Davide Frey

Rachid Guerraoui

Anne-Marie Kermarrec

Boris Koldehofe

Martin Mogensen

Maxime Monod

Vivien Quéma

Heterogeneous Gossip

Gossip-based information dissemination protocols are considered easy to deploy, scalable and resilient to network dynamics. Loadbalancing is inherent in these protocols as the dissemination work is evenly spread among all nodes. Yet, large-scale distributed systems are usually heterogeneous with respect to network capabilities such as bandwidth. In practice, a blind load-balancing strategy might significantly hamper the performance of the gossip dissemination. This paper presents HEAP, HEterogeneity-Aware gossip Protocol, where nodes dynamically adapt their contribution to the gossip dissemination according to their bandwidth capabilities. Using a continuous, itself gossip-based, approximation of relative bandwidth capabilities, HEAP dynamically leverages the most capable nodes by increasing their fanout, while decreasing by the same proportion those of less capable nodes. HEAP preserves the simplicity and proactive (churn adaptation) nature of gossip, while significantly improving its effectiveness. We extensively evaluate HEAP in the context of a video streaming application on a 270 PlanetLab node testbed. Our results show that HEAP significantly improves the quality of the streaming over standard homogeneous gossip protocols, especially when the stream rate is close to the average available bandwidth.

Introduction

Gossip protocols are particularly appealing in the context of large-scale dynamic systems. Initially introduced for maintaining replicated database systems [START_REF] Demers | Epidemic Algorithms for Replicated Database Maintenance[END_REF], they are particularly useful for effective dissemination [START_REF] Birman | Bimodal Multicast[END_REF].

In the context of decentralized live streaming, for instance, gossip protocols [START_REF] Bonald | Epidemic Live Streaming: Optimal Performance Trade-Offs[END_REF][START_REF] Li | FlightPath: Obedience vs Choice in Cooperative Services[END_REF][START_REF] Li | BAR Gossip[END_REF] constitute an appealing alternative to classical mesh-based techniques for large-scale dynamic systems. While efficient under steady state, meshbased solutions require sophisticated and sometimes expensive repair schemes to maintain, possibly several, dissemination paths in case of churn [START_REF] Li | Inside the New Coolstreaming: Principles, Measurements and Performance Implications[END_REF]. In the streaming context, the churn might be caused by failures, overloads, leaves and joins (e.g., users switching TV channels).

In a gossip protocol, each node periodically forwards every packet identifier it received to a subset of nodes picked uniformly at random. The size of this subset is called the fanout. Nodes subsequently request the packet whenever necessary. As no particular structure needs to be maintained, there is no need for a recovery protocol in case of churn, which is considered the norm rather than the exception. Robustness stems from the proactive and random selection of new communication partners. This proactiveness is a major difference with respect to mesh-based techniques, relying on a rather static neighborhood, which react to churn by having every node select new neighbors after noticing malfunctions [START_REF] Li | Inside the New Coolstreaming: Principles, Measurements and Performance Implications[END_REF][START_REF] Zhang | Understanding the Power of Pull-Based Streaming Protocol: Can We Do Better[END_REF]. In a sense, gossip-based protocols build extreme forms of mesh-based overlay networks with a continuously changing set of neighbors, and an ultimate splitting procedure where each packet is potentially disseminated through continuously changing dissemination paths, as opposed to explicit substreams creation leading to multi-trees [START_REF] Castro | SplitStream: High-Bandwidth Multicast in Cooperative Environments[END_REF][START_REF] Li | Inside the New Coolstreaming: Principles, Measurements and Performance Implications[END_REF][START_REF] Zhang | Understanding the Power of Pull-Based Streaming Protocol: Can We Do Better[END_REF].

Gossip in action. Consider a stream of 600 kbps produced by a single source and intended to be disseminated to 270 PlanetLab nodes in a decentralized manner. Our preliminary experiments revealed the difficulty of disseminating through a static tree without any reconstruction even among 30 nodes. The static nature of the tree exacerbates the loss rate of UDP packets particularly in the presence of heavily loaded nodes, which may see their upload capabilities change by 20% from one experiment to the other. One might consider sophisticated reactive mechanisms to cope with the dynamicity of the network but these are particularly challenging in highly dynamic environments. Instead, we could obtain a good quality stream using a simple gossip protocol over all 270 PlanetLab nodes. Figure 1 reports on our experiments (which we detail later in the paper) by conveying a high average delivery ratio (the number of stream packets received over the total number of stream packets produced), and a low stream lag (the difference between the time the stream is produced at the source and the time it is viewed): 50% of the nodes receive 99% of the stream with a stream lag of 1.3 s, 75% of the nodes receive the same amount after 2.4 s and 90% after 21 s. The fanout considered here is 7. In a system of size n, and assuming a uniformly random peer selection, a fanout of ln(n) is the theoretical threshold between a non connected and a well connected communication graph. By overestimating ln(n), theory [START_REF] Kermarrec | Probabilistic Reliable Dissemination in Large-Scale Systems[END_REF] and experiences [START_REF] Frey | Stretching Gossip with Live Streaming[END_REF] reveal that the graph gets fully connected with high probability.

This simple experiment, as well as the encouraging ones of [START_REF] Li | FlightPath: Obedience vs Choice in Cooperative Services[END_REF][START_REF] Li | BAR Gossip[END_REF], rely however on all nodes having uniform and high upload capabilities. Assuming nodes with limited and different upload capabilities (e.g., with ADSL users having heterogeneous bandwidths), the situation is less favorable as shown in Figure 2. Several fanouts are tested given two upload capabilities distributions having the same average capability of 691 kbps. Dist1 contains three classes of nodes with 512 kbps, 768 kbps, and 3 Mbps of upload bandwidth (more details about the distributions are provided in Section 3), while dist2 is a uniform distribution. Fig. 2: When constraining the upload capability in a heterogeneous manner (with an average upload capability of 691kbps -dist1), the stream lag of all nodes significantly deteriorates. Adjusting the fanout (e.g., between 15 and 20) slightly improves the stream lag but a blind fanout increase (e.g., if it goes over 25) degrades performance. Moreover, the good fanout range in this case (fanouts of 15, 20 in dist1) reveals bad with a different distribution (uniform distribution -dist2) having the same average upload capability. With dist2, a fanout of 7 is optimal and much more effective than fanouts of 15 and 20.

A case for adaptation. A major reason for the mixed behavior of gossip in heterogeneous setting is its homogeneous and load-balanced nature. All nodes are supposed to disseminate the same number of messages for they rely on the same fanout and dissemination period. However, this uniform distribution of load ignores the intrinsic heterogeneous nature of large-scale distributed systems where nodes may exhibit significant differences in their capabilities. Interestingly, and as conveyed by our experiments (and pointed out in [START_REF] Deshpande | CREW: A Gossip-based Flash-Dissemination System[END_REF]), a gossip protocol does indeed adapt to heterogeneity to a certain extent. Nodes with high bandwidth gossip rapidly, get thus pulled more often and can indeed sustain the overload to a certain extent. Nevertheless, as the bandwidth distribution gets tighter (closer to the stream rate) and more skewed (rich nodes get richer whereas poor nodes get poorer), there is a limit on the adaptation that traditional homogeneous gossip can achieve.

Heterogeneous gossip. Echoing [START_REF] Bishop | Considering Priority in Overlay Multicast Protocols under Heterogeneous Environments[END_REF][START_REF] Deshpande | CREW: A Gossip-based Flash-Dissemination System[END_REF][START_REF] Li | Inside the New Coolstreaming: Principles, Measurements and Performance Implications[END_REF][START_REF] Sung | Enabling Contribution Awareness in an Overlay Broadcasting System[END_REF][START_REF] Venkataraman | Chunkyspread: Heterogeneous Unstructured Tree-Based Peer to Peer Multicast[END_REF][START_REF] Vishnumurthy | On Heterogeneous Overlay Construction and Random Node Selection in Unstructured P2P Networks[END_REF], we recognize the need to account for the heterogeneity between peers in order to achieve a more effective dissemination. This poses important technical challenges in the context of a gossip-based streaming application. First, an effective dissemination protocol needs to dynamically track and reflect the changes of available bandwidth over time. Second, the robustness of gossip protocols heavily relies on the proactive and uniform random selection of new target peers: biasing this selection could impact the average quality of dissemination and the robustness to churn. Finally, gossip is simple and thus easy to deploy and maintain; sophisticated extensions that account for heterogeneity could improve the quality of the stream but would render the protocol more complex and thus less appealing. We propose a new gossip protocol, called HEAP (HEterogeneity-Aware Gossip Protocol ), whose simple design follows from two observations. First, mathe-matical results on epidemics and empirical evaluations of gossip protocols convey the fact that the robustness of the dissemination is ensured as long as the average of all fanouts is in the order of ln(n) [START_REF] Kermarrec | Probabilistic Reliable Dissemination in Large-Scale Systems[END_REF] (assuming the source has at least a fanout of 1). This is crucial because the fanout is an obvious knob to adapt the contribution of a node and account for heterogeneity. A node with an increased (resp. decreased) fanout will send more (resp. less) information about the packets it can provide and in turn will be pulled more (resp. less) often. Second, using gossip dissemination, one can implement an aggregation protocol [START_REF] Jelasity | Gossip-Based Aggregation in Large Dynamic Networks[END_REF][START_REF] Van Renesse | Astrolabe: A Robust and Scalable Technology for Distributed System Monitoring, Management, and Data Mining[END_REF] to continuously provide every node with a pretty accurate approximation of its relative bandwidth capability. Using such a protocol, HEAP dynamically leverages the most capable nodes by increasing their fanouts, while decreasing by the same proportion those of less capable nodes. HEAP preserves the simplicity and proactive (churn adaptation) nature of traditional (homogeneous) gossip, while significantly improving its effectiveness.

