Elicit: Tumor Immune Evasion and Pembrolizumab

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Tumor Immune Evasion and Pembrolizumab

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May 5, 2026

How does the PD-1/PD-L1 pathway contribute to tumor immune evasion, and how does pembrolizumab interrupt it?

The PD-1/PD-L1 pathway enables tumors to evade immune destruction by suppressing T cell cytolytic activity, inducing T cell exhaustion, and creating adaptive immune resistance through interferon-driven PD-L1 upregulation, while pembrolizumab interrupts this pathway by blocking PD-1/PD-L1 interaction to restore T cell proliferation and effector function, resulting in intratumoral CD8+ T cell expansion and durable tumor regression in 18-76% of patients depending on tumor type and baseline immune infiltration.

Abstract

The PD-1/PD-L1 pathway enables tumor immune evasion through multiple mechanisms: direct suppression of T cell function by inhibiting cytokine production and cytolytic activity, induction of T cell exhaustion, and adaptive immune resistance whereby interferon production by tumor-infiltrating CD8+ cells upregulates PD-L1 expression. This interaction dampens T cell proliferation and activation, blocks effector functions, and creates an immunosuppressive tumor microenvironment through upregulation of genes involved in extracellular matrix remodeling and metabolic changes favoring immune evasion. Some CD8+ T cells enter an irreversible dysfunctional state that cannot be rescued by pathway blockade.

Pembrolizumab interrupts this pathway by blocking PD-1 interaction with PD-L1 and PD-L2, removing the suppressive effect on T-cell proliferation and immune response. This blockade restores T cell function, leading to increased proliferation of intratumoral CD8+ T cells that directly correlates with tumor size reduction, expansion of memory CD8+ T cells, enhanced effector function marked by granzyme B expression, and development of a more clonal TCR repertoire. Clinical evidence across 10 sources demonstrates objective response rates of 18-76% depending on tumor type and treatment regimen, with responses lasting >1 year in most responders. Pre-existing CD8+ T cells at the invasive tumor margin with close proximity between PD-1 and PD-L1 expressing cells predict response, while higher PD-L1 expression (≥50%) and high tumor mutational burden correlate with improved response rates, though responses occur in PD-L1-negative tumors.

Methods

We analyzed 10 sources from an initial pool of 200, using 9 screening criteria. Each paper was reviewed for 9 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: PD-1/PD-L1 Pathway Focus, Clinical Relevance, Study Design, Relevant Outcomes, Pembrolizumab or PD-1/PD-L1 Inclusion, Beyond Pharmacokinetics, Adequate Sample Size, Human System Validation, Publication Type

n = 200

Papers screened out

n = 190

Papers included for extraction

n = 10

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Paper search

We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.

We ran this query: “How does the PD-1/PD-L1 pathway contribute to tumor immune evasion, and how does pembrolizumab interrupt it?”

The search returned 200 total results from Elicit.

We retrieved 200 papers most relevant to the query for screening.

Screening

We screened in sources based on their abstracts that met these criteria:

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

Extract study type and basic methodology for research investigating PD-1/PD-L1 pathway immune evasion mechanisms and pembrolizumab interruption, including:

Extract specific cancer types and patient populations studied in relation to PD-1/PD-L1 immune evasion and pembrolizumab treatment, including:

Extract all described mechanisms by which the PD-1/PD-L1 pathway contributes to tumor immune evasion, including:

Extract specific mechanisms by which pembrolizumab (and other anti-PD-1/PD-L1 antibodies) interrupt the PD-1/PD-L1 immune evasion pathway, including:

Extract detailed changes in immune cell populations and function during pembrolizumab treatment that demonstrate PD-1/PD-L1 pathway interruption, including:

Extract biomarker data that demonstrates PD-1/PD-L1 pathway activity and its interruption by pembrolizumab, including:

Extract clinical outcome data that serves as evidence of successful PD-1/PD-L1 pathway interruption by pembrolizumab, including:

Extract factors that predict response or resistance to pembrolizumab treatment in the context of PD-1/PD-L1 pathway interruption, including:

Extract safety and toxicity data related to pembrolizumab treatment and PD-1/PD-L1 pathway blockade, including:

Results

Characteristics of Included Studies

The review included 10 sources published between 2012 and 2020, comprising 5 primary clinical studies, 4 review articles, and 1 literature review. Four sources were available only as abstracts.

Study

Full text retrieved?

Study Type

Cancer Types

Sample Size

Key Methods

A. Ribas et al., 2016

No

Observational study

Melanoma

102 tumor biopsies from 53 patients

Multicolor flow cytometry with computational approaches

J. McDermott & A. Jimeno, 2015

No

Review

Melanoma, non-small cell lung cancer

N/A

Literature review of preclinical studies and early clinical trials

K. Mahoney et al., 2015

Yes

Review of clinical trials

Melanoma

10-418 patients across multiple trials

Review of clinical trial data and abstracts

J. Gong et al., 2018

Yes

Literature review

Melanoma, NSCLC, urothelial carcinoma, HNSCC, classical Hodgkin lymphoma, MSI-H/dMMR solid tumors

N/A

MEDLINE and manual literature search

P. Tumeh et al., 2014

Yes

Clinical trial and mechanistic study

Metastatic melanoma

46 patients with metastatic melanoma; validation cohort of 16 patients

Quantitative immunohistochemistry, multiplex immunofluorescence, next-generation TCR sequencing

J. Brahmer et al., 2012

Yes

Phase 1 clinical trial (anti-PD-L1)

Non-small-cell lung cancer, melanoma, colorectal cancer, renal-cell cancer, ovarian cancer, pancreatic cancer, gastric cancer, breast cancer

207 patients

Intravenous anti-PD-L1 antibody administration, immunohistochemistry, flow cytometry

S. Topalian et al., 2012

Yes

Phase 1 clinical trial (anti-PD-1)

Advanced melanoma, non-small-cell lung cancer, castration-resistant prostate cancer, renal-cell cancer, colorectal cancer

296 patients

Anti-PD-1 antibody administration, immunohistochemistry, flow cytometry

Z. Xu-Monette et al., 2017

No

Review

Hodgkin lymphoma, melanoma, advanced cancers

N/A

Review of functional and clinical studies

J. Miguel et al., 2015

No

Phase 1 clinical trial

Multiple myeloma (relapsed/refractory)

34 patients

Modified 3+3 dose-escalation design, pembrolizumab with lenalidomide and dexamethasone

K. Hudson et al., 2020

Yes

Review

Melanoma, breast, gastric, ovarian, liver, kidney, pancreatic, bladder, NSCLC, urothelial, Merkel cell carcinomas

N/A

Review of oncology models and clinical data

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Pageof

Disease stages across studies were predominantly advanced or metastatic. Patient populations included heavily pretreated individuals, with 47% receiving at least three prior regimens in one study, and patients who had failed at least two prior therapies in another.

Mechanisms of PD-1/PD-L1 Pathway in Tumor Immune Evasion

Suppression of T Cell Function

The PD-1/PD-L1 pathway serves as a critical immune checkpoint that tumors exploit to evade host immunity. PD-1/PD-L1 interaction suppresses immune responses by reducing T-lymphocyte function and inhibiting cytokine production and cytolytic activity of PD-1-positive, tumor-infiltrating CD4+ and CD8+ T cells. This interaction dampens T cell function and proliferation, blocks effector functions, and reduces T cell killing capacity. At the molecular level, PD-1 signaling inhibits glucose metabolism in T lymphocytes and involves inhibitory signaling downstream of the T-cell receptor, including recruitment of SHP1/2 phosphatases that dephosphorylate signaling molecules necessary for T cell activation.

Adaptive Immune Resistance

A key mechanism of immune evasion involves adaptive immune resistance, where PD-L1 expression is upregulated in response to immune attack. Production of interferons by tumor-infiltrating CD8+ cells can induce PD-L1 expression, creating a negative feedback loop. This upregulation of PD-L1 in response to interferon-induced JAK-STAT signaling, along with expression of indoleamine 2,3-dioxygenase (IDO), represents an adaptive mechanism by which tumors respond to immune pressure. PD-1 expression is induced by T-cell activation and can lead to an “exhausted” T-cell phenotype if the immune response is unsuccessful, with some CD8+ T cells entering an irreversible dysfunctional state that cannot be rescued by PD-1/PD-L1 blockade.

Tumor Microenvironment Effects

Tumors create an immunosuppressive microenvironment through multiple mechanisms. PD-L1 is abnormally expressed by tumor cells and lymphocytes in the tumor microenvironment, and its overexpression enhances tumorigenesis and makes tumor cells less susceptible to T-cell mediated lysis. The tumor microenvironment undergoes changes including upregulation of genes involved in cell adhesion, extracellular matrix remodeling, mesenchymal transition, angiogenesis, and wound healing. Additionally, increased lactate production and hypoxia favor tumor growth and immune evasion. PD-L1 expression on both cancer cells and myeloid-derived cells contributes to the creation of a suppressive environment.

