Role of perforin in controlling B-cell hyperactivity and humoral autoimmunity

Abstract

To determine the role of perforin-mediated cytotoxic T lymphocyte (CTL) effector function in immune regulation, we studied a well-characterized mouse model of graft-versus-host disease (GVHD). Induction of acute GVHD using perforin-deficient donor T cells (pfp-->F1) initially resulted in features of acute GVHD, e.g., engraftment of both donor CD4(+) and CD8(+) T cells, upregulation of Fas and FasL, production of antihost CTL, and secretion of both Th1 and Th2 cytokines. Despite fully functional FasL activity, pfp donor cells failed to totally eliminate host B cells, and, by 4 weeks of disease, cytokine production in pfp-->F1 mice had polarized to a Th2 response. Pfp-->F1 mice eventually developed features of chronic GVHD, such as increased numbers of B cells, persistence of donor CD4 T cells, autoantibody production, and lupuslike renal disease. We conclude that in the setting of B- and T-cell activation, perforin plays an important immunoregulatory role in the prevention of humoral autoimmunity through the elimination of both autoreactive B cells and ag-specific T cells. Moreover, an ineffective initial CTL response can evolve into a persistent antibody-mediated response and, with it, the potential for sustained humoral autoimmunity.

Figure 1

Long-term survival of host B cells in pfp GVHD is similar to that of chronic GVHD mice. Splenic host B cells were quantitated by flow cytometry in acute GVHD (B6wt→F1), chronic GVHD (DBA→F1), and pfp GVHD (B6pfp→F1) mice at 9 and 12 weeks after parental cell transfer. Values represent group mean cells per spleen × 10^–6 ± SE (n = 4–5 mice per group). Similar results were seen at 6 and 8 weeks after parental cell transfer.

Figure 2

Splenocytes from B6pfp→F1 mice exhibit Fas-dependent antihost CTL activity. Antihost CTL activity from control BDF1 (open circles), B6wt→F1 (closed circles), and B6pfp→F1 (closed triangles) mice is shown for (a) L1210 Fas-positive, (b) P815 Fas-dull, and (c) L1210 Fas-negative targets at four successive E/T ratios. Target expression of murine Fas is shown in d–f. Isotype-matched irrelevant Ig was used for background staining (shaded histograms). Similar results were obtained in an additional independent experiment.

Figure 3

Kinetics of cytokine gene expression. (a) Cytokine gene expression from acute (B6wt→F1) and pfp (B6pfp→F1) GVHD mice at 10 and 28 days after parental cell transfer. (b) Cytokine gene expression from pfp GVHD mice and chronic GVHD (DBA→F1) mice at 6 weeks after cell transfer. Results are shown as fold increase over uninjected F1 mice + SE (n = 4–5 mice per group). ^AP < 0.01. ^BP < 0.05. Similar results have been seen in two additional experiments at day 10 after parental cell transfer.

Figure 4

Kinetics of spontaneous IFN-γ and IL-10 production in control F1 and GVHD mice. Splenocytes were tested for spontaneous in vitro IFN-γ or IL-10 production by acute and pfp GVHD mice at 1 week of disease (upper) and by chronic and pfp GVHD mice at 6 weeks of disease (lower). Values are shown as mean ng/mL ± SE for IFN-γ and mean U/mL ± SE for IL-10 at a supernatant dilution of 1:2 (n = 5 mice per group). ^AP < 0.05 compared with control F1 mice.

Figure 5

Kidneys from both pfp GVHD and chronic GVHD mice exhibit glomerulonephritis and deposition of IgG and C. Kidney sections from chronic GVHD (a, d, and g), pfp GVHD (b, e, and h), and control F1 (c, f, and i) mice were stained with anti-murine IgG2a (a, b, and c) and complement (d, e, and f) or H&E (g, h, and i) 12 weeks after donor cell transfer as described in Methods. Photographs were taken at × 200. Representative results are shown from a single mouse from each group (n = 3 per group).