Applying HEAP in the PlanetLab context of Figure 2, i.e., assuming a heterogeneous bandwidth distribution conveying users using ADSL, we significantly improve the streaming delay and quality using HEAP (Figure 3) with an average fanout of 7: 50% of nodes receive 99% of the stream with 13.3 s lag, 75% with 14.1 s and 90% with 19.5 s. More generally, we report on an exhaustive evaluation which shows that, when compared to a standard gossip, HEAP: (i) better matches the contribution of nodes to their bandwidth capabilities; (ii) enables a better usage of the overall bandwidth thus significantly improving the stream quality of all nodes and; (iii) significantly improves the resilience to churn.

Summary of contributions.

We present HEAP, an information dissemination protocol that preserves the simplicity of standard gossip protocols, while significantly outperforming them with respect to the efficiency of the streaming and the resilience to churn. We also report on a full implementation of a P2P video streaming application using a proactive gossip protocol over a 270 PlanetLab node testbed with constrained and heterogeneous bandwidth distribution.

Roadmap. The rest of the paper is organized as follows. Section 2 gives some background on gossip-based content dissemination protocols and describes HEAP in detail. We report on the results of our experiments on PlanetLab in Section 3. Related work is covered in Section 4. Concluding remarks are given in Section 5.

HEAP

This section presents HEAP, HEterogeneity-Aware Gossip Protocol, a gossip protocol for collaborative content distribution in heterogeneous environments. We start this section by giving a short background on gossip-based content dissemination.

Background: gossip-based content dissemination

Consider a set of n nodes, and an event e to be disseminated in the system: e typically contains a series of application blocks (e.g., stream packets in a streaming application), as well as control information. Gossip-based content dissemination generally follows a three-phase push-request-push protocol as depicted in Algorithm 1. The use of a three-phase mechanism is essential when dealing with high payloads in that it guarantees that a packet may never be delivered more than once to the same node, thus causing the average data rate induced by the protocol to be less than or equal to the stream rate.

The protocol operates as follows. Each node periodically contacts a fixed number, f (fanout), of nodes chosen according to the selectNodes() function and proposes them a set of event identifiers (ids) with a [Propose] message (line 5 for the broadcaster and 6 for other nodes). A node receiving such a message pulls the content it has not yet retrieved by sending a [Request] to the proposing peer. The peer being pulled sends back the actual content (the payload) in a [Serve] message that contains the requested events. This procedure is then iterated according to an infect-and-die model [START_REF] Eugster | Lightweight Probabilistic Broadcast[END_REF]. Each node proposes each event id, exactly once, to f other peers, thus avoiding the need to deal with time-to-live.

Algorithm 1 Standard gossip protocol

Initialization:

1:

f := ln(n) + c
{f is the average fanout} 2: eToPropose := eDelivered := eRequested := ∅ 3: start(GossipTimer(gossipPeriod))

Phase 1 -Push event ids procedure publish(e) is 4: deliverEvent(e) As discussed in the introduction, standard gossip-based content dissemination works very well in unconstrained or otherwise homogeneous network environments, in which the load-balancing features of gossip provide the greatest benefit. Nevertheless, it becomes inefficient in constrained [START_REF] Frey | Stretching Gossip with Live Streaming[END_REF] and heterogeneous scenarios. In these, the standard homogeneous gossip described in Algorithm 1 stabilizes at a state in which low capability nodes saturate their bandwidth, while high capability ones have plenty of available capability. This results in congested queues and increases the transmission delays introduced by low capability nodes, impacting the overall performance experienced by all nodes of the system. Key to HEAP's adaptation mechanism is the fact that, in a non-congested setting, each [Propose] message has roughly the same probability, p, to be accepted (thereby generating a subsequent [Serve] message) regardless of the bandwidth capability of its sender 6 . HEAP exploits this fact to dynamically adapt the gossip fanouts of nodes so that their contribution to the stream delivery remains proportional to their available bandwidth. Specifically, because the average number of proposals accepted in each gossip round can be computed as p • f , f being the fanout of the proposing node, we can derive that the fanout f A of node A should be b A /b B times the fanout of node B.

Adapting contribution

f A = b A b B • f B (1)
Preserving reliable dissemination. Interestingly, Equation [START_REF] Birman | Bimodal Multicast[END_REF] shows that determining the ratios between the fanouts of nodes is enough to predict their average contribution as the three phases of Algorithm 1 guarantee that the average upload rate7 over all nodes is less than or equal to the stream rate. However, simply setting the fanouts of nodes to arbitrary values that satisfy Equation 1 may lead to undesired consequences. On the one hand, a low average fanout may hamper the ability of a gossip dissemination to reach all nodes. On the other hand, a large average fanout may unnecessarily increase the overhead resulting from the dissemination of [Propose] messages.

HEAP strives to avoid these two extremes by relying on theoretical results showing that the reliability of gossip dissemination is actually preserved as long as a fanout value of f = ln(n) + c, n being the size of the network, is ensured on average [START_REF] Kermarrec | Probabilistic Reliable Dissemination in Large-Scale Systems[END_REF], regardless of the actual fanout distribution across nodes. To achieve this, HEAP exploits a simple gossip-based aggregation protocol (see Algorithm 2) which provides an estimate of the average upload capability b of network nodes. A similar protocol can be used to continuously approximate the size of the system [START_REF] Jelasity | Gossip-Based Aggregation in Large Dynamic Networks[END_REF]. But, for simplicity, we consider here that the initial fanout is computed knowing the system size in advance. The aggregation protocol works by having each node periodically gossip its own capability and the freshest received capabilities. We assume a node's capability is either (i) a maximal capability given by the user at the application level (as the maximal outgoing bandwidth the user wants to give to the streaming application) or (ii) computed, when joining, by a simple heuristic to discover the nodes upload capability, e.g., starting with a very low capability while trying to upload as much as possible in order to reach its maximal capability as proposed in [START_REF] Zhang | Universal IP multicast delivery[END_REF]. Each node aggregates the received values and computes an estimate of the overall average capability. Based on this estimate, each node, p i , regulates its fanout, f pi , according to the ratio between its own and the average capability, i.e.,

f pi = f • b pi /b.

Evaluation

We report in this section on our evaluation of HEAP in the context of a video streaming application on a ∼270 PlanetLab nodes testbed. This includes a head to head comparison with a standard gossip protocol. In short, we show that, when compared to a standard gossip protocol: (i) HEAP adapts the actual load of each node to its bandwidth capability (Section 3.3), (ii) HEAP consistently improves the streaming quality of all nodes (Section 3.4), (iii) HEAP improves the stream lag from 40% to 60% over standard gossip (Section 3.5), (iv) HEAP resists to extreme churn situations where standard gossip collapses (Section 3.6). Before diving into describing these results in more details, we first describe our experimental setup.

Experimental setup

Video streaming application. We generate stream packets of 1316 bytes at a stream rate of 551 kbps on average. Every window is composed of 9 FEC coded packets and 101 buffered stream packets resulting in an effective rate of 600 kbps.

Gossiping parameters. The gossiping period of each node is set to 200 ms, which leads to grouping an average of 11.26 packet ids per [Propose]. The fanout is set to 7 for all nodes in the standard gossip protocol, while in HEAP, the average fanout is 7 across all nodes. The aggregation protocol gossips the 10 freshest local capabilities every 200 ms, costing around 1 KB/s and is thus completey marginal compared to the stream rate.

Message retransmission and bandwidth throttling. Given the random nature of its gossip-based dissemination process, HEAP does not attempt to establish stable TCP connections, but rather combines UDP datagrams with a retransmission mechanism. To further reduce message losses, HEAP also exploits a bandwidth throttling mechanism. This guarantees that nodes never attempt to send bursts of data that exceed their available bandwidth. Excess packets resulting from bursts are queued at the application level, and sent as soon as there is enough available bandwidth. To guarantee a fair comparison in our evaluation, we also integrated both retransmission and bandwidth throttling into the standard gossip protocol.

PlanetLab and network capabilities. PlanetLab nodes, located mostly in research and educational institutions, benefit from high bandwidth capabilities. As such, PlanetLab is not representative of a typical collaborative peer-to-peer system [START_REF] Spring | Using Planetlab for Network Research: Myths, Realities, and Best Practices[END_REF], in which most nodes would be sitting behind ADSL connection, with an asymetric bandwidth and limited upload/download capabilities. We thus artificially limit the upload capability of nodes so that they match the bandwidth usually available for home users. We focus on upload as it is a wellknown fact that download capabilities are much higher than upload ones. As we rely on UDP, we implemented, at the application level, an upload rate limiter that queues packets which are about to cross the bandwidth limit. In practice, nodes do never exceed their given upload capability, but some nodes (between 5% and 7%), contribute way less than their capability, because of high CPU load and/or high bandwidth demand by other PlanetLab experiments. In other words, the average used capabilities of nodes is always less or equal to their given upload capability limit.