Mechanisms by Which Pembrolizumab Interrupts the PD-1/PD-L1 Pathway

Antibody-Mediated Blockade

Pembrolizumab is a highly selective, humanized IgG4 monoclonal antibody designed to block the interaction of PD-1 with its ligands PD-L1 and PD-L2. By preventing PD-1 from binding to PD-L1, pembrolizumab removes the suppressive effect on T-cell proliferation and immune response. This blockade prevents PD-L1 from inhibiting T cell function and promotes immune responses against tumors. The antibody achieves high receptor occupancy, with median PD-L1 receptor occupancy exceeding 65% at doses of 1-10 mg/kg on CD3+ peripheral-blood mononuclear cells.

Restoration of Immune Cell Function

Following PD-1 blockade, significant changes occur in immune cell populations and function. CD8+ T cells show increased proliferation within tumors, with responding patients demonstrating increased Ki67 positivity indicating active proliferation and elevated granzyme B expression indicating enhanced effector function. Memory CD8+ T cells were the most prominent phenotype that expanded intratumorally on therapy, representing the major T-cell subset expanded in patients with a response to therapy. The frequency of intratumoral B cells and monocytic myeloid-derived suppressor cells significantly increased in patients’ biopsies taken on treatment.

Treatment leads to proliferation of intratumoral CD8+ T cells that directly correlates with radiographic reduction in tumor size. There is significant expansion of T cell clones post-treatment in responders, with development of a more clonal TCR repertoire within the T cell population directed against the tumor. Expression of pSTAT1, a marker of interferon signaling, increases in responders, indicating enhanced immune activation.

Spatial and Temporal Dynamics

Pre-treatment samples from responding patients showed higher numbers of CD8-, PD-1-, and PD-L1-expressing cells at the invasive tumor margin and inside tumors, with close proximity between PD-1 and PD-L1 cells at the invasive tumor margin. These pre-existing CD8+ T cells distinctly located at the invasive tumor margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and predict response to therapy. Following treatment, increased CD8+ T cell density occurs at the invasive margin and inside tumors, demonstrating changes in immune cell trafficking and infiltration.

Clinical Evidence of Pathway Interruption

Response Rates Across Cancer Types

Clinical trials demonstrated substantial objective response rates across multiple cancer types, providing evidence of successful PD-1/PD-L1 pathway interruption. In melanoma, response rates ranged from 26-45.2%, with one study reporting 28% objective response rate and another reporting 41% overall response with 9% complete responses. For non-small-cell lung cancer, objective response rates were 18-21%. Renal-cell cancer showed 27% response rate, while pembrolizumab in combination with lenalidomide and dexamethasone for relapsed/refractory multiple myeloma achieved a 76% objective response rate.

The anti-PD-L1 antibody trial reported objective responses in 9 of 52 patients with melanoma, 2 of 17 with renal-cell cancer, 5 of 49 with non-small-cell lung cancer, and 1 of 17 with ovarian cancer. Disease control rates showed prolonged stabilization in 12-41% of patients at 24 weeks.

Duration and Durability of Responses

Responses to PD-1/PD-L1 blockade demonstrated remarkable durability. In the anti-PD-1 trial, 20 of 31 responses lasted 1 year or more in patients with 1 year or more of follow-up. Similarly, the anti-PD-L1 study found that responses lasted for 1 year or more in 8 of 16 patients with adequate follow-up. Median progression-free survival exceeded 7 months in one pembrolizumab melanoma study, with overall survival not reached at the time of reporting. In gastric cancer, the median duration of response was 8.1 months, while overall survival reached 25.9 months in one KEYNOTE-001 cohort.

Biomarker Correlations

PD-L1 expression emerged as a predictive biomarker, though with limitations. Higher PD-L1 expression levels were associated with better response rates to pembrolizumab. In one study, 36% of patients with PD-L1-positive tumors had an objective response, compared to none with PD-L1-negative tumors (P=0.006). Pembrolizumab showed improved response rates and progression-free survival in PD-L1-positive patients, with 45.2% objective response rate in patients with PD-L1 ≥50%. However, PD-L1 expression proved inadequate as a definitive biomarker, as responses occurred in some PD-L1-negative tumors.

Beyond PD-L1 expression, baseline CD8+ T cell infiltration at the invasive tumor margin predicted response. Responding patients had higher numbers of CD8+, PD-1+, and PD-L1+ cells at the invasive margin and inside tumors, with close proximity between PD-1 and PD-L1 expressing cells. A more clonal TCR repertoire correlated with clinical response, and higher pSTAT1 expression was observed in responding patients both before and during treatment. High tumor mutational burden and POLE mutations predicted benefit from PD-1 blockade, with MSI-H or dMMR status associated with better responses.

Safety Profile

Immune-Related Adverse Events

PD-1/PD-L1 blockade demonstrated a more favorable safety profile compared to previous immunotherapies. Grade 3 or 4 drug-related adverse events occurred in 14% of anti-PD-1-treated patients, 9% of anti-PD-L1-treated patients, and 13% in one pembrolizumab melanoma study. In the multiple myeloma combination trial, 58% experienced grade 3/4 treatment-related adverse events, though no treatment discontinuation for toxicity occurred.

Common immune-related adverse events included pneumonitis (3%), with 1% experiencing Grade 3 or 4 severity. Other immune-related toxicities encompassed vitiligo, colitis, hepatitis, hypophysitis, and thyroiditis. In the anti-PD-L1 trial, 39% of patients experienced immune-related events such as rash, hypothyroidism, hepatitis, sarcoidosis, endophthalmitis, diabetes mellitus, and myasthenia gravis. The multiple myeloma study reported thrombocytopenia (47%), neutropenia (41%), fatigue (29%), anemia, hyperglycemia, and muscle spasms (23% each) as the most frequent treatment-related adverse events.

Three deaths from pulmonary toxicity occurred in the anti-PD-1 trial. Serious adverse events related to treatment occurred in 5% of anti-PD-L1-treated patients. Treatment discontinuation rates due to adverse events were 5% in the anti-PD-1 trial and 11% (6% treatment-related) in the anti-PD-L1 study.

Management Strategies

Management of immune-related toxicities involved glucocorticoids and replacement therapy. Events such as hypothyroidism and adrenal insufficiency were managed with replacement therapy, while hepatic or gastrointestinal events required treatment interruption and glucocorticoids. Education regarding recognition and management of immune-related effects proved essential for maximizing clinical benefit.

Synthesis

The body of evidence demonstrates a coherent mechanistic and clinical narrative for how pembrolizumab interrupts the PD-1/PD-L1 immune evasion pathway. The mechanistic studies provide direct cellular and molecular evidence linking antibody-mediated pathway blockade to restoration of anti-tumor immunity, while clinical trials confirm these mechanisms translate into durable clinical responses.

The apparent variability in response rates across studies (18-76%) reflects genuine biological heterogeneity rather than conflicting findings. Response rates correlate with specific tumor characteristics: Hodgkin lymphoma and melanoma show higher responses due to high PD-L1/L2 expression and high tumor mutational burden respectively, while most advanced cancers show approximately 20% response rates. The 76% response rate in multiple myeloma occurred with combination therapy (pembrolizumab plus lenalidomide and dexamethasone), not monotherapy, explaining the higher efficacy. Similarly, combination pembrolizumab with chemotherapy achieved 55% response rate in NSCLC, demonstrating that combination approaches enhance efficacy beyond single-agent blockade.

PD-L1 expression serves as an imperfect but useful biomarker. While PD-L1-positive tumors show higher response rates (36-45.2%), responses occur in PD-L1-negative tumors, suggesting PD-L1 expression identifies a responsive subset but does not fully explain all responses. This paradox resolves when considering that pre-existing CD8+ T cells at the invasive margin—regardless of PD-L1 status—predict response, indicating that both adaptive immune resistance (PD-L1 upregulation) and pre-existing T cell infiltration contribute to responsiveness. The close proximity between PD-1 and PD-L1 expressing cells creates a microenvironment where blockade can immediately restore T cell function, explaining why spatial distribution matters as much as expression levels.

The durability of responses (>1 year in most responders) contrasts with the CD8+ T cell exhaustion described in the mechanistic studies, suggesting pathway blockade catches tumors before irreversible T cell dysfunction occurs. The observation that some CD8+ T cells enter an irreversible dysfunctional state explains primary resistance in non-responders, while the expansion of memory CD8+ T cells in responders provides a mechanism for durable immunity. The more clonal TCR repertoire in responders indicates that effective therapy amplifies pre-existing tumor-specific T cell clones rather than generating entirely new responses.

