Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues
Introduction
PD-1 has been shown to be one of the inhibitory receptors expressed on immune cells like T and B cells [1]. Gene disruption studies suggest that the PD-1 molecule is involved in the control of auto-immune responses, since PD-1−/− B6 mice develop lupus-like auto-immune diseases and PD-1−/− BALB/c mice suffer from autoimmune cardiomyopathy [2], [3]. These phenotypes, however, become explicit late in life. On the contrary, mice lacking CTLA-4, another inhibitory receptor expressed on T-cells, develop a massive polyclonal lymphoproliferation as early as 5–6 days after birth [4], [5]. This strongly suggests that PD-1 has a physiological role distinct from CTLA-4, even though both receptors deliver similar inhibitory signals in immune cells.
CTLA-4 exerts an inhibitory signal in CD4 positive T-cells upon engagement of its ligands, B7-1 and B7-2 [6], [7]. Since PD-1 is functionally and structurally related to CTLA-4, it was postulated that ligands for PD-1 might be members of the B7 gene family. In a search for candidates in the B7 family proteins, two membrane proteins related to B7 molecules containing two immunoglobulin-fold domains, PD-L1 and PD-L2, were demonstrated to interact directly with the soluble PD-1/Fcγ chimera protein in an in vitro binding assay [8], [9]. Engagement of PD-1 by PD-L1 or PD-L2/Fcγ fusion protein was shown to inhibit proliferation and cytokine production in T-cells [8], [9]. CTLA-4 contains an amino acid stretch like an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail, which recruits src homology 2-domain-containing tyrosine phosphatase, SHP-2 [10], [11], [12], [13]. Although, PD-1 also contains an amino acid stretch like an ITIM, the N-terminal tyrosine residue in the putative ITIM motif is not involved in the delivery of negative signal and rather another C-terminal tyrosine residue plays a pivotal role in the execution of an inhibitory signal cascade through recruitment of SHP-2 [14].
In order to determine the physiological roles of PD-1-PD-L1/PD-L2 interaction, it is necessary to examine the distribution and expression profiles of the PD-L1 and PD-L2 proteins. In the present study, we established monoclonal antibodies specific for mouse PD-L1 and PD-L2 molecules, and examined the distribution and expression patterns of the ligands in the cells of lymphohematopoietic tissues.
Section snippets
Mice
BALB/c mice (6–9-week-old, female) were purchased from Japan Clea and maintained in our animal facility.
Preparation of PD-L1 molecule
A cDNA encoding His-tagged murine PD-L1 was digested with EcoRI and NotI and incorporated into an expression plasmid, pVL1393 (Clontech). The resultant vector together with Bsu36I-digested BacPAK6™ DNA (Clontech) was introduced into SF9 insect cells (Invitrogen) for packaging according to the manufacturer's protocol. HiFive insect cells (Invitrogen) were infected with the viral particles at 27
Monoclonal antibodies specific for PD-L1 and PD-L2
Northern blot analysis indicated that mouse P815 and A20 cell lines exhibited no detectable PD-L1 and PD-L2 transcripts, respectively (data not shown). We, therefore, established a PD-L1-expressing clone of P815 (P815-PD-L1) and a PD-L2-expressing clone of A20 (A20-PD-L2) by cDNA transfection, the expression of each transcript being confirmed by Northern blotting (data not shown). Rats were immunized with purified His-tagged PD-L1 protein and PD-L2/human Ig chimera protein as described in
Discussion
In the present study, we have developed monoclonal antibodies specific to ligands for the PD-1 receptor, PD-L1 and PD-L2, and systematically examined their expression profiles in normal lymphohematopoietic cells. The results indicated the following findings. First, PD-L1 was expressed on almost all the types of lymphohematopoietic cells at varying levels, while PD-L2 was not at all on any cell types examined. Previous studies indicated that PD-L1 transcripts were detected not only in the
References (17)
- et al.
Trends Immunol.
(2001) - et al.
Immunity
(1999) - et al.
Immunity
(1997) - et al.
Curr. Opin. Immunol.
(1997) - et al.
Biochem. Biophys. Res. Commun.
(2000) - et al.
Science
(2001) - et al.
Science
(1995) - et al.
Annu. Rev. Immunol.
(2002)
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PRESTO, JST.