We consider three different distributions of upload capabilities, depicted in Table 1 and inspired from the distributions used in [START_REF] Zhang | Understanding the Power of Pull-Based Streaming Protocol: Can We Do Better[END_REF]. The capability supply ratio (CSR, as defined in [START_REF] Zhang | Understanding the Power of Pull-Based Streaming Protocol: Can We Do Better[END_REF]) is the ratio of the average upload bandwidth over the stream rate. We only consider settings in which the global available bandwidth is enough to sustain the stream rate. Yet the lower the capability ratio, the closer we stand to that limit. The ms-691 distribution was referred to as dist1 in Section 1. Each distribution is split into three classes of nodes. The skewness of an upload distribution is characterized by the various percentages of each class of nodes: in the most skewed distribution we consider, most nodes are in the poorest category and only 15% of nodes have an upload capability higher than the stream rate.

Fraction of nodes

Evaluation metrics

In the following, we first show that HEAP adapts the contribution of nodes according to their upload capability, and then we show that HEAP provides users with a good stream. We consider two metrics. The first is the stream lag and is defined as the difference between the time the stream was published by the source and the time it is actually delivered to the player on the nodes. 8 The second is the stream quality, which represents the percentage of the stream that is viewable. A FEC-encoded window is jittered as soon as it does not contain enough packets (i.e., at least 101) to be fully decoded. A X% jittered stream therefore means that X% of all the windows were jittered. Note that a jittered window does not mean that the window is entirely lost. Because we use systematic coding, a node may still receive 100 out of the 101 original stream packets, resulting in a 99% delivery ratio in a given window. We therefore also assess the quality of the jittered windows by giving the average delivery ratio in all jittered windows.

Adaptation to heterogeneous upload capabilities

We considered all three configurations. In ref-691, ref-724 and ms-691, resp. 60%, 54% and 15% of the nodes have an available bandwidth higher than the one required on average for the stream rate. As we observed similar results in ref-691 and ref-724, we only report on ref-691 in Figure 4a. Results on ms-691 are reported on Figure 4b.

Figure 4a depicts the breakdown of the contributions among the three classes of nodes. For example, the striped bar for standard gossip means that nodes having an upload capability of 768 kbps use 76.42 % of their available bandwidth. It is interesting to observe that nodes contribute somewhat proportionally to their upload capabilities even in standard gossip. This is because of the correlation between upload capability and latency: packet ids sent by high-capability nodes are received before those sent by lower capability ones. Consequently, the former are requested first and serve the stream to more nodes than the latter. In addition, nodes with low capabilities are overloaded faster and therefore naturally serve fewer nodes (either because they are slower or because they are subject to more packet drops). Yet, despite this natural self-adaptation, we observe that high capability nodes are under utilized in standard gossip. To the contrary, HEAP homogeneously balances the load on all nodes by correctly adapting their gossip fanouts: all nodes approximately consume 70% of their bandwidth. This highlights how the similar results of standard gossip and HEAP on Figure 4a Figure 4b conveys the limits of the self-adaptation properties of standard gossip with an upload distribution in which only 15% of the nodes have an upload capability higher than the stream rate (ms-691). We observe that with standard gossip, the 15% nodes with high capabilities only use 40.80% of their bandwidth because their limited fanout does not allow them to serve more nodes. In HEAP, on the other hand, the 15% high capability nodes can serve with up to 71.13% of their bandwidth, lowering the congestion of the low-capability nodes and providing an overall much better performance than standard gossip in terms of quality as we show in next section.

Stream quality

Our next experiment compares the percentages of jitter-free windows received by nodes in the three considered scenarios. Results are depicted in Figures 5, 6a and6b. For instance, the black bar in Figure 5 for standard gossip indicates that nodes with low capabilities in ref-691 have only 18% of the windows that are not jittered (considering packets received with a stream lag of up to 10 s.). The same figure also shows that HEAP significantly improves this value, with low capability nodes receiving more than 90% of jitter-free windows. This reflects the fact that HEAP allows high capability nodes to assist low capability ones.

Results in Figure 6a are even more dramatic: high capability nodes receive less than 33% of jitter-free windows in standard gossip, whereas all nodes receive more than 95% of jitter-free windows with HEAP.

Figure 6b clearly conveys the collaborative nature of HEAP when the global available bandwidth is higher (ref-724). The whole system benefits from the fact that nodes contribute according to their upload capability. For instance, the number of jitter-free windows that low capability nodes obtain increases from 47% for standard gossip to 93% for HEAP. These results are complemented by Table 2, which presents the average delivery ratio in the jittered windows for both protocols, for each class of nodes in the three considered distributions. Again, results show that HEAP is able to provide good performance to nodes regardless of their capability classes. It should be noted, however, that the table provides results only for the windows that are jittered, which are a lot more in standard gossip than in HEAP. This explains the seemingly bad performance of HEAP in a few cases such as for high-bandwidth nodes in ref-724. jitter that is less than or equal to 10%. Note that in this figure, we do not differentiate between capability classes. We consider standard gossip and HEAP in two settings: offline and with 10 s lag. We present offline results in order to show that, with standard gossip, nodes eventually receive the stream. However, with a 10 s lag, standard gossip achieves very poor performance: most windows are jittered. In contrast, HEAP achieves very good performance even with a 10 s lag. With HEAP and a stream lag of 10 s, 93% of the nodes experience less than 10% jitter.

Stream lag

Next, we compare the stream lag required by HEAP and standard gossip to obtain a non-jittered stream. We report the results for ref-691 and ms-691 on Figures 8a and8b, respectively. In both cases, HEAP drastically reduces the stream lag for all capability classes. Moreover, as shown in Figure 8b, the positive effect of HEAP significantly increases with the skewness of the distribution. Figures 9a and 9b depict the cumulative distribution of nodes viewing the stream as a function of the stream lag, without distinguishing capability classes. We compare standard gossip and HEAP in two configurations: without jitter and with less than 1% of jitter. Sporadically, some PlanetLab nodes seem temporarily frozen, due to high CPU load and/or suffer excessive network problems explaining why neither protocol is able to deliver the stream to 100% of the nodes. 9 Still, both plots show that HEAP consistently outperforms standard gossip. For instance, in ref-691, HEAP requires 12 s to deliver the stream to 80% of the nodes without jitter, whereas standard gossip requires 26.6 s.

Table 3 complements these results by showing the percentage of nodes that can view a jitter-free stream for each bandwidth class and for the three described distributions. In brief, the table shows that the percentage of nodes receiving a clear stream increases as bandwidth capability increases for both protocols. However, HEAP is able to improve the performance experienced by poorer nodes without any significant decrease in the stream quality perceived by high-bandwidth nodes.

Resilience to catastrophic failures

Finally, we assess HEAP's resilience to churn in two catastrophic-failure scenarios where 20% (resp. 50%) of the nodes fail simultaneously 60 s after the beginning of the experiment. The experiments are based on the ref-691 bandwidth distribution, while the percentage of failing nodes is taken uniformly at random from the set of all nodes, i.e., keeping the average capability supply ratio unchanged. In addition, we configure the system so that surviving nodes learn about the failure an average of 10 s after it happened. Figure 10a depicts, for each encoded window in the stream, the percentage of nodes that are able to decode it completely without any jitter. The plot highlights once more the significant improvements provided by HEAP over standard gossipbased content dissemination. The solid line showing HEAP with a 12 s lag shows that the percentage of nodes decoding each window is always close to 100% (or to the 80% of nodes remaining after the failure) except for the stream packets generated immediately before the failure. The reason for the temporary drop in performance is that the failure of a node causes the disappearance of all the packets that it has delivered but not yet forwarded. Clearly, windows generated after the failure are instead correctly decoded by almost all remaining nodes. The plot also shows two additional lines depicting the significantly worse performance achieved by standard gossip-based dissemination.

The number of nodes receiving the stream with a 20 s lag in standard gossip is, in fact, much lower than that of those receiving it with only 12 s of lag in HEAP and only after 30 s of lag is standard gossip able to reach a performance that is comparable to that of HEAP after 12 s. The figure also highlights that the number of packets lost during the failure is higher in standard gossip than in HEAP (the width of the drop is larger). The reason is that in standard gossip upload queues tend to grow larger than in HEAP. Thus packets that are lost as a result of nodes that crash span a longer time interval in standard gossip than they do in HEAP. Finally, The 20 s lag line for standard gossip shows that the delay experienced by packets in standard gossip increases as time elapses: this is a clear symptom of congestion that is instead not present in HEAP.

Figure 10b provides similar information for a scenario in which 50% of the nodes fail simultaneously. HEAP is still able to provide the stream to the remaining nodes with a lag of less than 12 s. Conversely, standard gossip achieves mediocre performance after as many as 20 s of lag.

Related work

When contrasting HEAP with related work, we distinguish two classes of contentdissemination protocols: (i) proactive protocols that continuously change the dissemination topology, namely gossip-based dissemination schemes, and (ii) reactive protocols which only change the dissemination topology (possibly in a random manner) in case of malfunctions (e.g., churn). This latter set includes tree and mesh-based protocols.

Proactive protocols

Several proactive protocols have incorporated some adaptation features, but none does so by dynamically adapting the fanout of the nodes according to their relative (bandwidth) capabilities. The protocol of [START_REF] Garbinato | An Adaptive Algorithm for Efficient Message Diffusion in Unreliable Environments[END_REF] aims at increasing the reliability of a spanning tree, by having each node in the tree dynamically adapt its number of children using a global knowledge of the reliability of nodes and network links.

