C-V2X (Cellular Vehicle-to-Everything) is a state-of-the-art wireless technology used in autonomous driving and intelligent transportation systems (ITS). This technology has extended the coverage and blind-spot detection of autonomous driving vehicles. Economically, C-V2X is much more cost-effective than the traditional sensors that are commonly used by autonomous driving vehicles. This cost-benefit makes it more practical in a large scale deployment. PC5-based C-V2X uses an RF (Radio Frequency) sidelink direct communication for low latency mission-critical vehicle sensor connectivity. Over the C-V2X radio communications, the autonomous driving vehicle’s sensor ability can now be largely enhanced to the distances as far as the network covers. In 2020, 5G is commercialized worldwide, and Taiwan is at the forefront. Operators and governments are keen to see its implications in people’s daily life brought by its low latency, high reliability, and high throughput. Autonomous driving class L3  or L4 (Highly Automation) are good examples of 5G’s advanced applications. In these applications, the mobile networks with URLLC (Ultra-Reliable Low-Latency Communication) are perfectly demonstrated. Therefore, C-V2X evolution and 5G NR (New Radio) deployment coincide and form a new ecosystem. This ecosystem will change how people will drive and how transportation will be managed in the future. In this paper, the following topics are covered. Firstly, the benefits of C-V2X communication technology. Secondly, the standards of C-V2X and C-V2X applications for automotive road safety system which includes V2P/V2I/V2V/V2N, and artificial intelligence in VRU (Vulnerable Road User) detection, object recognition and movement prediction for collision warning and prevention. Thirdly, PC5-based C-V2X deployment status in global, especially in Taiwan. Lastly, current challenges and conclusions of C-V2X development. V2V (Vehicle-to-Vehicle): between vehicle and vehicle, e.g., for keeping safe distance, speed, and lane changing.V2I (Vehicle-to-Infrastructure): between vehicle and road infrastructure, e.g., road sign, traffic light, toll gantry.V2P (Vehicle-to-Pedestrian): between vehicle and pedestrian, e.g., sensing nearby person or cyclist.V2N (Vehicle-to-Network): communication between vehicle and network, e.g., infotainment from the Internet, sending vehicle mechanical performance to its automaker.According to the statistics, 1.35 million people die in road accidents worldwide every year, equivalent to 3700 deaths per day . C-V2X iC-V2X uses 3GPP The 3rd Generation Partnership Project) 4G (The fourth-generation) LTE (Long-Term Evolution) or 5G (The fifth generation) NR (New Radio) connectivity to transmit and receive signals. It uses two complementary transmission modes. The first is direct communications to vehicles, infrastructures, and road pedestrians. In this mode, C-V2X works independently of the cellular networks. It uses a PC5 interface for communication. The second mode is cellular network communications, in which C-V2X employs the conventional mobile network to enable vehicles to receive information about road and traffic conditions in the area. It uses Uu interface for communication.With the evolution and deployment of C-V2X technology, fatal accidents due to human errors or road conditions and massive traffic jams due to special occasions or accidents will no longer be a problem. Detecting risks by C-V2X V2V and V2P before they become a threat and noticing traffic jams by C-V2X V2I and V2N before you see them will be coming soon. This vision of safer roads and more efficient travel can be realized by C-V2X, Intelligent Transport Systems, and 5G together.In this article, we first describe the benefits of C-V2X and why it has been gaining popularity in recent years. We also describe the specifications of C-V2X in LTE and NR defined by 3GPP and introduce a few application scenarios to show that C-V2X can provide safer roads, more efficient travel, and better driving experiences. Moreover, we investigate worldwide C-V2X deployment progress. This work contains many technical research works and insights from a C-V2X technical expert from Nokia who designed a series of workable system integration solutions that have been deployed in some pilot and commercial C-V2X projects. This pioneering study discusses C-V2X technology applied in practice, and all the test results and conclusions come from real C-V2X communication system integration, not simulation tools. These projects are to be introduced one by one in a later chapter. Finally, the C-V2X challenges in the current ecosystem are discussed. We expect that through this work, we can promote C-V2X technology and take it forward.C-V2X with low latency and high-reliability allows vehicles to communicate with other vehicles (V2V), pedestrians (V2P), roadside infrastructure (V2I), and networks (V2N) with or without a cellular network  and addrC-V2X can aggregate information collected in the cooperative perception, update the map with exact road structure information, and distribute the localized HD (High Definition) map to vehicles based on their locations. These enhanced and advanced services such as blind-spot detection, long-range perception, remote driving and platooning, etc. C-V2X can increase road capacity, driver safety, and comfort. All the C-V2X benefits for autonomous driving are summarized in Early V2X technologies, like DSRC (Dedicated Short Range Communication) in the USA and ETSI (European Telecommunications Standards Institute) ITS-G5 in Europe, are based on IEEE  802.11p, which adds wireless access in vehicular environments (WAVE) at the physical layer and the medium access control (MAC) layer. Currently, 802.11bd is under development as the next-generation standard. DSRC has since been used in many countries for V2V, V2P, and V2I applications. But it has not been commercialized on a large scale for almost two decades . FurtherWorldwide forecast for the number of vehicles equipped with V2X technology is shown  in FigurIt is worth mentioning that DSRC and C-V2X technologies are addressing similar use-cases. They have an identical network, security, and application layers; the only difference is in the access layer. Therefore, some companies ,9 are deThe evolution of C-V2X standardization in the 3rd Generation Partnership Project (3GPP) and deployment milestone in 5G Automotive Association (5GAA)  with chiPlatooning: Vehicles dynamically form a platoon traveling together. All vehicles in the platoon exchange information that enables them to maintain a short distance safely.Extended Sensors: Exchange of raw or processed sensor data among vehicles, roadside units, devices of pedestrians, and V2X application servers, enhancing environment perception beyond what sensors can detect .Advanced Driving: Enable semi-automated or fully-automated driving. Perception data obtained from local sensors and driving intention exchanged with vehicles in proximity to synchronize and coordinate.Remote Driving: Remote driver or V2X application operates remote vehicle .Completed in March 2017, phase 1 of 3GPP Rel-14 provided the initial standards supporting V2V services and the additional V2X services leveraging the cellular infrastructure. The primary safety features of C-V2X under 3GPP Rel-14 were delivered through cellular networks or sidelink communications via the PC5 interface. A new LTE (Long-Term Evolution)-V2X band 47 (with 10 and 20 MHz bandwidth) was introduced to support C-V2X by unlicensed 5.9 GHz spectrum. 3GPP Rel-14 also introduced two new physical channels for PC5-based C-V2X communication, namely the Physical Sidelink Shared Channel (PSSCH) and Physical Sideline Control Channel (PSCCH). As the name discloses, PSSCH carries data, and PSCCH has control information for decoding the data channel in the physical access layer. To accelerate the LTE-V2X development, LTE-V2X uses the centralized scheduling mode (Mode 3), and decentralized scheduling mode (Mode 4)  of LTE-D5G NR (New Radio)-V2X as phase 3 of 3GPP V2X is backward compatible at upper layers of LTE-V2X. To meet the low latency and reliability requirements for the advanced V2X services, NR-V2X is designed to support these applications. As a V2N type application, 5G URLLC (Ultra-Reliable Low-Latency Communication) network slicing can provide autonomous driving advanced functions for L3  and L4 (Highly Automation) of Some of these advanced application scenarios require the transmission of periodic traffic. So besides broadcast, two new communication types, unicast and groupcast, are introduced in 5G NR-V2X. Similar to LTE-V2X, 5G NR-V2X  also defRSU (Road Side Unit) and OBU (On Board Unit) are sufficient to deploy C-V2X V2I/V2V/V2P application scenarios:RSU: Wireless transmitting device can provide direct sildelink communication via PC5 interface without cellular networks. Road signs, traffic lights, and IP camera information can be broadcasted in real-time to the vehicle via RSU in the predefined area. Another useful scenario is that RSU can be equipped with a SIM card to transfer road information via cellular networks to create additional applications for public safety.OBU: Installed in a vehicle as a wireless communication device to enhance autonomous driving vehicles’ sensor function by direct communication with RSU and other OBUs. OBU is responsible for broadcasting vehicle’s location, direction, and speed information to other defined devices while receiving other vehicle’s data as input for its internal algorithms to avoid possible accidents.Since PC5-based C-V2X Mode4  does notSPAT : a V2I service that integrates traffic signal controller (light color and remaining time) with long-range wireless transmission device RSU, which broadcasts them to the OBU. Depending on this information, the driver or autonomous driving control unit can decide whether to change the route or speed up.TSP : a V2I service that allows high-priority vehicles, such as ambulances, fire trucks, and police cars approaching the signal control intersection to send a priority signal so that the vehicles can pass through.VRUCW (Vulnerable Road User Collision Warning): a V2P service that alerts the driver or autonomous driving control unit when a vehicle is sensing a potential pedestrian collision threat via IP Camera and RSU on the roadside.ICW (Intersection Collision Warning): a V2V service that warns the host vehicle driving towards an intersection if there is a risk of collision.EBW (Emergency Brake Warning): another V2V service, which warns the host vehicle if the front remote vehicle is making a sudden brake. The host vehicle receives an alert from the front vehicle and determines if a collision will happen.DNPW (Do Not Pass Warning): a V2V service used when a host vehicle is planning to overtake the front vehicle via opposite direction lane. The host vehicle will alert the nearby vehicle traveling in the opposite direction. The host vehicle’s OBU will receive a DNPW message to decide if it is safe to overtake.HLW (Hazardous Location Warning): a V2I service that warns a host vehicle of potentially hazardous situations, such as deep waters after a storm, deep pits on the road surface, or slippery roads.To utilize C-V2X applications, RSU/OBU devices must be equipped with 3GPP C-V2X  is from All the above-described application scenarios can be deployed by using PC5-based C-V2X direct communication technology. However, the 4G LTE cellular network can’t support advanced C-V2X applications due to limited performance. 