UNIVERSIDAD AUTÓNOMA DE MADRID
PROGRAMA DE DOCTORADO EN DEPARTAMENTO DE BIOQUÍMICA
PD-L2 mediates immune evasion of chemotherapy-treated tumours and protects from tissue injury
DOCTORAL THESIS
Selim Chaib
M.Sc. Biomedical Sciences B.Sc. Biology
MADRID, 2019
DEPARTAMENTO DE BIOLOGÍA MOLECULAR
FACULTAD DE MEDICINA
UNIVERSIDAD AUTÓNOMA DE MADRID
PD-L2 mediates immune evasion of chemotherapy-treated tumours and protects from tissue injury
Selim Chaib
The work presented in this Thesis has been carried out at the Tumor Suppression Group in the Spanish National Cancer Research Centre (CNIO, Madrid) and at the Cellular Plasticity and Disease Group in the Institute for Research in Biomedicine (IRB Barcelona, Barcelona), under the direction and supervision of Dr. Manuel Serrano Marugán.
Madrid, 2019
Dr. Manuel Serrano Marugán, Head of the Cellular Plasticity group (IRB Barcelona)
CERTIFIES:
That the Doctoral Thesis entitled “PD-L2 mediates immune evasion of chemotherapy- treated tumours and protects from tissue injury” developed by Selim Chaib meets all requirements to obtain the degree of Doctor of Philosophy (PhD) in Molecular Biosciences, that will, with the aforementioned objective, be defended at the Universidad Autónoma de Madrid. This thesis has been carried out under my supervision and I authorize it to be presented to the Thesis Tribunal accordingly.
I hereby issue this certification in Madrid on December 4th, 2019.
Manuel Serrano, PhD ICREA Professor
Head of Cellular Plasticity and Disease Group
Institute for Research in Biomedicine (IRB Barcelona) C/ Baldiri Reixac 10-12
Parc Científic de Barcelona-01C43 E-08028 Barcelona, Spain
Acknowledgements
Acknowledgements
First and foremost, I would like to thank Manuel for his continuous support and mentoring. I truly appreciate the privilege working with you. Thank you also for all the opportunities and advice.
I also would like to thank the rest of the cellular plasticity and disease lab. In particular, I would like to thank Maribel for all the help. Susana for all her work on this project.
Kathleen for all the help and input. Federico providing crucial reagents and help. Noelia, Raquel, Dafni and Miguel for help with the University matters. Mar for all the little favours.
Furthermore, I would like to thank…
Ana for her time and patience.
Neus, Monica and Alicia and the rest of the IHC team for all their work.
Maria, Alena, Olga and Andrea for their input, work and crucial contributions.
Fatima and Hector for all their important work.
Eiji, Shimpei, Ken, Ayako and Kumiko for all their help there.
Paco, Elena, Alvar, Rosa, Sandra for their all their effort working on this project.
Pablo, thanks for being such an outstanding mentor. I am grateful for all your time, help and advice. Nicole, Lucy, Anne thank you so much for your help there.
Marianne and Brad, thanks for your time and mentoring. I am very grateful to have had that experience. Steve, Kitty, Dan, Ryan and Elizabeth thank you so much for your help there.
Dr. Jaehde, thank you very much for all you support throughout these years. Carina and Verena thank you so much for your help there.
Christine, thank you for the opportunity to work with you. Peggy, Neil, Sabine and Michel thank you so much for your help there.
I am grateful beyond words to my family for the ever-present support. Thanks to all my friends for their support, patience and understanding. It was an unforgettable journey.
Abstract
Abstract
PD-L2 mediates immune evasion of chemotherapy-treated tumours and protects from tissue injury
Immune checkpoint blockade has been shown to be very effective in some cancer patients. Significant research and developments are centred around PD-1 and its ligand PD-L1; however, little is known about PD-1 in relation to its alternative ligand PD-L2. We have found that PD-L2 is upregulated by multiple cells in response to a large variety of cellular stresses leading to senescence, including chemotherapy. PD-L2 is highly upregulated in mouse tumours upon treatment with chemotherapy. Interestingly, we show that inactivation of PD-L2 in cancer cells, renders them more susceptible to immune clearance after chemotherapy. On the other hand, we also found that PD-L2 knockout mice are abnormally sensitive to tissue injury. Based on our results, we hypothesize that PD-L2 is important to create an immunosuppressive protection for damaged cells, including cancer post-chemotherapy and normal tissues post-injury. These observations lead to a better understanding of how damaged cells evade their immune clearance and gives further context for potential combinations of immune checkpoint blockade with standard chemotherapy. Moreover, we also illustrate the crucial role of immunosuppression in tissue protection upon injury.
Abstract (Español)
Abstract (Español)
PD-L2 media la evasión inmune de tumores tratados con quimioterapia y protege contra lesiones tisulares
El bloqueo de la inmunosupresión ha demostrado ser muy efectivo en algunos pacientes con cáncer. La investigación y los desarrollos terapéuticos más importantes se centran en PD-1 y su ligando PD-L1; sin embargo, se sabe poco sobre PD-1 en relación con su ligando alternativo PD-L2. Esta investigación muestra por primera vez que PD-L2 se activa en respuesta a una gran variedad de daños celulares, incluida la quimioterapia.
Demostramos que la inactivación de PD-L2 en células cancerosas las hace susceptibles a su eliminación por el sistema inmune en combinación con quimioterapia. Además, los ratones en los que se ha delecionado PD-L2 son anormalmente sensibles a los daños tisulares. En base a estos resultados, proponemos que PD-L2 es importante para crear una protección inmunosupresora para las células dañadas, lo cual aplica a las células cancerígenas tratadas con quimioterapia y a los tejidos normales después de una lesión.
Estas observaciones permiten una mejor comprensión de cómo las células dañadas evaden su eliminación por el sistema inmune y proporcionan un contexto adicional para posibles combinaciones de bloqueo de la inmunosupresión con la quimioterapia.
También ilustramos el papel crucial que juega la inmunosupresión mediada por PD L2 en la protección de los tejidos ante una lesión.