In Smart Gossip [START_REF] Kyasanur | Smart Gossip: An Adaptive Gossipbased Broadcasting Service for Sensor Networks[END_REF], nodes of a wireless network may decide not to gossip depending on the number of nodes in their surrounding. In CREW [START_REF] Deshpande | CREW: A Gossip-based Flash-Dissemination System[END_REF], a threephase gossip protocol (similar to that of Section 2.1) is used to disseminate large content in the context of file sharing. Nodes locally decide, when their bandwidth is exhausted, to stop offering data.

In Gravitational Gossip [START_REF] Jenkins | A Gossip Protocol for Subgroup Multicast[END_REF], the fanin of nodes (i.e., the number of times a node is chosen as a gossip target) may be adjusted based on the quality of reception they expect. This is achieved by biasing the node selection such that certain nodes have a higher probability to be selected for gossip than others. The technique is however static and focuses on the incoming traffic that nodes receive. Because of the three-phase nature of HEAP, nodes have a payload fanin of 1.

Reactive protocols

Some tree-and mesh-based protocols do have nodes dynamically adapt their neighborhood sets. However, such adaptation is only achieved after churn or malfunctions, and as such it is not proactive as in HEAP, or in any gossip dissemination protocol.

Multi-tree schemes such as Splitstream [START_REF] Castro | SplitStream: High-Bandwidth Multicast in Cooperative Environments[END_REF] and Chunkyspread [START_REF] Venkataraman | Chunkyspread: Heterogeneous Unstructured Tree-Based Peer to Peer Multicast[END_REF] split streams over diverse paths to enhance their reliability. This comes for free in gossip protocols where the neighbors of a node continuously change. In a sense, a gossip dissemination protocol dynamically provides different dissemination paths for each stream packet, providing the ultimate splitting scheme. Chunkyspread accounts for heterogeneity using the SwapLinks protocol [START_REF] Vishnumurthy | On Heterogeneous Overlay Construction and Random Node Selection in Unstructured P2P Networks[END_REF]. Each node contributes in proportion to its capacity and/or willingness to collaborate. This is reflected by heterogeneous numbers of children across the nodes in the tree.

The approaches of [START_REF] Bishop | Considering Priority in Overlay Multicast Protocols under Heterogeneous Environments[END_REF][START_REF] Sung | Enabling Contribution Awareness in an Overlay Broadcasting System[END_REF] propose a set of heuristics that account for bandwidth heterogeneity (and node uptimes) in tree-based multicast protocols. This leads to significant improvements in bandwidth usage. These protocols aggregate global information about the implication of nodes across trees, by exchanging messages along tree branches, in a way that relates to our capability aggregation protocol.

Mesh-based systems [START_REF] Chu | A Case for End System Multicast[END_REF][START_REF] Li | Inside the New Coolstreaming: Principles, Measurements and Performance Implications[END_REF][START_REF] Liang | Is Random Scheduling Sufficient in P2P Video Streaming?[END_REF][START_REF] Magharei | PRIME: Peer-to-Peer Receiver-drIven MEsh-based Streaming[END_REF][START_REF] Pai | Chainsaw: Eliminating Trees from Overlay Multicast[END_REF][START_REF] Picconi | Is There a Future for Mesh-Based live Video Streaming?[END_REF][START_REF] Zhang | Understanding the Power of Pull-Based Streaming Protocol: Can We Do Better[END_REF] are appealing alternatives to treebased ones. They are similar to gossip in the sense that their topology is unstructured. Some of those, namely the latest version of Coolstreaming [START_REF] Li | Inside the New Coolstreaming: Principles, Measurements and Performance Implications[END_REF] and GridMedia [START_REF] Zhang | Understanding the Power of Pull-Based Streaming Protocol: Can We Do Better[END_REF] dynamically build multi-trees on top of the unstructured overlay when nodes perceive they are stably served by their neighbors. Typically, every node has a view of its neighbors, from which it picks new partners if it observes malfunctions. In the extreme case, a node has to seek for more or different communication partners if none of its neighbors is properly operating. Not surprisingly, it was shown in [START_REF] Li | Inside the New Coolstreaming: Principles, Measurements and Performance Implications[END_REF][START_REF] Liang | Is Random Scheduling Sufficient in P2P Video Streaming?[END_REF] that increasing the view size has a very positive effect on the streaming quality and is more robust in case of churn. Gossip protocols like HEAP are extreme cases of these phenomena because the views they rely on keep continuously changing.

Finally, [START_REF] Wang | Network Overlay Construction under Limited End-to-End Reachability[END_REF] adresses the problem of building an optimized mesh in terms of network proximity and latency, in the presence of guarded peers, i.e., peers that are behind a NAT or firewall. This work led to mixing application level multicast with IP multicast whenever possible [START_REF] Zhang | Universal IP multicast delivery[END_REF]. The core of this research is now commercially used in [START_REF] Zattoo | [END_REF] but little is known on the dissemination protocol. At the time the prototype was used for research, some nodes were fed by super peers deployed on PlanetLab and it is reasonable to think that those super peers are now replaced by dedicated servers in the commercial product. It is for instance known that the dissemination protocol of PPLive [START_REF] Pplive | [END_REF] substantially relies on a set of super peers and thus does not represent a purely decentralized solution [START_REF] Hei | A Measurement Study of a Large-Scale P2P IPTV System[END_REF].

Concluding remarks

This paper presents HEAP, a new gossip protocol which adapts the dissemination load of the nodes to account for their heterogeneity. HEAP preserves the simplicity and proactive (churn adaptation) nature of traditional homogeneous gossip, while significantly improving its effectiveness. Experimental results of a video streaming application on PlanetLab convey the improvement of HEAP over a standard homogeneous gossip protocol with respect to stream quality, bandwidth usage and resilience to churn. When the stream rate is close to the average available bandwidth, the improvement is significant.

A natural way to further improve the quality of gossiping is to bias the neighbor selection towards rich nodes in the early steps of dissemination. Our early experiments reveal that this can be beneficial at the first step of the dissemination (i.e., from the source) but reveals not trivial if performed in later steps.

We considered bandwidth as the main heterogeneity factor, as it is indeed crucial in the context of streaming. Other factors might reveal important in other applications (e.g., node interests, available CPU). We believe HEAP could easily be adapted to such factors by modifying the underlying aggregation protocol accordingly. Also, we considered the choice of the fanout as the way to adjust the load of the nodes. One might also explore the dynamic adaptation of the gossip targets, the frequency of the dissemination or the memory size devoted to the dissemination.

There are some limitations to adaptation and these provide interesting research tracks to pursue. While adapting to heterogeneity, a natural behavior is to elevate certain wealthy nodes to the rank of temporary superpeers, which could potentially have an impact in case of failures. Moreover, an attacker targeting highly capable nodes could degrade the overall performance of the protocol. Likewise, the very fact that nodes advertise their capabilities may trigger freeriding vocations, where nodes would pretend to be poor in order not to contribute to the dissemination. We are working towards a freerider-tracking protocol for gossip in order to detect and punish freeriding behaviors [START_REF] Guerraoui | On Tracking Freeriders in Gossip Protocols[END_REF].

Finally, since gossip targets are periodically changing and because sent messages are very small, it is quite natural to transfer them via UDP. Nevertheless, doing so can have a negative impact on other applications competing for bandwidth. In other words, our protocol is not TCP-friendly as it might simply take priority over other applications, similar to most commercial voice-over-IP protocols. Making protocols using multiple incoming streams TCP-friendly was quite difficult [START_REF] Ma | Congestion Control in Distributed Media Streaming[END_REF][START_REF] Widmer | Extending Equation-based Congestion Control to Multicast Applications[END_REF] assuming the serving nodes were static. Doing the same for ever changing neighbors such as in a gossip is therefore a problem on its own and needs further research.

  nodes (cumulative distribution) Stream Lag (s) Percentage of nodes receiving at least 99% of the stream 99% delivery

Fig. 1 :

 1 Fig. 1: Without constraining upload capabilities, a gossip with fanout 7 provides a stream of high quality and low lag to a large number of PlanetLab nodes.

  receiving at least 99% of the stream 99% delivery

Fig. 3 :

 3 Fig.3: With the same constrained distribution (dist1), HEAP significantly improves performance over a homogeneous gossip.

Fig. 4 :

 4 Fig. 4: Bandwidth consumption.

Fig. 5 :

 5 Fig. 5: Stream Quality (ref-691).

Figure 7 Fig. 6 :

 76 Figure 7 conveys the cumulative distribution of nodes that view the stream as a function of the percentage of jitter. For instance, the point (x = 0.1, y = 85) on the HEAP -10 s lag curve indicates that 85% of the nodes experience a

Fig. 7 :

 7 Fig. 7: Cumulative distribution of experienced jitter (ref-691). With HEAP and a stream lag of 10 s, 93% of the nodes experience less than 10% jitter.

Fig. 8 :

 8 Fig. 8: Stream lag by capability class.

Fig. 9 :

 9 Fig. 9: Cumulative distribution of stream lag values.

  of 20% of the nodes at t=60s HEAP -12s lag standard gossip -20s lag standard gossip -30s lag (a) 20% of nodes crashing.

  60 70 80 90 100 110 120 130 140 150 160 170 180 Percentage of nodes receiving each window Stream time Failure of 50% of the nodes at t=60s HEAP -12s lag standard gossip -20s lag standard gossip -30s lag (b) 50% of nodes crashing.