5G NR has opened an opportunity for time-sensitive applications. The future is inspiring for C-V2X once full 5G coverage is available.Even 802.11p based V2X technology was early adopted by some global automotive manufacturers as an electronic toll collection technology because C-V2X demonstrates a forward evolutionary path towards 5G. 5GAA  started Considering the current 3GPP releases and supply chain readiness, 5GAA even developed a long-term blueprint of traffic efficiency and basic safety C-V2X use cases worldwide in September of 2020  as shownAs a result, C-V2X has gained momentum in the United States, Europe, Australia, China, Japan, Korea, and other markets. C-V2X is dominating globally, and Taiwan is one of the countries at the forefront of C-V2X development supported by the government in the intelligent transportation systems programs .In this chapter, some Taiwan field tests and commercial projects, whose C-V2X networks are implemented by network solution provider Nokia, are introduced. As a C-V2X technical expert, the author from Nokia provides a sequence of PC5-based C-V2X system integration solutions. (1) Translating traffic light control signaling into C-V2X internal massages recognized by RSU/OBU to realize SPAT application. Normally, autonomous driving vehicles are equipped with cameras and artificial intelligence that can recognize traffic light information. However, recognition accuracy is easily affected by bad weather or occlusions. Our proposed solution is designed to be more robust against any conditions that can hinder visual recognition. (2) Utilizing AI technology, which has shown state-of-the-art performance in many problem domains ,25,26, fSolution1: Traffic Light Control System IntegrationTo realize SPAT application locally, we have designed a system architecture shown in We can collect traffic light information from the light controller directly.Light signaling collection program is responsible for receiving roadside light signal information, including light phase, color and remaining time, etc. All this information is sent to RSU.RSU reads and encapsulates them to C-V2X protocol messages.RSU broadcasts C-V2X messages to OBU via PC5 interface.OBU installed in the autonomous driving vehicle analyzes and filters these messages and then sends them to IPC  of autonomous driving system for slow or stop control.User Interface (UI) displays light information for intuitive C-V2X technology presentation.Solution2: C-V2X VRUCW Application System IntegrationFor the PC5-based C-V2X VRUCW application, we have developed a network diagram shown in The VRUCW application can be regarded as P2I2V service ( Pedestrian to Infrastructure to Vehicle). The IP cameras must be built on road areas for both line-of-sight (LOS) and non-line-of-sight (NLOS) monitoring purposes.It uses an AI server equipped with a series of deep learning technologies such as CNN  and SSD (Single Shot Detection). Objects will be detected if any pedestrian passes an area under camera coverage.The AI server transfers the analysis result with object recognition and movement prediction to RSU, which broadcasts this information to all the OBUs under the coverage of this RSU.OBU is responsible for combining vehicle information such as speed, heading, and location to determine if a collision is approaching. We use the target classification algorithm to determine the pedestrian’s direction for subsequent warning possibility calculation.Supposing that there is a risk of collision between pedestrian and host vehicle, for example, their distance is within 50 meters and driving speed of vehicle is more than 10km/hour. We use below Algorithm 1 for collision warning trigger.Definition 1.Algorithm 1ifthen   the target pedestrian is heading in the same direction as the host vehicle, no collision warning is sentelse ifthen   the target pedestrian is heading in the conflict direction with the host vehicle, collision warning is sentelse ifthen   the target pedestrian is crossing on the left side of the host vehicle, collision warning is sentelse ifthen   the target pedestrian is crossing on the right side of the host vehicle, collision warning is sentend if Solution3: Automated Driving System IntegrationWe have designed and implemented the system integration between PC5-based C-V2X and automated driving system, shown below in RSU receives information from traffic light controllers or AI servers. Then, it broadcasts this information by using a predefined message format in its coverage field.OBU receives broadcasting messages via PC5-based C-V2X communication.OBU is connected with IPC  of the autonomous driving system via TCP/IP. OBU receives GSNN  and CAN (Controller Area Network) messages from the vehicle.OBU uses advanced internal algorithms to judge if the situation is dangerous or not. Then a warning message will be sent accordingly to the IPC of the autonomous driving system.Finally, C-V2X technology is integrated with the automated driving system as expected.Based on the above solutions that were discussed, the following chapters will introduce which projects are implemented.Cellular infrastructure provider Nokia and its Partner research institutes in Hsinchu have been in cooperation to test C-V2X since 2018. Our contribution is to integrate all the systems with the most advanced technologies to prove C-V2X technology workable. The test involves autonomous driving vehicles RSU/OBU, which were both provided by Nokia. RSU and OBU were developed based on Qualcomm 9150 Chipset, which gives optimum performance. PC5-based C-V2X network topology is showed in One RSU is installed to extend the test vehicle’s field of vision. The RSU transmits predefined road information such as pedestrian detection to OBU by PC5-based C-V2X.OBUs are installed in the test vehicles to receive RSU information as well as between OBUs via PC5-based V2V application.RSU/OBU information is transferred back to “Traffic Control Centre” by 4G LTE cellular network as a V2N application.The test steps are described below:V2V PC5 interface communication performance is evaluated below in From the V2V test result in This project  is initiA commercial C-V2X project  is impleC-V2X can improve road safety, traffic efficiency, and distribution of road information. It is relatively low cost and highly effective compared to traditional onboard sensors. Standardization of LTE-V2X and NR-V2X has been actively promoted by 3GPP, which has motivated many organizations to develop C-V2X technology. However, there are still some challenges in PC5-based C-V2X deployment.C-V2X is an eco-system. There must be robust engagements from industry stakeholders, like road transport authority, autonomous driving developers, network transmission operators, and government. To improve C-V2X, the government needs to push road transport facility development and unify its standards. For example, traffic light control systems need to be upgraded from traditional equipment to higher processing devices. To transmit the traffic information promptly, it requires a traffic light control system to send signal changing information with a predefined frequency of at least 10HZ. But existing devices across Taiwan can’t meet this requirement. A mediation conversion process must be added. The disadvantage of this process is that it increases the message transport latency. So, there are delays between the traffic light control console and traffic lights, which is against the ITS standard. This problem causes difficulties in obtaining the correct timing information for C-V2X devices to synchronize in the SPAT application. To overcome these problems, the government must develop a unified standard to push the upgrade of traffic light control systems.Technical application layer specifications are not standardized in C-V2X. Some organizations follow up European Standard, while some use American Standard or combine both as a national standard. It is still unclear which standard will be adopted globally. Unifying and balancing all the pros and cons should be part of the government’s smart city agenda.In some regions such as Taiwan, 5G is still a year away from island-wide coverage, though it has completed LTE-V2X service testing and trials. So far, it is only focused on applications with limited KPI (Key Performance Indicator) performance. C-V2X will reach a new level once 5G is introduced, where bandwidth, low latency, and high throughput are critical for the use case. However, the deployment of 5G NR-V2X still needs the entire eco-system to be fully adopted.Plenty of privacy and confidential information from vehicles, roadside infrastructure, and IP cameras are broadcast in the public space. Protecting information security is an essential topic for PC5-based C-V2X deployment, as mentioned in ,31,32. TAutonomous Driving is defined as Level 0–Level 5 according to international specification SAE J3016 . HoweverRegulations and insurance claims for ITS road accident are yet to be defined.In this work, we have introduced the standardization of C-V2X in 3GPP. The critical difference between LTE-V2X mode 3 and mode 4, NR-V2X mode 1 and mode 2 are discussed. Operators and governments around the world are excited to see 5G technology applied in C-V2X. Countries with early adoption of LTE-V2X are certainly at the forefront. As our contribution, we have proposed some workable solutions for ITS, AI, and ADS integration. In our lives today, digitization is everywhere, and people need what 5G provides now more than ever. Our purpose in this article is to verify C-V2X technology feasibility and accessibility as proof of concept, then revise technology flaws to accumulate academic improvement. The field tests have been done to conclude that the technology is promising. The success of the field tests illustrated in this paper will motivate manufacturers to invest and the government to define favorable legislation towards the development of C-V2X. Taiwan is a world-leading electronic manufacturer and is in an advantageous position to develop intelligent transportation systems and utilize autonomous driving with C-V2X technology. However, there are still some research challenges and limitations that must be addressed. For instance, C-V2X deployment requires the cooperation of different industries. A nationwide C-V2X network requires millions of RSUs and OBUs. Full 5G coverage in the country isn’t available yet. Our future research direction includes network coverage, latency, reliability, and security policies. Because many of the V2I, V2V, and V2P applications are mission-critical, any gaps or glitches would mean life or death.


A high proportion of PD-L1 and IDO-1 expressing TAMs from all TAMs (CD68+), or from CD163+ TAMs, is associated with inferior outcome. In multivariate analysis with age and stage, high proportions of PD-L1+ and IDO-1+ TAMs remain independent prognostic factors for freedom from treatment failure . In contrast, proportions of PD-L1+ tumor cells, all TAMs or PD-L1− and IDO-1− TAMs are not associated with outcome. The findings implicate that adverse prognostic impact of TAMs is checkpoint-dependent in cHL.Tumor microenvironment and immune escape affect pathogenesis and survival in classical Hodgkin lymphoma (cHL). While tumor-associated macrophage (TAM) content has been associated with poor outcomes, macrophage-derived determinants with clinical impact have remained undefined. Here, we have used multiplex immunohistochemistry and digital image analysis to characterize TAM immunophenotypes with regard to expression of checkpoint molecules programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO-1) from the diagnostic tumor tissue samples of 130 cHL patients, and correlated the findings with clinical characteristics and survival. We show that a large proportion of TAMs express PD-L1 (CD68 Classical Hodgkin lymphoma (cHL) is characterized by unique cell composition of the tumor, where typically low numbers of neoplastic Hodgkin and Reed–Sternberg cells (HRS cells) are surrounded by abundant reactive inflammatory cells . HRS cel+ HRS cells [+ leukocytes [Apart from HRS cells, PD-L1 and PD-L2 are expressed in the tumor microenvironment (TME) , and PD-RS cells . MacrophRS cells . IDO-1 aRS cells ,9, whichukocytes  and IDO ukocytes  have bee+ or CD163+ TAMs translate to unfavorable survival in cHL [Several studies have demonstrated that high numbers of either CD68l in cHL ,15,16,17l in cHL ,19,20,21The main multiplex immunohistochemistry (mIHC) cohort consisted of 130 cHL patients whose demographics and outcome are described in CD68 and CD163) and selected checkpoint molecules CD274 (gene encoding PD-L1), as well as IDO-1 from 88 diagnostic cHL samples. Then, we examined whether their gene expressions in the tumor tissue correlated with each other. CD274 expression correlated positively with CD68  and to a lesser extent with CD163 expression . IDO-1 expression correlated with CD68 expression , whereas no correlation with CD163 expression was found. Expressions of CD68 and CD163 correlated with each other   . Interes outcome .+ cells, CD163+ cells, IDO-1+ cells, and PD-L1+ cells in the mIHC analysis correlated with the gene expression of CD68 , CD163 , CD274  and IDO-1 , respectively  and CD163+ cells . Furthermore, CD68+ and CD163+ cells correlated with each other   . Finallypression . The prosurvival .