STEM I

Course Overview

This course focuses on scientific research and engineering. During the first part of the year, we conduct 5-month long independent research projects, advised by Dr. Kevin Crowthers, where we learn how to review scientific literature, develop methodology, and communicate findings.

PD-L1 Expression is Upregulated in Tumor Infiltrating Myeloid Cells of Young Tumor-Bearing Hosts

The project aim is to understand the potential mechanisms which are responsible for the lack of success of PD-1/PD-L1 blockade treatment in the younger population.

Supporting Documents

Abstract

Over the past decades, research has shown that Programmed Death-1 (PD-1)/Programmed Ligand Death-1 (PD-L1) blockade is one of the most revolutionary cancer immunotherapies. The affinity binding of PD-1 to its ligand, PD-L1, triggers a wide range of intracellular signaling and compromises antitumor responses by promoting self-tolerance through regulating the activity of T-cells. Although PD-1/PD-L1 blockade treatment is well-studied in adults, the experience in the young population is not effective. Therefore, the improvement of these therapies is a priority for younger patients. The project aim is to understand the potential mechanisms which are responsible for the lack of success of PD-1/PD-L1 blockade treatment in the younger population. We hypothesize that PD-L1 is differentially regulated and expressed in the cellular components of tumor microenvironment in young and adult patients. By using a dataset published by Moustaki et al (2020), PD-L1 was specifically upregulated in the tumor-infiltrating myeloid cells rather than the tumor cells in young mice. The JAK/STAT signaling pathways have intensively shown involvement in PD-L1 upregulation. Here we show that some components of the JAK/STAT pathways, including Ptpn18, Il18, Zeb2 and Il17r, were conjunct to PD-L1 upregulation in the tumor infiltrating myeloid cells in the young tumor host. First, our findings give a deeper insight of the compromised PD-1/PD-L1 blockade treatment in young patients. The genes conjunct to PD-L1 upregulation in myeloid cells provide potential targets to optimize the PD-1/PD-L1 blockade therapy in young cancer patients.

Keywords: PD-1, PD-L1, tumor infiltrating myeloid cells, JAK/STAT pathway, young tumor

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Researchable Question: PD-1/PD-L1 blockade is a revolutionary cancer immunotherapy. However, this therapy has poor response rates in young patients. This study addresses the differential response rate in young patients.

Hypothesis: We hypothesize that PD-L1 is differentially regulated and expressed in the cellular components of tumor microenvironment in young and adult patients.

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Introduction

In the last decades, cancer immunotherapies have achieved astonishing success in different tumor types and has led to an increased interest in the field (Moreno-Vincente et al, 2019). The current consensus of cancer immunology suggests that the host immune system is capable of surveying against cancerous cells at an early stage of development, recognizing and eliminating emerging malignant cells. During this control phase, immune cells exert a selective pressure that favors the outgrowth of less immunogenic clones, which can eventually escape immune recognition and lead to cancer progression (Dunn et al, 2002). In order to combat this, cancer immunotherapies target the restoration of the host's immune function and the rescue of anti-tumor responses. Of all the immunotherapeutic approaches explored in adult cancers, targeting the programmed cell-death protein (PD)-1 and its ligand, PD-L1, with monoclonal antibodies has perhaps emerged as the most promising strategy, showing therapeutic benefit in a wide range of cancer types (Topalian et al, 2012; Brahmer et al, 2012).