Fig. 10 :

 10 Fig. 10: Resilience in presence of catastrophic failures.

  Consider two nodes A and B with upload capabilities b A and b B . HEAP adapts the contribution of each node to its capability and thus causes the upload rate resulting from node A's [Serve] messages to be b A /b B times as large as that of node B.

	Algorithm 2 HEAP protocol details	HEAP addresses the limitations of standard gossip by preventing conges-
		tion at low capability nodes through
		the adaptation of each node's work-
		load.

Initialization:

1: capabilities := ∅ 2: b := own available bandwidth 3: stdGossip.Initialization 4: start(AggregationTimer(aggPeriod)) Fanout Adaptation function getFanout() returns Integer is 5: return b/b • f Retransmission upon receive [Propose, eProposed] do 6: stdGossip.receive [Propose, eProposed] 7: start(RetTimer(retPeriod, eProposed)) upon receive [Serve, events] do 8: stdGossip.receive [Serve, events] 9: cancel(RetTimer(retPeriod, events)) upon (RetTimer mod retPeriod) = 0 do 10: receive [Propose, eProposed] Aggregation Protocol upon (AggregationTimer mod aggPeriod) = 0 do 11: commPartners := selectNodes(f ) 12: for all p ∈ commPartners do 13: fresh = 10 freshest values from capabilities 14: send(p) [Aggregation, fresh] upon receive [Aggregation, otherCap] do 15: merge otherCap into capabilities 16: update b using capabilities

Table 1 :

 1 The reference distributions ref-691 and ref-724, and the more skewed distribution ms-691.

	Name CSR Average 2 Mbps 768 kbps 256 kbps
	ref-691 1.15 691 kbps 0.1	0.5	0.4
	ref-724 1.20 724 kbps 0.15	0.39	0.46
	Name CSR Average 3 Mbps 1 Mbps 512 kbps
	ms-691 1.15 691 kbps 0.05	0.1	0.85

  are due to opposite reasons: congestion of low capability nodes in standard gossip and fanout adaptation, which prevents congestion, in HEAP.

		Average Bandwidth Usage by Bandwidth Class			Average Bandwidth Usage by Bandwidth Class
	2Mbps	256kbps				2Mbps	3Mbps	512kbps					3Mbps
	1.5Mbps	768kbps 2Mbps	55.76%		72.05%		2Mbps 2.5Mbps	1Mbps 3Mbps				71.13%
	512 kbps 1Mbps	88.77%	76.42%	68.07%	73.07%	256kbps 768kbps	512 kbps 1Mbps 1.5Mbps	88.34%	79.70%	40.80%	79.02%	74.71%	512kbps 1Mbps
	0						0
		Standard-Gossip		HEAP			Standard-Gossip		HEAP
			(a) ref-691				(b) ms-691

Table 2 :

 2 Average delivery rates in windows that cannot be fully decoded.

	Standard gossip	HEAP
	upload capability 256 kbps 768 kbps 2 Mbps 256 kbps 768 kbps 2 Mbps
	ref-691 63.4% 87.1% 89.3% 80.4% 77.1% 89.8%
	ref-724 75.6% 88.6% 89.6% 87.9% 87.7% 64.4%
	upload capability 512 kbps 1 Mbps 3 Mbps 512 kbps 1 Mbps 3 Mbps
	ms-691 42.8% 56.5% 64.5% 83.7% 80.7% 90.9%

Table 3 :

 3 Percentage of nodes receiving a jitter-free stream by capability class.

		Standard gossip			HEAP
	bandwidth 256 kbps 768 kbps 2 Mbps 256 kbps 768 kbps 2 Mbps
	ref-691 (10 s lag)	0	29.80 86.67	65.93	79.61 96.55
	ref-724 (10 s lag)	0	67.52 97.73	61.95	74.34 93.02
	bandwidth 512 kbps 1 Mbps 3 Mbps 512 kbps 1 Mbps 3 Mbps
	ms-691 (20 s lag)	0	0	0	84.58	89.66 85.71

In reality, proposals from low capability nodes incur in higher transmission delays and thus have a slightly lower probability of acceptance, but this effect is negligible when dealing with small [Propose] messages in a non-congested setting.

Not counting the overhead of [Propose] and other messages.

A different and complementary notion, startup delay, is the time a node takes to buffer the received packets until they are sent to the video player. Note that in a gossip protocol like HEAP the startup delay of all nodes is similar because of the unstructured fashion of the gossip itself and its dynamicity.

Note that when running simulations without messages loss,

100% of the nodes received the full stream.

Acknowledgements

The authors would like to thank Ken Birman, Pascal Felber, Ali Ghodsi and Dahlia Malkhi for useful comments.

Maxime Monod has been partially funded by the Swiss National Science Foundation with grant 20021-113825.


MS Lynda Zeboudj

MS Mikael Maître

Léa Guyonnet

MS a ; Ludivine Laurans

Jérémie Joffre

Jérémie Lemarié

MD a ; Simon Bourcier

Wared Nour-Eldine

PhD b ; Coralie Guérin

MS c ; Jonas Friard

PhD Lea Guyonnet

MD, PhD a ; Jeremie Joffre

MD, PhD Jeremie Lemarie

MS a ; Wared Nour- Eldine

PhD c ; Abdelilah Wakkach

MD d ; Elizabeth Fabre

PhD Alain Tedgui

MD, PhD Ziad Mallat

MD, PhD Pierre-Louis Tharaux

MD, PhD Hafid Ait-Oufella
email: hafid.aitoufella@inserm.fr

Selective EGF-Receptor Inhibition in CD4+ T Cells Induces Anergy and Limits Atherosclerosis

Keywords: Atherosclerosis, immunity, inflammation, lymphocyte. Condensed CD, Cluster of Differentiation EGF, Epidermal Growth factor IL-, Interleukin LDL, Low Density Lipoprotein Th, T Helper TKI, tyrosine kinase inhibitors

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Epidermal Growth Factor Receptor (EGFR) is a cell membrane-bound receptor with tyrosine kinase activity involved in the control of major signaling pathways, including cell survival, proliferation and migration. EGFR overexpression, autocrine ligand stimulation, or constitutively active receptor mutants (1,[START_REF] Gridelli | Erlotinib in non-small-cell lung cancer[END_REF] can lead to dysregulation of this fine-tuned signaling system, resulting in a variety of pathophysiological disorders and promoting cancer development. Six EGFR ligands have been described, including Epidermal Growth Factor (EGF), Heparin Binding-Epidermal Growth Factor (HB-EGF), Amphiregulin and Transforming Growth Factor-. Extracellular ligand binding causes dimerisation of EGFR, which becomes autophosphorylated at distinct tyrosine residues. In addition, EGFR could be transactivated in the absence of a specific ligand through G-protein-coupled receptor activation [START_REF] Guo | Ligand-Independent EGFR Signaling[END_REF].

EGFR has been extensively explored in cancer. Human and experimental studies showed that EGFR activation on tumor cells ultimately leads to cell proliferation, invasion, and migration, as well as promoting angiogenesis and inhibiting apoptosis [START_REF] Krakstad | Survival signalling and apoptosis resistance in glioblastomas: opportunities for targeted therapeutics[END_REF]. Targeting of EGFR by either neutralizing monoclonal antibodies or small-molecule tyrosine kinase inhibitors (TKI) have been shown during the last ten years to be a successful therapeutic strategy in cancer setting [START_REF] Bareschino | Erlotinib in cancer treatment[END_REF][START_REF] Ciardiello | EGFR antagonists in cancer treatment[END_REF]. However, EGFR expression and functions have been poorly investigated in non-tumoral cells. Some authors described an expression in circulating leucocytes [START_REF] Chan | Activation of EGFR on monocytes is required for human cytomegalovirus entry and mediates cellular motility[END_REF][START_REF] Zaiss | Amphiregulin enhances regulatory T cellsuppressive function via the epidermal growth factor receptor[END_REF] but little is known about its role in the modulation of the immune response.

Atherosclerosis is an inflammatory disease driven by innate and adaptive immunity, in which CD4 + T cells play a pathogenic role. Interestingly, EGFR ligands, including HB-EGF have been detected in human atherosclerotic plaques [START_REF] Dreux | The epidermal growth factor receptors and their family of ligands: their putative role in atherogenesis[END_REF]. The aim of this study was to ascertain the expression of EGFR in human and mouse CD4 + T cells, and to investigate the effects of EGFR blockade on CD4 + T cell functions using pharmacological inhibitors and cell-specific genetic deletion in mouse models of atherosclerosis.

Methods

Animals

Experiments were conducted according to the guidelines formulated by the European Community for experimental animal use (L358-86/609EEC), and were approved by the Ethical Committee of INSERM and the French Ministry of Agriculture (agrement no. A75-15-32). To generate a cell-specific knockout of Egfr in CD4 + T cells, we crossbred mice carrying a Cd4Cre allele with mice carrying a floxed Egfr allele. All animals have been backcrossed more than ten generations on C57bl/6 background. Ten-week old male C57BL/6 Ldlr -/-mice were put on a high fat diet for 8 weeks and were treated with the oral (daily gavage), specific EGFR tyrosine kinase inhibitor, erlotinib (15mg/kg/day). For bone marrow transplantation experiments, 10-week old male C57bl/6 Ldlr -/-mice were subjected to medullar aplasia by lethal total body irradiation (9.5 grays). The mice were repopulated with an intravenous injection of bone marrow cells isolated from femurs and tibias of sex-matched C57BL/6 Cd4Cre Egfr +/+ mice or Cd4Cre Egfr lox/lox littermates. After 4 weeks of recovery, mice were fed a pro-atherogenic diet containing 15% fat, 1.25% cholesterol and 0% cholate for 4, 6, and 12 weeks.

