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.

SummaryFas ligand (FasL) is a type II integral membrane protein homologous with tumor necrosis factor (TNF) . Recent studies indicate that TNF is processed to yield the soluble cytokine by metalloproteinases at the cell surface of activated macrophages and T cells . In the present study, we investigated whether FasL is also released by metalloproteinases . Treatment with hydroxamic acid inhibitors of matrix metalloproteinases specifically led to accumulation of membrane-type FasL

Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of vascular cell injury in Takayasu's arteritis. To clarify the immunological mechanisms involved, we examined the expression of a cytolytic factor, perforin in infiltrating cells of aortic tissue samples from seven patients with Takayasu's arteritis. We also examined the expression of a 65-kD heat-shock protein

PD-1, a member of the Ig superfamily, was previously isolated from an apoptosis-induced T cell hybridoma 2B4.11 by subtractive hybridization. Expresson of the PD-1 mRNA is restricted to thymus in adult mice. Using an anti-PD-1 mAb (J43), we examined expression of the PD-1 protein during differentiation of thymocytes in normal adult, fetal and RAG-2(-/-) mice with or without anti-CD3 mAb stimulation. While PD-1 was expressed only on 3-5% of total normal thymocytes, approximately 34% of the CD4(-)CD8(-) double-negative (DN) fraction are PD-1(+) cells with two distinct expression levels (low and high). PD-1(high) thymocytes belonged to TCR gammadelta lineage cells. In the DN compartment of the TCR alphabeta lineage, PD-1 expression started at the low level from the CD44(+)CD25(+) stage and the majority of thymocytes expressed PD-1 at the CD44(-)CD25(-) stage in which the thymocytes express TCR beta chains. The anti-CD3epsilon antibody administration augmented the PD-1 expression as well as the differentiation of the CD44(-)CD25(+) DN cells into the CD44(-)CD25(-) DN stage, not only in normal mice but also in RAG-2-deficient mice. The fraction of the PD-1(low) cells in the CD4(+)CD8(+) double-positive (DP) compartment was very small (<5%) but increased by stimulation with the anti-CD3 antibody, although the total number of DP cells was drastically reduced. The results show that PD-1 expression is specifically induced at the stages preceding clonal selection.

ABSTRACT

To verify the physiological role of the pore-forming protein perforin in vivo, its expression in subpopulations of human peripheral blood lymphocytes was examined by immunocytochemical staining and their cytolytic potentials compared. In addition to NK cells and gamma delta T cells, which uniformly expressed abundant perforin in their cytoplasmic granules, only a small subpopulation of CD8+ alpha beta T cells contained perforin, namely the CD11b+ subset. However, in vitro activation with an anti-CD3 antibody and IL-2 induced perforin expression in approximately 50% of the CD8+CD11b- T cells and also in a small subset of CD4+ T cells. A distribution of perforin in CD8+ and CD4+ T cells, similar to in vitro activated T cells, was observed in fresh peripheral blood lymphocytes from infectious mononucleosis patients. In all instances, the expression of perforin correlated with the cytolytic potential of these subpopulations. The results strongly suggest that perforin plays a role in the manifestation of cytotoxic activity in vivo.

No abstract

Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this paper, we report rat mAbs against mouse perforin established by immunization with a recombinant mouse perforin fragment. These mAbs reacted with purified mouse perforin prepared from cytoplasmic granules of an NK-like cell line in ELISA and Western blot analysis. However, none of these mAbs blocked the hemolytic activity of mouse perforin or absorbed it when fixed in the solid phase. These results indicate that all of these mAbs react with denatured but not with native mouse perforin. By using a combination of the mAbs, we established a sandwich ELISA, for quantitating the cellular contents of perforin. These mAbs were also useful for immunohistochemical staining analysis, and perforin was detected in the cytoplasmic granules of CTL and NK cell lines. Perforin was also detected in a minor population of lymphocytes of the spleen, liver, and lymph node. In normal spleen cells of 5- to 8-week-old mice, 12-15% of asialo GM1+ cells and 7-21% of CD8+ T cells were perforin-positive, but CD4+ T cells, B cells, and macrophages were totally negative. These data clearly show that perforin is expressed in cells of a cytotoxic character in normal mice, in the same way as in primed mice.