References

J. McDermott, A. Jimeno\ (2015).Pembrolizumab: PD-1 inhibition as a therapeutic strategy in cancer. Drugs of Today

K. Mahoney, G. Freeman, D. McDermott\ (2015).The Next Immune-Checkpoint Inhibitors: PD-1/PD-L1 Blockade in Melanoma. Clinical Therapeutics

J. Gong, Alexander Chehrazi-Raffle, S. Reddi, R. Salgia\ (2018).Development of PD-1 and PD-L1 inhibitors as a form of cancer immunotherapy: a comprehensive review of registration trials and future considerations. Journal of Immunotherapy for Cancer

P. Tumeh, C. Harview, J. Yearley, I. Shintaku, E. Taylor, and 21 more\ (2014).PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature

J. Brahmer, S. Tykodi, L. Chow, W. Hwu, S. Topalian, and 21 more\ (2012).Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. New England Journal of Medicine

S. Topalian, F. Hodi, J. Brahmer, S. Gettinger, David C. Smith, and 25 more\ (2012).Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. New England Journal of Medicine

Z. Xu-Monette, Mingzhi Zhang, Jianyong Li, K. Young\ (2017).PD-1/PD-L1 Blockade: Have We Found the Key to Unleash the Antitumor Immune Response?. Frontiers in Immunology

J. Miguel, M. Mateos, J. Shah, E. Ocio, P. Rodríguez-Otero, and 8 more\ (2015).Pembrolizumab in Combination with Lenalidomide and Low-Dose Dexamethasone for Relapsed/Refractory Multiple Myeloma (RRMM): Keynote-023. Blood

K. Hudson, N. Cross, N. Jordan-Mahy, R. Leyland\ (2020).The Extrinsic and Intrinsic Roles of PD-L1 and Its Receptor PD-1: Implications for Immunotherapy Treatment. Frontiers in Immunology

A. Ribas, D. Shin, J. Zaretsky, J. Frederiksen, A. Cornish, and 12 more\ (2016).PD-1 Blockade Expands Intratumoral Memory T Cells. Cancer immunology research

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The Extrinsic and Intrinsic Roles of PD-L1 and Its Receptor PD-1: Implications for Immunotherapy Treatment

K. Hudson, N. Cross, N. Jordan-Mahy, R. Leyland

Frontiers in Immunology·

2020·

188 citations

SourceDOI

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Study Design

Not applicable (the paper is a review and does not describe a specific study design or methodology)

Cancer Types

- Primary cancer types: Melanoma, breast, gastric, ovarian, liver, kidney, pancreatic, bladder, non-small-cell lung cancer (NSCLC), urothelial carcinomas, Merkel cell carcinomas - Disease stage: Advanced, metastatic - Prior treatment status: Not specified - Cancer-specific characteristics: PD-L1-positive tumors, high mutational burden

PD-1/PD-L1 Immune Evasion Mechanisms

- PD-1/PD-L1 interaction suppresses immune responses by inhibiting cytotoxic T lymphocyte effector function. - Effects on T cell function include inhibition of activation, cytokine production, and promotion of anergy or apoptosis. - Role in adaptive immune resistance involves promoting T cell exhaustion and immune evasion. - Impact on other immune cell types includes potential effects on B cells and myeloid cells, although specific mechanisms are not detailed. - Molecular pathways involved include the recruitment of SHP1/2, which dephosphorylates signaling molecules necessary for T cell activation. - Tumor microenvironment changes include increased lactate production and hypoxia, which favor tumor growth and immune evasion. - Pembrolizumab interrupts this pathway by blocking PD-1, preventing PD-L1 from inhibiting T cell function and promoting immune responses against tumors.

Pembrolizumab Interruption Mechanisms

Pembrolizumab interrupts the PD-1/PD-L1 immune evasion pathway by blocking the interaction between PD-1 and PD-L1, restoring T cell function and activation, increasing the proliferation of tumor-infiltrating lymphocytes, and enhancing the T cell receptor repertoire. This leads to the reversal of immune suppression mechanisms and promotes durable anti-tumor immune responses. The paper does not provide specific molecular or cellular changes or differences between anti-PD-1 vs anti-PD-L1 approaches.

Immune Cell Changes

- Increased proliferation of tumor-infiltrating lymphocytes - Development of a more clonal TCR repertoire within the T cell population directed against the tumor - No specific details on changes in CD8+ and CD4+ T cell numbers, phenotypes, and function - No specific details on memory T cell expansion and activation status - No specific details on changes in regulatory T cells, B cells, NK cells, myeloid cells - No specific functional assays showing restored immune activity

Biomarker Evidence

- PD-L1 expression levels: Elevated expression correlates with advanced disease and poor prognosis in various cancers. - Spatial distribution: Not specifically detailed in the paper. - Other immune checkpoint molecules: CTLA-4 mentioned but no detailed expression data. - Inflammatory markers and cytokine profiles: Interferon gamma upregulates PD-L1. - Tumor mutational burden: High mutational burden is a criterion for PD-1/PD-L1-targeted therapy eligibility. - Molecular signatures: Not specifically detailed in the paper.

Clinical Evidence

- Objective response rates: ∼15% in NSCLC, ∼20% in urothelial carcinomas, ∼30% in Merkel cell carcinomas. - Duration of responses and progression-free survival: Not specifically mentioned for pembrolizumab. - Overall survival data: Not specifically mentioned for pembrolizumab. - Disease control rates and stable disease: Not specifically mentioned for pembrolizumab. - Time to response and durability metrics: Not specifically mentioned for pembrolizumab. - Correlation between clinical outcomes and mechanistic evidence of pathway interruption: PD-1/PD-L1-targeted therapies reactivate the immune system to induce immune-mediated tumor eradication.

Predictive Factors

- Baseline PD-L1 expression as predictor of response: PD-L1-positive tumors are considered eligible for PD-1/PD-L1-targeted therapy. - Immune cell infiltration patterns predicting response: Lack of immune stimulatory cells can lead to unresponsiveness despite high PD-L1 expression. - Genetic or molecular features associated with response/resistance: High mutational burden and PD-L1 promoter methylation status are relevant. - Patient characteristics affecting pathway blockade efficacy: Presence of circulating PD-1 positive/CD8+ T cells is relevant for eligibility. - Mechanisms of primary or acquired resistance to PD-1/PD-L1 blockade: Primary resistance includes inadequate T cell infiltration and impaired IFNγR signaling; acquired resistance involves loss of T cell function and disrupted antigen processing. - Biomarkers that correlate with successful pathway interruption: PD-L1 promoter methylation status is a biomarker.

Safety Profile

Not mentioned (the paper does not provide specific safety and toxicity data related to pembrolizumab treatment and PD-1/PD-L1 pathway blockade)

Programmed death-ligand 1 (PD-L1) is an immune checkpoint inhibitor that binds to its receptor PD-1 expressed by T cells and other immune cells to regulate immune responses; ultimately preventing exacerbated activation and autoimmunity. Many tumors exploit this mechanism by overexpressing PD-L1 which often correlates with poor prognosis. Some tumors have also recently been shown to express PD-1. On tumors, PD-L1 binding to PD-1 on immune cells promotes immune evasion and tumor progression, primarily by inhibition of cytotoxic T lymphocyte effector function. PD-1/PD-L1-targeted therapy has revolutionized the cancer therapy landscape and has become the first-line treatment for some cancers, due to their ability to promote durable anti-tumor immune responses in select patients with advanced cancers. Despite this clinical success, some patients have shown to be unresponsive, hyperprogressive or develop resistance to PD-1/PD-L1-targeted therapy. The exact mechanisms for this are still unclear. This review will discuss the current status of PD-1/PD-L1-targeted therapy, oncogenic expression of PD-L1, the new and emerging tumor-intrinisic roles of PD-L1 and its receptor PD-1 and how they may contribute to tumor progression and immunotherapy responses as shown in different oncology models.