Extent and composition of atherosclerotic lesions

Plasma cholesterol was measured using a commercial cholesterol kit (Biomérieux). The heart of mice was removed and successive 10-µm transversal sections of aortic sinus were obtained. Lipids were detected using Red Oil staining. The presence of T cells was studied using specific antibodies, as previously described (polyclonal anti-CD3, DAKO) [START_REF] Mallat | Rho-associated protein kinase contributes to early atherosclerotic lesion formation in mice[END_REF]. Egfr was detected in cells and lesions using a rabbit polyclonal anti-Phospho-Egfr (Cell Signaling, Boston, USA). For human staining, an anti-EGFR antibody (clone 31G7, AbCys S.A.) was used. At least 4 sections per mouse were examined for each immunostaining, and appropriate negative controls were used. Morphometric studies were performed using Histolab software (Microvisions) [START_REF] Mallat | Rho-associated protein kinase contributes to early atherosclerotic lesion formation in mice[END_REF].

Spleen cell recovery and purification

Spleen cells were purified according to standard protocols as follows. CD4 + T cells were negatively selected using a cocktail of antibody-coated magnetic beads from Miltenyi Biotech (anti-CD8a, anti-CD11b, anti-CD45R, anti-DX5, anti-ter 119), according to manufacturer's instructions, yielding CD4 + cells with >95% purity. CD11c + cells were positively selected with biotin-conjugated anti-CD11c mAb (7D4, PharMingen), streptavidin microbeads (Miltenyi Biotec), followed by 2 consecutive magnetic cell separations using LS columns (Miltenyi Biotec), yielding CD11c + cells with >80% purity.

CD4 + T cell culture and cytokine assays

Cells were cultured in RPMI 1640 supplemented with Glutamax, 10% FCS, 0.02 mM mercaptoethanol and antibiotics. For cytokine measurements, CD4 + T cells were cultured at 1x10 5 cells/well for 48 hours in anti-CD3-coated microplates (10 g/ml) or with Concanavalin A (Sigma, 10 g/ml). In some experiments, CD4 + T cells were stimulated with purified soluble CD3-specific antibody (1 g/ml, Pharmingen) in the presence of antigen-presenting cells purified on CD11c-coated magnetic beads (Miltenyi Biotech). IL-2, IL-4, IL-10 and IFN- productions in the supernatants were measured using specific ELISAs (R&D Systems).

Other methods (Cell culture, proliferation assays, cytosolic calcium recording, flow cytometry and western Blot) are available in the Online Appendix.

Statistical analysis

Values are expressed as median and percentiles (25 th -75 th ) in the text. Differences between values were evaluated using non-parametric Mann-Whitney or Kruskal-Wallis test.

Values of p<0.05 were considered significant. All these analysis were performed using GraphPad Prism version 5.0b for Mac (GraphPad Software). No adjustments were made for multiple pairwise comparisons.

Based on preliminary experiments, we assumed that EGFR blockade induces a 40% reduction of atherosclerosis. With a standard deviation of plaque size estimated at 30%, the inclusion of 8 mice/group was sufficient to detect a significant difference between groups with 80% power.

Results

EGFR in mouse CD4 + T cells: expression and signaling pathways

Using immunocytostaining, we detected EGFR expression by splenocytes and found that EGFR co-localized, non-exclusively, with CD4+ T cells (Figure 1A). In addition, EGFR was present in mouse atherosclerotic lesions and co-localized with CD4 + T cells (Figure 1B). In vitro, anti-CD3-and concanavalin A-induced activation of purified CD4 + T cells caused EGFR phosphorylation after 60 minutes of stimulation (Figure 1C). AG-1478, a pharmacological inhibitor of tyrosine kinase, blocked EGFR phosphorylation in stimulated T cells (Figure 1C) and significantly reduced ERK1/2 phosphorylation (Figure 1D, Online Figure 1), but had no effect on AKT (Online Figure 1). In addition, we found that EGFR inhibition blocked cytoplasmic calcium increase following anti-CD3/CD28 stimulation (Figure 1E). In summary, anti-CD3 stimulation of CD4 + T cells leads to EGFR transactivation that was efficiently inhibited by AG-1478.

Effects of pharmacological inhibition of EGFR on atherosclerosis

To address the role of EGFR on T cell functions, splenic CD4 + T cells were purified and stimulated in vitro. EGFR activity was inhibited using AG-1478 at different concentrations.

EGFR inhibition significantly reduced T cell proliferation following non-antigen-specific (Figure 2A) and antigen-specific (Figure 2B) stimulation in a dose-dependent manner without any effect on T cell apoptosis (Figure 2C) or nucleus-cytoplasm organization (Online Figure 2). Pharmacological inhibition of EGFR significantly reduced intracellular IFN- production by CD4 + T cells in response to anti-CD3 (Figure 2D) and concanavalin stimulation (Figure 2E). This was confirmed by ELISA in the supernatants of cultured splenic CD4 + T cells, showing that EGFR inhibition significantly reduced the production of IFN- (Figure 2 F,G), reduced the production of IL-2 (supplemental figure 3), as well as that of IL-4 (Figure 2H), in a dosedependent manner, but had no effect on IL-10 production that was very low (Figure 2I).

To investigate the in vivo consequences of EGFR pharmacological inhibition, Ldlr -/-male mice were put on a high fat diet during 8 weeks and were orally treated with erlotinib (15mg/kg/day) (Figure 3A). At sacrifice, animal weight (Online Figure 4A) and plasma cholesterol level (Figure 3B) were not different between groups, but there was a significant reduction of spleen size (supplemental figure 4B) and splenocyte number (Figure 3C) in the erlotinib-treated group. Splenic CD4 + T cells from erlotinib-treated group were characterized by a significant decrease in CD25 (Online Figure 5A) and CD44 high expression (Supplemental figure 5B), suggesting reduced in vivo activation. Erlotinib had no effect on CD4 + CD25 high Foxp3 + regulatory T cell population (Online Figure 5C). Splenic purified CD4 + T cells from erlotinib-treated group were characterized by a reduction of proliferation ex vivo (Figure 3D) and a reduction of Th1 (IFN-) and Th2 (IL-4) cytokine production with no effect on IL-10 (Figure 3E). Immunohistochemistry analysis revealed that erlotinib treatment induced a 70% reduction of T cell infiltration within atherosclerotic lesions (P=0.01) (Figure 3F), which was associated with 37% reduction of atherosclerotic lesion size in the aortic sinus (167 [91-190] x10 3 in treated mice versus 267 [180-333] x10 3 m 2 in control mice, P<0.05) (Figure 3G).

In the vast majority of cases, patients in need of an anti-atherosclerotic therapy already have established atherosclerotic plaques. Thus, we examined the effects of EGFR blockade on the progression of established atherosclerotic plaques in mice. Six-week old Ldlr -/-female mice were put on a high fat diet during 8 weeks and then were orally treated with a placebo or erlotinib (15mg/kg/day) during 8 weeks (Figure 3H). At sacrifice, animal weight (data not shown) and plasma cholesterol levels (Online Figure 6C) were not different between groups. In 3I). More importantly, atherosclerosis plaque size increased between baseline 14-week old mice and 22-week old mice in the placebo group, but did not progress in the group receiving Erlotinib (Figure 3I and Online Figure 6), suggesting that Erlotinib treatment blocked atherosclerosis progression.

EGFR in human CD4 + T cells: expression and functions

To evaluate the clinical relevance of our findings, we investigated the expression of EGFR in human blood T cells. We performed immunocytostaining of purified blood CD4+ T cells and found that EGFR was expressed and clustered in the membrane of CD4 + T cells after Concanavalin-induced activation (Figure 4A). To investigate the functions of human EGFR, we purified circulating CD4+ T cells from healthy donors and performed in vitro proliferation tests.

CD3/CD28-coated beads stimulation induced T cell proliferation and EGFR inhibition using AG-1478 or Cetuximab, an EGFR neutralizing mAb, significantly decreased T cell proliferation (Figure 4B &4C). This result was confirmed in vivo in patients with lung cancer. Circulating T cells were isolated from 3 patients before and one month after oral erlotinib treatment. Interestingly, T cell proliferation was lower after erlotinib treatment (Figure 4D).

Cell-specific genetic invalidation of Egfr in CD4 + T cells

Erlotinib administration induced T cell anergy and reduced atherosclerosis development.

