Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

Abstract

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.

Figure 1

Effects of immunoablative therapy. Shown are the effects of immunoablative therapy on rheumatoid arthritis (RA)-specific autoantibody responses, physiological antibody responses and total immunoglobulin levels. The individual disease course of patients was split in four consecutive time periods: before immunoablative therapy ('pre-TX'), the lowest antibody level after immunoablative therapy ('nadir'), the period after immunoablative therapy without disease-modifying antirheumatic drug (DMARD) therapy ('DMARD free'), and the time of overt clinical flare of synovitis ('relapse'). Because the duration of the DMARD-free period varied for each patient (see Table 1), the presented levels are a mean of all observations during that period. Each symbol corresponds to a different patient. (a) Titers of antibodies against anti-cyclic citrullinated protein (ACPA)-IgG. The cut-off for ACPA-IgG is 25 arbitrary units (AU)/ml. In patient 1 a long-lasting undetectable level of ACPA-IgG was observed after high-dose chemotherapy plus haematopoietic stem cell transplantation. Levels below the detection limit were arbitrarily assigned a value of 4 AU/mL to optimize the graphical representation. (b) Titres of rubella of the IgG isotype (RL-IgG). (c) Titres of total circulating IgG. (d) Titres of rheumatoid factor (RF)-IgM.(e) Titres of anti-phosphorylcholine of the IgM isotype (PC-IgM). (f) Titres of total circulating IgM.

Figure 2

Associations between synovial inflammation and circulating ACPA-IgG autoantibodies. (a) Semiquantitative scores of individual patients before immunoablative therapy, scoring synovial infiltration for CD68 expression (macrophages), CD138 expression (plasma cells), CD20 expression, CD79a expression (B cells), CD3 expression (T-cells), Ki-67 expression (proliferation marker) and total inflammation. (b) Correlation of synovial inflammation scores with serum levels of antibodies against anti-cyclic citrullinated protein (ACPA)-IgG (open symbols) and avidity of ACPA-IgG (closed symbols) before immunoablative treatment. Each symbol corresponds to a distinct patient. (c) Correlation of serum levels of ACPA-IgG with C-reactive protein (CRP) levels (r = 0.56, P < 0.001) during complete follow up in the three patients whose ACPA-IgG levels are susceptible to immunoablative therapy ('ACPA responders'). (d) Correlation of serum levels of ACPA-IgG with CRP levels (r = 0.027, P = 0.86) during complete follow up in the three patients whose ACPA-IgG levels remained stable after immunoablative therapy ('ACPA nonresponders'). AU, arbitrary units; NaSCN, sodium thiocyanate.

Figure 3

Regeneration after immunoablative therapy of ACPA-IgG autoantibodies with low avidity. (a) Low avidity of antibodies against anti-cyclic citrullinated protein (ACPA)-IgG (solid lines) before and after immunoablative therapy when autoimmunity is reactivated with rising levels of ACPA-IgG (dotted lines) in the three 'ACPA responders'. Levels below the detection limit were arbitrarily assigned a value of 4 arbitrary units (AU)/ml to optimize the graphical representation. Thick lines represent mean values. (b) Avidity of ACPA-IgG (solid lines) before and after immunoablative therapy in the three 'ACPA nonresponders', in whom, despite relatively stable ACPA-IgG levels (dotted lines), reactivation of autoimmunity was accompanied by the avidity maturation of ACPA-IgG autoantibodies. Thick lines represent mean values. (c) Avidity of tetanus toxoid (TT)-IgG (solid lines) before and after immunoablative therapy in four patients. Increasing levels of TT-IgG (dotted lines) after repeated TT immunizations (see Materials and methods) were accompanied by stably high avidity of TT-IgG, indicative of an intact humoral recall response despite immunoablative therapy. Thick lines represent mean values. DMARD, disease-modifying antirheumatic drug; NaSCN, sodium thiocyanate.