INTRODUCTION

Cancer immunotherapies work to re-establish immune-mediated tumor eradication (1). Despite the advancements in immunotherapy over the past decade, the interactions of cells within the tumor microenvironment continue to mediate immune evasion and tumor progression (2). The tumor consists of extracellular matrix components and diverse cell populations such as T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells, fibroblasts and endothelial cells (3). Although immune cells such as NK, CD8+, and CD4+ T cells which migrate to the tumor display anti-tumor activity, over time the tumor microenvironment becomes immunosuppressive, favoring the emergence of tumor promoting cells such as regulatory T-cells (T-regs), myeloid derived suppressor cells (MDSCs) and M2 macrophages (1). This is known as the phenomenon cancer immuno-editing which involves three phases: elimination, equilibrium, and escape (Figure 1 ). Besides tumor cells acquiring the ability to escape immune recognition, they also employ immune-inhibitory mechanisms to evade the immune systems defenses (4). Immuno-editing FIGURE 1 | The process of cancer immuno-editing: elimination, equilibrium and escape. Normal healthy cells transform into tumor cells through acquiring mutations that allows for uncontrolled growth of the cell. In the elimination phase, immune cells can recognize and eliminate tumor cells by inducing apoptosis via granule and/or receptor-mediated mechanisms. Some tumor cells avoid immune destruction and enter dynamic equilibrium with immune cells whereby the immune system elicits a potent enough response to contain the tumor cells but not enough to eradicate them. During this phase tumor cells develop increased genetic instability and undergo immune selection, whereby the immune cells eliminate those tumor cells susceptible to immune-mediated killing, whilst selecting those tumor cells with mechanisms to evade the immune system. These selected tumor cells can now proliferate freely and expand leading to immune escape. PD-L1 expression is one of the many mechanisms employed by tumors to facilitate immune evasion and tumor development. Tumor-intrinsic mutations can induce PD-L1 expression and influence tumor cell-immune cell interactions within the tumor microenvironment to favor tumor growth (as discussed later in this review). Interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), interleukin 10 (IL-10), interleukin 6 (IL-6), transforming growth factor beta (TGF-β), hypoxic inducible factor 1/2 alpha (HIF-1/2α), vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs).

occurs in patients with advanced cancers and this process can be influenced by therapies targeting immuno-inhibitory proteins (5, 6). One such mechanism is targeting of programmed deathligand 1 (PD-L1) (7, 8).

Abbreviations: NK, Natural killer cells; Tregs, Regulatory T cells; MDSCs, Myeloid-derived suppressor cells; PD-L1, Programmed death-ligand 1; PD-1, Programmed death 1; FDA, USA Food and Drug Administration; NSCLC, Non-small cell lung cancer; CTLA-4, Cytotoxic T lymphocyte antigen-4; TCR, T cell receptor; APCs, Antigen presenting cells; PD-L2, Programmed deathligand 2; SHP1/2, Src-homology 2 domain-containing phosphatase 1/2; miRNA, microRNA; 3'UTR, 3' untranslated region; GSK3β, Glycogen synthase kinase 3β; S/T, Serine/Threonine; ccRCC, Clear cell renal cell carcinoma; ER, Endoplasmic reticulum; 2D, Two-dimensional; CSC, Cancer stem cell; EMT, Epithelial to mesenchymal transition; 3D, Three-dimensional; MDR1/P-gp, Multidrug resistant 1/P-glycoprotein.

PD-L1, also known as B7-H1 and CD274, is an immune checkpoint inhibitor that binds to its receptor PD-1 expressed by T cells, B cells, dendritic cells, and monocytes (7). PD-L1 is expressed by T cells, B cells, NK cells, dendritic cells, macrophages, MDSCs, and many other cell types such as epithelial and endothelial cells (9, 10). The PD-1/PD-L1 signaling axis regulates immune responses to prevent exacerbated activation and autoimmunity (8, 10). Many tumors exploit this mechanism by overexpressing PD-L1 (11-13). Recently, tumors have also been shown to express PD-1 (14, 15). PD-L1 binding to PD-1 on immune cells induces inhibitory responses which in turn can promotes immune evasion and tumor progression.

Elevated expression of PD-L1 on tumors has been reported to strongly correlate with advanced disease state and unfavorable prognosis in melanoma (11), breast (13), gastric (16), ovarian (12), liver (17), kidney (18), pancreatic (19), and bladder (20) cancer. Immunotherapies targeting the PD-1/PD-L1 signaling axis have become the first-line treatment for some cancers due to their ability to promote durable anti-tumor immune responses in select patients with advanced cancers (21-23), leading to their approval by the USA Food and Drug Administration (FDA). Although PD-1/PD-L1-targeted therapies have demonstrated clinical benefits across a broad range of cancers, some patients are unresponsive, hyperprogressive, or develop resistance (24). The objective response rate of anti-PD-L1 monoclonal antibodies alone is ∼15% in non-small-cell lung cancer (NSCLC) (21, 25), ∼20% in urothelial carcinomas (23, 26) and ∼30% in Merkel cell carcinomas (27, 28) . Consequently, novel therapeutic strategies are required to enhance patient response rates through combining PD-1/PD-L1-targeted therapy with other immune approaches, small molecule inhibitors, chemotherapy, or other modalities. Patients considered eligible for PD-1/PD-L1-targeted therapy are those that present with PD-L1-positive tumors, circulating PD-1 positive/CD8+ T cells and/or tumors with high mutational burden (23, 29). However, patient tumors that have shown to lack PD-L1 have also responded positively to PD-1/PD-L1-targeted therapy (10, 30), suggesting that either blocking PD-L1 expression on tumors is not required for anti-tumor responses and inhibition of PD-L1 on immune cells alone may be sufficient or that more sensitive approaches to detecting PD-L1 expression on tumors is required. Conversely, some tumors with high PD-L1 expression have shown to be unresponsive to PD-1/PD-L1-targeted therapy (31), likely due to the lack of immune stimulatory cells present in the tumor microenvironment to elicit an effective anti-tumor immune response, but reasons for this remain to be fully elucidated. However, patients that are intrinsically unresponsive to PD-1/PD-L1-targeted therapy can also demonstrate "primary resistance" whereby their tumors display inadequate T cell infiltration, T cell exclusion, impaired IFNγR signaling, and/or local immune suppression (2, 31) . Patients that initially respond to PD-1/PD-L1-targeted therapy also show "acquired resistance" whereby their tumors display loss of T cell function and/or disrupted antigen processing and presentation (31, 32). In recent years, approximately 10% of cancer patients have experienced pseudoprogression in response to PD-1/PD-L1-targeted therapy, whereby patients temporally exhibit rapid progression of their condition before responding successfully to treatment (33). On the other hand, some patients have experienced hyperprogression in response to PD-1/PD-L1targeted therapy, which is characterized by rapid deterioration of their condition upon initialization of treatment without a successful response; giving patients <2 months to live from onset (33). The reasons for pseudoprogression and hyperprogression in response to PD-1/PD-L1-targeted therapy remain speculative and need to be explored. It is also important for clinicians to be able to distinguish between the two types of responses to inform patient selection for therapy. Further insight into the role of PD-L1 and PD-1 in the tumor microenvironment could allow the identification of more appropriate biomarkers predictive of clinical efficacy to PD-1/PD-L1-targeted therapy necessary to ensure patients receive the maximum clinical benefit whilst avoid immune-related adverse effects (24, 29), pseudoprogression and hyperprogression (33).

IMMUNE CHECKPOINT SIGNALING IN CANCER

Immune checkpoint molecules expressed on T cells such as cytotoxic T lymphocyte antigen 4 (CTLA-4) and PD-1 regulate immune responses by dampening T cell activation to prevent exacerbated activation and autoimmunity (4, 34) . During cancer development anti-tumor immunity is suppressed and immunotherapies targeting CTLA-4 and PD-1 signaling axes have been developed to reactivate T cells to induce immunemediated tumor eradication (35). Normally, T cell activation requires two signals. Signal one is the T cell receptor (TCR) recognizing and binding to an antigenic peptide presented on an MHC molecule on antigen presenting cells (APCs) or tumor cells. The second is a co-stimulatory signal through CD28 on T cells binding to CD80/CD86 on APCs. CTLA-4 prevents T cell activation by competing with the co-stimulatory molecule CD28 for the CD80/CD86 on APCs (36). Ipilimumab is a CTLA-4 inhibitor approved for the treatment of advanced or unresectable melanoma (36, 37). Unlike CTLA-4 expression restricted to T cells, PD-1 is expressed by activated T cells, B cells and monocytes. PD-1 binds to its two ligands, PD-L1 and PD-L2, expressed primarily by APCs and tumor cells (7). The function of PD-L2 however is not as widely known as PD-L1 (38). Activated PD-1 on T cells through PD-L1 binding counteracts the downstream signaling of the TCR and CD28 co-stimulatory signal by phosphorylating the cytoplasmic immunoreceptor tyrosine-based switch motif leading to the recruitment of Src homology region 2 domain containing phosphatases 1 and 2 (SHP1/2) and slam-associated protein (38, 39). SHP1/2 dephosphorylate the TCR and CD28 proximal signaling molecules including ZAP70 and PI3K, respectively, inhibiting T cell activation, cytokine production and promoting pro-apoptotic molecule expression, ultimately resulting in T cell anergy or apoptosis (2) (Figure 2 ). The overexpression of PD-L1 in many cancers causes functionally exhausted and unresponsive T cells, promoting immune evasion and tumor progression (7, 11-13) and abrogating PD-L1 expression on tumor cells can enhance sensitivity to T cell killing (40, 41). Signaling of PD-L1 intrinsically within immune cells has not been as well studied as PD-1, however macrophage treatment with anti-PD-L1 antibodies has been shown to upregulate mTOR pathway activity, and RNA-Seq analysis revealed upregulation of multiple macrophage inflammatory pathways (42).