As the expression of EGFR is ubiquitous, we next assessed the specific role of EGFR activation in CD4 + T cells in erlotinib-induced atheroprotection. We bred mice carrying a Cd4-Cre allele with mice carrying a floxed Egfr allele and generated Cd4-Cre/Egfr lox/lox mice. The deletion of EGFR specifically in CD4 + T cells was confirmed by immunocytostaining (Online Figure 7). We purified splenic CD4 + T cells from control Cd4-Cre/Egfr +/+ and Cd4-Cre/Egfr lox/lox mice and performed functional tests. In vitro, in agreement with experiments using AG-1478, proliferation of CD4 + T cells from Cd4Cre Egfr lox/lox mice was significantly decreased compared with wildtype cells (Figure 5A), and their production of Th1 (Figure 5B) and Th2 (Figure 5C) cytokines was reduced. There was no difference in IL-10 production (Figure 5D). Similar reduction in cell proliferation and cytokine production was observed when EGFR-deficient CD4 + T cells were coincubated with CD11c + antigen presenting cells (Figure 5A-D). Intracellular staining by flow cytometry confirmed the specific reduction of IFN- production by CD4 + (Figure 5E) but no by CD8 + T cells. Apoptosis susceptibility was significantly lower in EGFR-deficient CD4 + T cells (Online Figure 8). To address the in vivo role of EGFR in CD4 + T cell proliferation, we transferred into Apoe -/-/Rag2 -/-mice 20.10 6 CD4 + T cell-depleted splenocytes resupplemented with 8.10 6 purified CFSE-labeled CD4 + T cells from either Cd4-Cre/Egfr +/+ or Cd4-Cre/Egfr lox/lox mice. The proliferation of adoptively transferred cells was visualized by flow cytometric analysis of CFSE-labeled CD4 + T cells. At day 10 after transfer, we found that CD4 + T specific deletion of EGFR limited T cell proliferation in the spleen and lymph nodes (Figure 5F).

Impact of CD4 + T cell specific invalidation of Egfr on atherosclerosis

To address the consequences of these findings in the context of atherosclerosis, we performed bone marrow transplantation experiments using either Cd4-Cre/Egfr +/+ or Cd4-Cre/Egfr lox/lox littermate bone marrow to repopulate lethally irradiated Ldlr -/-mice. After 4 weeks of recovery and additional 4 weeks on high fat diet, animals were sacrified. We did not observe any difference in animal or spleen weights, but a 32% reduction in the number of splenocytes in chimeric Cd4-Cre/Egfr lox/lox group (P=0.05). Leukocyte populations (neutrophils, monocytes, B cells, CD8 + T cells) were not different between groups, either in the blood or in the spleen (data not shown). Splenic CD4 + T cell subset was not different between chimeric Cd4-Cre/Egfr +/+ /Ldlr -/-and Cd4-Cre/Egfr lox/lox /Ldlr -/-mice (Figure 6A) but the CD4 + CD25 high FoxP3 + regulatory T cell (Tregs) population was slightly reduced in the group with CD4 specific deletion of EGFR (-14%, P<0.05; Figure 6B). Splenic CD4 + T cells from Cd4-Cre/Egfr lox/lox /Ldlr -/-were characterized by a significant decrease in CD69 (Figure 6C) and CD44 high expression (Figure 6D), suggesting reduced in vivo activation. Functional tests were performed ex vivo in purified splenic CD4 + T cells. The suppressive function of Tregs was preserved in Cd4-Cre/Egfr lox/lox /Ldlr -/-mice (Figure 6E) but the proliferation of CD4 + T cells in response to CD3 stimulation was reduced compared to cells from control group (figure 6F). Finally, we also observed a > 2-fold decrease in IFN- and IL-4 production in the supernatant of anti-CD3stimulated CD4 + T cells from Cd4-Cre/Egfr lox/lox /Ldlr -/-mice compared with Cd4-Cre/Egfr +/+ /Ldlr -/-mice (Figure 6G).

As shown in Figure 7A, after 4 weeks on high fat diet, Egfr invalidation in CD4 + T cells led to a 36% decrease in atherosclerotic lesion size in the aortic sinus compared with controls (25 [16-30] x10 3 m 2 vs 34 [31-57] x10 3 m 2 in Cd4-Cre/Egfr lox/lox /Ldlr / and Cd4-Cre Egfr +/+ /Ldlr -/-, respectively, P=0.03). The reduction of atherosclerosis was confirmed after 6 (-39%, P=0.02) and 12 weeks (-43%, P=0.02) of high fat diet (Figure 7A). There were no significant differences in plasma cholesterol levels between the groups (Online Figure 9).

We finally analyzed T cell infiltration within the lesions. We found a decrease in T cell number in lesions of Cd4Cre Egfr lox/lox  Ldlr -/-compared to Cd4Cre Egfr +/+  Ldlr -/-mice (Figure 6B) but no difference in macrophage infiltration (Online Figure 10).

Discussion

Using several complementary approaches, we identified a critical role of EGFR in CD4 + T cell homeostasis, in both mice and humans. EGFR genetic invalidation or pharmacological blockade impaired T cell activation, proliferation, cytokine production and reduced atherosclerosis development (Central Illustration).

Using immunocytostaining and immunohistochemistry, we found that splenic CD4 + T cells and blood human T cells express EGFR, especially in response to CD3 or concanavalin A stimulation. Immunofluorescent staining showed, after anti-CD3 stimulation, that EGFR clustered on the cell membrane and was phosphorylated. In addition, tissue T cells within atherosclerotic plaques express EGFR. Our results are in line with those of Zaiss et al. who detected both Egfr mRNA expression and EGFR protein in purified CD4 + T cells [START_REF] Zaiss | Amphiregulin enhances regulatory T cellsuppressive function via the epidermal growth factor receptor[END_REF].

Our findings indicate that EGFR signaling is crucial in CD4 + T cell homeostasis in both human and mouse. Pharmacological inhibition using TKIs (AG-1478 or erlotinib), neutralizing antibodies or CD4 + T cell specific genetic invalidation of Egfr markedly reduced in vitro and in vivo cell proliferation and Th1/Th2/Th17 cytokine production. A similar observation has been reported in a mouse model of graft versus host disease with a reduction of Th1 and Th2 cytokine production in erlotinib-treated animals [START_REF] Morin | Inhibition of EGFR Tyrosine Kinase by Erlotinib Prevents Sclerodermatous Graft-Versus-Host Disease in a Mouse Model[END_REF]. In addition, we observed that EGFR inhibition/invalidation reduced T cell infiltration within atherosclerotic lesions suggesting a modulation of T cell migration. This might be accounted for reduced chemokine production as described in a model of skin inflammation, in which TKI treatment decreased Ccl-17, Ccl-21 and Ccl-27 production [START_REF] Morin | Inhibition of EGFR Tyrosine Kinase by Erlotinib Prevents Sclerodermatous Graft-Versus-Host Disease in a Mouse Model[END_REF]. Reduction of T cell infiltration in the vascular wall of Cd4Cre Egf-r lox/lox animals might also be due to altered cell motility resulting from impairment of cytoskeleton reorganization [START_REF] Dudu | Role of Epidermal Growth Factor-Triggered PI3K/Akt Signaling in the Migration of Medulloblastoma-Derived Cells[END_REF]. A large body of evidence in cancer highlighted the role of EGFR signaling in epithelial-mesenchymal transition and invasion/migration of normal and malignant epithelial cells [START_REF] Camorani | Aptamer targeting EGFRvIII mutant hampers its constitutive autophosphorylation and affects migration, invasion and proliferation of glioblastoma cells[END_REF]. Recently, Tai et al. showed that EGFR/Src-signaling triggers the tyrosine phosphorylation of β4 integrin, which, in turn, activated FAK, a kinase involved in cytoskeleton reorganization [START_REF] Tai | An EGFR/Src-dependent beta4 integrin/FAK complex contributes to malignancy of breast cancer[END_REF]. In our study, we did not observe any side effect due to EGFR blockade, including survival of Erlotinib-treated or Cd4 Cre Egfr lox/lox mice. In addition, we did not find any difference in weight or infection susceptibility between groups. These observations suggest that EGFR inhibition modulates the immune response but does cause full immunosuppression. EGFR inhibition/invalidation did not affect cell death susceptibility but induced a global CD4 + T cell anergy. The mechanisms of anergy induced by EGFR inhibition likely involved MAP kinase signaling pathway as suggested by reduced Erk phosphorylation in CD4 + T cells treated with AG 1478. This is an agreement with studies by Luo et al. showing that erlotinib caused G0/G1 arrest and suppressed the phosphorylation of c-Raf, Erk in activated T cells [START_REF] Luo | Erlotinib inhibits T-cell-mediated immune response via down-regulation of the c-Raf/ERK cascade and Akt signaling pathway[END_REF].

We also showed that EGFR pharmacological blockade negatively impacted on intracellular calcium signaling in T cells confirming previous reports on cancer context. Bryant et al. showed on glioma tumor cell lines that tyrosine kinase inhibitors including erlotinib and gefinitib limited the acute cytoplasmic release of calcium from the endoplasmic reticulum in response to EGF [START_REF] Bryant | EGF activates intracellular and intercellular calcium signaling by distinct pathways in tumor cells[END_REF].

A recent study suggested that EGFR and one of its ligands, amphiregulin, play a specific role in Treg suppressive functions. In mouse models of atherosclerosis, Treg deficiency, obtained by Foxp3, Cd28 or Cd80/86 genetic invalidation, increased T cell activation and accelerated vascular disease [START_REF] Subramanian | Treg-mediated suppression of atherosclerosis requires MYD88 signaling in DCs[END_REF][START_REF] Ait-Oufella | Adaptive (T and B cells) immunity and control by dendritic cells in atherosclerosis[END_REF][START_REF] Ait-Oufella | Natural regulatory T cells control the development of atherosclerosis in mice[END_REF]. However, in our study, the genetic invalidation of Egfr in Ldlr -/- chimeric mouse model had no effect on the suppressive function of Tregs. We only observed a slight reduction in Treg pool in chimeric Cd4-Cre/Egfr lox/lox /Ldlr -/-compared with Cd4-Cre/Egfr +/+ /Ldlr -/-mice. Our results show that EGFR inhibition had a predominant effect on T cell anergy and reduced atherosclerosis development. In vitro, we challenged T cells with anti-CD3 antibody or concanavalin A, and observed EGFR phosphorylation probably through transactivation. Transactivation of EGFR is well documented for G-protein-coupled receptor like ATR-1, the receptor for angiotensin II [START_REF] Mehta | Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system[END_REF]. This transactivation is mediated by metalloproteinase-dependent release of EGFR ligands, including EGF, TGF- and HB-EGF, from their cell membrane-bound precursors and intermediary signaling molecules, including intracellular Ca 2+ , Protein Kinase C, and cytosolic tyrosine kinases such as Src kinases [START_REF] Higuchi | Angiotensin II signal transduction through the AT1 receptor: novel insights into mechanisms and pathophysiology[END_REF].