+ and CD163+ M2-like TAMs, 32% (range 0.2–89%) and 22% (range 0.1–94%) expressed PD-L1, whereas fewer CD68+  and CD163+ TAMs  were characterized as IDO-1+  translated to inferior survival when analyzed as continuous variables  and IDO-1+ TAMs  from all macrophages in comparison to patients with low ratios. When the distribution of the baseline characteristics was compared between the high and low subgroups, no significant differences in gender, age and IPS were observed  and IDO-1  was expressed in macrophages c. Of thes IDO-1+ d. Both hariables . In cont+ ratios . Accordiobserved . However+ HRS cells with PD-L1 positivity. As expected, the median proportion of CD30+ HRS cells from the whole tumor cellularity was low , and about half of CD30+ HRS cells were PD-L1+  (+ HRS cells (PD-L1+CD30+/CD30+) correlated with the proportion of PD-L1+ macrophages . While high CD30+ cell content in the tumor tissue translated to inferior DSS, neither the proportion of PD-L1+CD30+ cells from all cells, nor the PD-L1+CD30+/CD30+ cell ratio correlated with survival  c. The prsurvival .+ and IDO-1+ TAM ratios with clinical risk factors, Cox regression analyses were performed with categorical variables  and stage (IIB-IV) had adverse prognostic impact on FFTF in our mIHC cohort. High PD-L1+CD68+/CD68+ cell ratio and high age (≥60 years) also had adverse prognostic impact on DSS and OS, whereas EBV positivity and high IPS (4–7) were associated only with poor OS. Gender or cHL subtype did not have any association with survival. High PD-L1+CD68+/CD68+ ratio remained as an adverse prognostic factor for FFTF, DSS and OS when adjusted for cHL subtype, and also for FFTF and DSS when adjusted for stage or EBV status, whereas the association with OS remained significant only in the EBV negative cases , p = 0.043; EBV+, HR = 1.860 (95% CI 0.306–11.296), p = 0.500) and was more evident in the patients with advanced than limited stage , p = 0.062; stage I-IIA, HR = 0.043 (95% CI 0.000–2.971 ×1011), p = 0.835). Furthermore, when IDO-1+CD68+/CD68+ content was adjusted for stage, cHL subtype or EBV status, a high ratio remained as an adverse prognostic factor for FFTF. In multivariate analysis, both PD-L1+CD68+/CD68+ ratio and IDO-1+CD68+/CD68+ ratio predicted FFTF independently of age and stage , nor the PD-L1− or IDO-1− TAMs translated to survival. These results indicate that only distinct TAM immunophenotypes mediate significant adverse impact on survival. The aim of our study was to quantify and characterize immunophenotypes of TAMs and investigate whether PD-L1-tumoral . On the + and IDO-1+ macrophages from all macrophages. Furthermore, despite the finding that only a very small proportion of CD163+ TAMs were IDO-1+ (median 1.4%), these few cells had a notably adverse impact on the treatment outcome, highlighting the importance of the subgroup of macrophages as immunomodulators in the TME. Furthermore, considering the variation of checkpoint molecule expression on TAMs, our findings may potentially provide explanation for the opposite results in previous studies, which have investigated the prognostic impact of CD68+ and/or CD163+ macrophages [Our data emphasize the diversity of the TME, since the proportions of different cell types varied significantly between the patients, as did the cell ratios of PD-L1rophages . + cells was associated with inferior outcome, neither the proportion of PD-L1+CD30+ cells nor the ratio of PD-L1+CD30+/CD30+ cells translated to inferior survival. This finding is important by demonstrating that the adverse prognostic impact of PD-L1 expression is TME-dependent, and, particularly, TAM-dependent. Interestingly, two previous studies also showed that neither the expression of PD-L1 nor PD-L2 on HRS cells is associated with outcome [+ TAMs are more abundant than IDO-1+ TAMs. The overall proportion of PD-L1+ cells is also higher than IDO-1+ cells in the tumor tissue, indicating variation between the expression of these two molecules on macrophages. Although the overall proportion of PD-L1 outcome ,23. Unfo outcome . Togethe+ TAMs on survival in patients with primary testicular lymphoma [+CD68+/CD68+ and IDO-1+CD68+/CD68+ were associated with EBV positivity, which is in line with previous studies showing higher expression of macrophages [+ and IDO-1+ TAMs with interferon γ gene expression in turn suggests an interaction between TAMs and cytotoxic T lymphocytes. We have previously demonstrated the importance of PD-L1lymphoma . To our lymphoma ,25. Furtrophages ,26, PD-Lrophages  and IDO rophages  in EBV p+ and PD-L1+ macrophages remains unclear, and further studies are needed to resolve this question. However, PD-L1+ TAMs have been shown to be in contact both with PD-1+CD4+ T-cells and PD-1+CD8+ T-cells in the close vicinity of HRS cells [+ T-cells in promoting immunosuppression. Interestingly, suppression of PD-1+ natural killer (NK) cells has also been recently shown to occur via PD-L1 expressing CD163+ TAMs, and more prominently in cHL than in diffuse large cell lymphoma (DLBCL) [The mechanism for immune evasion via IDO-1RS cells , possibl (DLBCL) . + and PD-L1+ TAMs could be potential targets for novel immunotherapies. They might also be useful biomarkers to stratify treatments in cHL. The patients with high proportions of IDO-1+ or PD-L1+ macrophages at the time of diagnosis may define subgroups that particularly benefit from PD-1 and/or IDO-1 blockade.PD-1 blockade with monoclonal antibodies has demonstrated promising response rates 65–87%) and long-term remissions in a subgroup of poor prognosis patients with relapsed and refractory cHL ,29,30,317% and lo+ TAMs are both associated with unfavorable outcomes in cHL patients treated with standard chemotherapy. The results should be confirmed prospectively in an independent cohort of cHL patients. Nevertheless, they provide rationale for studying PD-1 and IDO-1 inhibitors in combination with standard chemotherapy for patients with high PD-L1⁺ and IDO-1+ TAM content in their tumor tissue.In this study we have demonstrated that high proportions of PD-L1⁺ and IDO-1The study material included clinical data and diagnostic formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from the patients with primary cHL. All patients were diagnosed between the years 1993–2012 and were treated or followed in Helsinki University Hospital. Patients and corresponding clinical data were retrospectively extracted from electronic and/or paper-based medical records. Gene expression data from 88 patients enriched in elderly and relapsed/refractory (R/R) cases were used for screening. The main study cohort consisted of 130 patients, who were selected based on the availability of representative tumor tissue for tissue microarray (TMA) and which was named as “mIHC cohort”. Seventy-eight patients were overlapping between the gene expression dataset and the mIHC cohort. Detailed description of staging procedures, response evaluation and treatment is provided in the Patient data were handled according to Good Scientific Practice (GSP) Guidelines. The study was approved by the Ethics Committee in Helsinki University Hospital , and by the Finnish National Authority for Medicolegal Affairs (9505/06.01.03.01/2013), which waived the requirement to obtain informed consent.CD68 and CD163, INFG (gene encoding interferon γ), and immunosuppressive molecules CD274 (gene encoding PD-L1) and IDO-1 were measured from 88 samples using digital gene expression analysis with NanoString nCounter  [Gene expression levels of macrophage markers WA, USA) .+ , CD163+  and IDO-1+ cells , respectively, emphasizing the reliability and repeatability of the method.TMA was constructed from one to six replicate spots of the same FFPE tumor tissue. Core selection on the TMA was based on the evaluation of a hematopathologist. TMA sections were stained with panels of primary antibodies for CD68, CD163, PD-L1, IDO-1 and CD30. CD68 was used as universal macrophage marker, CD163 as marker for M2-polarized macrophages, CD30 as a marker to recognize HRS-cells, and PD-L1 and IDO-1 for immunosuppression. The quantities of different immunophenotypes were counted as proportion from all cells or from specific cells on the whole TMA spot. The mean value of the cell proportions from the same tissue samples replicate spots were used. The mIHC analysis was performed digitally using the open-source platform CellProfiler . A descrEBV status was determined as either negative or positive using Epstein–Barr virus encoded RNA (EBER) in situ hybridization (ISH) for the TMAs. Cases with positive nuclear staining of HRS-cells were qualified as EBV positive.FFTF was defined as the time between the date of the diagnosis and disease progression, including progression during primary therapy and later relapses. OS was defined as time between the date of the diagnosis and death from any cause, and DSS as the time between the date of the diagnosis and death due to cHL. +CD68+/CD68+ and IDO-1+CD68+/CD68+ cell ratio subgroups, respectively.Statistical analyses were processed with IBM SPSS v.25.0 . The chi-square test was used to assess differences in the frequency of individual prognostic factors. Univariate and multivariate analyses were performed according to Cox proportional hazards regression model. Survival rates were estimated with the Kaplan–Meier method and the differences were compared using a log-rank test. In Kaplan–Meier analysis the highest fifth (50%) and the median (5.5%) values were used as cut-off levels to divide the patients to high and low PD-L1Correlation analyses were performed with Spearman rank analysis. A level of probability below 0.05 was considered significant. All comparisons were two-tailed.+ and IDO-1+ TAMs in pretreatment tumor samples translates to poor survival in patients treated with standard chemo- and radiotherapy. Our findings indicate that PD-L1+ and IDO-1+ TAMs play important roles as immunomodulators in the TME and are potential new biomarkers for treatment stratification as well as potential new targets for novel immunotherapies. Earlier studies have highlighted the important role of TAMs in the pathogenesis of cHL, as their high proportion has been associated with inferior outcomes. In this study we demonstrate for the first time that the adverse prognostic impact of TAMs on survival is checkpoint dependent, and more specifically PD-L1 and IDO-1 expression-dependent; high content of PD-L1


The 8th edition American Joint Committee on Cancer (AJCC) proposed a prognostic stage (PS), which included not only anatomical factors, but also biological factors. We aimed to investigate the clinicopathological significance of the PS and to compare PS and anatomical stage (AS) that has been established by the Union for International Cancer Control (UICC).Between 2002 and 2017, 800 patients were included in the study. Patients were classified using pathological UICC AS and pathological AJCC PS. The usefulness of PS in comparison with AS was validated using the Akaike information criterion (AIC) and Harrell concordance index (C-index).A total of 401 (50.1%) patients had pathological AS I, 324 (40.5%) had AS II, and 75 (9.4%) had AS III. Meanwhile, 535 (66.8%) had pathological PS I, 163 (20.4%) had PS II, and 102 (12.8%) had PS III. The number of AS II cases was 1.99-fold higher than that of PS II cases. For each stage, these survival curves were almost similar between AS and PS classification. Therefore, many patients to be classified into stage I and stage III were included in AS II group, while many patients to be classified into stage II were included in AS I group. To trichotomize the survival groups, PS appeared to be more specific than AS, and AIC and C-index confirmed the speculation.For the prognostication of primary breast cancer patients, AJCC PS appeared to be able to stratify the cases more appropriately than UICC AS.The online version of this article (10.1007/s12282-020-01115-x) contains supplementary material, which is available to authorized users. Breast cancer is the most frequent malignant disease, the number of the patients who were newly diagnosed with breast cancer was 1.8 million, and 471,000 died of breast cancer in 2013 . The RFS curves (c) and OS curves (d) for 324 patients with UICC AS II breast cancer stratified by PS-NG. RFS curves and OS curves were significantly different between PS-NG I subgroup and PS-NG II + III subgroup . The RFS curves (e) and OS curves (f) of 75 patients with UICC AS III breast cancer stratified by PS-NG. (e) Curves for PS-NG I + II subgroup and PS-NG III subgroup tended to differ but were not significant (P = 0.127). (f) Curves did not differ significantly (P = 0.299) (TIF 583 kb)Supplementary file3 (DOCX 17 kb)Below is the link to the electronic supplementary material.