Index
Index
1 LIST OF ABBREVIATIONS ... 10
2 INTRODUCTION ... 12
2.1 RELEVANCE OF RESEARCH ON SENESCENCE ... 12
2.2 CELLULAR SENESCENCE ... 12
2.3 SENESCENCE-ASSOCIATED SECRETORY PHENOTYPE (SASP) ... 14
2.4 STRATEGIES TO ELIMINATE SENESCENT CELLS ... 14
2.5 SENESCENCE AND CANCER ... 16
2.6 SENESCENCE AND THE IMMUNE RESPONSE ... 17
2.7 PD-1 IMMUNE CHECKPOINT BLOCKADE ... 18
2.8 PD-L2 ... 20
3 OBJECTIVES ... 22
4 MATERIALS AND METHODS ... 23
4.1 MAMMALIAN TISSUE CULTURE ... 23
4.2 SENESCENCE INDUCTION IN VITRO ... 23
4.3 GENE EXPRESSION ANALYSIS ... 23
4.4 GENERATION OF CELL PELLETS FOR IHC ... 25
4.5 SAΒGAL STAINING ... 25
4.6 IMMUNOHISTOCHEMISTRY ... 25
4.7 FLOW CYTOMETRY ... 26
4.8 MOLECULAR BIOLOGY TECHNIQUES ... 26
4.8.1 PD-L2 ORF ... 26
4.8.2 CRISPR ... 27
4.8.3 PCR of genomic DNA ... 27
4.9 TRANSFECTIONS ... 28
4.10 GENERATION OF LENTIVIRUSES ... 28
4.11 B16-OVA AND OT1 CO-CULTURE ... 28
4.12 PATIENT DERIVED CO-CULTURE MODEL ... 29
4.13 DRUG CLASS ENRICHMENT ANALYSIS ... 29
4.14 ANIMAL EXPERIMENTATION ... 30
4.14.1 Folic acid induced kidney fibrosis ... 30
4.14.2 Unilateral ureteral obstruction (UUO) ... 30
4.14.3 Bleomycin induced lung fibrosis ... 30
4.14.4 Xenografts assays ... 30
4.14.5 Syngeneic tumour mouse models ... 31
4.14.6 PD-L2 KO mice ... 31
4.14.7 Aged animals ... 31
4.15 STATISTICAL ANALYSIS ... 31
5 RESULTS ... 32
5.1 PD-L2 GENE EXPRESSION IS UPREGULATED IN SENESCENT/DAMAGED CELLS IN VITRO INDEPENDENTLY FROM PD-L1 .. 32
5.2 CHEMOTHERAPY INDUCES PD-L2 GENE EXPRESSION IN VIVO ... 37
5.3 GENERATION OF PD-L2 KNOCKOUT CELLS USING CRISPR TECHNOLOGY ... 39
5.4 PD-L2 PROTEIN IS INDUCED IN DAMAGED/SENESCENT CELLS ... 41
5.5 PD-L2 MEDIATES IMMUNE EVASION OF SENESCENT CELLS ... 45
5.6 PD-L2 MEDIATED IMMUNE EVASION OF TUMOURS POST CHEMOTHERAPY ... 48
5.7 PD-L2 EXPRESSION INCREASES IN FIBROSIS ... 51
5.8 PD-L2 PROTECTS FROM ACUTE TISSUE DAMAGE ... 54
5.9 PD L2 AND PD-L1 GENE EXPRESSION INCREASES DURING AGING ... 55
6 DISCUSSION ... 56
6.1 SENESCENCE-INDUCING THERAPIES UPREGULATE PD-L2 EXPRESSION AND NOT PD-L1 ... 56
Index
6.2 PD-L2 MEDIATES IMMUNE EVASION OF DAMAGED CELLS ... 59
6.3 PD-L2 PROTECTS FROM ACUTE TISSUE INJURY ... 60
7 CONCLUSIONS ... 63
8 REFERENCE LIST ... 65
List of Abbreviations
1 List of Abbreviations
ANOVA Analysis of variance
BclXL B-cell lymphoma-extra large
Bleo Bleomycin
Bp Base pair
BSA Bovine serum albumin Bw Body weight
CAF Cancer-associated fibroblast CD Cluster of differentiation CDK4/6 Cyclin-dependent kinase 4/6 Col1a1 Collagen, type I, alpha 1
CRISPR Clustered regularly interspaced short palindromic repeats Ct Threshold cycle
Ctrl Control
D Days
DNA Deoxyribonucleic acid Doxo Doxorubicin
EDTA Ethylenediaminetetraacetic acid
FA Folic acid
FACS Fluorescence activated cell sorting FBS Fetal bovine serum
GAPDH Glyceraldehyde 3-phosphate dehydrogenase HDAC Histone deacetylase
HNSCC Head and neck squamous cell carcinoma I.p. Intraperitoneal injection
I.v. Intravenous injection IFNg Interferon Gamma
IgC-like Immunoglobulin like constant domain IgV-like Immunoglobulin like variable domain IHC Immunohistochemistry
IL Interleukin
IPF Idiopathic pulmonary fibrosis
KO Knockout
LINCS Library of Integrated Network-Based Cellular Signatures MAPK Mitogen-activated protein kinase
mRNA Messenger RNA
mTOR Mammalian target of rapamycin
NFkB Nuclear factor kappa-light-chain-enhancer of activated B cells NK Natural Killer
NKG2D Natural-killer group 2, member D OCT Optimal cutting temperature
List of Abbreviations
ORF Open reading frameOVA Ovalbumin
Palbo Palbociclib
PAM Protospacer adjacent motif PCR Polymerase chain reaction
PD-1 Programmed cell death protein 1 PD-L1 Programmed death-ligand 1 PD-L2 Programmed death ligand 2 PI3K Phosphoinositide 3-kinases RGMb Repulsive guidance molecule B RNA Ribonucleic acid
RPMI Roswell Park Memorial Institute
RT-qPCR Reverse transcription quantitative polymerase chain reaction SAbGAL Senescence-associated beta-galactosidase
SASP Senescence Associated Secretory Phenotype SD Standard deviation
SEM Standard error of the mean SgRNA single guide RNA
SpCas9 Streptococcus pyogenes CRISPR associated protein 9 STAT6 Signal transducer and activator of transcription 6 TBP TATA-Box Binding Protein
TGFb1 Transforming Growth Factor Beta 1 TIL Tumor-infiltrating lymphocytes UUO Unilateral ureteral obstruction
WT Wildtype
Introduction
2 Introduction
2.1 Relevance of research on senescence
The accumulation of senescent cells in multiple organs of the body is one of the hallmarks of aging (López-Otín et al., 2013). Moreover, the increase of these cells has been shown to be associated with various age-associated diseases, such as, but not limited to, lung fibrosis and kidney fibrosis (Muñoz Espín & Serrano, 2014). Recently, this field leaped forward by reports showing the causal role that senescent cells play in aging and progression of age-associated diseases, using sophisticated genetic models to specifically eliminate senescent cells. These results illustrated, that elimination of senescent cells leads to health-span extension and alleviate age-associated pathologies in mice (Baker et al., 2011). Those remarkable results led to development of a new class of drugs that specifically eliminates senescent cells, namely senolytics (Y. Zhu et al., 2015).
Noteworthy, the first preliminary results of clinical trials have been recently reported showing promising results in humans (Justice et al., 2019; Hickson et al., 2019). Various strategies have been developed and are still in development to eliminate senescent cells and translate these finding to humans.
2.2 Cellular Senescence
Senescence is a cellular stress response characterized by an inability to proliferate and by their abundant pro-inflammatory secretome (Hernandez-Segura et al., 2018).
Senescence can be induced by various means, such as DNA-damage, telomere attrition, or oncogene-expression to name a few (Hernandez-Segura et al., 2018). In general, the induction of senescence is regarded as a tumour suppressor mechanism to prevent the uncontrolled proliferation of pre-malignant cells that have accumulated damage or gained the expression of oncogenes (Serrano et al., 1997). The induction of the senescence program is mediated and executed by the cyclin-dependent kinase inhibitors p16Ink4a and p21Cip1/Waf1 as well as p53, but other mediators are also important,
Introduction
particularly for the pro-inflammatory phenotype, such as NFkB, and the kinases p38 and mTOR (Hernandez-Segura et al., 2018).
There are various methods to induce senescence in vitro and in vivo.
Chemotherapy induced-senescence using DNA-damaging agents, such as doxorubicin and bleomycin have been shown to be strong inducers of senescence (Schafer et al., 2017;
Demaria et al., 2017). Additionally, highlighting the important role of p16Ink4a in mediating senescence, CDK4/6 inhibitors mimicking the function of p16 have been developed and approved for cancer therapy, such as palbociclib (Leontieva &
Blagosklonny, 2013; Chen et al., 2018). Furthermore, drugs stabilizing p53, such as nutlin, also efficiently induce senescence (Efeyan et al., 2007). Therefore, treatment of mammalian cells with these compounds potently triggers senescence in vitro.
