Elsevier

Cellular Immunology

Volume 235, Issue 2, June 2005, Pages 109-116
Cellular Immunology

Alternative splice variants of the human PD-1 gene

https://doi.org/10.1016/j.cellimm.2005.07.007Get rights and content

Abstract

PD-1 is an immunoregulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. We describe four alternatively spliced PD-1 mRNA transcripts (PD-1Δex2, PD-1Δex3, PD-1Δex2,3, and PD-1Δex2,3,4) in addition to the full length isoform. PD-1Δex2 and PD-1Δex3 are generated by alternative splicing where exon 2 (extracellular IgV-like domain) and exon 3 (transmembrane domain) respectively are spliced out. PD-1Δex3 is therefore likely to encode a soluble form of PD-1. PD-1Δex2,3 lacks exon 2 and 3. These three variants have unaffected open reading frames. PD-1Δex2,3,4 lacks exon 2, 3, and 4 (intracellular domain) and contains a premature stop codon in exon 5. Activation of human PBMCs with anti-CD3 + anti-CD28 monoclonal antibodies induces an increased level of each PD-1 transcript. A parallel increase in the expression of PD-1Δex3 and flPD-1 upon activation suggests an important interplay between the putative soluble PD-1 and flPD-1 possibly involved in maintenance of peripheral self-tolerance and prevention of autoimmunity.

Introduction

Programmed death 1 (PD-1) is a type I transmembrane protein, structurally belonging to the CD28/CTLA-4 subfamily of the Ig superfamily [1]. Engagement of PD-1 with its two ligands, PD-L1 (B7-H1) [2], [3] or PD-L2 (B7-DC) [4], [5], opposes lymphocyte activation and attenuates cytokine production by recruitment of SHP-2 to the phosphorylated tyrosine residue in the cytoplasmatic region [6]. In contrast to the T cell-restricted expression of other CD28 superfamily members, PD-1 is induced on T cells, B cells, and monocytes after activation [6], suggesting that PD-1 is a costimulatory receptor family member with unique functions.

PD-1 is considered to play an important inhibitory role in immune responses as PD-1-deficient animals develop autoimmune conditions such as lupus-like glomerulonephritis and proliferative arthritis in mice with a C57BL/6 background [7] and fatal autoantibody-mediated dilated cardiomyopathy in BALB/c mice [8]. Because these phenotypes differ markedly compared from the phenotypes of CTLA-4-deficient mice, this suggests differential regulatory roles for PD-1 and CTLA-4 [9], [10].

An important function of PD-1 in maintaining the peripheral self-tolerance and prevention of autoimmunity is further supported by the reported association between single nucleotide polymorphisms (SNPs) in the human PD-1 gene with susceptibility to systemic lupus erythematosus (SLE) [11], type 1 diabetes [12], rheumatoid arthritis [13], and the presence of nephropathy in SLE patients [14].

Recently, an alternative CTLA-4 mRNA splice variant lacking the entire transmembrane-encoding exon has been identified. This alternative mRNA transcript is expressed in non-stimulated T cells, and encodes a soluble form of CTLA-4 (sCTLA-4). sCTLA-4 is translated, secreted and present in human serum, and represents an intact functional receptor for the B7.1 ligands with downregulatory function like the membrane bound CTLA-4 molecule in vitro [15], [16], [17]. Both serum protein and mRNA levels of sCTLA-4 have been associated with the development of several human autoimmune diseases [16], [18], [19].

The aim of this work was to search for alternative splice variants of PD-1 using cDNA from both non-stimulated and stimulated human peripheral blood mononuclear cells (PBMCs). In the present study, we report the identification of four PD-1 splicing variants, one of which is likely to encode a soluble form of the PD-1 receptor, and show that the mRNA expression of these is increased during activation of PBMCs.

Section snippets

Materials and methods

Preliminary cDNA PCR analyses with primers (forward 5′-GCGGCCAGGATGGTTCTTA-3′ and reverse 5′-TACTCCGTCTGCTCAGGGA-3′ corresponding to positions 125–143 and 793–811, respectively, of the PD-1 mRNA, GenBank Accession No. NM-005018) designed to amplify the entire coding sequence of the human PD-1 mRNA revealed the expression of five different transcripts in stimulated human T cells (Fig. 1). Amplification was carried out in a Peltier Thermal Cycler using the following specific temperature cycling

Identification of alternatively spliced variants of PD-1 mRNA

PCR amplification of the human PD-1 coding sequence revealed the expression by human PBMCs of five splice variants: flPD-1, PD-1Δex2, PD-1Δex3, PD-1Δex2,3, and PD-1Δex2,3,4 (Fig. 2). The flPD-1 transcript showed complete homology with the published membrane PD-1 sequence (GenBank Accession No. NM_005018), while the PD-1Δex2 and PD-1Δex3 transcripts, according to the genomic organization of the human PD-1 gene, are generated by alternative splicing of the PD-1 mRNA, where exon 2 and exon 3 are

Discussion

To compare mRNA levels (obtained by RT-PCR), the expression of a reference gene is commonly assessed in parallel with the expression of the gene of interest. Fundamental to a reference gene is that its expression should be at a constant level among different tissues, at all stages of development and should be unaffected by the experimental treatment [20]. Commonly, a housekeeping gene is selected, but numerous studies have shown that these do not necessarily behave as usable reference genes in

Acknowledgments

This work was supported by grants from a combined grant from The Danish Medical Research Council and the counties of Southern Denmark. The experiments performed comply with the current laws of Denmark.

References (25)

  • T. Okazaki et al.

    PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine

    Proc. Natl. Acad. Sci. USA

    (2001)
  • H. Nishimura et al.

    Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice

    Science

    (2001)
  • Cited by (170)

    • Strategies for developing PD-1 inhibitors and future directions

      2022, Biochemical Pharmacology
      Citation Excerpt :

      Being highly specific and of high affinity against antigens and relatively easy to develop, antibodies are limited by high manufacturing cost, harsh transportation/storage conditions, and inconvenience of administration through injection, low capability to penetrate tissues.6.2 Non-classic-antibody based PD-1/PD-L1 targeting protein drugs. Lillevang and colleagues noted some splicing variants of PD-1 gene, most probably encoding soluble extracellular part of PD-1 [53]. Considering that it functions as equivalent of PD-L1 antibody, soluble PD-1 was explored for its potential as PD-L1 inhibitor [54,55] (Fig. 5A).

    View all citing articles on Scopus
    View full text