The classical type of programmed cell death is characterized by its dependence on de novo RNA and protein synthesis and morphological features of apoptosis. We confirmed that stimulated 2B4.11 (a murine T‐cell hybridoma) and interleukin‐3 (IL‐3)‐deprived LyD9 (a murine haematopoietic progenitor cell line) died by the classical type of programmed cell death. Assuming that common biochemical pathways might be involved in the deaths of 2B4.11 and LyD9, we isolated the PD‐1 gene, a novel member of the immunoglobulin gene superfamily, by using subtractive hybridization technique. The predicted PD‐1 protein has a variant form of the consensus sequence found in cytoplasmic tails of signal transducing polypeptides associated with immune recognition receptors. The PD‐1 gene was activated in both stimulated 2B4.11 and IL‐3‐deprived LyD9 cells, but not in other death‐induced cell lines that did not show the characteristic features of the classical programmed cell death. Expression of the PD‐1 mRNA in mouse was restricted to the thymus and increased when thymocyte death was augmented by in vivo injection of anti‐CD3 antibody. These results suggest that activation of the PD‐1 gene may be involved in the classical type of programmed cell death.

A mAb J43 has been produced against the product of the mouse PD-1 gene, a member of the Ig gene superfamily, which was previously isolated from an apoptosis-induced T cell hybridoma (2B4.11) by using subtractive hybridization. Analyses by flow cytometry and immunoprecipitation using the J43 mAb revealed that the PD-1 gene product is a 50-55 kDa membrane protein expressed on the cell surface of several PD-1 cDNA transfectants and 2B4.11 cells. Since the molecular weight calculated from the amino acid sequence is 29, 310, the PD-1 protein appears to be heavily glycosylated. Normal murine lymphoid tissues such as thymus, spleen, lymph node and bone marrow contained very small numbers of PD-1(+) cells. However, a significant PD-1(+) population appeared in the thymocytes as well as T cells in spleen and lymph nodes by the in vivo anti-CD3 mAb treatment. Furthermore, the PD-1 antigen expression was strongly induced in distinct subsets of thymocytes and spleen T cells by in vitro stimulation with either anti-CD3 mAb or concanavalin A (Con A) which could lead T cells to both activation and cell death. Similarly, PD-1 expression was induced on spleen B cells by in vitro stimulation with anti-IgM antibody. By contrast, PD-1 was not significantly expressed on lymphocytes by treatment with growth factor deprivation, dexamethasone or lipopolysaccharide. These results suggest that the expression of the PD-1 antigen is tightly regulated and induced by signal transduction through the antigen receptor and do not exclude the possibility that the PD-1 antigen may play a role in clonal selection of lymphocytes although PD-1 expression is not required for the common pathway of apoptosis.

Activated mature B cells in which the DNA-binding activity of E-proteins has been disrupted fail to undergo class switch recombination. Here we show that activated B cells overexpressing the antagonist helix-loop-helix protein Id3 do not induce expression of the murine Aicda gene encoding activation-induced deaminase (AID). A highly conserved intronic regulatory element in Aicda binds E-proteins both in vitro and in vivo. The transcriptional activity of this element is regulated by E-proteins. We show that the enforced expression of AID in cells overexpressing Id3 partially restores class switch recombination. Taken together, our observations link helix-loop-helix activity and Aicda gene expression in a common pathway, in which E-protein activity is required for the efficient induction of Aicda transcription.

Class switch recombination (CSR) at the antibody immunoglobulin locus is regulated by germline transcription (GLT)-coupled modifications in the accessibility of the switch region, where CSR takes place. Here we show that histone acetylation of switch regions is linked to CSR but that histone acetylation cannot alone promote CSR or GLT. Activation-induced cytidine deaminase (AID) specifically associates with the CSR target chromatin in a GLT-coupled manner, which may occur potentially by means of physical interaction between AID and the transcription machinery. These data indicate an important role of GLT in the regulation of chromatin accessibility, strongly suggesting that the target of AID is chromatin DNA. Our results give insights on the role of AID and the regulatory mechanism of CSR.