The best and most widely used assay to determine senescence is the senescence-associated beta-galactosidase staining (SAbGAL) (Dimri et al., 1995).
Senescence cells show a higher lysosomal content compared to non-senescent cells and thereby feature higher lysosomal enzymatic activities that can be used to detect senescence. Other assays to detect senescence consists of assessing unrepaired DNA-damage by gamma-H2AX and evaluating the lack of proliferative capacity (Hernandez-Segura et al., 2018). The induction of the DNA-damage response directly or indirectly constitutes a central event at the time of senescence induction and development leading to a persistent DNA-damage response which cannot be repaired.
Besides the permanent cell cycle arrest, senescent cells remain metabolic active and secrete a plethora of cytokines collectively called the senescence-associated secretory phenotype (SASP) (Coppé et al., 2010). The expression of these pro-inflammatory cytokines has led to the concept of chronic inflammation as a driver of senescence-associated pathologies (Franceschi & Campisi, 2014). The SASP is multifaceted and has been shown to depend on the cell type, senescence inducing stimulus and time (Coppé et al., 2010).
Senescence has been shown to be associated with various disease models and pathologies in vivo such as, but not limited to, kidney and lung fibrosis (Muñoz Espín &
Serrano, 2014). Upon acute damage, senescent cells also play a crucial role in orchestrating tissue repair, as shown by their role in optimal wound healing (Demaria et al., 2014). However, if the damage persists or cannot be adequately repaired senescent cells accumulate and contribute to disease progression. Therefore, senescence plays an
Introduction
important role in various contexts. Starting from the embryo, senescent cells are important in ensuring its optimal organ development (Muñoz Espín et al., 2013). In the adult, senescence is an important tumour suppressor mechanism and is crucial for optimal tissue regeneration (Demaria et al., 2014).
2.3 Senescence-associated secretory phenotype (SASP)
The composition of the senescence associated secretory phenotype is very complex. The SASP can be roughly divided into three arms important for the biological functions that senescent cells have in tissue regeneration. The first arm induces senescence in the neighbouring cells in a paracrine manner, thereby amplifying the biological response and signalling cascade. The second arm consists of pro-inflammatory cytokines and chemokines. This leads to the attraction of immune cells to resolve the damage and starts the cascade of the inflammatory response. The third arm is responsible for tissue remodelling to allow progenitors and stem cells to contribute to tissue regeneration upon injury. This cascade of tissue regeneration is tightly regulated and if it goes awry it impairs optimal organ development in the embryo, and in the adult, this could promote the delay in regeneration for example in wound healing (Demaria et al., 2014). Another illustration of the importance of senescence in tissue remodelling is the observation that the induction of reprogramming is tightly regulated by factors that senescent cells secrete as part of the SASP (e.g. IL-6) (Mosteiro et al., 2016). This model can be seen as a surrogate model of tissue repair fostering the hypothesis that senescent cells directly participate in tissue regeneration facilitating cellular plasticity and de-differentiation of neighbouring cells.
2.4 Strategies to eliminate senescent cells
Accumulation of senescent cells has been shown to impair the function of neighbouring cells (Coppé et al., 2010). Also, the persistence of the cells secreting pro-inflammatory cytokines has detrimental effects and signals a sterile inflammation (Tchkonia et al., 2013). Giving the role that senescence plays in aging and various pathologies, elimination
Introduction
of senescent cells is a highly desirable and recently has been pointed out as a promising strategy to treat age-related disorders and extend health-span. In this regard, in recent years two mouse models have been developed to specifically eliminate p16 expressing cells. (Baker et al., 2011; Demaria et al., 2014). The specific elimination of senescent cells leads to an increase in health span and alleviated age-related pathologies, illustrating the causal role that senescent cells play in disease initiation and disease progression of many age-related pathologies (Baker et al., 2011).
Two main therapeutic strategies have been developed to combat senescence. The first strategy is to target the SASP of senescent cells to reduce the sterile inflammation that these cells are signalling. Multiple pathways have been shown to regulate the SASP such as the NFkB, p38 MAPK, mTOR and JAK/STAT signalling (Chien et al., 2011; Laberge et al., 2015; Xu et al., 2015; Freund et al., 2011). Treatment of mice with inhibitors of these pathways alleviates the pro-inflammatory cytokine secretion of senescent cells.
However, such interventions require a continuous treatment and giving the crucial roles that these pathways have in tissue homeostasis and preventing infections, this therapeutic approach might not be ideal. Alternatively, an approach that would not require chronic treatments is to eliminate the underlying cause and induce the elimination of these senescent cells pharmacologically (Y. Zhu et al., 2015). These drugs are collectively known as senolytics, due to their ability to specifically destroy senescent cells. These drugs have been shown in mice to alleviate age-related diseases and to extend health-span to similar extend to the genetic mouse models of senescence cell elimination (Baker et al., 2011; Yousefzadeh et al., 2018; Y. Zhu et al., 2016; Y. Zhu et al., 2015). In recent years several vulnerabilities of senescent cells have been discovered and exploited. One therapeutic strategy takes advantage of the upregulation of the anti- apoptotic machinery that is characteristic of senescent cells. The BclXL inhibitor navitoclax induces apoptosis preferentially in senescent cells and has been shown to be effective in mouse models (Y. Zhu et al., 2016). Another approach is the inhibition of pro- survival pathways which renders senescent cells sensitive to apoptosis. An example of this approach is the combination of the plant flavonoid quercetin and the multi tyrosine kinase inhibitor dasatinib, which are thought to inhibit the pro-survival PI3K pathway (Y.
Zhu et al., 2015). Both approaches are currently in clinical trials and the first promising results have been published recently. The administration of quercetin and dasatinib in IPF patients leads to an increase in exercise performance (Justice et al., 2019). In a phase
Introduction
I trial of diabetic kidney disease quercetin and dasatinib treatment led to a decrease of senescent cells and proinflammatory cytokines (Hickson et al., 2019). An alternative approach to eliminate senescent cells takes advantage to high lysosomal load and beta- galactosidase activity in senescent cells to deliver cytotoxic drugs (Muñoz Espín et al., 2018). Our laboratory developed nanoparticles that are coated with oligosaccharides which get preferentially digested and their cargo released in senescent cells. This approach showed promising results in mouse models of cancer and pulmonary fibrosis (Muñoz Espín et al., 2018).
2.5 Senescence and cancer
A very important function of cellular senescence is to act as a tumour suppressor.
Severely damaged cells are prevented from dividing by activating the senescence program. Furthermore, the senescence program gets activated in cells that acquire oncogenic mutations and, in this manner, senescence prevents the outgrowth of pre-cancerous cells and the eventual formation of full-blown cancer (Serrano et al., 1997). Interestingly, as we age and more senescent cells accumulate the incidence of cancer increases as well. It is speculated that besides the cell-autonomous tumour suppressive role that senescence has, the accumulation of senescent cells may also facilitate tumour growth and invasiveness in a paracrine manner (Chan & Narita, 2019;
Krtolica et al., 2001).
