An affinity/avidity model of peripheral T cell regulation

Abstract

We show in these studies that Qa-1-dependent CD8+ T cells are involved in the establishment and maintenance of peripheral self tolerance as well as facilitating affinity maturation of CD4+ T cells responding to foreign antigen. We provide experimental evidence that the strategy used by the Qa-1-dependent CD8+ T cells to accomplish both these tasks in vivo is to selectively downregulate T cell clones that respond to both self and foreign antigens with intermediate, not high or low, affinity/avidity. Thus, the immune system evolved to regulate peripheral immunity using a unified mechanism that efficiently and effectively permits the system to safeguard peripheral self tolerance yet promote the capacity to deal with foreign invaders.

Figure 1

Qa-1–dependent CD8⁺ T cells are involved in the establishment and maintenance of peripheral self tolerance to HEL in HEL Tg mice. (A) The unresponsiveness to HEL in HEL high Tg mice could be broken by treatment with anti-CD8 and anti–Qa-1 mAbs. HEL immunization and in vivo mAb treatment were performed and CD4⁺ T cells were purified from pooled draining lymph node cells from different groups of mice and assayed in a T cell proliferation assay as described in Methods. Data are representative of 4 separate experiments with 2–4 mice per group. (B) CD8⁺ T cells regulate immune response to self antigen HEL in HEL low Tg mice. Experiments were performed as described in Methods. Data are representative of 6 separate experiments with 2–4 mice per group. (C) CD8⁺ T cells downregulate the primary immune responses to HEL in HEL low Tg mice when adoptively transferred. CD8⁺ T cells were injected i.v. into recipient mice, and the mice were immunized with HEL 1 day later. The CD4⁺ T cells were isolated from pooled lymph node cells of recipient mice 7–9 days after the immunization, and T cell proliferation assays were performed. Data are representative of 4 separate experiments with 2–4 mice per group. Control, no transfer; n/LTg, CD8⁺ T cells transferred from naive HEL low Tg mice; HEL/LTg, CD8⁺ T cells transferred from 2– HEL-immunized HEL low Tg mice; n/HTg, CD8⁺ T cells transferred from naive HEL high Tg mice; HEL/HTg, CD8⁺ T cells transferred from 1– HEL-immunized HEL high Tg mice. ³HTdr, ³H-thymidine.

Figure 2

Qa-1–dependent CD8⁺ T cells selectively downregulate T cell clones of intermediate affinity/avidity for HEL. (A) In this representative example, the CD8⁺ T cells differentially downregulate a susceptible but not a nonsusceptible clone in a dose-dependent manner. The CD8⁺ T cells were directly mixed with CFSE-labeled testing clones as described in Methods. In this type of setting, we used non–CFSE-labeled activated clones to set up the cutoff line for undivided or less divided and more divided fractions for each clone individually. Since the autofluorescent background of each unlabeled activated clone in the FITC channel differs among clones, slightly different cutoff lines sometimes appear among different clones. Only 2 of 5 E/Ts for 2 representative clones, 13C9 (intermediate affinity/avidity) and 9E4 (high affinity/avidity), are shown. (B) Qa-1–dependent CD8⁺ T cells selectively downregulate HEL-specific T cell clones based on their affinity/avidity to HEL. In this set of tests, CD8⁺ T cells isolated from naive WT or HEL low Tg mice, which showed no effect on target T cells, were routinely used as control for the regulatory CD8⁺ T cells. (C) The downregulation of the intermediate–affinity/avidity clones by the CD8⁺ T cells is blocked by antibodies against TCR, Qa-1, and CD8, but not by the control antibody. The experiments were performed as described in Methods. Data represent experiments at E/Ts of 0.2:1 to 1:1, and final concentrations of mAbs were 12.5–25 μg/ml.

Figure 3

Qa-1–dependent CD8⁺ T cells facilitate maturation of affinity for HEL in WT mice. (A) Regulatory CD8⁺ T cells are involved in the increase of the overall affinity of HEL-reactive CD4⁺ T cells during the secondary immune response to HEL in WT BALB/c mice. CD4⁺ T cells were purified from pooled lymphocytes from draining lymph nodes of different groups of mice and assayed in a T cell proliferation assay as described in Methods. Data are representative of 3 separate experiments with 2–4 mice per group. (B) CD8⁺ T cells selectively downregulate certain HEL Vβ8.2 clones in the secondary HEL-reactive repertoire in WT BALB/c mice. Mice were prepared as described in Methods. CD4⁺ T cells were isolated and purified from the draining lymph nodes on days 7–9 after the secondary HEL immunization and stimulated in vitro for a week. Analysis of distribution of the TCR CDR3 length of CD4⁺ T cells isolated from the different groups of mice was performed as previously described (4, 47). Data are representative of 5 separate experiments with 2–4 mice per group.

Figure 4

Actual CDR3 sequences of TCR Vβ8.2 HEL-reactive repertoires in WT mice, representative 1 of the 5 experiments described in Figure 3B. CDR3 sequencing was performed as described in Methods.

Figure 5

An affinity/avidity model of peripheral T cell regulation. (A) The selective downregulation of intermediate–affinity/avidity T cells by the CD8⁺ T cells shapes the peripheral TCR repertoire to both self and foreign antigens during the evolution of immune response. (B) Cellular events involved in the Qa-1–dependent CD8⁺ T cell–mediated regulatory pathway.