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. 2006 May 1;176(9):5255-66.
doi: 10.4049/jimmunol.176.9.5255.

Interleukin-2-dependent mechanisms of tolerance and immunity in vivo

Paul A Antony  1 , Chrystal M Paulos, Mojgan Ahmadzadeh, Akgül Akpinarli, Douglas C Palmer, Noriko Sato, Andrew Kaiser, Christian S Hinrichs, Christopher A Klebanoff, Yutaka Tagaya, Nicholas P Restifo
Affiliations

Interleukin-2-dependent mechanisms of tolerance and immunity in vivo

Paul A Antony et al. J Immunol. .

Erratum in

  • J Immunol. 2006 Jun 15;176(12):7788. Heinrichs, Christian [corrected to Hinrichs, Christian S]

Abstract

IL-2 is a critical T cell growth factor in vitro, but predominantly mediates tolerance in vivo. IL-2 is mainly produced by CD4(+) Th cells, but the role of Th cell-derived IL-2 in vivo is controversial. We demonstrate that during immunity to a tumor/self-Ag, the predominant role of Th cell-derived IL-2 was to maintain IL-2Ralpha (CD25) on CD4(+) T regulatory cells (T(reg)), which resulted in their maintenance of the T(reg) cell lineage factor, Forkhead/winged helix transcription factor (Foxp3), and tolerance. However, in the absence of T(reg) cells, Th cell-derived IL-2 maintained effector T cells and caused autoimmunity. IL-2R signaling was indispensable for T(reg) cell homeostasis and efficient suppressor function in vivo, but, surprisingly, was not required for their generation, because IL-2(-/-) and CD25(-/-) mice both contained Foxp3(+) T cells in the periphery. IL-2R signaling was also important for CD8(+) T cell immunity, because CD25(-/-) tumor-reactive CD8(+) T cells failed to affect established tumors. Conversely, IL-2R signaling was not required for Th cell function. Lastly, administration of anti-IL-2 plus exogenous IL-15 to tumor-bearing mice enhanced the adoptive immunotherapy of cancer. Therefore, Th cell-derived IL-2 paradoxically controls both tolerance and immunity to a tumor/self-Ag in vivo.