Senescent cells have been identified within tumours corresponding either to the cancer cells themselves or to cells of the tumour microenvironment such as cancer-associated fibroblasts (T. Wang et al., 2017; Kim et al., 2018). The induction of senescence in cancer cells is especially prominent subsequent to traditional chemotherapy treatments. Due to their rapid proliferative rate, cancer cells are especially sensitive to DNA-damage induced apoptosis. However, a fraction of these cells will rather enter a senescent state in which the cells remain alive and participate in remodelling the tumour microenvironment (Ewald et al., 2010). This induction of senescence protects remaining viable cancer cells from further damage and stimulate tumour “regeneration”.
This tumour contributing function has been shown to be mediated by the SASP which provides pro-survival/proliferating signals to neighbouring non-senescent cancer cells
Introduction
(Rao & Jackson, 2016; Krtolica et al., 2001). This has been shown by experiments co- injecting proliferating cancer and either non-senescent or senescent fibroblasts. Whereas non-senescent cells do not form a tumour, the co-injection with senescent cells resulted in tumour formation illustrating the detrimental function these cells have on cancer development and progression (Krtolica et al., 2001). Furthermore, due to the effectiveness of senescence in halting proliferation, p16 mimicking drugs such as palbociclib or abemaciclib have been developed and are approved for the treatment of breast cancer (Sobhani et al., 2019). The use of this targeted pro-senescence therapy takes advantage of the desirable pro-inflammatory effect upon induction of the senescence program and helps to induce a physiological clearance of senescent cancer cells (Rao & Jackson, 2016). However, it has also been shown that if the clearance is not effective the remaining senescent cells have a detrimental effect and it is speculated that these cells contribute to tumour growth due to their SASP (Rao & Jackson, 2016). Another cell type in which the occurrence of senescence has been described are, cancer-associated fibroblasts (CAFs). In some instances, CAFs acquire a senescence-like phenotype to support tumour growth, metastasis spreading and cancer cell survival (Alspach et al., 2013).
To summarize; cellular senescence is an important tumour suppressor mechanism. However, if senescent cells are not rapidly cleared, they may have detrimental consequences through their SASP by promoting tumour growth in a paracrine manner.
2.6 Senescence and the immune response
Due to their pro-inflammatory secretome, senescent cells play a pivotal role in coordinating the immune response upon injury. This coordination is mediated by the SASP. Core components of the SASP consist of cytokines and chemokines involved in immune cell attraction/migration and differentiation. Under “normal conditions”
senescent cells are cleared by immune cells as part of the process of restoring tissue homeostasis. Senescent cells play a major role in modulating and regulating tissue regeneration and interact with the cells of the immune system, to ensure their self- elimination. This intercellular communication happens by regulating membrane proteins
Introduction
and by secreting cytokines and chemokines to attract and modulate immune populations (Prata et al., 2019).
Immune cells that have been shown to participate in the clearance of senescent cell consist of natural killer cells (NK-cells), macrophages and CD4+ T-cells, depending on the context (Sagiv et al., 2016; Krizhanovsky et al., 2008; Egashira et al., 2017; Kang et al., 2011). It has been shown that senescent cells express NKG2D ligands which label these cells for NK-cell mediated clearance (Sagiv et al., 2016). Another immune cell type that has been reported to eliminate senescent cells are macrophages. Elimination of macrophages in a model of limb regeneration prevented the clearance of senescent cells (Yun et al., 2015). In addition, it has been shown that CD4+ T-cells instruct macrophages to eliminate senescent hepatocytes (Kang et al., 2011). However, when things go awry, senescent cells are able to escape elimination by NK cells and macrophages. This results in the accumulation of senescent cells, which in turn, disrupts tissue homeostasis and organ functionality. Moreover, senescent cells also accumulate during the course of aging and it is still unclear how this phenomenon occurs (C. Wang et al., 2009).
Is it that during aging the rate of senescence induction increases? Or does immune clearance of senescence fades with aging? Or is it a combination of both? Finally, the interaction of the immune system and senescence is still poorly understood but it is tempting to speculate that if we interfere with molecules expressed by senescent cells we could elicit immunosurveillance of senescent cells.
2.7 PD-1 immune checkpoint blockade
A therapeutic approach that is revolutionizing cancer therapy is immunotherapy. The premise of this therapeutic approach is to interfere with immunosuppressive signalling pathways, thereby eliciting an immune response against the tumour. In recent years various pathways have been identified that allow cancer cells to evade their immune clearance. One of the most promising strategies consists of neutralizing/blocking antibodies to hinder binding of inhibitory ligands expressed by cancer cells to suppressive receptors expressed by immune cells thus keeping the inflammatory response against the tumour in check. A prominent example is the PD-1 pathway. The premise is that cancer cells evade their immune clearance by upregulating immune
Introduction
checkpoint inhibitors like PD-L1, thus deactivating the cytotoxic program of CD8+ T-cells.
Dr. Tasuku Honjo discovered the expression of the PD-1 receptor on activated T-cells in 1992, and, about 20 years later, the anti-PD-1 therapy was approved for melanoma (Ishida et al., 1992; Alsaab et al., 2017). Interfering with this pathway has resulted in remarkable preclinical animal studies leading to a complete eradication of tumours.
Interestingly, PD-1 has two ligands; PD-L1 and PD-L2. PD-L1 is extensively studied and has been also pursued as a therapeutic target (Fig.1). PD-L1 has been shown to be ubiquitously expressed and can be induced by IFNg on cells of various origins. Besides binding to PD-1, PD-L1 has also been shown to bind and inhibit the T-cell immunostimulatory ligand B7-1 (CD80) (preventing CD80 from activating the immunostimulatory receptor CD28) (Chaudhri et al., 2018). This is in stark contrast to PD-L2, which has not been deeply studied (Fig.1).
Figure 1. Publications per year for PD-1, PD-L1 and PD-L2.
Interfering with the PD-1 pathway is very effective in 30% of the melanoma patients, but there are still many patients that do not benefit from this therapy (Topalian et al., 2012).
So, there is an ongoing need to further understand why some patients benefit from immunotherapy, and how the patients get resistant to it, and finally, what would be the rational combinatory treatments to increase the response rate in cancer patients.
1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 0
500 1000 1500 2000 2500 3000 3500 4000
Year
# of publications per year
PD-1 PD-L1 PD-L2
T. Honjo discovers PD-1
Nobel Prize in
Physiology or Medicine FDA
approval of anti-PD-1 therapy for melanoma
Introduction
2.8 PD-L2
Programmed Cell Death 1 Ligand 2 also known as CD273, B7-DC or short PD-L2 (encoded by the (PDCD1LG2 gene) was identified in 2001 as a ligand for the PD-1 receptor by Gordon Freeman and colleagues (Latchman et al., 2001). PD-L2 is a member of the B7 family of the ligands that signal co-inhibitory or co-stimulatory signals to T-cells to modulate the immune response and prevent autoimmune diseases. In particular, the PD-1 receptor has been shown to be one of the main co-inhibitory pathways that mediate T-cell exhaustion. This inhibitory signalling pathway gets triggered on activated T-cells once the PD-1 receptor binds to its ligands PD-L1 (also known as CD274) or PD-L2. By that means, cancer cells commonly exploit this regulatory mechanism and upregulate PD-L1 to evade their immune clearance. However, the role of PD-L2 is still elusive. PD-L1 is expressed in cells of hematopoietic and non-hematopoietic origin whereas PD-L2 has been thought to be restricted to be expressed on dendritic cells and macrophages only (Latchman et al., 2001). However, recent reports showed that PD-L2 expression also occurs on cancer-associated fibroblasts suggesting PD-L2 has a broader role than initially thought (Costa et al., 2018).