PD-1 was first discovered as a 288 amino acid protein expressed on the surface of T-cells and associated with apoptosis (Constantinidou et al, 2019; Ishida et al, 1992). Further study on the role of PD-1 in the immune response has been studied in PD-1 deficient mice. These mice exhibit autoimmunity, including lupus-like arthritis, glomerulonephritis and splenomegaly with predominantly IgG3 deposition (Nishimura et al, 1999). Homozygous PD-1 null 2C-TCR (anti-H-2Ld) transgenic mice in the H-2b/d background develop a chronic graft-versus-host-like disease. The B7-1/B7-2 - CD28/CTLA4 pathway is the best studied of the T-cell costimulatory pathways and plays an important role in T-cell activation or tolerance. In this pathway, a fine balance occurs between CD28 “co-stimulation” mediated activation of T-cells and CTLA4 immune-checkpoint mediated inhibition of T cells. PD-1 functions as an inhibitory receptor and maintains self-tolerance (Barber et al. 2006). These findings confirm that PD-1 is a molecule that limits T-cell activation and proliferation while it promotes self-tolerance. The ligand for PD-1 was initially identified as B7-H1, a molecule homologous to B7-1/B7-2 that did not interact with ICOS, CD28 or CTLA4 (Dong et al, 1999). It was later found to be the ligand for PD-1, hence named PD-L1 (Pdcd1lg1, CD274) (Freeman et al, 2000). The second PD-1 ligand, PD-L2 (Pdcd1lg2, CD273), was discovered a year later (Latchman et al, 2001). Malignant cells are able to evade immune destruction by modulation of the immune checkpoint pathways but also by increasing the co-inhibitory ligand expression (such as PD-L1) thereby rendering tumor-infiltrating lymphocytes ineffective against the tumor despite being present in the tumor microenvironment (TME) (Ahmadzadeh et al, 2009).

In the view of cancer immunotherapy, PD-L1 has been identified as a bio-marker of adult cancers. Initially, tumor cells were described as a major cell population where PD-L1 was expressed (Moreno-Vincente et al, 2019). Thereafter, PD-L1 expression on myeloid populations was rapidly recognized. Activated CD4 helper T cells at the tumor site may sustain PD-L1 expression on tumor-associated macrophages (TAMs) (Noguchi et al, 2017). Myeloid cells expressing PD-L1 in the TME and the tumor-draining lymph nodes may also decrease the activation of tumor-reactive T cells and compromise the therapeutic effect of PD-1/PD-L1 blockade (Tang et al, 2018). However, the dynamic contribution of PD-L1 expression from tumor cells and myeloid cells is not yet clear.

The regulation of PD-L1 expression is complex as it varies between different tumor types and occurs at the genetic, transcriptional and post-transcriptional levels. Copy number alterations of PD-L1 locus have been reported with varying frequency in several tumor types (Cader et al, 2020). At the transcriptional level, a number of transcriptional factors seem to regulate PD-L1 expression including HIF-1, STAT3, NF-κΒ, and AP-1 (Zerdes et al, 2018). Growing studies have suggested that activation of common oncogenic JAK/STAT pathway affect tumoral PD-L1 expression. However, the role of each member in the JAK/STAT signaling pathway, specifically their role in tumor-infiltrating myeloid cells, is not clear.

Current research has proven PD-1/PD-L1–targeted immunotherapy to be an effective strategy for treating multiple adult malignancies (Xin et al, 2020). The success of these treatments has been shown to depend on the immune cell composition of tumors, as well as the immune receptors present on both immune and cancer cells that may be relevant for immune evasion. However, the impacts of the pediatric immune contexture remains largely unknown, and this lack of knowledge has created a gap in the evidence-based development of pediatric cancer immunotherapy trials (Thakur et al, 2022). In this manuscript, we derived the single-cell RNA-sequencing data which was generated from young and adult MHC I-deficient tumor model (Moustaki et al, 2022). Using bioinformatic analysis, we explored the expression of PD-L1 in tumor infiltrating (TME) myeloid cells and computed the intracellular pathways which are potentially related to PD-L1 expression.

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Methodology

Animal model

As described in the original paper (Moustaki et al. 2022), C56Bl/6J mice were purchased from The Jackson Laboratory (Jax; Stock No: 000664). Mice allocated to different experimental groups were sex-, age-, and housing-matched. Female mice were used throughout the study. All animal studies were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (the Guide) of the National Institutes of Health. Animal protocols were approved on an annual term by the Institutional Animal Care and Use Committee (IACUC; protocol #: 598-100569) at St. Jude Children’s Research Hospital.

The mouse colon adenocarcinoma cell line, MC38 (H-2b haplotype), was maintained in high glucose DMEM medium (Gibco) supplemented with 10% FBS (Hyclone) and antibiotics (Gibco, penicillin 100U/mL, streptomycin 100 μg/mL). The immunogenic cell lines were generated by lentiviral transduction and subsequent FACS sorting of mCherry+ cells spreading within a narrow window of fluorescence intensity (low CV). All transgenic cell lines used in this study are polyclonal to dilute-out potential artifacts due to insertional mutagenic effects.

