Background/Purpose:  

Rheumatoid factor (RF) is an autoantibody directed against the Fc portion of IgG antibodies.  It is found in ~80% of patients with rheumatoid arthritis (RA). It has been postulated that RF potentiates immune complex disease in RA by enhanced fixation of complement. Rituximab (RTX), an antibody targeting CD20 on B cells, exhibits increased efficacy in seropositive patients with refractory RA.  We hypothesized that this improved efficacy was due to the ability of RF to­­ enhance the ability of RTX to mediate complement dependent cytotoxicity, leading to enhanced synovial depletion of B cells.

Methods:

We developed a model assay system of RTX to mediate complement dependent cytotoxicity (CDC) using the Daudi human B cell line modified to grow in serum-free media. CDC was determined by propidium iodide staining 30 minutes after combining Daudi cells with RTX 10 μg/ml and human sera (1%; as a source of complement) from healthy donors and patients. The effect of RF on RTX-CDC was determined by the following methods:  i) Comparison of CDC by RF+ sera vs RF- sera; ii) Addition of monoclonal IgA or IgM RF to seronegative sera; iii) Mixing studies of RF+ and RF- sera. 

Results:

In the presence of 1% human sera, RTX resulted in rapid (minutes) and profound (>50%) Daudi cell death. This effect was complement dependent as proven by a lack of Daudi cell death by RTX in serum free media, heat-inactivated serum, and C5 deficient serum.  A surprising variation in the ability of human sera to mediate RTX-CDC was observed. The mean percent of RTX-CDC ranged from healthy donors 54% (n=15), RF+ RA patients 47% (n=40), RF- RA patients 88% (n=15), non-RA patients 83% (n=15). Remarkably, sera with an IgM RF >250 IU/ml resulted in a mean CDC of 13% compared to sera with IgM RF 9-100 IU/ml having a mean CDC of 74%.  A similar effect of increasing IgA RF concentration was seen but was not as profound an effect as IgM RF.  Mixing of RF+ sera with RF- sera demonstrated the reduced RTX-CDC, indicating the presence of an inhibitor. The identity of RF as the inhibitory factor was demonstrated by the ability of either purified monoclonal IgM RF or monoclonal IgA RF added to RF- sera to mediate near complete inhibition of CDC at concentrations of 50 μg/ml and 10 μg/ml, respectively.  The inhibitory effect of RF could be blocked by excess IgG.  In addition, we observed that RF did not alter RTX binding to CD20 on B cells.  Therefore, we conclude that RF blocks the ability of early complement components to be recruited to the IgG Fc portion of RTX.

Conclusion:

Contrary to our original hypothesis, RF inhibits RTX induced CDC in vitro. Thus, the enhanced efficacy of RTX in seropositive RA patients cannot be easily attributed to modulation of B cell depletion through CDC. This result is surprising given the roles of RF in immune complex clearance and complement activation. Not only does this generate a new set of insights into the biologic role of RF, it indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody.

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« Back to 2012 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-rheumatoid-factor-paradox-inhibition-of-rituximab-induced-complement-dependent-cytotoxicity-of-b-cells/