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Under the first heading (IgM and IgG purification from sera) in the Materials and Methods, the third sentence should read “Column-purified IgM was not absorbed with protein G but repassaged through Sephacryl S-300 to remove contaminating proteins.”

We did not absorb IgM with protein G, as after the absorption procedure residual protein G in the IgM preparation enhanced lymphocyte activation and proliferation.

The corrected full method is as follows:

Sera from WT-C57BL/6 (referred to as WT-B6) were heat treated (56°C for 1 h) to inactivate complement. CaCl2 (30 μl 1 M solution) and dextran sulfate (40 ml 10% solution) were added to each milliliter sera, which was then incubated at room temperature for 30 min to precipitate out fibrin and lipids from the serum. Serum was then diluted 1:2 with PBS and centrifuged at 12,000 × g to remove the precipitate. Approximately 15 ml of this pretreated, diluted serum was loaded on a column (2.5 cm diameter × 100 cm length) containing Sephacryl S-3000 HR (GE Health Care Biosciences, Upsalla, Sweden) suspended in PBS (Gibco, Grand Island, NY) to separate serum proteins by size. All effluent from the first protein peak was saved for IgM whereas effluent from the peak portion of the second larger protein peak was saved for IgG. Effluents were filtered to remove large particles and then concentrated on a centrifugal concentrator (100-kDa pore size; Amicon Ultra, EMD Millipore, Cork, Ireland) to achieve IgM and IgG levels of 1.0–1.2 mg/ml as quantitated by ELISA assays. Approximately 10 mg of concentrated IgM and IgG was then repassaged through the same column to further purify these preparations, after which effluents were reconcentrated (1 mg/ml), dialyzed against RPMI 1640, and then millipored using a 0.45 μm filter. There was <5% contaminating IgG in the IgM preparation. With this procedure, we obtained 6–8 mg IgM from 30 ml mouse serum. Purified IgM or IgG was aliquotted and stored at 4°C and not frozen. IgM forms aggregates when frozen. The effect of IgM on in vitro cultures was dose dependent and maximal functional effects were observed using a physiological dose of 5–15 μg/250 × 103 cells/0.5 ml culture.

IgM was not isolated by dialyzing in water or by ammonium chloride precipitation as both these techniques yielded IgM with impaired functional activity. Additionally, we did not use protein G to remove contaminating IgG from IgM as residual protein G in the IgM preparation activated T cells to proliferate.