Decay-accelerating factor modulates induction of T cell immunity

Abstract

Decay-accelerating factor (Daf) dissociates C3/C5 convertases that assemble on host cells and thereby prevents complement activation on their surfaces. We demonstrate that during primary T cell activation, the absence of Daf on antigen-presenting cells (APCs) and on T cells enhances T cell proliferation and augments the induced frequency of effector cells. The effect is factor D- and, at least in part, C5-dependent, indicating that local alternative pathway activation is essential. We show that cognate T cell-APC interactions are accompanied by rapid production of alternative pathway components and down-regulation of Daf expression. The findings argue that local alternative pathway activation and surface Daf protein function respectively as a costimulator and a negative modulator of T cell immunity and explain previously reported observations linking complement to T cell function. The results could have broad therapeutic implications for disorders in which T cell immunity is important.

Figure 1.

Daf deficiency/blockade augments cellular immunity in vitro. (A) IFNγ ELISPOT assays for Daf1 ^{− / −} splenocytes (± reconstitution using lipid-tailed murine rDaf) or Daf1 ^+/+ splenocytes versus allogeneic H-2 ^k spleen cells. (B) Flow cytometry of Daf expression levels on cells in A. (C) WT H-2 ^k T cells stimulated with allogeneic H-2 ^b spleen cells (left) or Mar TCR transgenic T cells stimulated with HYDby peptide-loaded H-2^bAPCs (right) with or without anti-Daf mAb 2C6 or control IgG in IFNγ ELISPOT assays. Means and SD (n = 3–5/group) shown are representative of 2–4 independent experiments; *, P < 0.05.

Figure 2.

Deficiency of Daf protein augments cellular immunity in vivo. (A) IFNγ ELISPOT assays were performed at rejection of male to female skin grafts (+/+, Daf1 ^{+ / +}; −/−, Daf1 ^{− / −}) to assess reactivity to male peptides HYDby (left) and HYUty (right). Each symbol represents an individual animal (mean of triplicate wells, SD < 15%). –, means for all animals per group (n = 4–8); *, P < 0.05 versus Daf1 ^+/+ to Daf1 ^+/+. No response (<10 IFNγ producers/500,000 cells) was detected in any animal to control antigens OVA_323-339 and β-gal_96-103, and no responses were detected in naive mice (not depicted). (B) Spleen cell responses by IFNγ ELISPOT 12 d (top) or 30 d (bottom) after immunization of Daf1 ^{− / −} or Daf1 ^+/+ mice with HYDby peptide plus CFA. No response was detected to control antigens (not depicted). Means plus SD for three to five animals/group are shown. The results are fully representative of two to four independent experiments. *, P < 0.05.

Figure 3.

Daf1 ^−/− APCs augment T cell immunity in vitro and in vivo via alternative complement activation. (A) H-2 ^k T cells mixed with allogeneic H-2 ^b splenic stimulators with or without rDaf were tested in IFNγ ELISPOT assays (means plus SD; n = 3 replicates/group). (B) Proliferation of CFSE-labeled WT H-2 ^k T cells versus allogeneic Daf1 ^{− / −} or Daf1 ^+/+ macrophages on d 5. The precursor frequencies of responding T cells were 1.01% (4.1% underwent more than three divisions) against Daf1 ^{− / −} cells versus 0.07% against Daf1 ^+/+ cells (1.1% underwent more than three divisions). (C) Proliferation of splenic CFSE-labeled H-2 ^k T cells on day 5 after injection into allogeneic hosts. The mean numbers of transferred cells that underwent more than three cell divisions (n = 3 animals/group) are shown. (D) IFNγ ELISPOT production by Mar T cells stimulated with 10 μM HYDby plus spleen cells. (E) Proliferation of CFSE-labeled Vβ6⁺ Mar T cells 3 d after adoptive transfer into indicated mice (3 × 10⁶ cells/mouse). (Left) representative plots gated on CFSE⁺ Vβ6⁺ cells (inset: % Mar cells/spleen). (Right) quantified means (n = 3 animals/group). (F, left) Daf1 ^+/+ or Daf1 ^−/− mice were immunized with OVA_323-339 plus anti-C5 mAb or control (1 mg i.p. every other day), and recall assays to OVA_323-339 (0.1 μM) were performed by ELISPOT on d 16. *, P < 0.05 versus the response in Daf1 ^{− / −} mice. Right: 3-d in vitro IFNγ ELISPOT assays of H-2 ^k T cells versus Daf1 ^+/+ or Daf1 ^{− / −} APCs plus 100 μg/ml anti-C5 mAb or control (top) or H-2 ^k T cells versus Daf1 ^+/+ spleen cells plus recombinant C5a (bottom). *, P < 0.05 versus control. Each result is representative of three individual experiments.

Figure 4.

Daf deficiency on T cells enhances T cell responsiveness. (A) Purified T cells were mixed with WT allogeneic H-2 ^k splenic stimulator cells in IFNγ ELISPOT assays. (B) T cell–depleted WT Thy1.1 (H-2 ^b) females were given adoptive transfers of 7.5 × 10⁶ unfractionated, purified splenic T cells isolated from Thy1.2⁺ Daf1 ^+/+ or Daf1 ^{− / −} mice. The reconstituted hosts were immunized with HYDby plus CFA, and recall IFNγ ELISPOT assays (means plus SD; n = 3/group) were performed 21 d later. Flow cytometry confirmed that all IFNγ derived from the Thy1.2⁺ cells (not depicted). *, P < 0.05.

Figure 5.

T cells and APCs produce complement components during cognate interactions. (A) H-2 ^k T cells were mixed with allogeneic peritoneal macrophages, flow sorted at predetermined time points, tested for complement expression by quantitative RT-PCR, and compared with unstimulated controls. (B) Electron micrograph of immunostaining using gold–anti-C3 of Mar T cells mixed with female H-2 ^b peritoneal macrophages plus HYDby peptide (top) or OVA_323-339 (bottom). Note gold particles (arrowheads) only in the HYDby peptide-stimulated sample. T, T cell; M, macrophage. Bar, 200 μm. (C) C3, factor B, and factor D in serum-free culture supernates of Mar T cells mixed with HYDby-loaded APCs overnight before (top) and after (middle) cell sorting and reculturing for an additional 3 h. Data are plotted as relative increase over OVA_323-339-stimulated controls. C3, factor B, and factor D in serum-free culture supernates of H-2 ^k T cells stimulated overnight with anti-CD3 (bottom panel), expressed as fold increase over controls. (D) Western blots for factor B (fB, top) and C3 (bottom) in culture supernatants of Mar T cells stimulated with WT or C3 ^{− / −} splenic APCs and HYDby peptide or OVA_323-339. Densitometry revealed 20% increase in C3 and 175% increase in factor B with HYDby peptide versus OVA_323-339. +, positive control (normal mouse serum); —, negative control (serum from fB ^{− / −} /Crry ^{− / −} or C3^−/− mice). E. Plots of Daf expression on macrophages at 0, 24, and 48 h after mixing with Mar T cells plus HYDby peptide or OVA_323-339. All results are representative of experiments performed at least two or three times.