PD-1/PD-L1-targeted therapies yield remarkable anti-tumor immune responses with limited side effects in select patients with advanced cancers (24). They have shown to increase the proliferation of tumor-infiltrating lymphocytes and develop a more clonal TCR repertoire within the T cell population directed against the tumor (32). Currently there are six approved PD-1/PD-L1-targeted therapies for the treatment of multiple cancers as single agents (Table 1 ); some of which have gained accelerated approval and emerged as front-line treatments for some cancers (35). In 2014, the FDA approved the first anti-PD-1 monoclonal antibody, Nivolumab, for treatment of patients with unresectable or metastatic melanoma and disease progression following Ipilimumab (anti-CTLA-4), based on the CA209037 clinical trial data where Nivolumab alone achieved 31.7% objective response rate (43). Subsequently, Nivolumab was approved for the first-line treatment of metastatic melanoma (44) and second-line treatment for NSCLC (45) and renal cell carcinoma (46) following successful phase I-III clinical trials. Nivolumab has also been approved for classic Hodgkin lymphoma (47), head and neck squamous cell carcinoma (48), bladder cancer (49), and colorectal cancer with microsatellite instability or mismatch repair deficiency (50). Similarly, the anti-PD-1 monoclonal antibody Pembrolizumab is approved for the first-line treatment of metastatic melanoma (51) and NSCLC (25) and second-line treatment for metastatic head and neck squamous cell carcinoma (52) and refractory classical Hodgkin's lymphoma (53). In addition, it has also been approved for gastric/gastroesophageal junction adenocarcinomas (16), cervical cancer (54), and primary mediastinal large B-cell lymphoma (55). Pembrolizumab is also the first therapy to be approved for the treatment of all solid tumors with high mutational burden (56). More recently, Nivolumab and Pembrolizumab have gained accelerated approval for many more cancers (Table 1 ). Cemiplimab represents a newly approved anti-PD-1 monoclonal antibody for the treatment of metastatic cutaneous squamous cell carcinoma (57). Atezolizumab was the first anti-PD-L1 monoclonal antibody to be approved for treatment of advanced NSCLC and metastatic urothelial carcinoma. Atezolizumab promoted a tolerable and durable objective response rate of 23% and 15% in NSCLC (21, 58) and in metastatic urothelial carcinoma, respectively (22, 59), whilst anti-PD-L1 monoclonal antibodies Avelumab and Durvalumab are approved for the treatment of Merkel cell carcinoma (27, 28) and NSCLC (60), respectively and are both approved for treatment of metastatic urothelial carcinoma (26, 61). Furthermore, these PD-1/PD-L1-targeted therapies are also being investigated for treatment of colorectal, bladder, prostate and breast cancer as well as hematological malignancies and have shown promising results in the early clinical trials.

As single agents, immunotherapies targeting the PD-1/PD-L1 signaling axis have demonstrated unprecedented capabilities to elicit anti-tumor immune responses in some patients with advanced cancers (21-23), however the fact remains; there are a large percentage of non-responders or initial responders that acquire resistance (29, 68). Most research associated with PD-L1 and PD-1 has been focused on their extrinsic role to inhibit the immune system, but more recently a tumor-intrinsic role of PD-L1 and PD-1 is emerging in some cancer types; however these roles remain to be fully characterized in all cancers. Important questions to be addressed are the contribution of tumorigenic expression of PD-L1 and PD-1 to intrinsic signaling, whether monoclonal antibodies targeting the PD-1/PD-L1 signaling axis work sufficiently to block this new and emerging role of PD-L1 and PD-1 and whether the intrinsic roles of these proteins are contributing significantly to resistance, relapse to treatment, and hyperprogressive responses in patients.

MECHANISMS AFFECTING PD-L1 EXPRESSION IN TUMORS

The tumor-intrinsic PD-L1 pathway is aberrantly activated in many cancers (11-13, 69). There are several intrinsic and extrinsic mechanisms responsible for PD-L1 regulation in tumor cells, including genetic alterations, epigenetic modifications, oncogenic and tumor suppressor signals, inflammatory cytokines and other factors (Figure 3 ) (68-80).

Genetic Aberrations of PD-L1

Several tumors harbor genetic aberrations of the chromosome 9p24.1 which CD274, the gene for PD-L1, resides ultimately affecting the expression of PD-L1 (72, 81,82). Increased copy number alterations on chromosome 9p correlates directly with increased PD-L1 expression (72) and frequently occurs in primary mediastinal B-cell lymphoma (63%) (83), classical Hodgkin lymphoma (40%) (47), triple-negative breast cancer (29%) (84), and soft tissue carcinomas (21.1%) (85). A recent study analyzing 9,771 tumor samples from 22 cancer types revealed a high frequency of copy number gains in bladder, cervical, colorectal, ovarian, and head and neck cancer (more than 15% of tumors), but only a low frequency in pancreatic, renal cell, and papillary thyroid carcinoma (<5% of tumors) (72).

In addition, copy number gains are also less frequently observed in gastric cancer (15%) (86), NSCLC (5.3%) (87), small cell lung cancer (1.9%) (88), and diffuse large B-cell lymphoma (3%) (89). PD-L1 copy number gains are associated with substantial therapeutic activity in some cancers due to the high levels of tumor PD-L1 and increased immune infiltrates that they have shown to promote (47, 90). There is some evidence to suggest that PD-L1 chromosomal translocations influence PD-L1 overexpression in certain diffuse large B cell lymphomas (81,89). Disruption of the 3' untranslated region (UTR) of PD-L1 is another mechanism by which some tumors such as adult T-cell leukemia/lymphoma, diffuse large B-cell lymphoma, and gastric cancer display marked elevation of aberrant PD-L1 transcripts that have become stabilized by truncation of the 3'UTR (69, 82, 91-93). PD-L1 deletions however are more frequently observed in tumors than copy number gains (31 vs. 12%); particularly in melanoma and NSCLC where >50% of tumors harbor PD-L1 deletions (72). PD-L1 deletions, like PD-L1 copy number gains, are associated with a high tumor mutational load and poor prognosis, but the clinical significance of PD-L1 deletions is not yet clear (72, 84). Recently, PD-L1 promoter methylation has been shown to negatively correlate with PD-L1 mRNA and/or protein expression in multiple cancer types including acute myeloid leukemia (78), glioblastoma (112), melanoma (113), head and neck cancer (114), colorectal cancer (115) and prostate cancer (116). The methylation status of the PD-L1 promoter has clinical significance for predicting the outcome of PD-1/PD-L1-targeted therapy (78,97,(113)(114)(115)(116). For example, in NSCLC patients, anti-PD-1 therapy enhanced PD-L1 promoter methylation and reduced PD-L1 expression which mediated resistance to anti-PD-1 immunotherapy Nivolumab in NSCLC patients (117). In addition, histone modifications including methylation and acetylation have been reported to modulate PD-L1 expression in some cancers (118)(119)(120)(121)(122)(123). The histone methyltransferase, enhancer of zeste 2 polycomb repressive complex 2 subunit has been shown to suppress PD-L1 expression through mediating trimethylation of the PD-L1 promoter in hepatoma cells (120). Moreover, histone deacetylases have been reported to regulate PD-L1 expression in melanoma cells (122,124,125).

Constitutive Oncogenic Signaling Regulates PD-L1 Expression

Oncogenic and tumor suppressor signaling pathways have been shown to regulate PD-L1 expression (126,127). Oncogenic signals derived from aberrant receptors, effector molecules and transcription factors leads to the overexpression of PD-L1 by tumors and are associated with poor prognosis and patient response to PD-1/PD-L1-targeted therapy (69, 70, 127, 128). PI3K-AKT-mTOR and RAS-MAPK pathway activation is evidently linked to constitutive PD-L1 regulation in many cancers (69, [129][130][131]. Loss of PTEN (a tumor suppressor that negatively regulates PI3K-AKT-mTOR signaling) or mutations in PIK3CA (a catalytic subunit of PI3K) leads to elevated PD-L1 expression via constitutive PI3K-ATK-mTOR pathway activation in squamous cell lung carcinoma (132,133), NSCLC (130), gliomas (134), colorectal cancer (135), prostate cancer (136), and breast cancer (137). Some tumors harbor mutations in RAS, BRAF, and EGFR and exhibit constitutive RAS-MAPK pathway activation and consequently overexpress PD-L1 (70, 128,129,138). BRAF and EGFR mutations correlate with PD-L1 expression, poor prognosis and low patient response to PD-1/PD-L1-targeted therapy in melanoma (70, 138) and NSCLC (128), respectively. Moreover, oncogenic transcription factors including MYC (139), STAT (140), NFκB (141,142), IRF-1 (143), AP-1 (144), and HIF (145, 146) have been reported to modulate PD-L1 expression at the transcriptional level. MYC expression is found elevated in 70% of cancers (147) and has recently been shown to bind to the PD-L1 promoter transcriptionally inducing PD-L1 expression (148). Similar to MYC, other oncogenic reprogramming factors have been implicated in PD-L1 regulation. OCT4 and SOX2 have both been shown to upregulate PD-L1 expression in cervical cancer (79) and hepatocellular carcinoma (149), respectively, highlighting the necessity of PD-L1 expression for tumor reprogramming functions.