Senegalese tonguesole, Cynoglossus senegalensis was determined by Illumina MiSeq platform. The complete mitochondrial genome of C. senegalensis was 16,519 bp in length. The mitochondrial genome of C. senegalensis showed a cynoglosidae-characteristic gene organization, in which translocation of control region to the position between ND1 and tRNA-Gln gene, and also inversions in tRNA-Gln gene from L-strand position to H-strand position. Phylogenetic analysis showed that C. senegalensis is most closely related to C. sinicus and S.bilineatus, which supports the previous result that genus Cynoglossidae is evolutionary paraphyletic.The complete mitogenome of the  Cynoglossus senegalensis, is commercially important species in western Africa, which is mainly distributed along the coastal waters from Mauritania to Congo , Cameroon, Africa. Species identification and frozen storage were conducted by Fisheries and Oceanographic Research Station (IRAD Batoke), Cameroon. Molecular identification of COI region showed 99% sequence identity to C.senegalensis (GenBank Number: EU513631). Genomic DNA was isolated by Accuprep Genomic DNA Extraction Kit . Two large PCR products amplified by PCR with sequence-specific primers targeting COX1 and ITS regions were further processed into the small-sized fragments (∼350 bp) by Covaris® M220 Focused-ultrasonicator . Library was constructed by TruSeq® RNA library preparation kit V2  and its quality and quantity were confirmed by 2100 Bioanalyzer . DNA sequencing was conducted by MiSeq sequencer (2 x 300 bp pair ends). The phylogenetic tree was constructed by MEGA7 with minimum evolution (ME) method , which contains 13 protein-coding genes, 22 tRNA genes, and 2 rRNA (12S and 16S), as well as two noncoding regions; control region (D-Loop) and origin of light-strand replication (OL). Non-canonical start condons were identified in two genes (GTG in COX1 and ATT in ND3) and incomplete stop codons were found in ND2, COX2, ND2, ND3, ND4, and ND5. The mitochondrial genome of C. senegalensis showed a cynoglosidae-characteristic gene organization; the translocation of control region to the position between ND1 and inversion of tRNA-Gln genes from L-strand to H-strand  and C. bilineatus (82%)  . This reus (82%) .


CTLA-4, PDCD1, PD-L1, BTLA, TIM-3, and LAG-3 genes in order to select SNPs which can be used as predictive biomarkers in personalized evaluation of cancer risk development and outcomes as well as possible response to immunotherapy.In recent years, immunotherapy has been revolutionized by a new approach that works by blocking receptors called immune checkpoints (IC). These molecules play a key role in maintaining immune homeostasis, mainly by suppressing the immune response and by preventing its overactivation. Since inhibition of the immune response by IC can be used by cancer to avoid recognition and destruction by immune system, blocking them enhances the anti-tumor response. This therapeutic approach has brought spectacular clinical effects. The ICs present heterogeneous expression patterns on immune cells, which may affect the effectiveness of immunotherapy. The inherited genetic variants in regulatory regions of ICs genes can be considered as potential factors responsible for observed inter-individual differences in ICs expression levels on immune cells. Additionally, polymorphism located in exons may introduce changes to ICs amino acid sequences with potential impact on functional properties of these molecules. Since genetic variants may affect both expression and structure of ICs, they are considered as risk factors of cancer development. Inherited genetic markers such as SNPs may also be useful in stratification patients into groups which will benefit from particular immunotherapy. In this review, we have comprehensively summarized the current understanding of the relationship between inherited variations of  Immune checkpoints (ICs) are key receptors that inhibit the immune response and prevent from its overactivation. Under normal conditions, this mechanism is responsible for maintaining tolerance to its own antigens; however; it can be used by cancer cells to avoid recognition and destruction . A discoRecently, a number of receptors belonging to the immune checkpoint family have been discovered. However, only two of them CTLA-4 and PD-1 are primarily and broadly explored and their blockade is used as a therapeutic procedure in the routine treatment of several cancers: among others, in the treatment of advanced melanoma, non-small cell lung cancer, non-Hodgkin’s lymphoma, and kidney cancer .In spite of the evidence of considerable clinical relevance of anti-CTLA-4 and anti-PD-1 antibodies there is still a need for deeper understanding of the relation between the host’s genetics and mechanisms involved in the regulation of these pathways; especially when considering that only some patients (20–40%) respond to therapy, while a small proportion of patients experience rapid progression or an increased risk of death . In addiTherefore, in recent years new inhibitory pathways have started to be intensively studied as promising targets for immune checkpoint blocking therapy. Several potential molecules as well as their ligands are considered , among tCTLA-4 and other immune checkpoints molecules have been studied in the context of variety types of cancers.The immune status of cancer is modulated by many factors including patients’ immune reactivity which may be affected by single nucleotide polymorphisms (SNPs) of immune related genes . These SCTLA-4, PDCD1, PD-L1, BTLA, TIM-3, and LAG-3 on cancer risk. In case of the CTLA-4, PDCD1 as well as PD-L1 genes, a considerable number of meta-analyses combining the results of numerous studies are available in the literature and in the next paragraphs their results will be preferentially analyzed. In the case of other IC molecules, the results of individual studies in particular cancers will be presented. Of note, we omitted those reports that analyzed only a small number of individuals in case-control groups.This review will summarize current knowledge about the impact of the genetic variants of eff) upon activation, and constitutively by the regulatory T cells (Tregs)  was discovered in 1987 by Brunet and colleagues Figure 1 (Tregs) . Althoug (Tregs) –15]. The (Tregs) , 17.CTLA-4 engagement, upon TCR activation, decreases T cells response through the inhibition of co-stimulatory signals coming from CD28. This molecule is vital for regulation of T-cell homeostasis and self-tolerance .From an “oncological” point of view, the most important feature of CTLA-4 is its engagement in evading immune surveillance by cancer cells, since cancer cells may employ mechanisms leading to upregulation of CTLA-4 and as consequence repression of immune response toward tumor cells . The disAfter more than decade later the idea of Prof. Allison’s team achieved an enormous success, when it was proved that antibody against CTLA-4 – ipilimumab, was active in patients with metastatic melanoma. Moreover, a long-term response to this treatment was observed in patients with prolonged stable disease . FinallyCTLA-4 gene may have impact on the ratio between these two isoforms and in consequence on T cell activity (The human CTLA-4 exists in two main isoforms: the full length CTLA-4 (flCTLA-4) and soluble CTLA-4 (sCTLA-4), which is devoid of the transmembrane domain . The flCactivity , 23.CTLA-4 gene is located between the CD28 and ICOS genes on chromosome 2q33.2. The structure of CTLA-4 gene is presented in The CTLA-4 gene expression is not fully understood. It is regulated by the NFAT TF, since it has been shown that NFAT binds to the CTLA-4 promoter region  and rs733618 (CTLA-4c.-1722T>C) are located in the upstream regulatory region and may impact the transcription-associated binding activity of CCAAT-enhancer binding protein C/EBPβ and nuclear factor 1 (NF-1) TFs, respectively. The in silico analysis suggested that the change of adenine to guanine at -1661 position may lead to creation of a new binding site for a C/EBPβ TF   . This as-1722 was predicted to destroy a binding site for a NF-1 TF. It has been suggested that the NF-1 TF and element surrounding -1772 position may regulate CTLA-4 alternative splicing, since CTLA-4c.-1722*C allele was associated with higher expression of sCTLA-4 .In contrast, the substitution of thymine to cytosine at position  sCTLA-4 . Hence, rs11571317C>T (CTLA-4c.-658C>T) is located in 5’ UTR region. In silico analysis revealed that this polymorphism is located in potential binding site for a SP1 TF , and that presence of thymine at this site may lead to disruption of the SP1 binding motif. This observation suggests that CTLA-4c.-658*T allele may be associated with lower expression of CTLA-4 molecule  may confer increased risk of cancer development.molecule . Hence, rs5742909C>T (CTLA-4c.-319C>T) is located in the promoter region. It has been demonstrated that this variant modified a binding site for a LEF1 TF. The presence of cytosine or thymine at position -319 has been shown to have an impact on regulatory potential of the LEF1, which led to differential activation of the -319C>T variant by LEF1. More precisely, it has been demonstrated in the reporter assay that in the presence of LEF1, the activity of the reporter gene was higher for thymine at -319 position in activated Jurkat cells  and CD4+ T cells, but not on CD8+ T cells  than PBMCs from individuals with CTLA-4c.-319 C/T or C/C genotypes. The authors concluded, that the carriers of CTLA-4c.-319*T allele “have stronger negative regulation of T-cell proliferation and function, which might be the underlying mechanisms conferring cervical cancer susceptibility”  is a non-synonymous SNP which leads to an amino acid change from threonine to alanine in position 17 of CTLA-4 leader peptide (Thr17Ala). The presence of the CTLA-4c.49 A/A (Thr17) genotype, as opposed to the G/G genotype (Ala17), was associated with significantly lower levels of T cells activation and lower proliferation of these cells. It has been also demonstrated that CTLA-4 molecule with (Thr17) had a higher capacity to bind CD80 and a stronger inhibitory effect on T cell activation compared to (Ala17)  . Moreove (Ala17) . It was fficking , 39. Takrs3087243G>A (CTLA-4CT60G>A) is situated in the 3’ UTR. The CTLA-4CT60 G/G genotype has been shown to be associated with lower expression of sCTLA-4 transcript  of healthy donors , CTLA-4c.-319C>T , CTLA-4CT60G>A , CTLA-4c.-1661A>G , and CTLA-4c.-1722T>C (in 14 studies). According to this analysis, three SNPs were significantly associated with overall cancer risk: CTLA-4c.-1661A>G ; CTLA-4c.-319C>T (T+ vs. C/C OR = 1.33); CTLA-4c.49A>G , while CTLA-4CT60G>A and CTLA-4c.-1722T>C were not associated with overall cancer risk. In analyses stratified by ethnicity, both CTLA-4c.49A>G and CTLA-4c.-1661A>G were significant susceptibility polymorphisms in Asians, but not in Caucasians, while CTLA-4c.-319C>T was associated with overall cancer susceptibility in Caucasians . When stratify by cancer type, the strong associations between the CTLA-4c.49A>G and bone, liver and pancreatic cancers  as well as breast and head and neck cancers  were observed. Additionally, CTLA-4c.-1661A>G was associated with significantly increased susceptibility to breast and head and neck cancers . Previous meta-analyses by Geng et al. (2014), Sun et al. (2008) and Zhang et al. (2011) . In a stratified analysis by ethnicity, a significant association was observed for CTLA-4c.-319*T allele in Asian, but not in Caucasian women. This meta-analysis confirmed the results of the previous studies . In the study by Xiong et al. (365 women - cases and controls) the carriage of CTLA-4CT60*G allele was associated with increased cervical cancer risk (OR = 1.92), and with more advanced stages of this disease   , 75. HowCTLA-4c.49A>G and breast cancer (BC) was evaluated in two meta-analyses . The most recent study examining relationship between CTLA-4c.49A>G and BC outcome was performed by Babteen and colleagues among Egyptian women .The n et al. , based on et al. , 91, 92.CTLA-4CT60G>A and the risk of BC. These studies revealed that A/A genotype was associated with lower risk of BC developing, while CTLA-4CT60*G allele increased the risk. Such association was not found in the study by Goske and colleagues   , howeverr growth . This grgenotype . Moreovegenotype  and by Cgenotype  revealedCTLA-4 SNPs: CTLA-4c.-1772T>C, CTLA-4c.-1661A>G, CTLA-4c.-319C>T, CTLA-4c.49A>G, CTLA-4CT60G>A and gastric cancer (GC) risk . This finding is inconsistent with results for other types of cancer, where the CTLA-4c.-319*T allele has been identified as a tumor risk factor. Additionally, this meta-analysis indicated that the carriage of the CTLA-4c.