The suppression of delayed-type hypersensitivity by CD8+ regulatory T cells requires interferon-gamma

Abstract

CD8(+) regulatory (suppressor) T cells are induced by complex cellular pathways in the spleens of mice that have received an injection of antigen into the anterior chamber (AC) of an eye, an immune-privileged site. Although these CD8(+) regulatory T cells perform an antigen-specific regulatory function for an immune response to self and non-self antigens, the mechanisms of the activation or function of these regulatory cells are not clear. Here, we describe a novel mechanism for the activation of splenic CD8(+) regulatory T cells induced by injection of antigen into the AC. Immunization of mice with trinitrophenyl and bovine serum albumin (TNP-BSA) amplified AC-induced splenic CD8(+) regulatory T cells that suppressed the initiation of contact sensitivity when transferred to immunized, challenged mice. These CD8(+) regulatory T cells were produced independently of perforin, indicating that they are not canonical cytotoxic T cells. Fas ligand (FasL)-deficient CD8(+) regulatory T-cell function was rescued by inclusion of exogenous interferon-gamma (IFN-gamma), demonstrating that the expression of FasL by CD8(+) regulatory T cells was dispensable, but IFN-gamma was not. Ultimately, we demonstrated that the generation of these CD8(+) regulatory T cells occurred independently of IFN-gamma, but their suppressor function required IFN-gamma receptor stimulation.

Figure 1

CD8⁺, CD4^– anterior chamber spleen (AC-SPL) cells effect the suppression of delayed-type hypersensitivity (DTH) in the modified local adoptive transfer (LAT) assay of suppression.(a)Scheme of modified LAT.(b)One week after trinitrophenyl/bovine serum albumin (TNP-BSA)-immunized BALB/c mice received an injection of TNP-BSA into the AC, spleen cell suspensions were prepared and separated with magnetic antibody cell sorting (MACS) beads, based on the expression of CD4 and CD8. Separated or unseparated spleen cells from AC-injected donors (AC-SPL), or naïve spleen cells, were injected subcutaneously into the challenged footpad minutes after the footpad of a TNP-BSA-immunized mouse received epicutaneous picryl chloride (PCl). Footpad swelling was measured 24 hr later. The data are representative of two experiments, one with BALB/c mice (shown) and one with C57Bl/6 mice, and show the mean swelling ± standard error of the mean (SEM) of four mice per group (*P < 0·01). CFA, Freund's complete adjuvant; DN, double negative; i.d., intradermal.

Figure 2

Immunization of anterior chamber spleen (AC-SPL) cell donors amplifies splenic CD8⁺ regulatory T cells. AC-SPL cell donors immunized with trinitrophenyl/bovine serum albumin (TNP-BSA), emulsified in Freund's complete adjuvant, 1 week previously, and naïve mice, received an injection of TNP-BSA into the AC. One week after the injection of TNP-BSA into the AC, spleens were removed and spleen cells from immunized (AC-SPL IMM) and naive AC-SPL donors were recovered. Either 5000 or 25 000 AC-SPL cells were injected into a footpad of TNP-BSA-immunized recipients immediately after the recipient was challenged with epicutaneous picryl chloride (PCl). Swelling was measured 24 hr later. The data represents the mean ± standard error of the mean (SEM) swelling of three mice per group. The data are representative of three experiments, with the same result obtained on each occasion. *P < 0·01; NS, not significant.

Figure 3

The suppression of delayed-type hypersensitivity (DTH) by anterior chamber spleen (AC-SPL) cells is perforin independent. Seven days after C57Bl/6 mice were immunized with trinitrophenyl/bovine serum albumin (TNP-BSA), a footpad of C57Bl/6-immunized or naïve recipients was challenged with epicutaneous picryl chloride (PCl). Immediately after challenge, some mice received an intradermal injection of AC-SPL cells from TNP-BSA-immunized C57Bl/6 perforin^+/+ or perforin^–/– donors. Swelling was measured 24 hr after challenge. The data represent the mean ± standard error of the mean (SEM) swelling of four mice per group. *P < 0·01.

Figure 4

Suppression by regulatory anterior chamber spleen (AC-SPL) cells requires interferon-γ (IFN-γ). Twenty-five thousand AC-SPL cells from trinitrophenyl/bovine serum albumin (TNP-BSA)-immunized (IMM) C57Bl/6 IFN-γ^+/+ and IFN-γ^–/– donors and 25 000 AC-SPL cells from the IFN-γ^–/– donors + 10 µg of IFN-γ, or IFN-γ only, were injected into the footpad of TNP-BSA-immunized mice minutes after epicutaneous challenge with picryl chloride (PCl). Swelling was measured 24 hr later and the data represent the mean swelling ± standard error of the mean (SEM) of four mice per group. The data are representative of two experiments, with three to four mice per group. *P < 0·05.

Figure 5

CD8⁺ anterior chamber spleen (AC-SPL) cells require an interferon-γ receptor (IFN-γR). C57Bl/6 IFN-γ^–/–, IFN-γR^–/– and wild-type mice were immunized (IMM) with trinitrophenyl/bovine serum albumin (TNP-BSA) and received an injection of TNP-BSA into the AC. AC-SPL cells were recovered 1 week after injection of TNP-BSA into the AC and were injected into a footpad without (left panel) or with (right panel) IFN-γ minutes after epicutaneous challenge with picryl chloride (PCl). Swelling was measured 24 hr later. Data represent the mean swelling ± standard error of the mean (SEM) of two separate experiments, with a total of six to eight mice per group. *P < 0·01.

Figure 6

Interferon-γ (IFN-γ) restores the suppression of delayed-type hypersensitivity (DTH) by Fas ligand^–/– (FasL^–/–) anterior chamber spleen (AC-SPL) cells. Twenty-five thousand AC-SPL cells from trinitrophenyl/bovine serum albumin (TNP-BSA)-immunized C57Bl/6 FasL^+/+ or FasL^–/– donors were injected, with or without IFN-γ, into the footpads of TNP-BSA-immunized C57Bl/6 mice immediately after the recipients were challenged with epicutaneous picryl chloride (PCl). Swelling of the footpads was measured 24 hr later. The data represents the mean swelling ± standard error of the mean (SEM) of three mice per group. The data are representative of three experiments, with similar results obtained on each occasion. *P < 0·01.