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scRNA sequencing

As described in the original study (Moustaki et al, 2022), single-cell gene expression libraries were generated using the Chromium Single Cell 3’ v2 kit (10X Genomics) with target outputs of 7,000-10,000 cells per library. Sequencing was performed on the Illumina NovaSeq platform with sufficient sequencing for at least 50,000 mean reads per cell. Raw sequencing data from each library were processed and aggregated using CellRanger (v3.0.2), normalizing for sequencing depth across libraries based on the median number of confidently mapped reads per cell. The subsequent feature/barcode matrices were then analyzed using Seurat (v3.1.0), controlling for potential variation owed to cell-specific variation including the number of unique RNA molecules, percent of mitochondrial gene expression, and inferred cell cycle state (Tirosh et al, 2016). Principal Component Analysis (PCA) was conducted on variable genes (detected using the standard ‘vst’ method), significant PCs were detected using random permutation, and those PCs were then leveraged for dimensional reduction and subsequent visualization using Uniform Manifold Approximation and Projection (UMAP) (McInnes et al, 2018). To compare cells from distinct age groups, differential gene expression analysis was conducted using default parameters, and significantly up- or down-regulated genes for each condition were assessed for GO biological processes (v2020-07-16) and Reactome (v65) pathway enrichment using the PANTHER Overrepresentation Test (Mi et al, 2019).

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Statistical Analysis

Statistical analysis was performed using Prism V8 (GraphPad). The normal distribution of variables was calculated using Shapiro-Wilk test. The continuous variables with normal distribution are presented as mean ± standard deviation (SD), while variables with skewed distribution as median with interquartile range (IQR). Population means were calculated within a 95% confidential interval. Null hypothesis between means with normal distribution between the groups was tested by Student’s t-test or one-way Anova parametric tests. Comparison of means without a normal distribution between the groups was done using Mann-Whitney U-test or Kruskal-Wallis nonparametric tests. Two-way Anova was performed to compare multiple categorical groups of 2 independent variables (factors). A two-tailed p-value of <0.05 is regarded as statistically significant, and different levels of significance are represented with asterisks: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

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Analysis

Unsupervised gene expression analysis of mCherry+ cells broadly revealed clusters that corresponded to tumor cells which expressed mCherry, neutrophils and myeloid cells which were identified by the expression of CD14, and effector T lymphocytes which were characterized by the expression of CD3e (Fig 2A). In agreement with the original publication (Nishimura et al, 1999), the proportions of cancer cells, neutrophils, lymphocytes and myeloid cells were 3.9%, 3.2%, 1.5% and 84.4% respectively. (Fig. 2B).

The cells obtained from young and adult hosts did not show significant difference among the tumor cells (Fig 2B). However, the myeloid cells demonstrated substantial heterogeneity in gene expression signatures that significantly related to host age (Fig 2B). Gene ontology analysis indicated that, relative to those from the adult host, myeloid cells from the young hosts upregulated processes involved the response to virus infection, the regulation of response to biotic stimulus, the immune response regulating signaling pathway, the negative regulation of immune system process, the positive regulation of response to external stimulus, the response to bacterium, the defense response to symbiont, the positive regulation of cytokine production and the negative regulation of viral genome replication (Fig 3A and B). This gene signature is consistent with the more stimulatory phenotype observed in the young MPCs (Nishimura et al, 1999).

To further investigate if the PD-1/PD-L1 axis possesses differential potential in young and adult hosts, we analyzed the expression of PD-L1 expression in the tumor-infiltrating myeloid cells. We found significantly greater expression of PD-L1 in myeloid cells (Fig 4A and B). However, PD-L1 expression did not show a significant difference in both tumor cells and neutrophils.

The previous studies have shown that JAK/STAT signaling pathways are positively involved in the upregulation of PD-L1 (Cader et al, 2020; Toshifumi et al, 2017). In this study, we analyzed the expression of 253 genes which have been identified as participants of JAK/STAT signaling pathways. Among these genes, Protein Tyrosine Phosphatase Non-Receptor Type 18 (Ptpn18), interleukin 18 (Il18), Zinc Finger E-Box Binding Homeobox 2 (Zeb2), interleukin 17 receptor (Il17r) and Signal Transducer And Activator Of Transcription 1 (Stat1) showed significant upregulation specifically in tumor-infiltrating myeloid cells (Fig 5). There expression also showed strong correlation to the upregulated PD-L1 expression in these cells.

