Antibody-enhanced cross-presentation of self antigen breaks T cell tolerance - PubMed (original) (raw)

. 2007 May;117(5):1361-9.

doi: 10.1172/JCI29470. Epub 2007 Apr 19.

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Antibody-enhanced cross-presentation of self antigen breaks T cell tolerance

Stephanie O Harbers et al. J Clin Invest. 2007 May.

Abstract

We have developed a model of autoimmunity to investigate autoantibody-mediated cross-presentation of self antigen. RIP-mOVA mice, expressing OVA in pancreatic beta cells, develop severe autoimmune diabetes when given OT-I cells (OVA-specific CD8(+) T cells) and anti-OVA IgG but not when given T cells alone. Anti-OVA IgG is not directly injurious to the islets but rather enhances cross-presentation of apoptotic islet antigen to the OT-I cells, leading to their differentiation into potent effector cells. Antibody-driven effector T cell activation is dependent on the presence of activating Fc receptors for IgG (FcgammaRs) and cross-priming DCs. As a consequence, diabetes incidence and severity was reduced in mice lacking activating FcgammaRs. An intact complement pathway was also required for disease development, as C3 deficiency was also partially protective. C3-deficient animals exhibited augmented T cell priming overall, indicating a proinflammatory role for complement activation after the T cell priming phase. Thus, we show that autoreactive antibody can potently enhance the activation of effector T cells in response to cross-presented self antigen, thereby contributing to T cell-mediated autoimmunity.

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Figures

Figure 1

Figure 1. OT-I cells and anti-OVA IgG synergistically induce diabetes.

Diabetes incidence: RIP-mOVA mice were injected with 5 × 106 OT-I cells alone or together with administration of rabbit anti-OVA IgG, control rIgG, an F(ab′)2 fragment of rabbit anti-OVA IgG, or murine polyclonal anti-OVA IgG. Anti-OVA refers to rabbit anti-OVA IgG unless otherwise noted. Fractions below bars indicate the number of diabetic mice over the total number treated. Diabetes was not induced by transfer of OVA-specific antibodies and rarely by OT-I cells alone but was synergistically induced by transfer of both OT-I cells and anti-OVA IgG.

Figure 2

Figure 2. Anti-OVA IgG drives OT-I cell infiltration into the pancreas.

(A) Anti-OVA IgG binds to the islets of Langerhans without causing acute inflammation. Staining for OVA (left), bound anti-OVA IgG (by detecting rIgG; middle), and H&E (right) in the pancreata of RIP-mOVA mice 24 hours after treatment with 1,000 μg anti-OVA IgG or 1,000 μg control rIgG (insets). Transferred anti-OVA IgG bound to islet cells (middle) but did not trigger infiltrative lesions (right). (B and C) Diabetes results from OT-I cell infiltration into the pancreas. H&E, CD8 (red), and OVA (green) staining on pancreatic sections (B) and flow cytometric analysis of total live pancreatic cells (C) from RIP-mOVA mice 7 days after transfer of either OT-I cells alone or together with 1,000 μg anti-OVA IgG. Vα2 and Vβ5 indicate the presence of OT-I TCR+ cells. Destructive cellular infiltrates were observed after the transfer of T cells and anti-OVA IgG, including massive intraislet CD8+ OT-I cell accumulation, whereas T cells transferred alone did not accumulate in the pancreas nor induced inflammatory responses.

Figure 3

Figure 3. Anti-OVA IgG enhances functional antigen presentation in the pancreatic lymph node.

(A) OT-I cell proliferative responses. Pancreatic lymph node cells isolated from RIP-mOVA mice 3 days after transfer of CFSE-labeled OT-I cells and anti-OVA IgG. Dot plots are gated on CD8+CFSE+ cells. Proliferation was enhanced 2-fold in recipients of OT-I cells and anti-OVA IgG. (B and C) OT-I cell effector differentiation. ELISPOT analysis of IFN-γ production by total lymph node and splenic cells (B) and intracellular IFN-γ staining of pancreatic lymph node OT-I cells (C) isolated from RIP-mOVA mice 5 days after treatment. Bars show mean ± SD for triplicate wells of 1 representative experiment out of 3. Dot plots are gated on CD8+Vα2+Vβ5+ cells. Differentiation of OT-I cells into IFN-γ–producing effectors was significantly (P = 1.06 × 10–6) enhanced in the presence of anti-OVA IgG.

Figure 4

Figure 4. Anti-OVA IgG enhances OT-I cell proliferation in response to apoptotic cell antigen.

Flow cytometric analysis of pancreatic lymph node cells isolated from ZVAD-treated and untreated RIP-mOVA mice 3 days after transfer of CFSE-labeled OT-I cells with or without anti-OVA IgG. Histograms show percentage of cells divided relative to total CD8+CFSE+ cells. Data represent 1 of 2 independent experiments. Antibody-enhanced OT-I cell proliferation was abolished in ZVAD-treated mice (right).