PD-L2 is a type I transmembrane protein and the human gene compromises of seven (mouse six) exons. Both orthologs consist of a 5′-untranslated region in the first exon and the second exon encodes for the signal peptide. The third and fourth exons encode for the IgV-like and IgC-like extracellular domain, respectively. Exon five encodes for the transmembrane domain and a short cytoplasmic tail (four amino acids) in mouse followed by the sixth exon containing the 3′-untranslated region. Exon five of the human ortholog consists of transmembrane domain and exon six to the beginning of exon seven consists of the cytoplasmic tail (30 amino acids) and the rest 3′-untranslated region.
Interestingly, two splice variants have been described for PD-L2 (He et al., 2004). The first one is lacking the IgC-like domain resulting in an intracellular localization. The second splice isoform lacks the IgC-like domain and transmembrane domain resulting in a soluble isoform.
Intriguingly, it has been shown that PD-L2 has two to six times higher binding affinity to PD-1 than PD-L1 to PD-1, indicating that PD-1 might preferentially bind to PD-L2 rather than PD-L1 (Youngnak et al., 2003). Furthermore, PD-L2 and PD-L1 do not concurrently
Introduction
bind to PD-1, rather these two ligands compete for PD-1 binding (Ghiotto et al., 2010).
The reason why and the physiological relevance of this regulatory interaction remains still mysterious.
A common inducer of PD-L2 expression has been shown to be IL-4 (Loke & Allison, 2003).
Downstream of this cytokine, the signal transducer and activator of transcription 6 (STAT6) pathway has been shown to regulate PD-L2 expression as inferred from the observation that STAT6 KO macrophages fail to induce PD-L2 post stimulation with IL-4 (Huber et al., 2010). Consistent with the involvement of IL-4, PD-L2 has been shown to be important and protective in various disease models, such as experimental autoimmune encephalomyelitis, oral tolerance and helminth infection (Zhang et al., 2006;
B. Zhu et al., 2006; Stempin et al., 2016).
However, it should be noted that besides the immune suppressive role of PD-L2, also an immune stimulatory function has been described (Tseng et al., 2001). This immunostimulatory function has been shown not to be mediated through PD-1 but rather second receptor that binds to PD-L2 namely RGMb (Nie et al., 2018).
Objectives
3 Objectives
1. Document the association between PD-L2 upregulation and senescence, both in vitro and in vivo
2. Determine the role of PD-L2 in the ability of the immune system to clear cancer cells after chemotherapy
3. Determine the role of PD-L2 in the immune reactions elicited by tissue injury
Materials and Methods
4 Materials and Methods
4.1 Mammalian tissue culture
SK-Mel-103 (human melanoma), U2OS (human osteosarcoma) and Saos-2 (human osteosarcoma), HEK293T (human embryonic kidney) and C2C12 (mouse myoblasts) cells were obtained from American Type Culture Collection. UT-SCC-38, UT-SCC-42B, UT-SCC-2 cells (human head and neck squamous cell carcinoma) were provided by Dr.
Reidar Grenman (University of Turku, Finland). 360RIO 008 (human hypopharyngeal carcinoma) cells were prived by Dr. Alena Gros and Dr. Maria Abad (Vall d'Hebron Institute of Oncology, Spain). B16F10 cells (mouse melanoma) were provided by Dr.
María Soengas (Spanish National Cancer Research Center, Spain). B16-OVA: B16 (mouse melanoma) expressing ovalbumin (OVA) were provided by Dr. Federico Pietrocola (Institute for Research in Biomedicine, Spain). HCmel3 cells (mouse melanoma) were provided by Dr. Thomas Tüting (University of Bonn, Germany). Cells were routinely tested for mycoplasma contamination.
HcMel3 and 360RIO 008 cells were maintained in Roswell Park Memorial Institute (Gibco). The rest of the cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco). All media were supplemented with 10% fetal bovine serum (Gibco) with 1%
penicillin/streptomycin (Gibco). All cell lines were cultured at 37 °C in a humidified atmosphere and 5 % CO2 and procedures were conducted under aseptic conditions in a biological safety cabinet according standard operating procedure.
4.2 Senescence induction in vitro
Senescence was induced using 5 µM palbociclib (Pfizer Inc.) or 1 µM nutlin (Sigma) for indicated times. For DNA-damage induced senescence using doxorubicin (Sigma) and bleomycin (Sigma), cells were treated for 48 hours with indicated concentrations.
4.3 Gene expression analysis
Total RNA from adherent cells or homogenized tissue biopsies were isolated using TRI Reagent (Sigma-Aldrich) according to manufactures instructions. A total of 3-4µg of total RNA was reverse transcribed using the iScript advanced cDNA synthesis kit (Bio-Rad) according to manufactures instructions. Quantitative PCR of target genes (Table 1) was
Materials and Methods
performed using SybrGreen (Applied Biosystems) and ran on QuantStudio™ 6 Flex Real- Time PCR System using QuantStudio™ 6 and 7 Flex Real-Time PCR software v1.0 (Applied Biosystems). Ct´s over 37 were considered as gene not expressed. Relative gene expression levels were quantified by the comparative Ct (ΔΔCt) method using b-actin, GAPDH or human TBP as housekeeping genes, as indicated.
Table 1. Primers used for human (h) and mouse (m) target genes:
hPD-L1 fwd 5´-CAGCTGAATTGGTCATCCCAG-3´
hPD-L1 rev 5´-TCAGTGCTACACCAAGGCATA-3´
hPD-L2 fwd 5´-ACCAGTGTTCTGCGCCTAAA-3´
hPD-L2 rev 5´-CCTGGGTTCCATCTGACTTTGA-3´
hTBP fwd 5´-ATCAGTGCCGTGGTTCGT-3´
hTBP rev 5´-TTCGGAGAGTTCTGGGATTG-3´
hβ-actin fwd 5´-CAAGGCCAACCGCGAGAAGAT-3´
hβ-actin rev 5´-CCAGAGGCGTACAGGGATAGCAC-3´
mCol1a1 fwd 5´-AAGAATGGCGATCGTGGTGA-3´
mCol1a1 rev 5´-CTGGAGACCAGAGAAGCCAC-3´
mGAPDH fwd 5´-TTCACCACCATGGAGAAGGC-3´
mGAPDH rev 5´-CCCTTTTGGCTCCACCCT-3´
mIL-6 fwd 5´-GTTCTCTGGGAAATCGTGGA-3´
mIL-6 rev 5´-GGTACTCCAGAAGACCAGAGGA-3´
mp21 fwd 5´-GTGGGTCTGACTCCAGCCC-3´
mp21 rev 5´-CCTTCTCGTGAGACGCTTAC-3´
mPD-L1 fwd 5´-AGTCAATGCCCCATACCGC-3´
mPD-L1 rev 5´-TTCTGGATAACCCTCGGCCT-3´
mPD-L2 fwd 5´-TCATTGACCCTCTGAGTCGG-3´
mPD-L2 rev 5´-GGAAGATCAAAGCGATGGTGC-3´
mTGFb1 fwd 5´-CATGACATGAACCGGCCCTT-3´
mTGFb1 rev 5´-CGCACACAGCAGTTCTTCTC-3´
mβ-actin fwd 5´-GGCACCACACCTTCTACAATG-3´
mβ-actin rev 5´-GTGGTGGTGAAGCTGTAGCC-3´
Materials and Methods
4.4 Generation of cell pellets for IHC
Cells were harvested using PBS + 10 mM EDTA at 37ºC. Harvested cells were then centrifuged at 300xg and washed once with PBS. Cell pellets were then overlaid with 10%
buffered formalin (Sigma) and fixed for 10 hours at 4 °C. Formalin was then removed and cell pellets processed for IHC following standard procedures.