-1661*A allele may predispose to GC development (OR = 1.78)  risk , 95–100. = 1.78) .CTLA-4 gene polymorphisms and risk of hepatocellular cancer (HCC) available in the literature. One of such study was published in 2018 by Wang et al.  risk was the subject of several studies.The evaluation of association between CTLA-4c.49A>G and CRC risk. Importantly, most of the studies included in this work were performed on Caucasians.The meta-analysis by Jiang et al. (2013)   ders11568821C>T  is located in intron 4, in an enhancer-like structure where four imperfect tandem repeats are placed. This region contains binding sites for TFs involved in hematopoietic differentiation and inflammation . According to Prokunina et al.  is located in exon 5. This synonymous variation (Ala268Ala) does not modify the amino acid structure of the protein. Therefore, it is speculated that this SNP may be in LD with another polymorphism, which may alter PD-1 expression (+CD4+ T cells) was significantly lower in individuals with PD-1.5 C/C genotype than those with the PD-1.5 C/T and PD-1.5 T/T genotypes  is located in the 3’UTR region, in a putative miRNA binding site. According to a study by Zhang et al. (PD-1.6 G/G genotype (but not in lymphocytes from patients with PD-1.6 A/A) miR-4717 mimic significantly decreased PD-1 expression and increased TNF-α and IFN-γ production, whereas miR-4717 inhibitor acted in opposite way  is a nonsynonymous SNP, located in exon 5, causing an amino acid substitution from alanine to valine in the extracellular domain of PD-1 leading to a different structure and possibly altering the function of PD-1  is located in intron 1, where a number of regulatory elements and splicing control elements exist. Due to the disruption of the splice site or alteration of the mRNA secondary structure, PD-1 rs7421861T>C may induce aberrant splicing, and lead to translational suppression (rs41386349G>A (C>T REV) is located in intron 4. According to data presented by Zheng et al. . A similar conclusion concerning PD-1.5 was drawn by the authors of three earlier meta-analyses   .PD-1.5, also PD-1.3 was associated with overall cancer risk. On the basis of 9 articles . Two earlier meta-analyses: by Zhang et al.  indicated an association of PD-1.3 G/A genotype with lower overall cancer risk.The findings of the meta-analysis by Hashemi et al.  revealedarticles , 167–170g et al.  and by Dg et al.  (in the T/C genotype of rs7421861T>C had an increased risk of cancer (OR = 1.16). Given the results of this study, it can be assumed that higher risk of cancer concerned carriers of rs7421861*C allele (OR = 1.14)   revealed = 1.14) . Despite = 1.14)  being si = 1.14)  significPD-1.1 and overall cancer risk was observed in two additional meta-analyses (PD-1.1 A/A genotype in the Asian population (OR = 1.15). Of note, taking into consideration the completely different genotype distribution among populations (PDCD1 gene (quite broadly examined in the context of cancers)—PD-1.9 —PD-1.9  and PD-1 . Whereas, in the study by Li et al.  . However, given the fact that in the study by Li et al. genotype distributions both in controls and cases were not in HWE, these results should be treated cautiously. In the study by Guzman et al. /IFNG (rs2430561AA)/PDCD1(rs2227981CT), although a major contribution to the observed association was suggested for CD28 and IFNG SNPs.As far as we know, only n et al. , based oontrols)  PD-1.5 Cn et al.  PD-1.5 sPDCD1 have been studied also in breast cancer. Two meta-analyses , indicated an association between PD-1.5 and susceptibility to BC. A decreased risk of BC (OR = 0.8) was observed for carriers of the PD-1.5*T allele (increased for PD1.5 C/C genotype). This observation is in line with the results obtained for overall cancer risk, however it is worth mentioning that two subgroup analyses for BC  were conducted on the same two studies. Two original research articles on the basis of which the analyses were made , whereas in the study by Haghshenas et al.  . A similar association was observed in a subgroup analyses by cancer type (combining the results of two above mentioned studies) performed by Hashemi et al., as part of meta-analysis   , which i21861T>C , 172, PDPDCD1 polymorphisms and ovarian cancer. The results of a case-control study  by Tan et al. demonstrated that possession of PD-1.9*T allele was associated with increased risk of ovarian cancer (OR = 1.67). The same allele was also associated with higher FIGO stage and higher differentiation grade , as in the case of overall cancer risk in Asians. Additionally, from data obtained by Li et al. it can be concluded that the PD-1.5*T allele was associated with a reduced risk of ovarian cancer (OR = 0.82)  .inter alia at evaluation of association between PDCD1 polymorphisms and the risk of gastrointestinal cancer . Just as in the case of overall cancer risk, decreased risk of gastrointestinal cancer was observed for individuals with the PD-1.5 T/T genotype (OR = 0.60), whereas increased risk was noted for subjects with rs7421861 T/C genotype (OR = 1.19)   . A highe = 1.16) . It is nPDCD1 SNPs as potential risk factors for esophageal cancer, more precisely esophageal squamous cell carcinoma (ESCC). In all three studies the PD-1.6 was suggested to be associated with susceptibility to esophageal cancer. However, two of them  . Moreover, in this study individuals with PD-1.6 G/G genotype and carriers of PD-1.6*G allele had, respectively, higher TNM stage (OR = 1.81) and higher risk of distant metastasis (OR = 1.67)  and higher TNM stage (OR = 1.37) for rs7421861*T allele carriers     . Moreove = 1.48) . This ob of ESCC , 175, hoPDCD1 polymorphisms  and risk of esophagogastric junction adenocarcinoma (EGJA) . Increased risk of EGJA  was noted for carriers of rs7421861*C allele (OR = 1.43)  and this observation was in accordance with the results obtained for gastrointestinal cancer (PD-1.1 C/C decreased risk of EGJA was noted (OR = 0.86)  . Higher l cancer , ESCC inl cancer  as well l cancer . Moreove = 0.86) . No asso (PD-1.6 .PD-1.5 and gastric cancer indicated that the presence of PD-1.5 C/T genotype may be a risk factor for GC (OR = 1.77) . Given the inconsistency with the results of other research and the fact that only 122 GC patients and 166 controls were enrolled to this study, further examination of this SNP in the context of GC will be required.Savabkar et al. in their study aimed at determining the potential association between  = 1.77) . From thPD-1.9, PD-1.6, and rs7421861T>C were studied in gastric cardia adenocarcinoma (GCA)  (The ma (GCA) , however = 2.04) .PD-1.3 and PD-1.6 may be considered as risk factors for HCC. There was no evidence of association between the PD-1.3 and HCC risk in the study carried out by Bayram et al. on 236 patients and the same number of control subjects   .PDCD1 were also investigated in colorectal cancer  by Mojtahedi et al. However, after patients subdivision for cancer location, it was observed that PD-1.5 C/T genotype was significantly more frequent in colon cancer patients as compared with healthy controls (OR = 1.74)   examined by Ge et al. , only rs7421861T>C was shown to be associated with susceptibility to CRC, with the rs7421861 T/C genotype increasing risk of CRC development (OR = 1.31)    . Moreove = 1.31) . Additio = 2.18) , however = 1.47) .PDCD1 polymorphisms with hematological malignancies was evaluated in several studies including multiple myeloma and leukemia.The potential association of PD-1.1, rs41386349, PD-1.9) individually were associated with multiple myeloma (MM). However, the authors observed higher frequency of the haplotype combination (PD-1.1 rs41386349 PD-1.9/PD-1.1 rs41386349 PD-1.9) G C C/G C C in the group of MM patients (N = 124) as compared with controls (N = 211). In the study by Grzywnowicz et al.  and susceptibility to chronic lymphocytic leukemia (CLL) were found in the group of 114 patients and 150 controls (PD-1.9 C/T genotype as associated with a decreased risk of leukemia (OR = 0.43)  , howeverPDCD1 SNPs and lung cancer was performed only in few studies. In a subgroup analysis performed on 1,058 patients and 1,103 controls , includi et al. , 197,  were examined in NSCLC in the study performed on 320 patients and 199 control individuals by Du et al.  as well as for rs4742098 A/G heterozygotes (OR = 1.60) (rs2297136 G/G genotype were less likely to exhibit LN metastasis (OR = 0.27) and more likely to exhibit distant metastasis (OR = 3.83) in comparison to subject with the rs2297136 A/A genotype. In case of rs4742098A>G, carriers of rs4742098*G allele had greater depth of tumor infiltration as compared to individuals with rs4742098 A/A (OR = 2.30). Taking into consideration the fact that this is the only study considering 3’UTR polymorphisms and NSCLC, further investigations are needed .Additionally, three SNPs in 3’UTR of u et al. . In the  = 1.60) . Furtherrs4143815G>C did not show the association with NSCLC risk and progression in the aforementioned study by Du et al.  had shorter overall survival as compared to rs4143815*C allele carriers. Similar effect was also observed for two promoter polymorphisms of PD-L1 gene—rs822336G>C and rs822337T>A. In details, subjects with rs822336 C/C as well as subjects with rs822337 A/A genotypes showed shorter overall survival as compared to rs822336*G and rs822337*T allele carriers, respectively. The above described associations between PD-L1 polymorphisms and cancer risk are summarized in Although u et al.  it was du et al.  that indBTLA was discovered in 2003 by Gavrieli et al.  as a thiSimilarly, to CTLA-4 and PD-1, BTLA  is a type 1 transmembrane glycoprotein comprising 289 amino acids Figure 4+ and CD8+ T cells, however upon T cell activation expression progressively decreased. In contrast to CTLA-4 and PD-1 which are highly expressed on Treg cells, BTLA expression is weak, while it is highly expressed on anergic T cells generated by chronic exposure to antigens  secretion   , 210, sytis (RA) . Below wBTLA genetic variants in different populations is presented in The frequency of the described below rs1844089C>T (G>A REV) and rs2705535C>T (G>A REV) are located in first intron, the exact functional role of these SNPs was not established yet, however Ge at al. postulated that rs2705535 may affect splicing of BTLA gene (TLA gene .rs9288953C>T (G>A REV) is situated in the first intron. Ge et al.  is located in exon 4, and leads to the exchange of asparagine to threonine at position 197 in the intracellular domain. Oki and colleagues  indicated that IL-2 production was strongly inhibited in cells expressing BTLA with BTLAc.590*A, whereas in cells with BTLAc.590*C the IL-2 production was enhanced. Moreover, transfected cells were stimulated with anti-CD3 and anti-BTLA antibodies (Ab). Anti -BTLA Ab inhibited IL-2 production only in Jurkat expressing BTLAc.590*A. The authors suggest that BTLAc.590*C had not ability to inhibit IL-2 production by Jurkat cells.lleagues  performeBTLAc.590*A allele was associated with decreased inhibitory activity of BTLA in Jurkat cells it may potentially constitute cancer risk factor.Even though the amino acid exchange is not within the ITIM motifs of BTLA, it is hypothesized that this substitution may alter the posttranslational modifications of BTLA, such as glycosylation of asparagine, or phosphorylation of threonine by serine/threonine kinase, which may influence the strength of BTLA signaling by SHP1/SHP2 . Hence, rs9288952T>C (BTLAc.800G>A REV) is a non-synonymous SNP causing an amino acid substitution from proline to leucine at position 267 in exon 5, which was initially described as associated with susceptibility to RA (ty to RA .rs1982809T>C (A>G REV) is situated in 3’ nearby gene region of BTLA (73 bp). In silico analysis with application of SNPinfo . Further studies are needed to established the functional role of this SNP. SNPinfo  and Fast SNPinfo  databasepatients . Althougrs2705511A>C is located in the intragenic region between genes encoding CD200 and BTLA (-97820bp||-3334bp). Rs2705511 and rs1982809 are in moderate linkage disequilibrium with each other  in relation to malignant BC risk  GTTTT was associated with the three times increased risk of BC in this population. These authors analyzed also association between these SNPs and some clinical features. The frequency of rs1844089 C/T genotype was higher in patients with tumor size over 5 cm, while  GTCTC haplotype was significantly more frequent in patients with metastasis to LNs  and esophageal squamous cell carcinoma was investigated by Cao et al.  risk of ESCC development in males. In a stratified analysis by age, BMI, smoking status, and alcohol consumption it was noticed that rs3112270 T/C genotype may protect lean individuals with BMI<24 from ESCC . Opposite effect was observed for rs3112270 C/C genotype in subjects with BMI>24. In this group C/C genotype increased 2 times risk of ESCC (C/C vs. T/T). Additionally, the G/A genotype of rs2171513G>A (G/A vs. A/A) was associated with lower risk of ESCC (OR = 0.61) in individuals’, who overused alcohol  and EGJA risk in 1,234 patients and 1,540 control subjects  TAAG increased three times risk of EGJA development. Moreover, after adjustment for gender, age, alcohol consumption and smoking, the A/A genotype of rs1982809A>G was associated with two times higher risk of EGJA in heavy smokers.Tang et al. examined relationship between subjects . None ofBTLA gene were investigated: rs1844089G>A, rs2705535C>T, and 9288953C>T. The T/T genotype of rs2705535C>T was associated with 2 times increased risk of rectal cancer development (in comparison to C/T+C/C), while the presence of rs9288953 T/T genotype decreased 1.4 times the risk of rectal cancer (in comparison to C/T+C/C)  .BTLA polymorphisms and hematological malignancies is concerned, such evaluation was performed only for CLL. The association of BTLA gene polymorphisms and CLL risk were evaluated by Karabon et al. . Additionally, the T/T genotype of rs9288953C>T was associated with 1.7 times higher risk of CLL.As far as potential association between n et al.  in 321 pBTLA SNPs rs1844089G>A, rs2705535A>C, rs9288953C>T, rs9288952A>G, rs16859633T>C, rs1982809A>G, rs2705511A>C, and renal cell carcinoma (RCC). The presence of rs1982809*G allele (G/G + A/G) was associated with 1.4 times higher risk of RCC, while G/G genotype was associated with higher risk (OR = 2.75) of the clear cell RCC (ccRCC) high grade tumors  , metastasis to LNs (OR = 3.71), and development of adenocarcinoma subtype of lung cancer (OR = 2.8) (BTLA polymorphisms and cancer risk is shown in The association of the following genetic variants of ontrols) . The posR = 2.8) . The sum+ (Th1), CD8+ (Tc1), and Th17 cells (T Cell Immunoglobulin And Mucin-Domain Containing-3 was discovered in 2002 (TIM-3) . TIM-3 a17 cells . TIM-3 e+ TIM-3+ CD8+ T cells are considered to be the most dysfunctional T cells—a “deeply” exhausted T cell subset  in a TIM-3/galectin-9 dependent manner . The PD-1l subset . CD8+ T gression . As mentt manner . TIM-3 ise cells .Several ligands have been proposed for TIM-3: phosphatidyloserine (PtdSer) on apoptotic cells; high-mobility group box1 (HMGB-1) - a damage associated molecular pattern protein that is secreted by stressed innate immune cells; and galectin-9. It has been demonstrated that interaction between galectin-9 and TIM-3 resulted in inhibition of Th1 and Th17 response including peripheral tolerance , 223.HAVCR2 (minus strand) gene located on chromosome band 5q33.3 together with genes HAVCR1 and TIMD4 for other members of the TIM family, TIM-1 and TIM-4 respectively  in different populations is presented in The frequency of the rs891246256G>A (-1541C>T REV) and rs10053538C>A (-1516G>T REV) are the 2 kb upstream SNPs. The function for rs891246256G>A has not been established yet. The rs10053538 is situated at the putative binding site of p300 TF  of this SNP has been shown to be associated with higher expression of TIM-3 on liver infiltrating lymphocytes in tumor tissue of HCC patients in comparison to -1516 G/G genotype as determined by immunohistochemistry ( p300 TF . The prehemistry rs1036199A>C (+4259T>G REV) is located in exon 3. This is a missense polymorphism causing substitution from arginine to leucine in position 140 (R140L). The functional consequence of this change has not been reported so far but it can be postulated that this variant may affect the mucin domain (rs1036199 and autoimmune diseases (ADs) mainly in Asian populations pointed to the rs1036199*G allele as a risk factor increasing susceptibility to ADs , -574*T (OR = 1.99), and +4259*G (OR = 2.21) were associated with a higher overall cancer risk  (OR = 1.79) and for the -574*T  (OR = 1.77) alleles . This allele was also associated with digestive system cancer risk (OR = 1.61)   . The authors concluded that their study clearly demonstrated involvement of HAVCR2 gene polymorphisms  in conferring higher risk for development of different human cancers   [based o = 1.61) ]. As forcontrols , the car = 2.21) . In the  = 2.39) . This ste system  which wae system . The ass cancers .HAVCR2 genetic variants and the risk of gastric cancer (GC) development was published in 2010 by Cao at al , -822 C/T (OR = 3.19), and -547 G/T (OR = 2.74)  in GC patients  of PC development in comparison to subjects with the G/G genotype  genotype and +4259 G/G (OR = 5.11) and an increased risk of CRC  . Additiopatients . Tong etpatients  conductegenotype . To the g et al.  examinedk of CRC .HAVCR2 gene polymorphisms and susceptibility to breast cancer were carried out on 560 BC patients and 583 control subjects of Northwest China descent  in comparison to the G/G genotype. Among patients, possession of -1516*T allele was associated with increased risk of LN metastasis (OR = 1.68)  (of note G/G genotype was not detected in this study) as well as with the LN or distant metastasis in BC patients (OR = 3.16). The two remaining genetic variants were not associated with BC risk or clinical parameters (The first study attempting to examine relations between the  descent . The aut = 1.68) . Additiogenotype . In anotgenotype  assessedrameters .1516G>T, -574G>T, and +4259T>G polymorphisms were investigated in non-Hodgkin lymphoma (NHL). A case-control study was performed on 496 NHL patients and 512 control subjects enrolled from the Han Chinese Population. The study demonstrated that subjects with the -574 G/T or +4259 T/G genotypes had more than two times higher risk for NHL development  in comparison to subjects possessing wild type genotypes -574 G/G and +4259 T/T  genotype was associated with increased risk of NSCLC (OR = 2.81)  (To the best of our knowledge so far only one report assessing the association of lleagues  examined = 2.81) . Moreovectively) .-1516G>T, -574G>T, and +4259T>G polymorphisms in 322 RRC patients and 402 control subjects of Chinese descent. Of the examined SNPs, association with increased RCC cancer risk was found for the -574 G/T (OR = 2.85) genotype and +4259 T/G genotype (OR = 3.34) . Additionally, the +4259*G allele was associated in RCC patients with metastasis (OR = 2.18) (HAVCR2 genetic variants and cancer risk is shown in Cai and colleagues  investig = 2.18) . The sumLymphocyte activation gene 3 is a heavily glycosylated type I transmembrane protein which shares significant homology with the CD4 molecule—in particular in its extracellular part, which consists of four immunoglobulin superfamily-like domains (D1-D4) Figure 6+ and CD8+ T cells, B cells, NK cells, but also on plasmacytoid DCs  MM, whereas carriers of rs3782735*A allele were less likely to develop MM (OR = 0.69) (Lee and colleagues  performe = 0.69) .LAG-3 genetic variants (described below) in different populations is presented in The frequency of the As was mentioned in the Introduction, the association between inherited variants in genes for ICs and cancer risk has been extensively investigated for CTLA-4 and broadly for PD-1/PD-L1 in a wide spectrum of cancers. Whereas, the potential targets for immune checkpoint blockers, like BTLA, TIM-3, and LAG-3 have been significantly less frequently investigated in such context. Moreover, there has been no publication to date that summarizes the knowledge on this subject. Therefore, our publication aims to fill this gap and collect currently available data and present the state of the art on that topic.CTLA-4c.49*A allele is associated with higher overall cancer risk alongside the risk of developing particular types of cancers, including breast, bone, and cervical cancers. Similarly, the CTLA-4c.-319*T increased an overall cancer risk in Caucasians, and predisposed to breast, cervical and hepatocellular cancers and bone cancer in Asian. The CTLA-4CT60*G allele was associated with higher risk of breast and hepatocellular cancers, while the CTLA-4c.-1661*G allele with overall, breast and gastric cancer risk.Our general impression derived from analysing of available literature survey is that SNPs may be considered as useful risk biomarkers in terms of cancer. From numerous studies it might be concluded that the PDCD1 gene polymorphisms, the PD1.1*A allele seems to be associated with susceptibility to overall cancer in Asians as well as with susceptibility to ovarian, breast, and EGJA cancers . The available data suggest that the PD-1.5*T allele may protect from cancer development in general, and specifically against ovarian and breast cancers. The rs7421861*C allele increased overall cancer risk as well as the development of colorectal and EGJA cancers. Finally, it may be suggested that the PD-1.9*T allele may confer higher risk of ovarian and EGJA cancers development.As for PD-L1 gene, namely rs4143815G>C has been shown to be associated with cancer risk, in particular the rs4143815*G allele increased risk of overall, hepatocellular, ovarian, and gastric cancers.Only one polymorphism in the BTLA gene polymorphisms and cancer risk is limited and since a meta-analysis of published results has not been performed yet, future research in this field is needed. On the basis of available literature, it can be however postulated, that rs1982809*G confer higher risk of lung cancer, CLL and renal cancer. The rs9288953 G/G genotype can be considered as a factor increasing risk of CLL and colorectal cancer, whereas rs1844089*T allele as a risk factor of breast cancer. Finally, rs2705535*A allele seems to be associated with higher risk of breast cancer and rs2705511*C with higher susceptibility to colorectal cancer and CLL.Since the number of studies concerning BTLA gene polymorphisms, genetic variants of the HAVCR2 gene for TIM-3 have also been rarely investigated so far, however two meta-analyses have been published. According to them, the minor alleles of -1516G>T, -822C>T, -574G>T, and +4259T>G can be considered as overall cancer risk factors. In particular, the +4259*G allele was associated with the increased risk of lung cancer, colorectal cancer, and NHL; the -574*T allele with NHL and gastric cancer; -822*T allele with gastric cancer and colorectal cancer and -1516*T with gastric cancer. The associations between variants of genes encoding LAG-3 and TIGIT and cancer risk has not been evaluated yet and this area is almost unexplored. Similarly, to A limitation of any conclusion arising from this literature survey is that most of the individual studies and meta-analysis were performed on Asian populations. It is worth mentioning that significant differences in distribution of genotypes between populations are observed and some of the presented associations are true only for individuals of Asian origin Table 1.As association between particular ICs’ SNPs and cancer risk seems to be relevant to specific population there is the urgent need to perform well designed studies on large scale in different populations in order to elucidate variants important in terms of risk estimation and selection of patients to the immunotherapy for particular ethnicities.The next issue concerns the quality of some studies, as well as numbers of patients and controls in compared cohorts. We observed the following negligence in carrying out case-control studies and meta-analyses which may influence the reliability of published data. In several reports we noted deviation from HWE in control subject which was not mentioned, and even worst the authors stated that such deviation was not observed. We came across a meta-analysis which was published in the same form in another journal. Another issue which created considerable difficulties in proper interpretation of analyzed data was serious inconsistency in describing SNPs and their alleles. The authors use the old, common names of SNPs without providing SNP reference numbers. Some of analyzed genes are located in minus orientation and we noticed that authors do not pay attention to that describing alleles in relation to plus or minus strand even within one work. This is particularly valid for SNPs causing G to C or C to G substitution if their frequency is similar in the population .As a conclusion from this study, we propose future directions of the research broadening and complementing presented topic.We think that in the future the researchers should analyze SNPs which will cover the whole genes of interest. The analysis of constellation of SNPs will allow to evaluate the phenotypic effect of particular haplotypes on immune cells which may allow evaluate if particular haplotype is associated with higher, lower or neutral risk of cancer development . MoreoveIt is also worth to analyze the impact of variation within genes encoding IC in context of tumor progression .Very important, but relatively poorly investigated is functional relevance of inherited variations not only in context of gene transcription, but also on gene translation. Little is also known about influence of SNPs on epigenetic gene regulation.TIGIT is considered as a new target for immunotherapy due to its important role in maintenance of immunosuppressive phenotype of T cells in TM. Nonetheless, to the best of our knowledge, in the literature there is no data concerning TIGIT polymorphisms in relation to cancer risk. The future research will need to fill this gap in our knowledge.CTLA-4, PDCD1, PD-L1 genes, while more studies are needed for BTLA, TIM3, LAG3, and TIGIT.In conclusion, the variants in genes encoding the molecules which regulate the immune surveillance might be considered as low-risk variants (OR<2) for cancer development, which has been well documented by numerous reports for 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.The authors participated equally in conception, writing, and revising the manuscript. MW prepared the figures. All authors contributed to the article and approved the submitted version.This work was supported by the National Science Centre, Poland, grant number 2019/33/B/NZ5/03029, as well as by the Hirszfeld Institute of Immunology and Experimental Therapy statutory program number 28/2020.


