PD-L1 Inhibitor

PD-L1 Inhibitor

PD-L1 checkpoint is operative in peripheral tissues and serves as a negative regulator of T-cells to help control local inflammatory responses and maintain self-tolerance. It is constitutively expressed on a subset of macrophages, but may be rapidly upregulated in a number of different tissue types and by tumors in response to interferon-gamma and other inflammatory mediators.

Many tumor cells constitutively express or are induced by interferon-γ (IFNγ) to express the inhibitory molecule PDL1. Additionally, loss of the tumor suppressor gene phosphatase and tensin homolog (PTEN) increases , T cell inhibition through PD1 is unfavorable; however in the absence of cancer, PD1 is crucial for maintaining immune tolerance. Mice lacking the PD1 gene have a higher prevalence of autoimmune disease. Moreover, blockade of PD1 or knocking out the gene in mice leads to accelerated autoimmune disease.

In the tumor microenvironment, tumor-associated PDL1 increases T cell apoptosis. Experiments comparing mock transfected 624mel cutaneous melanoma cells (624mel/mock) or PDL1-transfected 624mel (624mel/PDL1) cocultured with activated T cells identified that 624mel/PDL1 caused an increased prevalence of T cell-apoptotic death as compared to 624mel/mock. In agreement with tumor cell-expressed PDL1 causing apoptotic death of T cells, the use of blocking PDL1 monoclonal antibodies (mAb) decreased T cell death by >50%. Similar observations were seen in vivo, confirming the role of PD1-PDL1-mediated immunosuppression.

Until recently, the signaling cascade of PD1 was unknown. Only when PD1 is in close proximity to the TCR can PD1 inhibit T cell activation. PD1 has a 95 amino acid intracellular tail that contains two tyrosine molecules: a membraneproximal tyrosine in an immunoreceptor tyrosine-based inhibitory motif (ITIM) and a membrane-distal tyrosine in an immunoreceptor tyrosine-based switch motif (ITSM). Upon ligation with PD1, the two intracellular tyrosines become phosphorylated and recruit src homology 2-domain-containing tyrosine phosphatase-2 (SHP-2). Recruitment of SHP-2 results in dephosphorylation of proximal signaling kinases such as . , CTLA-4 signaling has no effect on PI3K. Blockade of the PI3K/Akt pathway prevents the expression of anti-apoptotic/pro-survival factors such as Bcl-xl, IL-2, and IFNγ.

In addition to PDL1 signaling through PD1 to inhibit T cell activation, PDL1 may reverse signal into tumor cells. P815 tumor cells modified to express PDL1 (P815/PDL1) co-cultured with allospecific T cells are resistant to CTL-mediated lysis, thus suggesting PDL1 serves as a molecular shield. When PDL1 blocking mAb were added to co-culture experiments, P815/PDL1 cells were lysed indicating the role of PDL1 to prevent CTL-mediated lysis. When T cells from PD1 knockout mice were co-cultured with P815/PDL1 tumor cells, P815/PDL1 lysis occurred indicating the need for PD1-PDL1 interactions to form the molecular shield. Furthermore when T cells from PD1 knockout mice were transfected with truncated PD1 lacking its cytoplasmic domain (ΔPD1) or with wildtype PD1 and co-cultured with P815/PDL1 tumor cells, P815/PDL1 tumor cells remained resistant to CTL lysis, indicating PD1 signaling does not contribute to formation of the molecular shield. To determine if PDL1 was reverse signaling into tumor cells, PDL1 lacking its cytoplasmic domain (ΔPDL1) was expressed in P815 tumor cells (P815/ΔPDL1) and P815/ΔPDL1 tumor cells were co-cultured with allospecific T cells. In the absence of PDL1’s cytoplasmic domain, P815/ΔPDL1 tumor cells loose the ability to function as a molecular shield and rendered tumor cells susceptible to CTL-mediated lysis, thus providing further evidence that PDL1 signals bidirectionally. To test whether the extracellular domains of PDL1 conferred resistance to CTL lysis, a chimera protein consisting of the extracellular domains of programmed death ligand 2 (PDL2, also known as CD273 or B7-DC) and the intracellular domain of PDL1 (PDL2/1) was overexpressed in P815 (P815/PDL2/1). P815/PDL2/1 when co-cultured with CD8+ CTL were resistant to lysis, further confirming the intracellular tail of PDL1 is required for molecular shielding.

In addition to serving as a molecular shield, PDL1 may prevent apoptosis of PDL1+ tumor cells following PD1 ligation. P815/PDL1 tumor cells express the death receptor Fas, which causes programmed cell death of tumor cells following ligation with Fas ligand (FasL) or agonistic Fas antibodies (ab). P815/PDL1 tumor cells were incubated with PD1 fused to the extracellular region of Ig (PD1-Fc) or control antibody, followed by incubation with Fas ab. Only when P815/PDL1 was pre-incubated with PD1-Fc were P815/PDL1 tumor cells resistant to Fas-mediatedapoptosis. Although PDL1 signaling through its 30 amino acid cytoplasmic tail is not known, it is speculated that PDL1 serves as an anti-apoptotic receptor.


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