Extrinsic Factors Promote PD-L1 Expression

Interferon gamma signaling in the tumor microenvironment is primarily responsible for PD-L1 upregulation by tumor cells in most cancer types (76, (150) (151) (152) (153) (154) . This may be due in part to secretion of IFNγ from tumor specific T-cells within the tumor microenvironment. A study investigating IFNγ-mediated PD-L1 upregulation in multiple cancers including melanoma, renal cell carcinoma, head and neck cancer, and NSCLC, found that IFNγ was able to induce mRNA and protein PD-L1 expression by tumor cells regardless of constitutive PD-L1 expression (76). Although, IFNγ is a dominant driver of PD-L1 expression in various tumors, the mechanism by which IFNγ mediates PD-L1 upregulation appears to be distinct among different cancer types. For example, transcription factors JAK/STAT1, IRF-1 and NFκB are responsible for IFNγ-induced PD-L1 expression in hematopoietic tumors (155), lung cancer (143), and melanoma (141), respectively. IFNγ signaling is often associated with a positive patient response to PD-1/PD-L1-targeted therapy in metastatic melanoma, NSCLC, head and neck cancer, gastric cancer, and urothelial carcinoma (29, 156, 157). Moreover, loss of function mutations in molecules involved in the IFNγ signaling pathway such as JAK1, JAK2, and β2-microglobulin have been identified to render tumor cells unresponsive to IFNγ signaling and mediate intrinsic or acquired resistance to PD-1-targeted therapy (158-160).

Other inflammatory cytokines shown to promote PD-L1 expression by tumor cells include: TNFα in breast (161), prostate, colorectal cancer (162) and hepatocellular carcinoma (152); IL-27 in lung, prostate and ovarian cancer (163); and TGFβ in breast (164) and lung cancer (165). Additionally, some cytokines have been shown to work synergistically to upregulate PD-L1 expression in tumors such as TNFα with IFNγ (166) and with IL-17 (162). Besides inflammatory cytokines extrinsically modulating PD-L1 expression, hypoxia in the tumor microenvironment selectively elevates PD-L1 expression via HIF-1α activation in melanoma, breast, lung, thyroid and prostate cancer (9, 146, 167). In recent studies, HIF-2α has also been shown to correlate with PD-L1 expression in clear cell renal cell carcinoma (168, 169).

Despite the tremendous efforts of scientific researchers to provide insight into the mechanisms behind PD-L1 signal activation in cancer, the regulation of PD-L1 expression by tumors remains to be fully elucidated in all cancer types. Understanding the mechanisms of tumorigenic PD-L1 expression and signaling in different cancer types may provide therapeutic opportunities to alleviate PD-L1-induced intratumoural immunosuppression and overcome resistance to PD-1/PD-L1-targeted therapy. For greater improvement in the efficacy of PD-1/PD-L1-targeted therapy, it is necessary to identify and target tumor-intrinsic mechanisms that are both responsible for controlling PD-L1 expression and promoting tumor progression.

TUMOR-INTRINSIC PD-L1 SIGNALING

To date, there are less than twenty publications investigating the intrinsic role of PD-L1 in tumors; predominantly using RNA interference approaches in two dimensional (2D)-cultured mouse or human cancer cell lines and immunocompromised mouse models. There is an emerging role of PD-L1 to send prosurvival signals within tumor cells to promote cancer initiation, metastasis, development, and resistance to therapy (Figure 4 ). However, how these emerging pro-survival signals are conveyed intracellularly from cell surface PD-L1 is largely unknown. There is accumulating evidence that intracellular regions of PD-L1 are responsible for transducing survival signals in tumor cells (170-172). Three conserved amino acid sequences including RMLDVEKC, DTSSK, and QFEET motifs have been reported and shown to be located in the intracellular domain of PD-L1. RMLDVEKC and DTSSK motifs were reported to be associated with regulating PD-L1 stability and signal transduction due to the discovery of two specific ubiquitination sites located in the motifs (161, 171). Gato-Cañas et al., demonstrated that the RMLDVEKC motif was required to inhibit IFN-mediated cytotoxicity toward tumor cells via directly preventing STAT3 phosphorylation and caspase-mediated apoptosis. Another study also demonstrated that tumor cells expressing PD-L1 were refractory to Fas-and protein kinase inhibitor Staurosporine-mediated apoptosis (170), which could suggest that the intracellular motifs of PD-L1 may be involved in crosstalk with other signaling pathways; in particular signaling pathways that control tumor cell survival. Other studies have shown that PD-L1 agonists can induce crosslinking between PD-L1 and CD80/CD86 to transduce reverse signaling (173-175). Recently, PD-L1 has been shown to form a heterodimer with CD80, a shared ligand with CTLA-4 and CD28, in cis on APCs and tumor cells. This heterodimer was reported to weaken CD80:CTLA4 interaction, but not CD80:CD28 binding indicating that PD-L1 may prevent CTLA-4 inhibitory signals (174, 175) . Furthermore, overexpression of CD80 on PD-L1 positive tumor cells was shown to blunt the pro-tumor role of PD-L1 (176). The above studies support the notation that PD-L1 reverse signaling exists in tumor cells. Research efforts should expand on this emerging concept of PD-L1 reverse signaling which has the potential to identify new mechanisms of PD-L1targeted immunotherapy.

Tumor-Intrinsic PD-L1 Is Associated With Cancer Initiation PD-L1 expression has been shown to correlate with the cancer stem cell (CSC)-like characteristics including the expression of CD44 and/or CD133 at high levels on tumor cells. Human head and neck (177), lung (178), and colorectal (179) cancer cells that have CSC-like characteristics (CD44 high /CD133 high ) were shown to preferentially express PD-L1 compared to CD44 low /CD133 low cancer cells in immunocompromised mouse models either inoculated with a patient-derived xenograft or human cancer cell lines mixed with Matrigel R , respectively. In breast and lung cancer cells CD44 was shown to be a key regulator of PD-L1 expression following shRNA-directed knockdown of CD44 in vitro and in vivo using a metastatic breast cancer xenograft mouse model (180). Additionally, primary tumor samples from breast and lung cancer patients expressed high levels of PD-L1 correlating with CD44 positivity (180), suggesting that CD44 regulation of PD-L1 expression observed in vitro could be similar to that of an in vivo human tumor.

OCT4 and Nanog are transcription factors critical for pluripotency and tumorigenesis (98). PD-L1 has been shown to promote OCT4 and Nanog expression via PI3K/AKT pathway in breast CSCs (131). PD-L1 knockdown compromised the capability of breast CSCs to self-renew themselves in vitro and in vivo using immune deficient nude mice. CSCs ability to self-renew and differentiate into heterogeneous lineages of cancer cells is thought to be responsible for drug resistance and relapse in cancer development and progression (98). A recent study showed that breast cancer stemness is regulated by miR-873 directly suppressing PD-L1 expression and thus PI3K/AKT and ERK1/2 signaling in breast cancer cells, which reduced CSC-like characteristics and enhanced chemosensitivity (181). Tumor PD-L1 has also been shown to promote the tumor-initiating cell generation in immunocompromised murine melanoma and ovarian cancer mouse models; a phenotype which was also verified in a human ovarian cancer cell xenograft mouse model (182,183). This mechanism of intrinsic PD-L1 to drive tumor stemness was associated with increased mTORC1 signaling (182). However, CSC-like characteristics including high aldehyde dehydrogenase activity, reduced production of reactive oxygen species and a dormant state in the cell cycle were favored following knockdown of PD-L1 in cholangiocarcinoma cell tumors inoculated into mice compared to high PD-L1 expressing tumors (184), indicating that intrinsically PD-L1 may have different roles in different cancer types. Moreover, the CSC-like phenotype is shown to be associated with epithelial to mesenchymal transition (EMT) (98). Chen et al. (100) indirectly knocked down PD-L1 via the microRNA-200/ZEB1 axis in lung adenocarcinoma cells and found that PD-L1 expression correlated with EMT. Low miRNA-200 expressing cells transplanted into a syngeneic immunocompetent mouse model exhibited decreased intratumoural CD8+ T cells and increased metastatic potential due to lack of control over PD-L1 regulation.

