Alternative splice variants of the human PD-1 gene
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.
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