4.5 SAβGAL staining
SAβGAL staining´s of adherent cell lines, tissue sections or whole mount organs were performed using the senescence β-galactosidase staining kit according manufactures instructions (Cell Signaling). For SAβGAL staining’s of tissue sections, fresh isolated tissue biopsies were embedded in OCT (Leica Biosystems), cryosectioned and processed according to manufactures instructions with the modification that tissue slides were incubated for four hours at 37 °C in the staining solution. Whole mount SAβGAL staining’s were processed with the modification that tissues were fixed for 45 minutes and incubated in staining solution for 16 hours at 37 °C.
4.6 Immunohistochemistry
Immunohistochemistry was performed following standard operating procedures of the histopathology facility at the institute for research in biomedicine (IRB Barcelona). For PD-L2 staining’s the following protocol was established. Tissues were fixed in 10%
buffered formalin and embedded in paraffin. Tissue sections were then deparaffinized, rehydrated and washed with EnVision FLEX wash buffer (Dako). Antigen retrieval was performed using Tris-EDTA buffer (pH 9) at 97ºC for 20 minutes. After blocking endogenous peroxidase, slides were blocked with 5 % goat serum + 2.5 % BSA for 60 minutes. Slides were then incubated with anti-PD-L2 (Cell Signaling, clone D7U8C) diluted 1:25 in EnVision FLEX antibody diluent (Dako) over night at 4ºC. Next day, slides were incubated with anti-Goat-HRP for 45 minutes and developed for 10 minutes adding DAB. Slides were then dehydrated and mounted with DPX.
IHC analysis of SAβGAL and sirius red staining’s was performed using Qupath (v0.1.2) using the positive pixel count feature with empirical parameters.
Materials and Methods
4.7 Flow cytometry
Cells were harvested in PBS + 10 mM EDTA at 37ºC. Collected cells were then stained with yellow live/dead dye solution (Invitrogen) for 15 minutes at 4ºC and washed. Cells were then stained with anti-PD-L2-biotin (Miltenyi Biotec, clone MIH18) diluted 1:11 in FACS buffer (0.5% BSA, 2mM EDTA in PBS) for 15 minutes after two washes the samples were incubated with anti-biotin-APCVio770 (Miltenyi Biotec), diluted 1:50 in FACS buffer for 15 minutes. After repeated washes cells were filtered using a 70 µM cell strainer and analysed on a Gallios flow cytometer and FlowJo 10.0.7.
4.8 Molecular biology techniques
4.8.1 PD-L2 ORF
Total RNA from C2C12 cells were isolated using TRI Reagent (Sigma) according to manufactures instructions. A total of 5 µg was reverse transcribed using the ProtoScript First Strand cDNA Synthesis Kit (NEB). cDNA was then used for PCR with 0.2 mM dNTP, 1 μM of fwd 5´-ATGCTGCTCCTGCTGCCGAT-3´ and rev 5´-CTAGATCCTCTTTCTCTGGAT-3´
primers and 1 U of BIOTAQ DNA polymerase (Ecogen). After 35 cycles of amplification, the PCR products were separated by electrophoresis on a 2 % agarose gel. PCR bands were gel purified using QIAquick gel extraction kit (Qiagen) according to manufactures instructions. Isolated PCR fragments were then sent for sequencing to the DNA sequencing facility at the CNIO.
Materials and Methods
4.8.2 CRISPR
For PD-L2 knockouts, mouse and human sgRNAs were designed (Table 2) using the CHOPCHOP web tool: (http://chopchop.cbu.uib.no) and cloned into pSpCas9(BB)-2A- Puro (PX459) (Addgene #48139) and lentiCRISPRv2 (Addgene #52961). Plasmids were isolated using Midi-preps (Qiagen) according manufactures instructions.
Table 2. sgRNAs targeting human (h) or mouse (m) PD-L2 gene
hPD-L2 sgRNA#1 fwd 5´-CACCGTTCTGGAATACTCACGTGA-3´
hPD-L2 sgRNA#1 rev 5´-AAACTCACGTGAGTATTCCAGAAC-3´
hPD-L2 sgRNA#2 fwd 5´-CACCGACTTGAGGTATGTGGAACG-3´
hPD-L2 sgRNA#2 rev 5´-AAACCGTTCCACATACCTCAAGTC-3´
hPD-L2 sgRNA#3 fwd 5´-CACCGCCAATGCATAATCATCTATG-3´
hPD-L2 sgRNA#3 rev 5´-AAACCATAGATGATTATGCATTGGC-3´
hPD-L2 sgRNA#4 fwd 5´-CACCGTTGCAGCTTCACCAGATAGC-3´
hPD-L2 sgRNA#4 rev 5´-AAACGCTATCTGGTGAAGCTGCAAC-3´
mPD-L2 sgRNA fwd 5´-CACC GAAGTGTACACCGTAGACGT-3´
mPD-L2 sgRNA rev 5´-AAACACGTCTACGGTGTACACTTC-3´
4.8.3 PCR of genomic DNA
Genomic DNA was isolated using the DNeasy blood and tissue kit (Qiagen) following manufactures instructions. 500ng of genomic DNA with 0.2 mM dNTP, 1 μM of primers hybridizing to intronic regions spanning exon 3 of PD-L2 (Table 3) and 1 U of BIOTAQ DNA polymerase (Ecogen). After 35 cycles of amplification, the PCR products were separated by electrophoresis on a 1 % agarose gel. PCR bands were gel purified using QIAquick gel extraction kit (Qiagen) according to manufactures instructions. Isolated PCR fragments were then sent for sequencing to Eurofins Genomics and analysed using Serial Cloner V2.6.1.
Table 3. Primers used for PCR of exon 3 of human and mouse PD-L2 gene
hPD-L2 exon3 fwd 5´-ATAAGACAGGTGCCTTTTGGAA-3´
hPD-L2 exon3 rev 5´-GGACTAATTTTCCTGGCTTCCT-3´
mPD-L2 exon3 fwd 5´-TTTTAAAGGCGGTAACAATGCT-3´
mPD-L2 exon3 rev 5´-TAGGGCCTGACTTTAATTCCAA-3´
Materials and Methods
4.9 Transfections
Cells were transfected with pSpCas9(BB)-2A-Puro (PX459) (Addgene #48139) using FuGENE6 (Promega) following manufactures recommendations. A total of 3 µg of sgRNA containing PX459 plasmid were transfected. After three days, successfully transfected cells were selected using puromycin (Merck). Cells were then either used in bulk or single clones were isolated by plating 0.5 cells per well in a 96 well plate until colony formation.
Successfully generated knockouts from single cell colonies were then assessed by sequencing of sgRNA targeted exons, IHC and flow cytometry for genome editing and PD- L2 protein expression respectively.
4.10 Generation of lentiviruses
Lentiviruses were produced by transfecting HEK293T cells with p8.91 (gag-pol expressor), pMDG.2 (VSV-G expressor) and and lentiCRISPRv2 plasmid (Addgene
#52961) following standard procedures. Virus batches were harvested 48, 72 and 96 hours after transfection. Cellular debris was removed by centrifugation and filtering.