Tumor-Intrinsic PD-L1 and the Promotion of Tumor Growth, Invasion and Metastasis

Besides EMT playing a key role in invasion and metastasis, it has the ability to alter the tumor immune microenvironment to immunosuppressive and influence response to PD-1/PD-L1-targeted therapies (185). PD-L1 knockdown in cultured human gastric cancer cell lines SGC-7901 and AGS reduced cell proliferation, migration, invasion and induced cell cycle arrest in vitro and reduced tumor growth and EMT phenotypic marker expression in immunocompromised mice in vivo compared to gastric tumors expressing PD-L1 (186). Similarly, in cultured human Jurkat lymphoid leukemia cells and Raji lymphoma cells, PD-L1 knockdown by lentiviral transduction reduced their invasive ability via downregulation of extracellular matrix-degrading enzymes, matrix metalloproteinase 2 and 9 (187). PD-L1 silencing in murine B16 melanoma cells has also been shown to slow tumor growth and reduce metastases to the lungs of immunocompetent mice as well as immunodeficient mice via mechanisms that increase autophagy and reduce mTORC1 signaling (188). These findings may be linked to the intrinsic functions of PD-L1 to promote tumor stemness via mTORC1 signaling (182,183). Tumor-initiating cells induced by intrinsic PD-L1 signaling are likely to show higher metastatic potential due to their self-renewal capabilities. Interestingly, the same therapeutic effect to reduce lung metastasis was absent in murine ovarian ID8agg cancer cells lacking PD-L1, in immunocompromised mice (188), suggesting the effects of intrinsic PD-L1 may be tumor specific and warrants further investigation. A recent study which knocked down PD-L1 in NCI-H1299 and Calu-1 cells showed enhanced proliferation in comparison to control cells, suggesting a tumor suppressor role of PD-L1 (15). Indeed, PD-L1 expression has been shown to correlate with EMT markers in many solid tumors including gastric, lung, breast, colon, and other common cancers (185,189). With consideration co-targeting of EMT vulnerabilities and PD-1/PD-L1 signaling axis may have the potential to improve clinical efficacy of immunotherapy by limiting the shift of the tumor microenvironment from immunostimulatory to immunosuppressive during tumor development.

Tumor-Intrinsic PD-L1 and Regulation of Metabolic Processes

Within the tumor microenvironment, nutrient competition between tumor cells and immune cells may regulate tumor progression and PD-L1 has been reported to directly regulate the metabolism of several cancer cell lines (190,191). Lactate derived from tumors can suppress the function of T cells by disrupting aerobic glycolysis, a process required for optimal T cell function (190). It has been reported that checkpoint blockade could induce an increase in the glucose concentration within a progressive tumor mouse model, which correlated with glycolytic capacity in tumor infiltrating lymphocytes and increased IFNγ production (191). Interestingly, treatment of B16 melanoma, MC38 colon cancer and sarcoma cancer cell lines in vitro with anti-PD-L1 antibodies was shown to reduce aerobic glycolysis mechanisms, including reduced glycolysis enzymes and Akt phosphorylation, indicating a tumor intrinsic role for PD-L1 in enhancing tumor glycolysis. The same results were achieved by shRNA mediated knockdown of PD-L1 (191), strongly suggesting that PD-L1 itself was the modulator of glycolysis in cancer cells. Hypoxic inducible factor, HIF-1α is a well-known modulator of glycolysis in cancer cells (192). The reduced glycolytic activity of cancer cells caused by PD-L1 blockade would subsequently induce an adaptive hypoxic response and stimulate the production of HIF-1α. HIF-1α also directly modulates immune cell activity in the tumor microenvironment to favor tumor growth and induces PD-L1 expression on tumor cells and immune cells; indirectly mediating immune escape and tumor progression (9). Under hypoxic conditions PD-L1 expression was directly induced by HIF-1α on MDSCs in B16-F10 tumor-bearing mice, and PD-L1 blockade increased MDSC-mediated T cell activation by downregulating IL-10 and IL-6 expression (193). Dual blockade of PD-L1 and HIF-1α could further reduce the glycolytic activity of cancer cells caused by PD-L1 blockade and enhance anti-tumor immunity, ultimately leading to cancer cell death.

Tumor-Intrinsic PD-L1 Facilitates Resistance to Anti-Cancer Therapies

PD-L1 exhibits an anti-apoptotic role in MDA-MB-231 breast cancer cells and silencing PD-L1 in these cells increased cancer cell apoptosis and enhanced cancer cell susceptibility to doxorubicin-induced apoptosis in vitro and in vivo (194), suggesting that PD-L1 not only prevents cancer cell apoptosis, but also promotes chemotherapy resistance. Likewise, CRISPR/Cas9 knockout of PD-L1 enhanced the sensitivity of human osteosarcoma KHOS and MNNG/HOS cells to doxorubicin and paclitaxel and compromised their ability to form three-dimensional (3D) spheroids in vitro (195). Further characterization of the role of PD-L1 in chemotherapy resistance in MDA-MB-231 breast cancer cells discovered that PD-L1 knockdown suppresses the expression of multidrug resistance 1/P-glycoprotein (MDR1/P-gp) via PI3K/AKT pathway in vitro (196); recognizing this has an additional therapeutic target. In fact, PD-1/PD-L1 interaction increased survival of breast cancer cells when exposed to doxorubicin (196), suggesting that PD-1/PD-L1-targeted therapy may increase chemotherapy efficacy by inhibiting MDR1/P-gp expression which usually confers resistance in breast cancer cells. Moreover, through culturing of breast (MDA-MB-231 and 4T1) and prostate (DU145) cancer cell lines with recombinant PD-1 or Jurkat T cells it has been shown how PD-1/PD-L1 interactions results in increased resistance to doxorubicin and docetaxel (197). Subsequent knockdown or blockade of PD-1 restored tumor cell chemo-sensitivity and reduced their metastatic potential in a synergistic breast cancer mouse model, suggesting blockade of intrinsic pathways is beneficial for therapy. Conversely, human colorectal cancer cells harboring a BRAF V600E mutation showed that the depletion of PD-L1 suppresses chemotherapy-induced apoptosis through the down regulation of BIM and BIK BH3-only proteins (198), even though depletion alone reduced tumor growth. The effect of PD-L1 on chemosensitivity was confirmed BRAF V600E mutant MC38 murine tumor xenografts, where PD-L1 knockout cells were less sensitive to chemotherapy due to the suppression of pro-apoptotic molecules, BIM and BIK, compared to parental cells expressing PD-L1. This study highlights the importance of understanding the role of PD-L1 in each cancer type and its subtypes to design effective treatment regimens that will benefit cancer patients.

The tumor-intrinsic role of PD-L1 appears to be similar across all cancer types investigated in the literature to date, with the exceptions of cholangiocarcinoma and contradictory evidence in lung cancer, in that PD-L1 promotes tumor growth and development. However, the molecular mechanisms of PD-L1 exerting pro-tumor activity appear to be distinct amongst different cancer types. Notably, in the studies that have investigated the intrinsic role of PD-L1 in lung cancer the cells utilized were mesenchymal lung cancer cell lines which harbored KRAS and/or p53 mutations, suggesting that the tumor cells metastatic capacity and mutational status may not be determining factors as to whether PD-L1 exhibits a pro-tumor or anti-tumor role in lung cancer. Furthermore, Wang et al. (15) reported that PD-L1 expression reduced lung cancer cell proliferation, suggesting that although PD-L1 expression may limit tumor cell proliferation it is still affecting other tumor characteristics that influence tumor progression. The reasons behind this potential role of PD-L1 in lung cancer warrants further investigation.

