We hypothesized that HO538-213 may have a similar mechanism of action. CD4 localizes to lipid rafts, and CD4-crosslinking activates signal transduction involving tyrosine kinases 27–29. Thus, we treated MOLT-4 cells with HO538-213, and the lipid raft fraction was isolated by a membrane floatation assay as verified by the raft markers glycosphingomyelin 1 and sphingomyelin (Fig. 3B, left panel). Tyrosine kinase activitiy was examined by

immunoblotting the lipid raft fractions using a PY20 anti-phosphotyrosine mAb (Fig. 3B, right panel, arrowhead). We detected a significant amount of tyrosine phosphorylation in the lipid raft fraction after HO538-213 treatment, indicating that HO538-213 can assemble cell surface CD4. This is consistent with our hypothesis that HO538-213 inhibits HIV-1 infection by decreasing Kinase Inhibitor Library cell assay the lateral movement of cell surface CD4. We then further characterized the donor from which the CD4-reactive Ab KPT-330 molecular weight was isolated. The donor serum did not show a strong reactivity to rhCD4 at 1:10 dilution, where the non-specific effect was

no longer detected. We analyzed the HIV-inhibition titer of the donor plasma. In a TZM-bl cell assay, the plasma did not block HIV replication at 1:50 dilution (data not shown). These data suggest that the CD4-reactive IgM circulates at very low titers in the donor and may not be sufficient to block HIV infection in vitro. However, it is possible that the CD4-reactive IgM may be able to limit HIV-1 propagation under in vivo conditions. We next investigated the immunological status of the donor. IgG and IgM levels were

within the normal range, Farnesyltransferase and the plasma was negative for rheumatoid factor, anti-DNA, and anti-ribonucleoprotein Ab. However, the donor serum reacted to nuclear Ag at a titer of 1:160 (1:40 or less is considered normal), and the staining patterns were nucleolar (1:160) and speckled (1:80). Consistent with these data, the frequency of auto-reactive Ab-producing cells from the same donor, namely against nuclear Ag and blood group i-glycolipid, was significantly higher than the other donors (Fig. 1A). In addition, we isolated anti-TNF-α IgG and IgM clones from this donor 16. Although clinical manifestations of autoimmune disorders were lacking, it is likely that the donor may have an immunological background that generates auto-reactive Ab and tolerates them. Moreover, the donor has been healthy for 29 years, at the time the CD4-reactive Ab was first isolated, suggesting that such CD4-reactive Ab may not disturb host immunity. Considering that the IgM-producing B cells we isolated went through positive/negative selection, their original target should not be CD4. It is thus likely that the IgM genes accumulated SHM that resulted in cross-reactivity to CD4 in the periphery after B-cell maturation.