PD-1 and its ligands: PD-L1 and PD-L2

The PD-1 gene was first cloned and its nucleotide sequence was reported by Professor Honjo’s group in Japan by a subtractive hybridization technique in their pursuit of a novel protein involved in programmed cell death.7,8 The group used a murine 2B4.11 and LyD9 cell line which had been shown to die by a programmed cell death. PD-1 gene was isolated with subtractive hybridization using cDNA derived from 2B4.11 and LyD9.

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As shown in Figure 1, PD-1 is a type I transmembrane glycoprotein with an estimated molecular weight of 50–55 kDa. Extracellular domain contains an immunoglobulin-like structure showing sequence homologies with those of other immune checkpoint inhibitors, like cytotoxic T-lymphocyte-associated protein 4 (CTLA4). Unlike other checkpoint inhibitors like CTLA4, PD-1 cannot homodimerize, since the membrane proximal cysteine residue does not exist in PD-1.

The PD-1 cytoplasmic domain is thought to transduce the negative signals into the cells through its immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). ITIM and ITSM can bind with Src homology 2 domain-containing phosphatases, Src homology region 2 domain-containing phosphatase (SHP)-1, and SHP-2.

To evaluate the function of tyrosines in ITIM and ITSM of PD-1, Okazaki et al made constructs containing amino acid replacements in tyrosine residues in ITIM and/or ITSM.9 They transfected B-lymphocyte cell lines with each expression vector with PD-1 mutants and found that a tyrosine residue in ITSM but not in ITIM is necessary for the inhibitory signaling cascade from intracellular PD-1. The amino acid sequences around the ITSM of PD-1 were identical between human and mouse, suggesting that structures around the tyrosine in ITSM are important in negative regulation by PD-1. Phosphorylation of tyrosine in ITSM induces the binding of SHP-2 to PD-1, which in turn dephosphorylates and down-modulates the function of ZAP70 in T-lymphocytes (Figure 1).9

The PD-1 is expressed in a quite limited series of cells. PD-1 expression is observed in activated T-lymphocytes and T-cells with chronic stimulations. PD-1 expression is not found in naïve T-lymphocytes. Upon stimulation through T-cell receptor and costimulation, nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) binds with DNA and induces the expression of PD-1 mRNA. IFN-α stimulation exerts IFN regulatory factor 9 binding with the promoter sequence of PD-1 gene and induces the PD-1 mRNA transcription.

In contrast to the limited expression of PD-1, PD-1 ligands – PD-L1 and PD-L2 – are widely expressed. PD-L1 and PD-L2 are type I transmembrane glycoproteins and belong to the B7 family. Other B7 family protein, CD80 and CD86, bind with CD28 and CTLA-4 and share sequence homology with PD-1. Extracellular domains of PD-L1 and PD-L2 have immunoglobulin-like structures. PD-L1 and PD-L2 have 40% amino acid homology between them. The functions of cytoplasmic regions of PD-L1 and PD-L2 are not well known.

PD-L1 is expressed in a wide variety of immune cells including activated T-lymphocytes, B-lymphocytes, dendritic cells, and macrophages. PD-L1 is also expressed in non-lymphoid cells or tissues, such as endothelial cells, lung cancer cells, and breast cancer cells.

The PD-L2 protein is usually expressed in lymphoid tissues; however, its expression is also observed in some cancer cell lines and tissues as discussed later.