purify NeuT TIDC is indicated (small upper left panel). Naive DUC18 T cells were incubated alone, or in the presence of tERK-pulsed NeuT TIDC, BALB/c spDCs, or both, at the indicated T cell: DC ratios. One assay, representative of 17, is shown. B, T cells, spDCs, and NeuT TIDC, were cultured at a 10:1:1 ratio. The mean percent inhibition of T cell proliferation, as compared to T cells plus spDCs alone, is shown +/− the SEM. For TIDC pulsed with tERK (black bars) n= 17 experiments, for spDCs pulsed with K^d FLU peptide (white bars) n= 5 experiments, for TIDC pulsed with K^d FLU peptide (grey bars) n= 8 experiments. C, T cells were cultured as in panel B, with either NeuT TIDC, or CMS5 TIDC, or 4T1 TIDC. The mean % inhibition of T cell proliferation is shown for results from 4–15 independent experiments.

Figure 5

NeuT TIDC use arginase to metabolize L-arginine. A, ³H-L-arginine incorporation of purified TIDC and spDCs was measured at 0, 0.5, 3, 18 and 24 hrs. The means +/− s.d. for triplicate wells are shown; error bars are too small to be seen. B, Arginase activity measured in lysates from spDCs, pooled from 12 mice, or TIDC, taken from 3 individual NeuT mice. Cells were cultured in medium alone or with IL-4 prior to harvest and lysis. TIDC versus spDC controls * indicates p < .001. NO production as measured by the concentration of total nitrite/ nitrate after culturing spDCs or TIDC for 24 hours, with or without IFNγ. Cumulative data from four experiments are shown. C, Increased expression of the phosphorylated form of eIF2a when T cells are cultured with TIDC. D, Downregulation of surface CD3ζ chain expression on DUC18 T cells stimulated with TIDC.

Figure 6

NeuT TIDC use arginase metabolism of L-arginine to suppress CD8 T cell proliferation. A, The mean percent restoration in T cell proliferation, relative to T cells plus spDC controls, is shown for T cells cultured with spDC plus TIDC +/− L-arginine, L-nil, L-NNA, or norvaline. Data are expressed as the mean +/− SEM for 9–17 independent experiments. B, Propidium idodide cell cycle analysis on naive DUC18 T cells cultured as indicated for 96 hours. The percentages of both dying and replicating cells are given for each condition.