Cpa4 with shRNA or overexpressed Cpa4 using adenovirus in neonatal rat cardiomyocytes and induced cardiomyocyte hypertrophy with ISO. We observed that Cpa4 overexpression promoted whereas Cpa4 knockdown reduced ISO-induced growth of cardiomyocyte size and overexpression of hypertrophy marker genes, such as myosin heavy chain β (β-Mhc), atrial natriuretic peptide (Anp), and brain natriuretic peptide (Bnp). Our further mechanism study revealed that the mammalian target of rapamycin (mTOR) signaling was activated by Cpa4 in cardiomyocytes, which depended on the phosphoinositide 3-kinase (PI3K)-AKT signaling. Besides, we showed that the PI3K-AKT-mTOR signaling was critically involved in the roles of Cpa4 during cardiomyocyte hypertrophy. Collectively, these results demonstrated that CPA4 is a regulator of cardiac hypertrophy by activating the PI3K-AKT-mTOR signaling, and CPA4 may serve as a promising target for the treatment of hypertrophic cardiac diseases.Carboxypeptidase A4 (CPA4) is a member of the metallocarboxypeptidase family. Current studies have identified the roles of CPA4 in cancer biology and insulin sensitivity. However, the roles of CPA4 in other diseases are not known. In the present study, we investigated the roles of CPA4 in cardiac hypertrophy. The expression of CPA4 was significantly increased in the hypertrophic heart tissues of human patients and isoproterenol (ISO)-induced hypertrophic heart tissues of mice. We next knocked down  In adult mammalian hearts, the proliferative capacity of the cardiomyocytes is limited. Upon injury or increased functional demand, the cardiomyocytes can not proliferate to repair or support the increased demand. Instead, cardiomyocytes can undergo hypertrophic growth, which can compensatorily support the functions of the myocardial tissues ,2. Cardi+-dependent protein deacylase Sirtuins, AMP-dependent protein kinase (AMPK), FOXOs, histone deacetylases (HDACs), and the mammalian target of rapamycin (mTOR) -leucine into proteins as described previously [Protein synthesis was monitored by incorporation of [eviously .t test was utilized. For analysis of the difference among more than two groups, two-way ANOVA followed by Tukey’s post-hoc test was applied. All the P-values less than 0.05 were considered significant. All statistical analyses were performed with the GraphPad Prism software version 8.0.All statistical values were shown as mean ± SD of three independent experiments if no other information was stated. To analyze the difference between the two groups, the standard Student’s ANP), brain natriuretic peptide (BNP), and myosin heavy chain β (β-MHC)  A. Next, controls B,C.Anp, Bnp, and β-Mhc)  for continuous 28 days using a protocol reported previously . ISO tred β-Mhc) F. Similal hearts G,H.loss-of-function (gene silence) and gain-of-function (gene overexpression) strategies were carried out by using the adenovirus system. Adenovirus-mediated shRNA targeting Cpa4 (shCpa4) significantly down-regulated the expression levels of Cpa4 in rat cardiomyocytes   B,C. Notamyocytes B,C. Cpa4s system D. By conal genes E,F. CollCpa4 knockdown repressed ISO-induced increase in phosphorylation of mTOR and S6K1 in rat cardiomyocytes (Cpa4 overexpression facilitated the increase in phosphorylation of mTOR and S6K1 in cardiomyocytes treated by ISO (Next, we investigated the underlying biological mechanisms. mTOR is a pivotal regulator for protein synthesis and cardiac hypertrophy via activating the ribosomal protein S6K1 . We hypomyocytes A. By cond by ISO B. Therefd by ISO C,D. Besid by ISO E,F. Of nd by ISO E,F. Therd by ISO G.Cpa4 deficiency (Then, we attempted to study how CPA4 regulates mTOR signaling to promote cardiomyocyte hypertrophy. A previous study showed that CPA4 activated the kinase AKT in cancer cells . Indeed,ficiency A. As thuficiency B. Importficiency C,D. Simificiency E–G. Takeficiency H.CPA4 is one of the members of the metallocarboxypeptidase family and it participates in cancer biology. Here we identified the roles of CPA4 in regulating PI3K-AKT-mTOR signaling and promotes cardiac hypertrophy. The expression of CPA4 was overexpressed in human and mouse cardiac hypertrophy. Gene silencing and gene overexpression experiments demonstrated that CPA4 promoted ISO-induced hypertrophy of cardiomyocytes. Further biological mechanism studies revealed that CPA4 promoted ISO-induced activation of the mTOR-S6K1 pathway through activating PI3K-AKT signaling.Gain-of-function and loss-of-function experiments collectively demonstrated that CPA4 promoted ISO-induced cardiomyocyte hypertrophy. Therefore, we identified the pro-hypertrophic roles of CPA4, which implicating that CPA4 is a critical regulator for the cardiovascular system. Since CPA4 was associated with adipogenesis and insulin sensitivity [CPA4 is a metallocarboxypeptidase that regulates peptide hormone activity and hormone-regulated tissue growth and differentiation . The physitivity , CPA4 made novo protein synthesis and controls cellular and organ size [The mTOR-S6K1 is critically involved in gan size . During gan size ,14. Besigan size ,13–16. TPI3K-AKT signaling is activated by neuroendocrine factors, insulin, and insulin-like growth factors . The actIn summary, using human samples and animal and cell models, we demonstrated the pro-hypertrophic roles of CPA4. CPA4 facilitates the development of cardiac hypertrophy by activating the PI3K-AKT-mTOR signaling pathway. Therefore, we identified CPA4 as a new upstream regulator of the mTOR pathway and CPA4 may serve as a potential therapeutic target for cardiac hypertrophy.