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Figures

FIGURE 1
FIGURE 1
Foxp3+ T cells suppress immunotherapy of established tumors. A, Treatment schematic and illustration of cell isolation for adoptive cell transfer into tumor-bearing recipients. B, Flow cytometry showing the percentage of Foxp3+ T cells in sorted CD45.1 Treg and CD45.2 Th cell populations at the single-cell level. C, Treg cells suppress and Th cells help immunotherapy of established tumors. Tumor-bearing RAG-1−/− hosts were treated with pmel-1 CD8+ T cells (1 × 106), rFP vaccine (2.0 × 107 PFU), and either 1 × 106 Th cells or 1 × 105 Treg cells and Th cells (1:10 ratio). Tumor graphs represent the average tumor measurement ± SEM with at least three mice per group. Experiments were repeated independently six times.
FIGURE 2
FIGURE 2
IL-2 and Treg cells control the expansion of CD4+ T cells in vivo. A, Adoptively transferred Treg and Th cells engraft the host at physiologic ratios. Flow cytometry of CD4+ T cells was performed 3–4 wk after adoptive cell transfer, showing the relative ratios of CD45.1 cells and Treg CD45.2 Th cells in spleen. B, Absolute numbers of CD45.1+ Treg (□) and CD45.2 Th cells (▪) from the indicated groups were enumerated 3–4 wk after adoptive cell transfer into tumor-bearing RAG-1−/− hosts. Cells were isolated from spleens of the respective groups.
FIGURE 3
FIGURE 3
IL-2 controls the frequency of Treg cells in vivo. A, Flow cytometry of phenotypic markers on Foxp3+ Treg cells from WT, IL-2−/−, CD25−/−, and human IL-2 transgenic mice. IL-2−/− and CD25−/− mice have Foxp3− T cells, but have more activated Foxp3− cells than WT mice. CD25, CTLA-4, and GITR are increased proportionally with Foxp3− T cells in human IL-2 transgenic mice. Cells were isolated from spleens and stained with the indicated Abs. Experiments were repeated independently more than three times. B, Absolute number of Foxp3+ T cells from WT, IL-2−/−, and CD25−/− mice 2 and 4 mo after birth.
FIGURE 4
FIGURE 4
Th cell-derived IL-2 controls CD25 and Foxp3 expression by Treg cells. A, CD25 expression on adoptively transferred CD45.1 Treg cells before (▪) and 3–4 wk after transfer (□) into tumor-bearing RAG-1−/− hosts with or without Th cells as indicated. Groups with p values are compared with Treg and Th (WT) cells. B, Foxp3 expression by Treg cells is controlled by Th cells. Foxp3 expression in adoptively transferred CD45.1 Treg cells alone or with WT, IL-2−/−, or CD25−/− Th cells 3–4 wk after treatment of tumor-bearing RAG-1−/− hosts with pmel-1 T cells and rFP vaccine. C, Flow cytometry of CD25 expression on CD45.1 Treg cells 3–4 wk after transfer into RAG-1−/− hosts. The numbers indicates the average mean fluorescence intensity for CD25 expression on CD45.1 Treg cells. Experiments were repeated independently seven times.
FIGURE 5
FIGURE 5
IL-2 signaling is required for the maintenance of Foxp3+ Treg cells in the periphery. A, CD25 expression on adoptively transferred CD45.2 Th cells before and 3–4 wk after transfer into tumor-bearing RAG-1−/− hosts. The p values are compared with Th (WT) cells (wk 3–4). B, Foxp3 expression at the single-cell level in CD4+ T cells 3–4 wk after transfer into tumor-bearing RAG-1−/− hosts. C, Foxp3 expression determined by RT-PCR in CD4+ T cells 3–4 wk after transfer into tumor-bearing RAG-1−/− hosts. Data are representative of three experiments.
FIGURE 6
FIGURE 6
IL-2 signaling is required for the efficient function of Foxp3+ Treg cells in the periphery. A, Foxp3 expression in CD25−/− T cells at the single-cell level before and 3–4 wk after transfer into tumor-bearing RAG-1-deficient hosts. Gates are based on WT controls. B, CD8+ T cells are amenable to suppression only by CD25+/+ Foxp3+ T cells. RAG-1−/− mice bearing 7- to 10-day B16 tumors were treated with pmel-1 CD8+ T cells, rFP vaccination, and Th (CD25−/−) cells (~5–10% Foxp3+) with and without CD25+/+ Foxp3+ T cells. Error bars represents ±SEM. C, CD25+/+ Foxp3+ Treg cells, but not CD25−/− Foxp3+ T cells, can suppress Th (CD25−/−) cells in vivo and prevent effective expansion of CD8+ T cells (pmel-1). The frequency of Th (CD25−/−) cells (upper panel) and pmel-1 T cells (lower panel) with CD45.1 Treg cells (right) or without (left) after adoptive transfer and rFP vaccination into tumor-bearing RAG-1−/− hosts is shown. The number above each density plot represents the absolute number of T cells. D, Treatment of established tumors is enhanced in CD25−/− hosts. Ten-day tumor-bearing CD25−/− and WT mice received 5 Gy TBI and pmel-1 T cells and rFP vaccine on the day of treatment. E, Flow cytometry of Foxp3+ T cells in tumor-bearing CD25−/− mice 4 wk after treatment.
FIGURE 7
FIGURE 7
IL-2 signaling is dispensable for Th cell function, but is indispensable for CD8+ T cell immunity. A, Treatment of established tumors in RAG-1−/− mice requires Th cell-derived IL-2, but not CD25 expression on Th cells. B, Th cells maintain the persistence of CD8+ T cells. The frequency of adoptively transferred CD8+Vβ13+ (pmel-1) T cells 3–4 wk after treatment with the indicated CD4+ T cells is shown. C, IL-2 sustains and Treg cells suppress CD8+ T cell numbers in vivo. The absolute number of pmel-1 CD8+ T cells 3–4 wk after adoptive transfer and vaccination with rFPVhgp100 into tumor-bearing RAG-1−/− hosts is shown. D, IL-2R signaling is important for CD8+ T cell function in vivo. Naive pmel-1 (WT) T cells or naive pmel-1 (CD25−/−) T cells were enriched from splenocytes by CD8 negative selection and transferred i.v. with rFP vaccine into tumor-bearing RAG-1−/− mice on day 7 after tumor inoculation. Error bars represent ±SEM.
FIGURE 8
FIGURE 8
IL-2 sustains and Treg cells suppress effector T cell numbers. A, IL-2 controls effector CD4+ T cells. Intracellular staining of IL-2 and IFN-γ in Th cells from the indicated treatment groups after stimulation with lymphocyte-activating mixture for 6 h. B, IL-2 maintains and Treg cells suppress effector CD8+ T cells. Intracellular staining of IFN-γ in pmel-1 T cells from the indicated treatment groups after stimulation with lymphocyte activating mixture for 6 h is shown. The average absolute number of CD8+Vβ13+IFN-γ+ T cells is indicated above each density plot. The absolute number was calculated by multiplying the cell count (total splenocytes) by the frequency of CD8+ T cells by the frequency of Vβ13+IFN-γ+ T cells. Data are representative of three experiments.
FIGURE 9
FIGURE 9
Anti-IL-2 therapy plus exogenous IL-15 enhances immu-notherapy of cancer. Tumor-bearing B6 mice were irradiated with 5 Gy and treated with pmel-1 T cells, rFP vaccine, and IL-2, IL-15, or anti-IL-2 as indicated. Anti-IL-2 (0.1 mg) was given every other day i.p. 4 days after therapy for 3 wk. IL-2 and IL-15 (36 μg/dose) were injected twice a day for 3 days after adoptive transfer. The tumor graph represents the average tumor measurement ±SEM for at least three mice per group. The experiment was repeated twice.
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