Evidence has gathered that CD28 costimulation facilitates T cell activation by potentiating TCR intrinsic-signaling. However, the underlying molecular mechanism is largely unknown. Here we show that, by enhancing T cell/APC close contacts, CD28 facilitates TCR signal transduction. Moreover, the signal supplied by CD28 does not lead to increased Zap-70 and Lat phosphorylation, but amplifies PLCgamma1 activation and Ca(2+) response. We provide evidence that the PTK Itk controls the latter function. Our data suggest that CD28 binding to B7 contributes to setting the level of TCR-induced phosphorylated Lat for recruiting signaling complexes, whereas the CD28 signal boosts multiple pathways by facilitating PLCgamma1 activation. These results should provide a conceptual framework for understanding quantitative and qualitative aspects of CD28-mediated costimulation.

The mechanism through which CD28 costimulation potentiates TCR-driven gene expression is still not clearly defined. Vav-1, an exchange factor for Rho GTPases thought to regulate, mainly through Rac-1, various signaling components leading to cytokine gene expression, is tyrosine phosphorylated upon CD28 engagement. Here, we provide evidence for a key role of Vav-1 in CD28-mediated signaling. Overexpression of Vav-1 in Jurkat cells in combination with CD28 ligation strongly reduced the concentration of staphylococcus enterotoxin E/MHC required for TCR-induced NF-AT activation. Surprisingly, upon Vav-1 overexpression CD28 ligation sufficed to activate NF-AT in the absence of TCR engagement. This effect was not mediated by overexpression of ZAP-70 nor of SLP-76 but necessitated the intracellular tail of CD28, the intactness of the TCR-proximal signaling cascade, the Src-homology domain 2 (SH2) domain of Vav-1, and SLP-76 phosphorylation, an event which was favored by Vav-1 itself. Cells overexpressing Vav-1 formed lamellipodia and microspikes reminiscent of Rac-1 and Cdc42 activation, respectively, for which the SH2 domain of Vav-1 was dispensable. Together, these data suggest that CD28 engagement activates Vav-1 to boost TCR signals through a synergistic cooperation between Vav-1 and SLP-76 and probably via cortical actin changes to facilitate the organization of a signaling zone.

Somatic hypermutation (SHM) of immunoglobulin genes is currently viewed as a two step process initiated by the deamination of deoxycytidine (C) to deoxyuridine (U), catalysed by the activation induced deaminase (AID). Phase 1 mutations arise from DNA replication across the uracil residue or the abasic site, generated by the uracil-DNA glycosylase, yielding transitions or transversions at G:C pairs. Phase 2 mutations result from the recognition of the U∶G mismatch by the Msh2/Msh6 complex (MutS Homologue), followed by the excision of the mismatched nucleotide and the repair, by the low fidelity DNA polymerase η, of the gap generated by the exonuclease I. These mutations are mainly focused at A∶T pairs. Whereas in activated B cells both G:C and A∶T pairs are equally targeted, ectopic expression of AID was shown to trigger only G:C mutations on a stably integrated reporter gene. Here we show that when using non-replicative episomal vectors containing a GFP gene, inactivated by the introduction of stop codons at various positions, a high level of EGFP positive cells was obtained after transient expression in Jurkat cells constitutively expressing AID. We show that mutations at G:C and A∶T pairs are produced. EGFP positive cells are obtained in the absence of vector replication demonstrating that the mutations are dependent only on the mismatch repair (MMR) pathway. This implies that the generation of phase 1 mutations is not a prerequisite for the expression of phase 2 mutations.

No abstract

During Plasmodium falciparum asexual intraerythrocytic development, the host's cell plasma membrane is modified by the insertion of parasite proteins. One or more of these modifications mediate the cytoadherence of infected erythrocytes to host vascular endothelium. However, these surface antigens can be the target of cytophilic antibodies which promote phagocytosis of the infected erythrocyte. It has been proposed that antibodies directed to epitopes rich in asparagine play an important role in this process, which has promoted efforts to isolate the corresponding gene(s). We describe here P. falciparum asparagine- and aspartate-rich protein 1 (PfAARP1), a new giant (circa 700-kDa) protein associated with the infected erythrocyte membrane which is rich in asparagine and aspartate residues due to the presence of nine blocks of repeats. Topology analysis predicts that PfAARP1 has multiple transmembrane domains and at least five external loops. Human antibodies immunopurified against a sequence composed exclusively of asparagine and aspartate amino acids derived from PfAARP1 label the surface of the infected erythrocyte, demonstrating that such motifs are exposed. Interestingly, external loop 4 of PfAARP1 contains repetitions of these residues, and their possible role as a target of cytophilic antibodies is discussed.