Despite the clear functional importance of CD28 costimulation, the signaling pathways transduced through CD28 have remained controversial. PI3K was identified early as a candidate for CD28 signaling, but conflicting data during the past decade has left the role of PI3K unresolved. In this report, we have resolved this controversy. We show that mutation of the PI3K interaction site in the cytosolic tail of CD28 site disrupts the ability of CD28 to recruit protein kinase C-θ to the central supramolecular activation cluster (c-SMAC) region of the immunological synapse, promote NF-κB nuclear translocation, and enhance IL-2 gene transcription. In contrast, mutation of the PI3K interaction site had no effect on the ability of CD28 to enhance IL-2 mRNA stability. These results suggest that two distinct pathways mediate CD28-induced up-regulation of IL-2 expression, a PI3K-dependent pathway that may function through the immunological synapse to enhance IL-2 transcription and a PI3K-independent pathway that induces IL-2 mRNA stability.

Objective To determine the prevalence anti-apoptotic cell antibodies with the 9G4+ idiotype (9G4+) and the relationship between this reactivity and other known 9G4+ specificities and disease activity. Methods Sera from 60 SLE patients and 40 healthy control donors were incubated with apoptotic Jurkat cells and assayed for binding of 9G4+ antibodies by flow cytometry. These samples were also tested for 9G4+ reactivity against naïve B cells and total IgG and IgM anti-apoptotic cell antibody (AACA) reactivity. Results 9G4+ antibodies bound late apoptotic cells in a majority of SLE patients (60%) but only 2 healthy control sera. Among samples with global IgM or IgG AACA, samples with 9G4+ AACA predominated. Patients with high levels of 9G4+ AACA were more likely to have active disease and this remained true even in patients with IgG AACA, or anti-dsDNA. Patients with lupus nephritis were also more likely to have 9G4+ AACA. While 9G4+ reactivity to apoptotic cells often coincided with anti-B cell reactivity, some samples had distinct anti-apoptotic cell or anti-B cell reactivity. Conclusion 9G4+ antibodies represent a major species of anti-apoptotic cell antibodies in SLE serum and this autoreactivity is associated with disease activity. The anti-apoptotic cell reactivity of 9G4+ antibodies can be separated from the germline VH4-34 encoded anti-B cell autoreactivity. Our results indicate that apoptotic cells are an important antigenic source in SLE that positively select B cells with intrinsic autoreactivity against other self-antigens. This selection of 9G4+ B cells by apoptotic cells may represent an important step in disease progression.

Somatic mutation of Ig variable regions occurs prominently in germinal centers, but it has been debated whether the mutation process initiates in germinal centers or is activated before germinal center entry of B cells. We have analyzed for the presence of somatic mutation in Ig gene rearrangements of the nonpolymorphic human VH6 gene in the X-linked HyperIgM syndrome, which is associated with defective CD40 ligand expression and absence of germinal centers and generation of memory B lymphocytes. IgM and rare IgG VH6 productive rearrangements were isolated from PBL of patients with X-linked HyperIgM syndrome. Although the majority of both the IgM and IgG VH6 rearrangements had a germline VH6 sequence, 7 of 102 VH6 IgM and 1 of 6 IgG rearrangements had a mutated VH6 gene. The mutation frequency (mutations/bp) was 1.4% with a range of 2-9 mutations per clone, a mutation frequency lower, however, than that observed in IgM (3.2%) and IgG (5.4%) VH6 rearrangements of normal individuals. These results suggest that somatic mutation may be initiated in a CD40 ligand-independent pathway before entry of B cells into germinal centers, but fails to achieve the high mutation frequency observed in the presence of germinal centers. (J. Clin. Invest. 1995. 95:1389-1393

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The extensive proliferation that B lymphocytes undergo in germinal centers could compromise generation of long term B cell memory if there occurs shortening of the telomeres of germinal center B cells with cell division. Telomere length, which is thought to act as a "mitotic clock" for somatic cells that dictates cellular senescence, can be preserved by the enzyme telomerase. Human tonsil germinal center B cells consistently expressed 100- to 1000-fold higher levels of telomerase activity than naive or memory B cells, which had no or very low detectable activity, as analyzed by the telomere repeat amplification protocol assay. In vitro stimulation of human memory B cells through surface Ig or CD40 was capable of up-regulating telomerase. The findings suggest that longevity of B cell memory is maintained, despite multiple cell divisions in the generation of a memory B cell, by up-regulation of telomerase in germinal center and activated memory B cells.

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