Virus containing supernatant was then used fresh or stored by snap-freezing aliquots supplemented with 8 μg/ml polybrene (Fisher Scientific) in ethanol/dry ice and stored at -80°C.
Recipient cells were incubated for eight hours with lenti-virus containing supernatant and selected with puromycin (Merck) three days later. Generated PD-L2 KO cells were then used as bulk.
4.11 B16-OVA and OT1 co-culture
B16-OVA cells were transfected and selected with pSpCas9(BB)-2A-Puro (PX459) (Addgene #48139) plasmid containing sgRNA targeting mouse PD-L2. After selection cells were harvested and genome editing confirmed by sequencing of exon 3. B16-OVA WT and B16 OVA PD-L2 KO bulk population was then used for co-culture experiments.
For doxorubicin pre-treatment 0.03x106 cells were plated in 24 well plates and treated the following day for 72hrs with 350 nM doxorubicin (Sigma). 0.03x106 cells control B16- OVA WT and B16 OVA PD-L2 KO bulk population were plated 24 hours before start of the co-culture. OT1 (C57BL/6-Tg(TcraTcrb)1100Mjb/J) mice were purchased from Jackson (Stock No:003831). OT1 splenocytes were activated in vivo by injecting 25 µg of OVA 257-
Materials and Methods
264 peptide (i.p.). Next day mouse was sacrificed and splenocytes isolated. Splenocytes were cultured 24 hours at 1x106 cells/ml in RPMI (Gibco) supplemented with 10% FBS (Gibco), 1% glutamine (Life Technologies), 50 µM 2-mercaptoethanol (Life Technologies) and 20ng/ml IL-2 (PeproTech). Next day serial dilutions of OT1 splenocytes were added to cells. After 24 hours co-culture, remaining adherent cells were stained with 0.5 % crystal violet staining solution (Sigma).
4.12 Patient derived co-culture model
Patient derived co-culture experiments were performed in Dr. Alena Gros´s and Dr. Maria Abad´s laboratory at the VHIO by Olga Boix Sanchez and Andrea Garcia Garijo. The 360RIO 008 cancer cell line and concurrently autologous reactive and non-reactive tumour infiltrating lymphocytes (TILs) were isolated and established from a hypopharyngeal carcinoma patient. The 360RIO 008 cancer cell line was then subsequently used to generate PD-L2 KO cells by lentiviral infection of lentiCRISPRv2 plasmid (Addgene #52961) containing sgRNA#2 against the human PD-L2 gene.
Successfully infected cells were selected using puromycin (Merck) and bulk population was tested for PD-L2 KO by flow cytometry and used for co-culture experiments. To induce senescence 360RIO 008 and 360RIO 008 PD-L2 KO cells were pre-treated for seven days with 6 mU bleomycin (Sigma). 360RIO 008 and 360RIO 008 PD-L2 KO control or bleomycin treated cells were co-cultured with reactive or non-reactive TILs at a ratio 1:0.4 (cancer cells : TILs). After 20 hours T-cell activation was assessed by flow cytometry using 4-1BB staining.
4.13 Drug class enrichment analysis
The drug class enrichment analysis has been performed by the group of Dr. Fátima Al- Shahrour by Dr. Héctor Tejero at the bioinformatic unit at CNIO. Datasets from the LINCS project (http://www.lincsproject.org/) were analysed and differential expression of PDCD1LG2 were detected by comparing control and treated samples introducing a correction by cell line and batch effect. 4690 drugs with common names were then further processed and categorized into drugs sets reflecting their mode of action. The resulting drug set enrichment analysis was then compared to a drugset consisting of random drugs.
Materials and Methods
4.14 Animal experimentation
Animal experimentation at the CNIO was performed according to protocols approved by the CNIO-ISCIII Ethics Committee for Research and Animal Welfare and the CEIyBA.
Animal experimentation at the IRB was performed according to protocols approved by the Comitè Ètic Experimentació Animal and the Generalitat de Catalunya.
4.14.1 Folic acid induced kidney fibrosis
Kidney fibrosis was induced in male C57BL6, PD-L2WT and PD-L2KO mice by injecting a single high dose of folic acid dissolved in NaHCO3 250ug/g of body weight (i.p.) or vehicle.
Animal welfare were monitored daily and animals sacrificed when humane endpoint was reached or 28 days post folic acid treatment.
4.14.2 Unilateral ureteral obstruction (UUO)
Kidney fibrosis was induced in male C57BL6 mice by the surgical obstruction of the left ureteral conduct (UUO). Mice were sacrificed 12-13 days post obstruction. The contralateral kidney was used as control.
4.14.3 Bleomycin induced lung fibrosis
Lung fibrosis was induced in male C57BL6, PD-L2WT and PD-L2KO mice by intratracheal inoculation of bleomycin (1.5 U/kg) or vehicle (PBS). Bodyweight and animal welfare were monitored daily and animals sacrificed when humane endpoint was reached or 14 days post bleomycin treatment.
4.14.4 Xenografts assays
SK-Mel-103 WT (1x106) or SK-Mel-103 PD-L2 KO cells were subcutaneously inoculated in female immunodeficient nu/nu mice. When tumours reached an average size of 200 mm 3, mice were treated with vehicle (50 mM sodium lactate, pH 4.0) or palbociclib 100 mg/kg by oral gavage on weekdays for up to 14 days. Tumour growth was monitored by caliper measurement and tumour volume calculated using the formula Volume = (width)2 x length/2.
Materials and Methods
4.14.5 Syngeneic tumour mouse models
0.4x106 HCmel3 cells were subcutaneously injected into male C57BL6 mice. Four weeks later mice were treated bi-weekly with 5mg/kg doxorubicin (i.v.) for a total of three times. 0.2x106 B16F10 cells were subcutaneously injected into male C57BL6 mice. Seven days later mice were treated bi-weekly with 5mg/kg doxorubicin (i.v.) for a total of three times.
0.2x106 B16OVA WT or B16OVA PD-L2KO cells were subcutaneously injected into male C57BL6 mice. Mice were treated then on day seven and day 10 with 5mg/kg doxorubicin (i.v.) and on day 17 with 5mg/kg doxorubicin (i.p). Tumour growth was monitored by caliper measurements and tumour volume calculated using the formula Volume = (width)2 x length/2. Mice were sacrificed two days after last treatment.
4.14.6 PD-L2 KO mice
PD-L2 knockout mice(B6N.129(Cg)-Pdcd1lg2tm2Dmp/J) were purchased from Jackson (Stock No: 017515). These mice had an unintendedly remaining neomycin cassette. This cassette was removed by crossing these mice with Sox2-Cre provided by Dr. Travis Stracker (IRB, Barcelona). The resulting PD-L2WT and PD-L2KO mice with a 129/B6 background were then subsequently used for experiments presented in this study.
4.14.7 Aged animals
Tissue samples from young (2-4 month) and old (18-24 month) female mice were collected and lysed in TRI Reagent (Sigma) according to according to manufacturer's instructions. Total RNA was then processed for gene expression analysis.
4.15 Statistical analysis
Data are represented as mean values and the error bars indicate ± standard deviation (SD) or standard error of the mean (SEM) as indicated. Statistical significance was assessed using unpaired two-tailed Welch’s t-test or ANOVA. Significance is indicated as
*P ≤ 0.05; **P ≤0.01; *** P ≤ 0.001; **** P ≤ 0.0001.
