Abortive activation precedes functional deletion of CD8+ T cells following encounter with self-antigens expressed by resting B cells in vivo

Abstract

InsHA mice express the haemagglutinin (HA) protein from influenza virus A/PR/8 H1N1 (PR8) as a self antigen on pancreatic islet beta cells. We have utilized these mice to investigate the ability of resting B cells expressing Kd to induce self-tolerance among naive KdHA-specific clone 4 CD8+ T cells. Adoptive transfer of KdHA-peptide-pulsed resting B cells into clone 4-->InsHA recipients resulted in the activation and proliferation of clone 4 CD8+ T cells throughout the peripheral lymphoid tissues. Significantly, proliferation was not associated with the acquisition of T cell effector function; as evidenced by a lack of interferon-gamma production and the complete absence of any autoimmune pathology even after immunization of recipient mice with PR8. These data demonstrate that resting B cells pulsed with self-epitopes can induce abortive activation of potentially self-reactive naive CD8+ T cells resulting in their functional deletion from the peripheral T-cell repertoire in the absence of any associated autoimmunity.

Figure 1

Naive clone 4 (CL4) CD8⁺ T cells proliferate in vivo following encounter with K^dHA peptide-pulsed resting B cells. Thy-1.2 InsHA mice were injected i.v. with 5 × 10⁶ CFSE-labelled purified Thy-1.1 clone 4 CD8⁺ T cells (a–i). After 24 hr mice were given 1200 HA units PR8 i.p. (a–c), 5 × 10⁶ purified unpulsed (d–f), or K^dHA peptide-pulsed B cells (g–i). Three days later peripheral lymphoid tissues were harvested, stained with phycoreythrin-conjugated anti-Thy-1.1 mAb and examined by flow-cytometry. Histograms show the level of CFSE (FL1) among Thy-1.1 (FL2) clone 4 CD8⁺ T cells obtained from spleen (a,d,g) peripheral lymph nodes (PerLN; from pooled cervical, axillary, brachial and inguinal nodes; b,e,h) and pancreatic lymph nodes (PLN; c,f,i). Autofluorescent cells were gated out in the FL3 channel. Data are representative of three separate experiments each containing four mice per group.

Figure 2

Abortive activation of naive clone 4 (CL4) CD8⁺ T cells by K^dHA peptide-pulsed resting B cells does not result in IFN-γ production. Thy-1.2 InsHA mice were injected i.v. with 5 × 10⁶ CFSE-labelled purified Thy-1.1 clone 4 CD8⁺ T cells (a–i). After 24 hr mice were given 1200 HA units PR8 i.p. (a–c), 5 × 10⁶ purified unpulsed (d–f), or K^dHA peptide-pulsed B cells (g–i). Three days later peripheral lymphoid tissues were harvested, stained with phycoreythrin-conjugated anti-Thy-1.1 and allophycocyanin-conjugated anti-IFN-γ mAb and examined by flow cytometry. Histograms show the level of CFSE (FL1) versus intracellular IFN-γ (FL4) among Thy-1.1⁺ (FL2) cells obtained from spleen (a,d,g), peripheral lymph nodes (Per-LN; from pooled cervical, axillary, brachial and inguinal nodes; b,e,h) and pancreatic lymph nodes (PLN; c,f,i). Autofluorescent cells were gated out in the FL3 channel. The percentage of Thy-1.1⁺ cells which are IFN-γ⁺ is shown. Data are representative of cells from two separate experiments each containing four mice per group.