B cell responses to a peptide epitope. VI. The kinetics of antigen recognition modulates B cell-mediated recruitment of T helper subsets

The ability of Ag-primed B cells to recruit distinct Th subsets was examined using two analogous synthetic peptides, G41CT3 and G28CT3, as model Ags. With sequence differences at only two positions, these peptides were identical both with respect to fine specificity of Abs induced and ability to prime T cells. Lymph node cell populations primed with peptide G41CT3, when challenged with the homologous Ag, yielded predominantly Th2 cytokines. In contrast, a challenge with the heterologous Ag, G28CT3, resulted in a markedly increased production of Th1 cytokines. These distinctions derived from altered APC function of Ag-primed B cells due to differential kinetics of recognition of the two Ags by surface Ig receptors, as confirmed by binding studies with a panel of anti-G41CT3 mAbs. A concentration-dependent circular dichroism study revealed differences in the nature of intermolecular associations for these two peptides. Furthermore, the on-rate of peptide G28CT3 binding to Ab also increased with increasing peptide concentration, implying a dependence on intermolecular interactions. This, in turn, correlated well with the ability of peptide G28CT3 to preferentially activate either Th1 or Th2 cells. Thus, the relative proportion of Th1 vs Th2 cells recruited by Ag-primed B cells is governed by the on-rate of Ag binding to surface Ig receptors, with higher on-rates promoting Th1 recruitment. Further, even subtle changes in solution behavior of an Ag can markedly influence the kinetics of recognition by B cells

Inhibition of CTLA-4 Function by the Regulatory Subunit of Serine/Threonine Phosphatase 2A

The catalytic subunit of the serine/threonine phosphatase 2A (PP2A) can interact with the cytoplasmic tail of CTLA-4. However, the molecular basis and the biological significance of this interaction are unknown. In this study, we report that the regulatory subunit of PP2A (PP2AA) also interacts with the cytoplasmic tail of CTLA-4. Interestingly, TCR ligation induces tyrosine phosphorylation of PP2AA and its dissociation from CTLA-4 when coligated. The association between PP2AA and CTLA-4 involves a conserved three-lysine motif in the juxtamembrane portion of the cytoplasmic tail of CTLA-4. Mutations of these lysine residues prevent the binding of PP2AA and enhance the inhibition of IL-2 gene transcription by CTLA-4, indicating that PP2A represses CTLA-4 function. Our data imply that the lysine-rich motif in CTLA-4 may be used to identify small molecules that block its binding to PP2A and act as agonists for CTLA-4 function

Production and characterization of pharmacologically active recombinant human phosphodiesterase 4B in Dictyostelium discoideum

Phosphodiesterase 4B (PDE4B) is an important therapeutic target for asthma and chronic obstructive pulmonary disease. To identify PDE4 subtype-specific compounds using high-throughput assays, full-length recombinant PDE4 proteins are needed in bulk quantity. In the present study, full-length human PDE4B2 was expressed in the cellular slime mould Dictyostelium discoideum (Dd). A cell density of 2 x 107 cells/mL was obtained and up to 1 mg/L recombinant PDE4B2 was purified through Ni-NTA affinity chromatography. The expressed protein was soluble and its activity was comparable to PDE4B2 protein expressed in mammalian cells (Km=1.7 μM). The functional significance of the Dd expression system is supported by the demonstration that, in concert with proteins expressed in mammalian systems, there are no major changes in the affinity for PDE4B2 inhibitors and substrates. These findings thus provide the first evidence that Dd can be utilized for the expression and purification of functionally active full-length human PDE4B2 in large amounts required for high-throughput screening of pharmacologically active compounds against this therapeutic target

An Autoimmune Disease-Associated CTLA-4 Splice Variant Lacking the B7 Binding Domain Signals Negatively in T Cells

AbstractCytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in downregulating T cell responses. A number of autoimmune diseases have shown genetic linkage to the CTLA-4 locus. We have cloned and expressed an alternatively spliced form of CTLA-4 that has genetic linkage with type I diabetes in the NOD mice. This splice variant of CTLA-4, named ligand-independent CTLA-4 (liCTLA-4), lacks exon2 including the MYPPPY motif essential for binding to the costimulatory ligands B7-1 and B7-2. Here we show that liCTLA-4 is expressed as a protein in primary T cells and strongly inhibits T cell responses by binding and dephosphorylating the TcRζ chain. Expression of liCTLA-4, but not full-length CTLA-4 (flCTLA-4), was higher in memory/regulatory T cells from diabetes-resistant NOD congenic mice compared to susceptible NOD mice. These data suggest that increased expression and negative signaling delivered by the liCTLA-4 may regulate development of T cell-mediated autoimmune diseases