Immune amplification of murine CD8 suppressor T cells induced via an immune-privileged site: quantifying suppressor T cells functionally

Abstract

Background: CD8(+) suppressor T cells exert antigen-specific suppression of the expression of hypersensitivity by activated T cells. Therefore, CD8(+) suppressor T cells serve a major regulatory role for the control of active immunity. Accordingly, the number and/or activity of CD8(+) suppressor T cells should be influenced by an immune response to the antigen. To test this hypothesis we used an adoptive transfer assay that measures the suppression of the expression of delayed-type hypersensitivity (DTH) by CD8(+) suppressor T cells to quantify the antigen-specific suppression of DTH by these suppressor T cells.

Methods: Suppressor T cells were induced in the spleens of mice by the injection of antigen into the anterior chamber of an eye. Following this injection, the mice were immunized by the same antigen injected into the anterior chamber. Spleen cells recovered from these mice (AC-SPL cells) were titrated in an adoptive transfer assay to determine the number of AC-SPL cells required to effect a 50% reduction of antigen-induced swelling (Sw50) in the footpad of immunized mice challenged by antigen.

Results: Suppression of the expression of DTH is proportional to the number of AC-SPL cells injected into the site challenged by antigen. The number of AC-SPL cells required for a 50% reduction in DTH-induced swelling is reduced by injecting a cell population enriched for CD8(+) AC-SPL cells. Immunizing the mice receiving intracameral antigen to the same antigen decreases the RSw50 of AC-SPL cells required to inhibit the expression of DTH.

Conclusions: The results provide the first quantitative demonstration that the numbers of antigen-specific splenic CD8(+) suppressor T cells are specifically amplified by antigen during an immune response.

Figure 1. The suppression of the expression of DTH in the LTS by intracamerally-induced AC-SPL spleen cells is proportional to the number of transferred AC-SPL cells.

(A) Reduction in antigen-induced swelling by AC-SPL cells is proportional to the number of AC-SPL cells transferred to the antigen challenge site. Spleen cells were recovered from donors that received an intracameral injection of TNP-BSA and were immunized to TNP-BSA. Unfractionated AC-SPL cells were quantified with a Coulter counter and the number of cells shown injected id into the footpads of TNP-BSA-immunized recipients immediately after the footpads were challenged with epicutaneous PCl. Open circles show naïve spleen cells. Swelling was measured 24 hr after challenge. The data is pooled from 3–4 separate experiments and is the mean swelling +/− SE of 8–12 mice/group. IMM: mean swelling of immunized mice that received no AC-SPL cells. NAÏVE: mean swelling of challenged, non-immunized mice. The straight line was generated by curve fit software. (B) The metric (RSw50) quantifies the suppression of DTH-induced swelling by AC-SPL cells. Suppression of DTH-induced swelling by AC-SPL cells was computed as the specific swelling (µm) of the challenged footpad receiving AC-SPL cells/ specific swelling of the challenged footpad that did not receive AC-SPL cells X 100. The straight line was generated by curve fit software. The data represents the pooled mean suppression of swelling +/− the standard error of the mean of five separate experiments, 15 mice/group. The number of AC-SPL cells providing 50% suppression of DTH swelling (RSw50) is shown and was computed based on the curve fit. (C) Only AC-SPL cells recovered from mice receiving an intracameral injection of TNP-BSA suppress TNP-induced swelling in TNP-immunized mice. The footpads of mice immunized with TNP-BSA (IMM) or naïve mice received 25,000 spleen cells recovered from TNP-BSA-immunized mice (IMM-SPL) TNP-BSA-immunized that received intracameral TNP-BSA (AC-TNP-BSA-SPL) or PBS only (AC-PBS-SPL) concomitant with epicutanteous PCl. Swelling was measured before and 24 hr after challenge. Data is the mean swelling in three experiments, 3–4 mice/group/experiment (total of 9–12 mice/group). * P<.01.

Figure 2. The RsW50 of regulatory spleen cells is decreased by enriching for CD8⁺ regulatory spleen cells.

The footpads of mice immunized with TNP-BSA and CFA 9 days previously received id CD8⁺ cells or unseparated AC-SPL cells prepared as in Fig. 1 immediately after the footpad was challenged with epicutaneous PCl. Swelling was measured 24 hr later. The data is pooled from two separate experiments with six mice /group. NAÏVE: non-immunized mice, IMM: immunized mice that did not receive regulatory spleen cells, CD8⁺: immunized mice that received CD8⁺ regulatory spleen cells. p<0.01. Straight lines were generated by curve fit software.

Figure 3. Immunization reduces the RSw50 of AC-SPL cells.

(A) BALB/c mice received an injection of TNP-BSA into the anterior chamber. Seven days after receiving an intracameral injection of TNP-BSA, some mice were immunized with TNP-BSA/CFA. Spleen cells were recovered from the immunized, AC-injected mice (AC-IMM) and mice that received an injection of antigen into the AC only (AC-ONLY) one week after the immunization of AC-injected mice or one week after intracameral injection only. Twenty-five thousand AC-SPL cells were injected into the footpads of TNP-BSA-immunized mice immediately after the footpads were challenged with epicutaneous PCl. (B) Immunization-induced increase in suppression is antigen-specific. Seven days after mice received an intracameral injection of TNP-BSA, the mice were immunized with TNP (AC-TNP), TNP-IMM or OVA (AC-TNP-OVA-IMM). Seven days after immunization, spleens were recovered from the mice and recovered spleen cells injected into the footpad of TNP-BSA-immunized mice immediately after the footpad was challenge with epicutaneous PCl. Swelling was measured 24 hr later. The data represents the mean RsW50 +/− the standard error of the mean for three experiments, 9 mice/group. *p<0.02.

Figure 4. The expansion of AC-SPL suppressor cells that suppress the expression of DTH is strain and antigen dose -dependent.

Seven days after mice were immunized with TNP-BSA, a footpad was challenged with epicutaneous PCl. Footpad thickness was measured before and 24 hr after challenge. Five thousand AC-SPL cells were injected into a footpad of mice immunized with TNP-BSA immediately after the footpad was challenged with epicutaneous PCl. Footpad thickness was measured before and 24 hr after challenge and swelling computed. (A): Effect of immunizing dose of antigen on DTH-induced swelling in BALB/c (A) C57BL/6 mice (C), Generation of suppressive AC-SPL cells is antigen dose dependent (B): BALB/c, D: C57BL/6. Data represents the mean swelling +/− S.E.M. of 12 mice/group , 3 experiments. * p<0.05,NS: not significant.

Figure 5. Durability of cell-mediated suppression OF DTH-mediated swelling.

The footpads of mice immunized with TNP-BSA were injected with 25000 spleen cells recovered from TNP-BSA-immunized mice that received an intracameral injection of TNP-BSA. The footpads were challenged with epicutaneous PCl immediately after receiving the spleen cells 0, 3 or 5 days after receiving the spleen cells. Footpad thickness was measured 24 and 48 hr after challenge with PCl. (A) Percent suppression is calculated as : 100- swelling (µm) immunized mice+regulatory spleen cells. Swelling (µm) immunized only. Data represents mean suppression +/− S.E.M. 9 mice/group, 3 experiments. (B) The footpads of mice immunized 11 or 13 days previously with TNP-BSA received id 25000 spleen cells recovered from TNP-BSA-immunized mice that received an intracameral injection of TNP-BSA and epicutaneous PCl. Swelling was measured 24 and 48 hr after challenge. Data shows the mean swelling (µm) of 8 mice/group, two experiments. * p<0.01.