T lymphocyte responses to anti-neutrophil cytoplasmic autoantibody (ANCA) antigens are present in patients with ANCA-associated systemic vasculitis and persist during disease remission

Abstract

ANCA with specificity for myeloperoxidase (MPO) and proteinase 3 (PR3) are present in patients with systemic vasculitis. The aim of this work was to determine whether such patients have T cell responses to these antigens and whether these responses are related to disease activity. Peripheral blood lymphocytes from 45 patients and 19 controls were cultured with ANCA antigens and proliferation measured. The antigens used were heat-inactivated (HI) MPO, HI PR3, native (non-HI) PR3, HI whole alpha-granules, and 25 overlapping peptides covering the entire PR3 sequence. Significant responses to both whole PR3 preparations were seen from patient and control groups, and to the alpha-granules from the patient group. Patients responded at all stages of disease: active, remitting, treated or untreated. Only two patients responded significantly to MPO. Responses were significantly higher with the patient group than the control group to all four whole ANCA antigens. Responses to those PR3 peptides containing epitopes known to be recognized by ANCA were detected from one patient. Thus, these studies demonstrate that T cells from vasculitis patients can proliferate to PR3 and occasionally to associated ANCA antigens. Further, responses may persist even after disease remission has been achieved.

Fig. 1

Silver-stained SDS–PAGE gel of proteinase 3 (PR3). Lane 1, whole α-granules; lane 2, DEAE-bound fraction (10 μg); lane 3, PR3 (10 μg).

Fig. 2

Dose response from a single patient to both heat-inactivated and native proteinase 3 (PR3): expressed as mean ct/min (of three wells) + 1 s.d. from the mean.

Fig. 3

Peripheral blood mononuclear cell (PBMC) proliferative responses to ANCA antigens from a single patient: expressed as mean ct/min (of three wells) + 1 s.d. from the mean.

Fig. 4

Peripheral blood mononuclear cell (PBMC) proliferative responses to ANCA antigens by ANCA⁺ vasculitis patients and controls (IgA nephropathy (IgAN) patients and normal controls). Proliferative responses are expressed as stimulation indices (SI), an SI = 1 is equivalent to no response, the bars represent the mean response. (a) PBMC proliferative responses to 10 μg/ml of heat-inactivated (HI) proteinase 3 (PR3). (b) PBMC proliferative responses to 10 μg/ml native PR3. (c) PBMC proliferative responses to 10 μg/ml HI myeloperoxidase (MPO). (d) PBMC proliferative responses to 25 μg/ml HI whole α-granules.

Fig. 5

Peripheral blood mononuclear cell (PBMC) proliferative responses to ANCA antigens and proteinase 3 (PR3) peptides from a single patient assayed at days 10–11. Proliferative responses are expressed as the mean ct/min (of three assays) + 1 s.d., peptides were assayed in pools of two at a final concentration of 10 μg/ml of each peptide, control responses were measured in the presence of DMSO.