Results
5 Results
5.1 PD-L2 gene expression is upregulated in senescent/damaged cells in vitro independently from PD-L1
How senescent cells interact with and modulate cells of the immune system remains elusive. It is well established that cells undergoing senescence are highly secretory (Coppé et al., 2008). The various cytokines and chemokines that senescent cells express could potentially modulate the immune cell compartment (Prata et al., 2019). However, an additional mechanism how senescent cells can interact with and instruct their microenvironment is through alterations of proteins at the cellular surface. One prominent example is the expression of NKG2D ligands which are expressed on hepatocytes undergoing oncogene-induced senescence. The expression of these ligands activates NK-cells leading to the elimination of senescent cells (Sagiv et al., 2016).
In order to deepen our understanding of how senescent cells can interact with their microenvironment a mass spectrometry analysis of membrane proteins was performed in Dr. Serrano´s laboratory. To find proteins differentially expressed by senescent cells; the human melanoma cell line SK-Mel-103 was treated with various pro-senescence stimuli that induce senescence potently and reliably. Seven days post-induction with palbociclib (a CDK4/6 inhibitor) or doxorubicin (a DNA-damaging agent) cells were harvested and protein lysates enriched for membrane proteins processed for analysis by mass spectrometry. This analysis of the cellular membrane composition of senescent and non-senescent control cells were performed independently by Susana Llanos, PhD and Federico Pietrocola, PhD, at the Proteomics Unit of the CNIO in Madrid. Among the various proteins differentially expressed by senescent cells, a particular protein attracted our attention. In both, independently performed experiments, PD-L2 was identified to be highly upregulated at the cellular membrane in response to senescence induction by using either palbociclib or doxorubicin (data not shown).
Whereas significant effort has been made to understand the biology of PD-1 and PD-L1, the alternative ligand PD-L2 is less studied and information is scarce. Moreover, PD-L1 was not differentially expressed at the cellular surface upon senescence induction
Results
(data not shown). So, we deemed it would be interesting to further investigate the role of PD-L2 in the context of cellular senescence.
To validated the observed upregulation of PD-L2 in senescent cells identified in the mass spectrometry analysis, we first assessed the transcriptional induction of PD-L2 and PD-L1 in human cancer cell lines using a variety of different cellular stressors that have been shown to induce senescence. Senescence was induced in the human melanoma cell line SK-Mel-103 using palbociclib, doxorubicin, bleomycin or nutlin. These drugs potently induce senescence as reflected by the positive SAβGal staining and flattened cell morphology (Fig. 2A). Seven days post-induction, we assessed PD-L2 and PD-L1 expression levels by RT-qPCR (Fig. 2B). These results show that pro-senescence therapies consistently induce PD-L2 whereas PD-L1 expression is marginally induced.
Furthermore, other human cancer cell lines such as head and neck cancer cell lines UT-SCC-38 and UT-SCC-42B which also undergo senescence with palbociclib (Fig. 2C) also strongly upregulate PD-L2 and to a minor extent PD-L1 after seven days of treatment (Fig. 2D).
Results
Figure 2. Chemotherapeutic drugs upregulate PD-L2 expression and not PD-L1 in human cancer cell lines.
(A) SK-Mel-103 cells were treated with 5 µM palbociclib, 10 nM doxorubicin, 1 µM nutlin or 6 mU bleomycin. Seven days post treatment start, cells were harvested for SAβGal staining. (B) SK-Mel-103 cells were treated as in (A) and PD-L2 and PD-L1 gene expression was evaluated compared to β-actin. (C) SAβGal staining of UT-SCC-38 and UT-SCC-42B treated for 7 days with 5 µM palbociclib. (D) PD-L2 and PD-L1 gene expression compared to β-actin in UT-SCC-38 and UT-SCC-42B treated for 7 days with 5 µM palbociclib.
Data represents mean ± standard deviation (SD). N=2-5. Where applicable: *P ≤ 0.05; **P ≤0.01; ***; P ≤ 0.001 (unpaired two-tailed Welch’s t-test)
To confirm the above data, we used a mouse cell line (C2C12 myoblasts) and a pair of PCR primers that amplify the entire open reading frame (ORF) of the PD-L2 transcript.
We did not see basal expression of the PD-L2 transcript in the murine C2C12 myoblast cell line (Fig. 3A). However, cells treated with doxorubicin for 48 hours presented amplification of two PCR products (Fig. 3A). The upper band at the expected size of 744 bp and a faint smaller band below. Sequencing of both PCR amplicons identified the upper band at the size of 744bp as the complete ORF of PD-L2 confirming our results generated by RT-qPCR. The bottom band at a shorter size was identified as a non-specific amplification.
Palbociclib
Bleomycin Nutlin Control
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Bleo n
Nutlin n
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PD-L1 PD-L2
SK-Mel-103
SAbGal ControlPalbociclib
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Palbo n
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PD-L2 PD-L2
PD-L1 PD-L1
Results
Giving the specific induction of PD-L2 (and not PD-L1) with DNA-damage we next wondered if a known inducer of PD-L1 would also stimulate PD-L2 expression. For this, we treated murine melanoma B16F10 cells with interferon gamma (IFNγ), a well-known inducer of PD-L1 expression (Loke & Allison, 2003). Consistent with other reports, IFNγ potently induces the expression of the PD-L1 transcript, however PD-L2 expression was not affected by IFNγ treatment (Fig. 3B). The opposite was true when we treated B16F10 cells with doxorubicin; PD-L2 mRNA was induced in response to DNA damage, whereas PD-L1 mRNA levels remained unaffected (Fig. 3B). Interestingly, non-chemotherapy treated cells do not express basal PD-L2 levels (Fig. 3A, 3B)
Figure 3. PD-L2 is induced by DNA-damage and its regulation is different from PD-L1.
(A) PCR of PD-L2 ORF in C2C12 treated with 500 nM doxorubicin for 48 hours. (B) B16F10 cells were treated with either 350 nM doxorubicin or 100ng/ml IFNγ for 48 hours. Cells were harvested and PD-L2 and PD-L1 gene expression compared to β-actin by RT-qPCR. Data represents mean ± standard deviation (SD). N=3. RT-qPCR Cts over 37 were considered as gene not expressed. **P ≤0.01; ***P < 0.001 (one-way ANOVA)
These results suggest that PD-L2 is regulated independently from PD-L1 activation. On one hand PD-L2 is upregulated as part of the DNA-damage response and during the time course of senescence development, PD-L1 on the other hand is not.
We then wondered whether the upregulation of PD-L2 can be generalized to other types of cellular damage. In order to do so, we collaborated with the team of Fátima Al- Shahrour, PhD, from the bioinformatic unit at the CNIO. Héctor Tejero, PhD, analysed the publicly available LINCS consortium datasets consisting of gene expression datasets of eight human cancer cell lines treated with 4690 drugs. These datasets were analysed and a drug set enrichment analysis performed. This kind of analysis allows to evaluate which classes of drugs significantly change PD-L2 expression levels. The analysis revealed that compared with a random drug-set, HDAC inhibitors and epigenetics as drug classes
Ctrl Doxo IFNg
0 10 20 30 40 50
PD-L１
Ctrl Doxo IFNγ mRNA levels normalized to b-actin
Ctrl Doxo IFNγ
PD-L1 PD-L2
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PD-L2 ORF
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