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Discussion

The first immune checkpoints described were CTLA-4 and PD-1. Although many other checkpoints have since been identified and are actively being investigated, CTLA-4 and PD-1 remain the only immune checkpoints that have been successfully targeted clinically. PD-1/PD-L1 blockade immunotherapy has become a major pillar of treatment for more than 15 types of adult cancers (Long et al, 2022). This success has prompted the exploration of PD-1/PD-L1 blockade immunotherapy even in pediatric malignances. However, the use of PD-1/PD-L1 blockade immunotherapy as individual agents has achieved disappointing response rates (Moreno-Vincente et al, 2019). To understand the limited response in pediatric cancers, many studies have been carried out to evaluate the role of PD-1/PD-L1 in pediatric cancers. Some of them described high PD-L1 expression across a broad range of pediatric cancers (Maude et al, 2014), while other studies reported low expression level (Majzner et al, 2017). In this study, we implanted colon cancer CD38 cells in young and adult hosts. The PD-1 expression did not show significant difference in adult and young CD3+ lymphocytes. The PD-L1 expression did not show significant difference in cancer cells from adult and young hosts either. The interesting result is that the PD-L1 expression is significantly upregulated in tumor infiltrating myeloid cells. This innovative finding suggests that PD-1/PD-L1 pathway plays a critical role in the pediatric colon cancer.

Early studies of PD-L1 expression mainly focused on tumor cells. More recent studies suggest that PD-L1 from myeloid populations is an undervalued immunosuppressant in tumor development and induces drug resistance to PD-1/PD-L1 blockade immunotherapy (Moreno-Vincente et al, 2019). As one of the most abundant cell types in solid tumors, myeloid cells contribute to T-cell dysfunction and exhaustion through the secretion of cytokines and metabolic products (Dong et al, 2021) and increase PD-L1 expression in tumor cells and other immunosuppressive cells (Zhang et al, 2017). In diagnosed cancers, high macrophage infiltration is often closely related to the occurrence of drug resistance to PD-1/PD-L1 immune suppressants (Pu et al, 2022). In this study, myeloid cells take 84.4% of the tumor infiltrating cells. More importantly, the expression of PD-L1 is upregulated in this population. Therefore, our findings suggest that tumor infiltrating myeloid cells should be an important target to reverse the resistance to anti-PD-1/PD-L1 therapy.

Many mechanisms have been demonstrated to regulate the expression of PD-L1 including signaling pathways, transcriptional factors, and post-transcriptional modulators (Zerdes et al, 2018). Among these regulators, accumulating evidence suggest that JAK/STAT signaling pathway plays crucial role. In nucleophosmin-anaplastic large-cell lymphoma kinase (NPM-ALK) positive anaplastic large-cell lymphoma (ALCL), STAT3 is activated by NPM-ALK oncoprotein through JAK3 activation, binds physically to the PD-L1 gene promoter, and induces its expression in vitro and in vivo (Marzec et al, 2008). Upon stimulation with IFNγ, another STAT family member, STAT1, was activated, resulting in PD-L1 upregulation and in reduction of NK-cell activity against tumor cells in multiple myeloma, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL) (Bellucci et al, 2015). In this study, we analyzed the expression of 253 genes which have been identified as participants of JAK/STAT signaling pathways. Among these genes, Ptpn18, Il18, Zeb2, Il17r and STAT1 showed significant upregulation specifically in tumor-infiltrating myeloid cells. Their expression also showed strong correlation to the upregulated PD-L1 expression in these cells. These findings not only add more support to the role of STAT1 regulating PD-L1 expression, but also indicate the potential roles of Ptpn18, Il18, Zeb2 and Il17r in PDL1-related drug resistance against PD-1/PD-L1 blockade immunotherapy in pediatric cancers. Better understanding of PD-L1 regulation may pave the way for combinational treatments with both immune checkpoint inhibitors and targeted therapies against kinases or transcription factors in pediatric oncology.

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Reference

Poster