Presentation of the candidate rheumatoid arthritis autoantigen aggrecan by antigen-specific B cells induces enhanced CD4(+) T helper type 1 subset differentiation

Abstract

Effective immune responses require antigen uptake by antigen-presenting cells (APC), followed by controlled endocytic proteolysis resulting in the generation of antigen-derived peptide fragments that associate with intracellular MHC class II molecules. The resultant peptide-MHC class II complexes then move to the APC surface where they activate CD4(+) T cells. Dendritic cells (DC), macrophages and B cells act as efficient APC. In many settings, including the T helper type 1 (Th1) -dependent, proteoglycan-induced arthritis model of rheumatoid arthritis, accumulating evidence demonstrates that antigen presentation by B cells is required for optimal CD4(+) T cell activation. The reasons behind this however, remain unclear. In this study we have compared the activation of CD4(+) T cells specific for the proteoglycan aggrecan following antigen presentation by DC, macrophages and B cells. We show that aggrecan-specific B cells are equally efficient APC as DC and macrophages and use similar intracellular antigen-processing pathways. Importantly, we also show that antigen presentation by aggrecan-specific B cells to TCR transgenic CD4(+) T cells results in enhanced CD4(+) T cell interferon-γ production and Th1 effector sub-set differentiation compared with that seen with DC. We conclude that preferential CD4(+) Th1 differentiation may define the requirement for B cell APC function in both proteoglycan-induced arthritis and rheumatoid arthritis.

Figure 1

Expression and antigen binding of transfected anti-aggrecan BCR. Flow cytometric analysis of (a) untransfected A20, A20 BCR transfectants [A20-agg 4C5; (b), and 5F10 (c)] showing BCR expression (left panels, shaded) and biotinylated aggrecan binding (right panels, shaded). Open histograms show staining following treatment with secondary reagent only. Analysis shown is representative of at least three independent experiments.

Figure 2

APC function of aggrecan-specific B cells. (a) Untransfected A20 (Δ), A20-agg (▾,▴), and A20- tetanus toxin C fragment (TTCF) (▪) transfectants were incubated with either 192 (anti-aggrecan; left, middle panels) or MC52 (anti-TTCF; right panel) T cell hybridomas in the presence of graded doses of aggrecan (left), p84–103 (middle), or TTCF (right panel) for 24 hr. (b) A20 (open) or A20-3A5 (filled) B cells were incubated with 192 T cell hybridomas in the presence of graded doses of p84–103 or aggrecan for 24 hr. (c) A20 (triangles) or A20-3A5 (circles) were transiently transfected with plasmids encoding the C7.1 BCR light (L) chain only, or with plasmids encoding the C7.1 BCR heavy and light chains (H+L). Twenty-four hours following transfection, B cells were incubated with 192 T cell hybridomas in the presence of graded doses of aggrecan for a further 24 hr. A20-agg transfectants [4C5 (▾)] were also included for comparison. Antigen presentation was assessed by proliferation of the interleukin-2 (IL-2) -dependent line CTLL-2 as described in Materials and methods. Data points show mean counts/min (cpm) values from triplicate wells (± SEM) and the data shown are representative of at least three independent experiments.

Figure 3

Aggrecan-specific B cells present antigen as efficiently as other APC: (a) 5 × 10⁴ or (b) 600 dendritic cells (DC) (•), macrophages (M) (▪), untransfected A20 (Δ) B cells or A20-agg transfectants (▴,▾) were incubated with 5 × 10⁴ 192 T cell hybridomas in the presence of graded doses of p84–103 (left panels) or aggrecan (right panels) for 24 hr. (c) APC were pulsed with 200 nm aggrecan for the times shown, fixed and washed. Then, 1 × 10⁴ cells were cultured with 5 × 10⁴ 192 T cells for 24 hr. Antigen presentation was assessed as in Fig. 1. Data points show mean counts/min (cpm) values from triplicate wells (± SEM) and the data shown are representative of at least two independent experiments.

Figure 4

Aggrecan-specific B cells use similar processing pathways to dendritic cells (DC) and macrophages (M). APC were pre-treated with graded doses of (a) brefeldin A or (b) ammonium chloride followed by further incubation in the presence of 200 nm aggrecan (•), 10 nm p84–103 (▪), or medium (▴). Cells were washed and fixed and 1 × 10⁴ co-cultured with 5 × 10⁴ 192 T cell hybridomas for 24 hr. Antigen presentation was assessed as in Fig. 1. Data points show mean counts/min (cpm) values from triplicate wells (± SEM) and are representative of at least three independent experiments.

Figure 5

Aggrecan presentation by antigen-specific B cells induces similar levels of transgenic CD4⁺ T cell activation compared with presentation by dendritic cells (DC). (a) Splenocytes (•) or purified CD4⁺ T cell receptor (TCR)-5/4E8 T cells (▴,⋆) were co-cultured in the presence of graded doses of p84–103 (▴,•) or 5 μg/ml concanavalin A (ConA) (⋆) for 72 hr. Insert panels show typical CD4/TCRβ staining of TCR-5/4E8 splenocytes before and after purification (numbers indicate typical % of CD4⁺/TCRβ⁺ cells/total cells). (b) DC (•), A20 (Δ) or A20-agg 4C5 B cells (▾) were incubated with APC-free, splenic TCR-5/4E8 CD4⁺ T cells for 72 hr in the presence of graded doses of p84–103 (left panel) or aggrecan (middle panel). T cell activation [mean (± SEM) counts/min (cpm) from triplicate wells] was measured as described in Materials and methods. Results are shown from a typical experiment. Right panels show statistical analysis of three independent experiments. Means (± SEM) T cell activation indices (T cell activation following culture with 100 nm antigen/T cell activation following culture with medium only, *P < 0.02, n = 3) are shown.

Figure 6

Aggrecan presentation by antigen-specific B cells induces enhanced interferon-γ (IFN-γ) production from CD4⁺ T cells compared with presentation by dendritic cells (DC). (a) DC (•), A20-agg 4C5 (▾) or A20 B cells (Δ) were incubated with APC-free, splenic T cell receptor (TCR)-5/4E8 CD4⁺ T cells for 72 hr in the presence of graded doses of p84–103 (left panel) or aggrecan (middle panel). Supernatant IFN-γ levels measured in a representative experiment are shown. The right panel summarizes data from three independent experiments showing IFN-γ levels measured following presentation of 100 nm antigen by DC (□) or A20-agg 4C5 (▪) (*P < 0.01, n = 3). (b) Purified TCR-5/4E8 CD4⁺ T cells were incubated with A20-agg 4C5 B cells or DC for 72 hr in medium only, or in the presence of either 100 nm p84–103 or aggrecan. Flow cytometric analysis of cell-associated IFN-γ on gated TCR-5/4E8 T cells is shown (see Materials and methods for gating strategy) from a representative experiment (numbers in top right quadrants represent % CD4⁺ IFN-γ⁺/total gated TCR-5/4E8 T cells. Right panel shows statistical analysis of data obtained from four independent experiments. Means (± SEM) % of IFN-γ⁺ CD4⁺ cells/total CD4⁺ cells (*P < 0.04, n = 4) are shown.