Elsevier

Immunology Letters

Volume 84, Issue 1, 21 October 2002, Pages 57-62
Immunology Letters

Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues

https://doi.org/10.1016/S0165-2478(02)00142-6Get rights and content

Abstract

PD-1 is a member of the immunoglobulin superfamily expressed on immune cells, including T and B cells, and is involved in the delivery of inhibitory signal upon engagement of its ligands, PD-L1 and PD-L2. While the expression profile of PD-1 has been well documented, the analysis of PD-L1 and PD-L2 distributions on a protein basis has not been carried out because of the lack of available monoclonal antibodies specific for the molecules. In this study, we established two monoclonal antibodies, 1-111A and 122, specific for murine PD-L1 and PD-L2, respectively, and examined their expression profiles. Based on flow cytometric analyses, the expression of PD-L1 was detected in a variety of lymphohematopoietic cell types, including a minor proportion of T and B cells in the spleen, majority of pre-B cells and myeloid cells in bone marrow and subsets of thymocytes, while the expression of PD-L2 was not observed in the lymphohematopoietic cells at all. Notably, a significant proportion of the most immature lineage-marker-negative and c-Kit-positive bone marrow cells containing stem cells did express PD-L1. Following mitogenic stimulation, essentially all lymphocytes expressed PD-L1. Furthermore, a variety of leukemic lines also expressed PD-L1, while none of them did PD-L2. Thus, present results demonstrate the distinct expression patterns of PD-L1 and PD-L2 with the cells of lymphohematopoietic tissues exclusively expressing the former.

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)

  • H. Nishimura et al.

    Trends Immunol.

    (2001)
  • H. Nishimura et al.

    Immunity

    (1999)
  • C.A. Chambers et al.

    Immunity

    (1997)
  • C.A. Chambers et al.

    Curr. Opin. Immunol.

    (1997)
  • H. Schneider et al.

    Biochem. Biophys. Res. Commun.

    (2000)
  • H. Nishimura et al.

    Science

    (2001)
  • P. Waterhouse et al.

    Science

    (1995)
  • B.M. Carreno et al.

    Annu. Rev. Immunol.

    (2002)
There are more references available in the full text version of this article.

Cited by (241)

View all citing articles on Scopus
1

PRESTO, JST.

View full text