Yaakov Naparstek

Homology of the NH2-terminal amino acid sequences of the heavy and light chains of human monoclonal lupus autoantibodies containing the dominant 16/6 idiotype.

The NH2-terminal amino acid sequences have been determined by automated Edman degradation for the heavy and light chains of five monoclonal IgM anti-DNA autoantibodies that were produced by human-human hybridomas derived from lymphocytes of two patients with systemic lupus erythematosus. Four of the antibodies were closely related to the idiotype system 16/6, whereas the fifth antibody was unrelated idiotypically. The light chains of the 16/6 idiotype-positive autoantibodies (HF2-1/13b, HF2-1/17, HF2-18/2, and HF3-16/6) had identical amino acid sequences from residues 1 to 40. Their framework structures were characteristic of VKI light chains. The light chain of the 16/6 idiotype-negative autoantibody HF6-21/28 was characteristic of the VKII subgroup. The heavy chains of the 16/6 idiotype-positive autoantibodies had nearly identical amino acid sequences from residues 1 to 40. The framework structures were characteristic of the VHIII subgroup. In contrast, the GM4672 fusion partner of the hybridoma produced small quantities of an IgG with a VHI heavy chain and a VKI light chain. The heavy chains of the lupus autoantibodies and the light chains of those autoantibodies that were idiotypically related to the 16/6 system had marked sequence homology with WEA, a Waldenstrom IgM that binds to Klebsiella polysaccharides and expresses the 16/6 idiotype. These results indicate a striking homology in the amino termini of the heavy and light chains of the lupus autoantibodies studied and suggest that the V regions of the heavy and light chains of the 16/6 idiotype-positive DNA-binding lupus auto-antibodies are each encoded by a single germ line gene.

In vitro production of an anti-DNA idiotype by lymphocytes of normal subjects and patients with systemic lupus erythematosus

We investigated whether normal B cells can synthesize antibodies with an idiotypic marker that occurs with high frequency in anti-DNA antibodies of patients with systemic lupus erythematosus (SLE). This idiotype, Id16/6, has been found in the serum of patients with active SLE and in monoclonal anti-DNA antibodies derived from unrelated patients with the disease. We found that cultured lymphocytes of all normal subjects tested produced Id16/6 when stimulated by pokeweed mitogen (PWM). By contrast, lymphocytes from SLE patients produced Id16/6 even without mitogenic stimulation, whether or not they were obtained from patients in remission or relapse. Relapsed patients lymphocytes spontaneously produced the highest levels of Id16/6 which was found in IgG and IgA, in addition to IgM. The majority of Id16/6 produced by PWM-stimulated lymphocytes from either normal subjects or patients in remission did not bind to nucleic acid. In relapse, however, the nucleic acid-binding proportion of Id16/6 rose substantially, indicating that the spontaneously activated B cells in active SLE differ from the subset of B cells that produce Id16/6 upon PWM stimulation. The findings suggest that the lupus Id16/6 family is conserved in normal individuals and it consists of two populations of antibodies with different antigenic specificities. The major set is not directed against nucleic acid antigens; its antigenic specificity is unknown and it dominates the Id16/6 family that appears after PWM stimulation. The other, minor set binds to nucleic acids and becomes prominent in clinically active lupus. These two sets of idiotypically related antibodies may be connected by an immunoregulatory network.

Effects of interferon-α on the expression of a lupus idiotype in normal humans☆

Interferon (IFN) has extensive immunoregulatory effects but its role in systemic lupus erythematosus (SLE) remains obscure. The observations that a high proportion of patients with active SLE have increased IFN levels in their sera, and that IFN injected to lupus-prone mice aggrevates their disease, led us to examine the effects of IFN on the production of 166—a high-frequency idiotype of monoclonal anti-DNA antibodies produced by human-human hybridomas derived from SLE patients. Peripheral blood mononuclear cells (PBMC) of healthy donors or of patients with SLE were incubated with IFN and pokeweed mitogen (PWM). Seven-day supernatants were assayed for total IgM, for IgM with 166 idiotype, and for IgM anti-DNA activity. PWM-stimulated PBMC of all healthy donors examined produced the 166 idiotype (mean 2.5 ng/ml). A significant increase of 166 in normals (above the level with PWM alone) was noted with 10–100 u/ml of IFN-α but not with 500 u/ml. In 310 normals the addition of IFN-α resulted in detectable anti-DNA activity. The IFN-induced increase in 166 idiotype was significantly more than the increase in IgM (335% vs 47% above baseline, with 10 u/ml of IFN). These effects of IFN could not be demonstrated in the absence of PWM nor in T-cell-depleted preparations. Recombinant IFN-γ had no augmenting effect on 166 production. Three SLE patients in remission had elevated levels of 166 in their PBMC supernatant (15–200 ng/ml) which could not be further augmented by IFN. Thus, we have demonstrated the potential of PWM-stimulated normal lymphocytes to generate a “lupus” idiotype and shown that production of this idiotype requires T cells and is preferentially enhanced by IFN-α. Further studies of the effects of IFN on the expression of anti-DNA antibodies may clarify a postulated role of IFN in autoimmune diseases.

Hereditary CD4+ T lymphocytopenia

Two siblings suffering from mental retardation, progressive bronchiectasis, extensive warts, and persistent hepatitis B are described. The propositus also had an unusual physiognomy and non-specific colitis. Both patients had a marked decrease in the population of CD4+ helper T cells.

Sequential anti-idiotypes define reciprocal idiotopes on the same anti-DNA antibody☆

Sequential anti-idiotypes have defined a pair of idiotypes with an idiotype-anti-idiotype relationship to each other in the variable region of the monoclonal anti-DNA antibody 16/6. A rabbit immunized with 16/6 made an antibody-1 (anti-Id-16/6R) and an antibody-2 (anti-anti-Id-16/6 or anti-anti-Id 16/6M). The latter bound to M-2, a monoclonal mouse anti-Id-16/6. M-2 was used to immunize a second mouse, which produced M-16. The variable region of M-16 mimics an antigenic surface on Id-16/6 and binds to a peptide contained within the first hypervariable region of the light chain of 16/6. The complementary idiotypes of 16/6 represent a reciprotope, an idiotype/anti-idiotype pair within the variable region of the same immunoglobulin molecule which may have functional importance.