Major peptide autoepitopes for nucleosome-specific T cells of human lupus - PubMed (original) (raw)

Major peptide autoepitopes for nucleosome-specific T cells of human lupus

L Lu et al. J Clin Invest. 1999 Aug.

Abstract

We tested 154 peptides spanning the entire length of core histones of nucleosomes for the ability to stimulate an anti-DNA autoantibody-inducing T helper (Th) clone, as well as CD4(+) T-cell lines and T cells, in fresh PBMCs from 23 patients with lupus erythematosus. In contrast to normal T cells, lupus T cells responded strongly to certain histone peptides, irrespective of the patient's disease status. Nucleosomal peptides in histone regions H2B(10-33), H4(16-39) (and overlapping H4(14-28)), H4(71-94), and H3(91-105) (and overlapping H3(100-114)) were recurrently recognized by CD4 T cells from the patients with lupus. Remarkably, these same peptides overlap with major epitopes for the Th cells that induce anti-DNA autoantibodies and nephritis in lupus-prone mice. We localized 2 other recurrent epitopes for human lupus T cells in H2A(34-48) and H4(49-63). All the T-cell autoepitopes have multiple HLA-DR binding motifs, and the epitopes are located in histone regions recognized by lupus autoantibodies, suggesting a basis for their immunodominance. Native nucleosomes and their peptides H4(16-39), H4(71-94), and H3(91-105) induced a stronger IFN-gamma response, whereas others, particularly, H2A(34-48), favored an IL-10- and/or IL-4-positive T-cell response. The major autoepitopes may reveal the mechanism of autoimmune T-cell expansion and lead to antigen-specific therapy of human lupus.

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Figures

Figure 1

Figure 1

IL-2 production by anti-DNA autoantibody–inducing T-cell clone DD2 in response to nucleosomal histone peptides (10 μM each) presented by autologous EBV B-cell APCs. Bars represent mean of triplicate values. Values higher than 3 SD (horizontal line) above the mean of background (DD2 + APCs without added peptide produced 132 pg/mL IL-2) were considered as stimulatory. IL-2 production by DD2 in response to anti-CD3 stimulation was 554 pg/mL.

Figure 2

Figure 2

(a) IL-2 production by a representative short-term CD4 T-cell line L-EB, with different concentrations of histone H449–63 peptide-pulsed autologous EBV B-cell APCs. Stimulation with 0.01–100 μM peptide showed significant differences (*P < 0.05 at 0.01 μM, Student’s t test) from background (T cells plus APCs cultured without peptide). (b) An example of MHC class II–dependent recognition of nucleosomal peptides. Presentation of H449–63 peptide by autologous EBV B cells to short-term T-cell line L-EB was inhibited mainly by anti-DR mAb.

Figure 3

Figure 3

Example of a pepscan of L-SC, a short-term CD4 T-cell line from a patient with lupus, with histone peptides presented by autologous EBV B-cell APCs. The baseline for y axis is set at 3 SD above the mean of background values obtained with T cells cultured with APCs alone. IL-2 production responses to peptides above that value are shown.

Figure 4

Figure 4

IL-2 production by N-JV, a short-term CD4 T-cell line from a representative normal donor to histone peptides using autologous EBV B-cell APCs. The horizontal line in each panel demarcates 3 SD above the mean of background values. None of the normal T-cell lines showed a response to any of the peptides that was 3 SD above their respective background values.

Figure 5

Figure 5

Summary of IL-2 production responses of short-term T-cell lines to nucleosomal histone peptides in 10 patients with lupus. The results are expressed as percentages of maximal responses of the T-cell lines stimulated by anti-CD3 mAb. Bars represent mean values from 10 lupus T-cell lines, with error bars in SEM. Values of T cells cultured with autologous EBV B-cell APCs alone were considered as background. The baseline for y axis is set at 22%, which is 3 SD above the mean of background values (14.5%). A peptide was considered stimulatory when it elicited a positive response above this baseline. For instance, the average of responses of all 10 T-cell lines to H416–30 peptide was 23 ± 2.6. All the stimulatory peptides in the panels elicited positive responses in all 10 lupus T-cell lines to levels greater than 3 SD above their respective background values. The extreme right bar in the bottom right (H4) panel was the mean value of responses of the 10 lines to whole nucleosome preparation.

Figure 6

Figure 6

A library of histone peptides that correspond to the stimulatory epitope regions identified with lupus T-cell lines and clone DD2. In addition, three 24-mer peptides that overlapped some of these epitopes were used for further studies: H2B10–33 (PKKGSKKAVTKAQKKDGKKRKRSR), H416–39 (KRHRKVLRDNIQGITKPAIRRLAR), and H471–94 (TYTEHAKRKTVTAMDVVYALKRQG). All peptide sequences are identical to those in human histones (GenBank).

Figure 7

Figure 7

Representative 2-color intracellular cytokine staining of gated CD4+ T cells freshly obtained from 2 patients with lupus who were in remission (R-WG and R-SC). IFN-γ and IL-10 response (a) and IL-2 and IL-4 production (b) in response to stimulation by anti-CD3, nucleosomes, or the histone peptide epitopes. Demarcation of the quadrants was set based on background staining of the T cells cultured in medium alone. Percentage values for the respective cytokine stainings can be found in Figure 8.

Figure 8

Figure 8

Flow cytometry results of intracellular cytokine staining of viable, CD4+-gated T cells from peripheral blood of all the patients with lupus. T-cell response to a peptide epitope was considered positive when the percentage of positive cells was twice the background (medium alone) and at least 0.2% of the total viable CD4+ T cells were stained positive. Positive response is shown in shaded and bold numbers. nd = not done.

Figure 9

Figure 9

Flow cytometry results of intracellular cytokine staining of CD4 T cells from all the normal subjects. A positive response is displayed, as in Figure 8. The normal T cells did not show a positive response to the longer (24-mer) peptides (data not shown).

Figure 10

Figure 10

Frequency of positive responders to the histone autoepitopes among the patients with lupus shown in Figure 8. Bold and shaded numbers highlight when 50% or more responded to a particular epitope; bold and italics represent at least 40% responders. These epitopes were also recognized by T-cell lines and clones from other lupus patients (Figures 1–6). AAssays could not be done in all cases owing to lack of sufficient PBMCs. The responses of certain lupus T cells were poor, judging from their anti-CD3 response, and those are also excluded from the total.

Figure 11

Figure 11

Multiple HLA-DR binding motifs in histone autoepitopes. The autoepitopes that are recurrently recognized by human lupus T cells (Figure 10) were aligned to various HLA-DR motifs published previously (–50): HLA-DR1, DR4, DR7: [ LIVMF ] XXXX [ STPALIVMC]; [RKHLIY] XXXX [YTNQCDERSW] XXX AMVKWLHYIFP; [RKHLIY] XXX [YTNQCDERSW] XXX AMVKWLHYIFP. HLA-DR3: [FILVY] XX [DNQT]. HLA-DR8: [FIVLY] XXX [HKR]. Any single amino acid inside the brackets is one of the most probable anchor residues at that position of the motif. In each of the histone autoepitopes, the corresponding residues are highlighted by bold and underlined letters. X indicates that any of the 20 amino acids can be present in that position, and also represents an individual amino acid position in the sequence. In addition to the motifs indicated here, H471–94 contains multiple motifs for HLA-DR1,3,4,7,8 and also HLA-DR5.

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