Our aim was to determine if polyunsaturated fatty acid (PUFA) metabolism can differentiate pre-symptomatic AD from normal aging and symptomatic AD. Using neuropsychology measures and Aβ42/T-tau in cerebrospinal fluid (CSF), we classify three groups of elderly study participants: cognitively healthy with normal Aβ42/T-tau (CH-NAT), cognitively healthy with pathological Aβ42/T-tau (CH-PAT), and AD individuals. We determined the size distribution and the concentration of CSF particles using light scattering and quantified PUFA composition in the nanoparticulate (NP) fraction, supernatant fluid (SF), and unesterified PUFA levels using gas chromatography combined with mass spectrometry. Four PUFAs  were enriched in NP of AD compared with CH-NAT. C20:3n-3 levels were higher in the NP fraction from AD compared with CH-PAT. When normalized to the number of NPs in CSF, PUFA levels were significantly higher in CH-NAT and CH-PAT compared with AD. In the SF fractions, only the levels of docosahexaenoic acid  differentiated all three clinical groups. Unesterified DHA was also higher in CH-NAT compared with the other clinical groups. Our studies also show that NP PUFAs in CH participants negatively correlate with CSF Aβ42 while C20:4n-6, DHA, and n-3 PUFAs in the SF fraction positively correlate with T-tau. The profile of PUFAs in different CSF fractions that correlate with Aβ42 or with T-tau are different for CH-NAT compared with CH-PAT. These studies show that PUFA metabolism is associated with amyloid and tau processing. Importantly, higher PUFA levels in the cognitively healthy study participants with abnormal Aβ42/T-tau suggest that PUFA enhances the cognitive resilience of the pre-symptomatic AD population. We propose that interventions that prevent PUFA depletion in the brain may prevent AD pathology by stabilizing Aβ42 and tau metabolism. Further studies to determine changes in PUFA composition during the progression from pre-symptomatic to AD should reveal novel biomarkers and potential preventive approaches.Alzheimer’s disease (AD) pathology is characterized by an early and prolonged decrease in the amyloid peptide (Aβ) levels concomitant with a later increase in phospho-tau concentrations in cerebrospinal fluid (CSF). We propose that changes in lipid metabolism can contribute to the abnormal processing of Aβ Lower licipants . These cer of AD . Howeversilience  or resersilience  in some fficking , we prop42 formation  compared with elderly CH study participants with pathological Aβ42/T-tau (CH-PAT) and with AD . Written informed consent was obtained from all study participants. Demographic data, medical, and diagnostic procedures have been described . ParticimRNA from peripheral blood lymphocytes was used for APOE genotyping and was performed using a polymerase chain reaction mixture of specific primers for E2, E3, and E4 . To dete42 and T-tau assays, were performed using CSF aliquots after a single thaw as previously described  were used as internal standards.to monitor PUFA extraction recovery and for quantification. Non-deuterated FA standards containing a mixture of 50 free fatty acids were purchased from NuChek Prep . Pentafluorobenzyl bromide (PFBBr) from Thermo Fisher Scientific  and NN-diisopropylethanolamine (DIPE) from Sigma-Aldrich were used for synthesizing PFBBr-derivatives of PUFAs.HPLC grade water, chloroform, methanol, formic acid, and anhydrous acetonitrile required for lipid extraction were purchased from VWR . Hydrochloric acid and butylated hydroxytoluene (BHT) were purchased from Sigma . Linoleic Acid-dCSF supernatant fluid (SF) or nanoparticle (NP) fractions were obtained by centrifugations as previously described . After t3 extract was washed using 2 mL NaCl (1 M) before the addition of 1 mL CH3OH containing 0.1 mg/mL BHT.We obtained the PUFA composition of each CSF fraction by first hydrolyzing aliquots (250 ul) of the lipid extracts, as previously described . The fat2 and then converted to pentafluorobenzyl esters using a mixture of PFBBr in acetonitrile solution  and DIPE in acetonitrile solution  for 20 min at 45°C with vortexing every 10 min   . The comWe obtained fatty acid levels in CSF fractions using GC negative ion chemical ionization MS . Single 2O) and continuously infused into the NS300 that had been calibrated with polystyrene beads . The sample temperature was maintained at 25°C, and recordings were obtained at 432 nm for 60 s (x5) with 10 s delay between recordings. The data were acquired and processed using the Nanoparticle Tracking Analysis software .Particle size number and distribution in CSF was determined using a NanoSight NS300 instrument . Briefly, freshly collected CSF was centrifuged , and 1 ml aliquots were frozen. CSF was diluted 10-fold (dd-HMassHunter Workstation Software (Agilent) was used to analyze GC-MS fatty acid data. Calibration and standard curves were obtained using deuterated fatty acid standards All CSF samples and standards were analyzed in replicates, followed by automatic peak integration for most fatty acids.U tests were performed to determine significant differences in fatty acid levels between CH-NAT, CH-PAT, and AD study participants. Spearman’s rank analyses were used to determine the correlation of PUFAs with Aβ42 or T-tau. Receiver operating characteristic (ROC curve) was performed to determine if larger or smaller values of fatty acids can classify AD or CH-NAT over the CH-PAT subjects. All statistical analyses were performed using GraphPad Prism software  or MetaboAnalyst software and data were considered significant if adjusted P for false discovery rate was <0.05  and another group (CH-PAT) with Aβ42/T-tau values similar to AD. We describe this group as a pre-symptomatic AD cohort because 4 years longitudinal follow-up study shows that 25% of these participants progress to MCI or AD while no CH-NAT participants deteriorate in the same period , supernatant fluid representative of interstitial metabolism (sfPUFA), and unesterified PUFA levels (uPUFA) resulting from lipolytic enzyme activities that increase in AD. The proportion of several n-6 and n-3 species varied between the various fractions . Our datThe concentrations of n-3 npPUFA , n-6 npPNanoparticle Tracking Analysis showed that the CSF is enriched with billions of nanoparticles. The average number of particles in CH-NAT and CH-PAT was lower than in AD. Levels of n-3 npPUFAs per billion particles trended lower in AD than in CH-NAT and CH-PAT . N-6 npPThe concentration of n-3 sfPUFA was significantly lower in AD than in CH-NAT and CH-PAT  while thThe concentration of n-3 uPUFA was significantly lower in AD than in CH-NAT and CH-PAT , while t42/T-tau in all study participants, probably due to the complex pathology of the three clinical groups. Therefore, we examined the CH, the CH-NAT, and CH-PAT groups separately.To further test our hypothesis that changes in PUFA composition can influence amyloid-beta metabolism, we performed Spearman ranked correlation analyses comparing fatty acids in each CSF fraction with CSF amyloid and T-tau levels in all study participants and in each subgroup. We did not find any correlation between CSF PUFA and AβCH– For all CH participants, C18:2n-6 and n-6 npPUFA in the NP fractions negatively correlated with Aβ42 levels , total PUFA, and n-3 PUFA inversely correlated with Aβ42. The ratio C20:4n-6/(C20:5n-3+C22:6n-3) positively correlated with Aβ42 levels. C20:4n-6 in NP and C22:6 in SF positively correlated with total T-tau. C18:2n-6 in the SF fraction negatively correlated with T-tau levels.CH-PAT. In the NP fraction, C18:3n-6, n-6/n-3 PUFA ratio, C20:4n-6/C22:6n-3 and C20:4n-6/(C20:5n-3+C22:6n-3) ratios were inversely correlated with CSF Aβ42 levels. C20:4n-6, C22:6n-3 and n-3 PUFAs positively correlated with T-tau in the SF fraction.P = 0.027) and 0.64 (P = 0.042) for C18.2n-6 and C20:4n-6, respectively.ROC analyses were performed between clinical groups to determine if PUFA levels in CSF fractions could differentiate the three clinical groups. Among all PUFAs analyzed, only two were able to classify between CH-NAT and CH-PAT subjects. Both were NP derived n-6 PUFA fatty acids and higher levels classified CH-PAT with AUC of 0.65  of the nine free PUFAs and levels of n-3 PUFAs were able to perform as binary classifiers of AD. In the SF fraction, two PUFAs , and levels of N-3 PUFAs performed as binary classifiers of AD subjects. Of the ten NP PUFAs analyzed, five were found to perform as binary classifiers of AD. ROC curve analysis showed that lower levels of C20:5n-3 classified AD while higher levels of C20:3 (n-3), C20:4, C22:4, and C22:5n-3 were shown to be classifiers of AD.p < 0.005), n-6 PUFA (p < 0.005), C22:4n-6 (p = 0.05), C30:4n-6 , C18:3n-6 (p < 0.005), and C18:2n-6 (p < 0.005) were binary classifiers of CH-PAT and AD (p < 0.005), C22:6n-3 (p < 0.05), and C20:3n-3 (p < 0.01) (We next examined whether PUFAs in CSF fractions distinguished CH-PAT from AD. None of the PUFAs in the NP fractions were classifiers of CH-PAT/AD (data not shown). However, the proportion of PUFA, n-6 PUFA, C18:3n-6, and C18:2n-6 were significant classifiers of CH-PAT and AD . More siT and AD . For PUF < 0.01) , were ac < 0.01) . In the  < 0.01) . When ex < 0.01) .42 is recognized as an early indicator of AD, it is known that this pathology can present for several decades before the onset of clinical symptoms, while p-Tau levels are closely linked with symptoms (42 similar to symptomatic AD (42 and T-tau ratio (CH-NAT) and a second group with pathological Aβ42/T-tau ratio (CH-PAT) similar to AD subjects. The aim of our study was to determine if PUFA differences in CSF fractions can classify these subgroups and isolate CH-PAT from AD that has similar CSF Aβ42/Tau ratios. The major findings of our study include: (1) No differences in plasma LDL and HDL, and no significant differences in self-reported consumption of fatty acids between the three clinical groups. (2) Significant differences in some PUFA levels in CSF fractions between our three clinical groups. (3) The progressive decrease in the amount of PUFA per billion nanoparticles in CH-NAT to CH-PAT to AD. (4) Correlation of some PUFA species with CSF Aβ42 or with CSF T-tau. (5). ROC analyses showing that some CSF PUFAs can perform as effective binary classifiers of our clinical subgroups. The implications of our study are that early changes in brain PUFA compositions are reliable indicators of AD pathology. Importantly, PUFAs contribute to the cognitive resilience in our pre-symptomatic AD population.As a neurodegenerative disease, AD is irreversible and progresses over many years before symptoms are evident . While Csymptoms . We idenmatic AD . Given tin vivo metabolism of fatty acids. Of all fatty acids detected, we found significant changes in several PUFAs, including AA (C20:4n-6) and DHA (C22:6n-3). These changes in PUFA metabolism may play an important role in the progression of AD pathology by not only influencing Aβ42 formation but also affecting mitochondrial energy homeostasis and the generation of inflammatory or pro-resolving and immune modulators.Since LDL/HDL levels and self-reported fatty acid consumption are similar in our clinical groups, it implies that changes we observe in CSF fractions are due to transport to the brain or 42 or with T-tau, respectively. In addition, whereas Aβ42 levels are similar in CH-PAT and AD, several PUFAs in CSF fractions are significantly lower in AD than in CH-PAT. While many n-3 fatty acids decreased, higher levels of two NP n-6 FAs (C20:4n-6 and C22:4n-6) classified AD participants (42 levels. However, the inverse correlation of PUFA with Aβ42 suggests there is an association between PUFA and the clearance of neurotoxic Aβ42 from the brain. Thus, changes in different PUFA types and ratios may impact brain structure and result in cognitive changes associated with AD pathology.PUFAs influence cognitive outcomes with several studies showing that n-3 supplementation improved cognitive measurements . Changesicipants . These d42 binds to BACE (42 and T-tau in our clinical groups confirm the role of fatty acids in AD severity and progression.We recently showed differences in fatty acid composition in CSF fractions from CH, MCI, and AD individuals . Fatty a to BACE . Similar42 in CH-NAT, and positively correlates with T-tau in SF from CH-NAT and CH-PAT. Given studies showing the non-amyloidogenic and the anti-amyloidogenic role of DHA and its product, neuroprotectin D1 (Fatty acids are also involved in posttranslational modification of membrane proteins. The palmitoylation of APP is known to influence its processing . We alsoectin D1 , these cModifications of PUFAs by enzyme oxidation or auto-oxidation can alter the physical properties of neuronal membranes, generate inflammatory mediators, or pro-resolving anti-inflammatory mediators . Auto-ox42. Although there is an increase in inflammatory n-6 fatty acids in CH-PAT, this is counteracted by increases in n-3 PUFAs that maintain pro-resolving mediators in the brain. This process protects the CH-PAT brain and maintains cognitive function, although Aβ42 levels would indicate otherwise.It is now recognized that neurological disorders are associated with metabolic syndromes . The braThese studies support our hypothesis that brain PUFA metabolism is altered in the early phases of AD. The balance in fatty acid metabolism in CSF fractions when comparing cognitively normal subjects with normal and abnormal amyloid to the T-tau ratio shows that PUFA metabolism is important in the clinical progression of dementia. We propose that dietary approaches that maintain normal PUFA levels, especially supplementation studies that favor pro-resolving fatty acids over inflammatory fatty acids may enhance cognitive function in an elderly population. The use of antioxidants or regulators of PUFA metabolism may maintain n-3 to n-6 balance and prevent cognitive decline in aging seniors. Further longitudinal studies to determine the rate and causes of PUFA changes are required to reveal novel biomarkers and validate potential preventive approaches.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the Huntington Memorial Hospital, Pasadena, California IRB #HMH-99-09. The patients/participants provided their written informed consent to participate in this study.AF and MH contributed to the conceptualization and study design, writing of original draft and manuscript preparation, project administration, resources, and funding acquisition. AF, MH, MC, AC, and SE contributed to the methodology. AF contributed to the validation and supervision. AC, SE, XA, and AF contributed to the formal analysis. AC, MC, and AF contributed to the data curation. XA, MH, and AF contributed to the manuscript review and editing.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.