TUMOR-INTRINSIC PD-1 SIGNALING

Similar to PD-L1, the expression of PD-1 on T cells and its role to inhibit the immune system is well characterized, but recent studies have found intrinsic expression of PD-1 in tumor cells including melanoma (199), hepatic carcinoma cells (200), ovarian (201), bladder (201), lung (15, 202), and colorectal (15) cancer cells. In melanoma B16 tumors, a subpopulation of PD-1 expressing cancer cells were identified to modulate downstream mTOR signaling and promote tumorigenesis independent of adaptive immunity, in an in vivo mouse model lacking an adaptive immune system (Figure 5A ) (199). This effect was abrogated with anti-PD-1 therapy, tumor-specific PD-1 knockdown and mutagenesis of intracellular signaling motifs downstream of PD-1, strongly suggesting an intrinsic function of PD-1 to promote tumorigenesis in melanoma. Similar to intrinsic PD-1 in melanoma cells, intrinsic PD-1 in liver cancer cells has been reported to mediate tumorigenesis in immunocompromised mice via regulating mTOR signaling (Figure 5A ) (200) and thus combined inhibition of PD-1 and mTOR may be a potential therapeutic strategy for melanoma and liver cancer. Moreover, anti-PD-1 therapy has been reported to reduce the cell growth of bladder RT4 cancer cells cultured in 2D in the absence of adaptive immunity (201), implying that PD-1 expression is potentially oncogenic. Interestingly in murine NSCLC M109 cells, intrinsic PD-1 exhibited an antitumor role in immunocompromised mice and when NSCLC cells were treated with anti-PD-1 therapy they demonstrated increased proliferation and tumor growth (Figure 5B ) (202). Consistent with this, silencing of PD-1 or therapeutic antibody blockade of PD-1 on the surface of NSCLC and colorectal cancer cells increased proliferation in vitro via activating PI3K and MAPK pathways (15), suggesting that PD-1 could be involved in development of resistance to immunotherapy blockade in NSCLC and could provide one explanation for why patients with NSCLC can display hyperprogressive disease following treatment with anti-PD-1 therapy (15, 203). The latter findings also suggest that the tumor suppressor role of PD-1 on cancer cells may not be limited to NSCLC. Although, Wang et al., demonstrated that PI3K and MAPK pathways were activated following anti-PD-1 therapy in NSCLC cells in vitro and in vivo, their study also showed that PD-1/PD-L1 dysfunction did not activate mTOR, illustrating that the mechanism behind tumorintrinsic PD-1 to either induce or inhibit tumor growth may be different. However, mTOR activation has been shown to occur in the only two studies investigating tumor-intrinsic PD-1 where PD-1 has a pro-tumor role, which may suggest that mTOR signal activation is necessary for PD-1 to exhibit tumorigenic activity. Therefore, the molecular mechanism behind tumorintrinsic PD-1 needs to be elucidated in other cancer types to confirm this potential role of mTOR in PD-1 signaling in tumor cells. Furthermore, studies investigating the role of tumor-intrinsic PD-1 in tumors have utilized tumor cell lines that exhibit invasive and metastatic potential. The metastatic potential of cells does not seem to be a factor in determining whether PD-1 is pro-or anti-tumorigenic and nor is it associated with enhanced tumor-intrinsic PD-1 activity (199). Additionally, studies have used both poorly-and well-differentiated tumor cells which have been shown to have the same PD-1-intrinsic function, implying that the differentiated state of the cell is also not a contributing factor to the role PD-1 in tumors. Yao et al., reanalyzed cancer transcriptomic and proteomic data from The Cancer Genomic Atlas Project and The Cancer Cell Line Encyclopedia Dataset to find that tumor-intrinsic PD-1 expression is widespread in many cancer types. This heterogeneity may explain the differential therapeutic effects of anti-PD-1 drugs and could provide crucial information required when selecting suitable patients for treatment dependent on the cancer cell type. However, further work in different cancers and tumor models could also shed more light into this area.

So far, most evidence for PD-L1 and PD-1 signaling in cancer cells is based on 2D cell culture models using murine and human cancer cells and immunodeficient mouse models that can fail to fully recapitulate the human in vivo tumor (24, 204). Therefore, more relevant models capable of recapitulating the heterogeneity of the tumor microenvironment during in vivo conditions could allow further predictive in vitro evaluation of the tumor-intrinsic role of PD-L1 and PD-1, and how these roles may be affected by immunotherapy treatment and influence immune cell function.

IMMUNOTHERAPY BLOCKADE OF INTRINSIC PD-L1 AND PD-1 SIGNALING

Recent reports discussed above suggest that the emerging intrinsic role of PD-L1 is largely pro-tumorigenic in a number of cancers, but that in lung cancer and cholangiocarcinoma, it may act as a tumor suppressor gene. Likewise, the new emerging tumor intrinsic role of PD-1 has also been reported to have differential roles in different cancer cell types and this remains to be further investigated. However, there are currently a limited number of reports investigating how immunotherapeutic drugs potentially modulate these intrinsic pathways. Theivanthiran et al. (205) demonstrated that PD-1 blockade on CTLs in a syngeneic mouse model was able to activate a PD-L1-NLRP3 inflammasome signaling pathway in tumor cells that promoted MDSC recruitment and infiltration into the tumor microenvironment. Intratumoural MDSCs can suppress T cell function (193) and thus may dampen the immune response and promote resistance to anti-PD-1 therapies. The effect of the immunotherapy drug Atezolizumab was measured on MDA-MB-231 breast cancer cells (206). In this study, RNA-Seq was utilized to assess the modulation of gene expression after treatment with Atezolizumab and it was reported that genes promoting cell migration, metastasis, EMT, cell growth, and hypoxia were downregulated whilst apoptosis genes were upregulated. This suggests that Atezolizumab may be able to modulate the signaling of PD-L1 in this cell line to some extent at the level of gene expression. In contrast, Wang et al. (15) , investigated the effects of anti-PD-1 antibodies Nivolumab and Pembrolizumab or the anti-PD-L1 antibody Atezolizumab on Calu-1, SW480, HT-29, BxPC-3, SK-BR-3, and U-2 OS cells. All immunotherapy drugs were shown to increase cell proliferation compared to isotype control in vitro. To verify these findings in vivo, human lung cancer cells were inoculated into immunocompromised mice. Similar to in vitro studies, monoclonal antibody administration to block PD-1 or PD-L1 activated PI3K and MAPK pathways by phosphorylating AKT and ERK1/2, respectively, promoting tumor cell growth in vivo. These small numbers of studies suggests that immunotherapeutic antibodies may be able to modulate the intrinsic function of PD-L1 and PD-1 and potentially highlights another mechanism by which tumors may develop resistance to PD-1/PD-L1 targeting therapy through co-expressing PD-L1 and its receptor PD-1. The ability of immunotherapy drugs to modulate the intrinsic PD-L1 and PD-1 pathway in other cancers in more heterogeneous tumor models could also provide further important insight into the mechanism of immunotherapy treatment.

FUTURE DIRECTION "MODELING TUMOR HETEROGENEITY" TO FURTHER ELUCIDATE INTRINSIC ROLES OF PD-L1 AND PD-1

Tumor heterogeneity makes it challenging to identify novel therapeutic targets and potential biomarkers of immunotherapy response that could substantially enhance therapeutic efficacy. The scientific basis for numerous clinical trials has derived from 2D cell culture models and animal models, which can fail to fully replicate the human tumor microenvironment due to lack of heterogeneity and species-to-species variability, respectively, which could account for lack of transferability of PD-1/PD-L1targeted antibodies into the clinic (24, 204). Furthermore, most evidence to date exploring the intrinsic role of PD-L1 and PD-1 has been based on 2D cell culture models using murine or human cancer cell lines or animal models, and thus limit the capacity to explore these roles in a relevant human tumor setting. Given the emerging intrinsic roles of PD-L1 and PD-1 and the differences between cancer types, utilizing models which closely mimic the heterogeneity of the human tumor microenvironment could allow a more predictive in vitro evaluation of the intrinsic role of PD-L1 and PD-1 in cancer and modulation by anti-cancer therapeutics. For example, human cancer cells implemented into different 3D cell culture models have shown to exhibit characteristics that more closely mimic in vivo human tumors, such as changes in morphology, proliferation, gene and protein expression, and response to treatment (204). Indeed the modulation of PD-L1 expression has been reported to be affected by the extracellular matrix stiffness of tumors in 3D culture (207) and a 3D model system utilizing patient-derived organoids that resembled the tumor immune microenvironment for the study of the PD-1/PD-L1 signaling axis has been developed (208). Furthermore, in a recent study a tumor-immune co-culture was utilized to assess the efficacy of immunotherapies Nivolumab and Durvalumab (209).

CONCLUSIONS

PD-1/PD-L1 checkpoint blockade is at the cutting edge of research offering cancer patients hope for new treatment regimens with potential to have substantial clinical benefit and prolong survival. PD-1/PD-L1-targeted therapies reactivate the immune system to induce immune-mediated tumor eradication, and although they have demonstrated success has single agents, they have also shown cooperation with conventional and targeted therapies in the clinic. Unfortunately, most patients are unresponsive or develop resistance to PD-1/PD-L1-targeted therapy. Further elucidating the tumor intrinsic role of PD-L1 and its receptor PD-1 in all cancer types will help understand the basis for or lack of response to immunotherapy and may allow the identification of novel therapeutic targets and biomarkers to enhance clinical efficacy.

AUTHOR CONTRIBUTIONS

KH and RL researched and wrote the manuscript. KH designed the figures. RL conceived the theme/direction. NC and NJ-M reviewed the manuscript. All authors contributed to the article and approved the submitted version.

Conflict of Interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgements

ACKNOWLEDGMENTSauthors wish to thank the Biomolecular Sciences Research Centre and Sheffield Hallam University for funding this work.

Funding

FUNDINGThis project was funded by the Biomolecular Sciences Research Centre at Sheffield Hallam University.Open access funds are supported by Sheffield Hallam University.

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