Figure 5

Figure 5. DCs are required for steady-state and antibody-enhanced OT-I cell proliferation in the pancreatic lymph node.

Flow cytometric analysis of pancreatic lymph node cells isolated from DT-treated RIP-mOVA and RIP-mOVA/CD11c-DTR mice 3 days after transfer of CFSE-labeled OT-I cells alone (left panels) or together with anti-OVA IgG (right panels). Dot plots are gated on CD8+CFSE+ cells and represent 1 of 3 independent experiments. OT-I cell proliferative responses were abolished in the absence of DCs (bottom panels).

Figure 6

Figure 6. Mice lacking activating Fcγ receptors and complement are protected from severe antibody-induced diabetes.

(A) Diabetes incidence. RIP-mOVA, RIP-mOVA FcγRγ–/–, RIP-mOVA C3–/–, and RIP-mOVA FcγRγ–/–C3–/– mice were injected with 5 × 106 OT-I cells alone or together with 1,000 μg anti-OVA IgG. Fractions below bars indicate the number of diabetic mice over the total number treated. Diabetes occurred in 100% of antibody-treated WT RIP-mOVA mice compared with 41% of FcγRγ–/–, 35% of C3–/–, and 0% of FcγRγ–/–C3–/– mice (P = 2 × 10–6, 5 × 10–7, and 8 × 10–10, respectively). (B) Survival of WT, FcγRγ–/–, and C3–/– mice. Kaplan-Meier curves of RIP-mOVA mice (black) compared with RIP-mOVA FcγRγ–/– mice (gray, left) and RIP-mOVA C3–/– mice (gray, right) after transfer of OT-I cells and anti-OVA IgG (***P = 1 × 10–4). Diabetes was severe and usually lethal in WT mice. In contrast, disease severity was attenuated in FcγRγ–/– and C3–/– mice, with no mice developing fatal diabetes.

Figure 7

Figure 7. Antibody-enhanced OT-I cell proliferation and effector cell differentiation is dependent on activating Fcγ receptors.

(A and B) OT-I cell proliferative responses in WT, FcγRγ–/–, and C3–/– mice. Flow cytometric analysis of pancreatic lymph node cells isolated from RIP-mOVA, RIP-mOVA FcγRγ–/–, and RIP-mOVA C3–/– mice (A) and ZVAD-treated and untreated RIP-mOVA C3–/– mice (B) 3 days after transfer of CFSE-labeled OT-I cells and anti-OVA IgG. The percentage of cells divided (A) and the numbers in the histogram (B) were calculated from FACS plots gated on CD8+CFSE+ cells found after the undivided peak. Bars show mean ± SD for at least 5 mice per treatment group. *P = 0.033; †0.0085; **P = 0.00040; #P = 0.00060; NS: P = 0.46. Histogram represents 1 of 2 independent experiments. Activating FcγRs were required for antibody-enhanced proliferation, whereas C3 negatively regulated T cell proliferation in a ZVAD-inhibitable manner. (C) OT-I cell effector differentiation in WT, FcγRγ–/–, and C3–/– mice. Intracellular IFN-γ staining of pancreatic lymph node cells isolated from mice 5 days after treatment. The percentage of cells producing IFN-γ was calculated from IFN-γ–positive cells among CD8+Vα2+Vβ5+-gated populations. Bars show mean ± SD for at least 3 mice per group. ***P = 1.06 × 10–6; ††P = 0.00048; ##P = 0.016; ζ_P_ = 0.037; NS: P = 0.28. Antibody enhancement of IFN-γ production was intact in C3–/– but not FcγRγ–/– mice.

Figure 8

Figure 8. FcγRIIB negatively regulates antibody-mediated OT-I cell priming.

(A) Diabetes incidence. RIP-mOVA or RIP-mOVA FcγRIIB–/– mice were injected with 5 × 106 OT-I cells plus either 1 mg murine polyclonal anti-OVA IgG or murine monoclonal anti-OVA IgG. For the murine monoclonal anti-OVA IgG, 6 WT and 5 FcγRIIB–/– mice were transferred with 18 mg anti-OVA IgG2b, and 5 WT and 3 FcγRIIB–/– mice were transferred with 42 mg anti-OVA IgG1. Fractions below bars indicate the number of diabetic mice over the total number treated. Both the murine polyclonal antibodies and mAbs showed increased diabetogenicity in mice lacking FcγRIIB. (B) OT-I cell effector differentiation. Intracellular IFN-γ staining on pancreatic lymph node OT-I cells isolated from mice 5 days after transfer of OT-I cells alone or together with 1 mg murine polyclonal anti-OVA IgG. Dot plots are gated on CD8+Vα2+Vβ5+ cells.

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