The expression of EGFR and its ligands have been detected in experimental and human atherosclerosis [START_REF] Dreux | The epidermal growth factor receptors and their family of ligands: their putative role in atherogenesis[END_REF][START_REF] Nakata | Localization of heparin-binding epidermal growth factor-like growth factor in human coronary arteries. Possible roles of HB-EGF in the formation of coronary atherosclerosis[END_REF]. Wang et al. recently reported that EGFR engagement is important for macrophage proatherogenic activity, as its pharmacological inhibition reduced proinflammatory cytokine production, lipid uptake and oxidative stress [START_REF] Wang | Inhibition of epidermal growth factor receptor attenuates atherosclerosis via decreasing inflammation and oxidative stress[END_REF]. In the present study, we reported that global EGFR inhibition and CD4 + T specific deletion of EGFR reduced both atherosclerosis development and progression, and induced a less inflammatory plaque phenotype.

Our findings indicate that EGFR inhibitors, widely used in patients with cancer, are unlikely to worsen the risk of cardiovascular disease, and even more suggest that EGFR may constitute a novel therapeutic target in atherosclerotic disease. The recent positive results of the CANTOS trial [START_REF] Ridker | Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease[END_REF] highlighted that modulating the immune system could be a promising approach to treat atherosclerosis-related cardiovascular diseases.

Conclusions

Figure Legends

Central Illustration: EGFR blockade limits pro-atherogenic activity of CD4+ T Cells.

EGFR engagement orchestrates CD4+T cell proliferation, cytokine production and recruitment within atherosclerotic plaques. Pharmalogical blockade of EGFR signaling may constitute an attractive novel approach for the treatment of atherosclerosis.

Western Blot

After extraction from isolated CD4 + T cells with RIPA buffer, proteins were quantified using a BCA protein assay kit (iNtRON Biotechnology). Equal amounts of proteins were loaded onto sodium dodecyl sulfate-polyacrylamide electrophoresis gels (4%-12% Bis-Tris Criterion XT gels, Biorad) for separation and transferred onto polyvinylidene difluoride membranes.

Membranes were blocked with milk and probed with different antibodies: anti-Akt (Cell Signaling Technology, C67E7), anti-Phospho Akt (Cell Signaling Technology, D9E), anti-ERK1/2 (Cell Signaling Technology, 137F5), anti-Phospho ERK T202/Y204 (Cell Signaling Technology, D13.14.4E) and anti-TUBA/a-tubulin (Abcam, ab6160). Protein loading was monitored using an anti-tubulin antibody (Abcam, Inc.). The membranes were then probed with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, 7074 and 7076) and the bands were visualized by enhanced chemiluminescence (Clarity Western ECL substrate; Bio-Rad, 170-5061). Antigens were revealed by enhanced chemiluminescence (Supersignal West Pico; Pierce) and detected on a LAS-4000 imaging system (Fuji, LAS4000, Burlington, NJ, USA). Densitometric analysis with ImageJ software was used for quantification.

Flow cytometry

Cells were labeled with APC-conjugated anti-CD3 (145-2C11), FITC-or PE-Cy7conjugated anti-CD4 (RM4-5), APC-conjugated anti-CD25 (PC61.5), PE-Cy7-conjugated anti-CD11b (M1/70), PE-conjugated anti-Foxp3 (PFJK-16s), eFluor 450-conjugated anti-CD62L (MEL-14) and APC-conjugated anti-CD44 (IM7) mAbs from eBioscience. PE-conjugated anti-Ly6G (1A8), Alexa Fluor 488-conjugated anti-CD11b (M1/70), APC-conjugated F4/80 (BM8) FITC-conjugated anti-CD19 (1D3), Alexa Fluor 700-conjugated anti-CD8 (53-6.7), APCconjugated anti-IFN (XMG1.2), PE-conjugated anti-IL10 (JES-16E3), Alexa Fluor 647conjugated anti-ERK1/2 (pT202/pY204) were from BD Biosciences. Alexa fluor 647-conjugated anti-7/4 was from Serotec. For blood staining, erythrocytes were lyzed using BD FACS lysis solution (BD Biosciences). For intracellular cytokine staining, lymphocytes were stimulated in vitro with leukocyte activation cocktail (BD Biosciences) according to manufacturer's instructions for 4 h. Surface staining was performed before permeabilization using Foxp3 staining buffer kit (eBioscience) and intracellular staining. Forward scatter (FSC) and side scatter (SSC) were used to gate live cells excluding RBC, debris, and cell aggregates in total splenocytes. Cells were analyzed using a BD CantoII or BD LSRII flow cytometer (BD Biosciences).

Apoptosis susceptibility of purified CD4 + T cells was determined using the apoptosis detection kit Annexin V-(FITC) and 7AAD (APC) (BD Biosciences, San Jose, CA, US) according to the manufacturer's instructions.

Online Figure Legends

  Erlotinib-treated group, we observed a marked reduction in atherosclerotic lesion size both in the aortic sinus (176 [159-218] x10 3 versus 269 [193-343] x10 3 m 2 , P<0.05) (Online Figure 6A & 6B) and along the thoracic aorta (14.0 [12.4-14.9] versus 20.7 [15.2-22.3] %, P<0.05) (Figure

  EGFR is expressed in human and mouse CD4 + T cells. EGFR pharmacological blockade or CD4 + T specific invalidation induced T cell anergy and reduced both atherosclerosis development and progression in mice.PerspectivesCOMPETENCY IN MEDICAL KNOWLEDGE: EGFR is identified as a critical regulator in CD4 + T cell activity both in mouse and in human. EGFR pharmacological blockade using Erlotinib or selective invalidation in CD4 + T cells limited the development and the progression of experimental atherosclerosis. TRANSLATIONAL OUTLOOK: Pharmacological inhibition of EGFR may represent a promising new immunomodulatory treatment of cardiovascular diseases preventing the progression and complications of atherosclerosis.

Figure 1 :

 1 Figure 1: EGFR receptor expression and activation in mouse T cells. A, EGFR is expressed by splenocytes (green) and co-localizes with CD4 + T cells (Red). B, EGFR is expressed within mouse atherosclerotic lesions (Red) and co-localizes with CD4 + T cells (green). Staining was performed on atherosclerotic plaques (aortic sinus) from Ldlr -/-mouse fed a high fat diet during 6 weeks. C, stimulation of purified CD4 + T cells by coated anti-CD3 during 60 minutes induced

Figure 2 .

 2 Figure 2. Effects of EGFR pharmacological inhibition on mouse T cell functions. A, in vitro AG-1478 induced a dose-dependent reduction of CD4 + T cell proliferation in response to soluble anti-CD3 stimulation, in the presence of CD11c + dendritic cells. B, in vitro AG-1478 induced a dose-dependent reduction of OT-II CD4 + T cell proliferation after OVA stimulation in the

Figure 3 .

 3 Figure 3. EGFR pharmacological inhibition limited T cell activation in vivo and reduced atherosclerosis. A, protocol of pharmacological blockade of EGFR in male Ldlr -/-mice

Figure 4 :

 4 Figure 4: EGFR expression and functions in human T cells. A, Blood human CD4 + T cells

Figure 5 .

 5 Figure 5. Selective EGFR deletion in CD4 + T cells induced anergy. A, In vitro, purified CD4 + T cells from control Cd4-Cre/Egf-r +/+ (white) or Cd4-Cre/Egf-r lox/lox mice (black) were stimulated by soluble anti-CD3 with or without co-incubation with CD11c + dendritic cells

Figure 6 .

 6 Figure 6. Selective EGFR deletion in CD4 + T cells in chimeric Ldlr -/-mouse model of atherosclerosis. A, representative examples and FACS quantification of CD4 + T subset. B,

Figure 7 .

 7 Figure 7. Selective EGFR deletion in CD4 + T cells reduced experimental atherosclerosis

Figure 1 .Online Figure 3 .Online Figure 4 .Online Figure 7 .Online Figure 8 .

 13478 EGFR pharmacological inhibition reduced ERK phosphorylation Online Figure 2. No effect of EGFR pharmacological inhibition on CD4+ T cell morphology. EGFR pharmacological inhibition reduced IL-2 production by CD4+ T cells. Erlotinib treatment had no effect on animal weight but induced a reduction of spleen size. Online Figure 5. Erlotinib treatment reduced CD4+ T cell activation Online Figure 6. Erlotinib treatment blocked atherosclerosis progression. Absence of EGFR expression on CD4+ T cells from Cd4 Cre EgfrLox/lox mice. EGFR deficiency reduced CD4+ T cell apoptosis. Online Figure 9. EGFR deficiency had no impact on cholesterolemia. Online Figure 10. EGFR deficiency in CD4+ T cells had no impact on macrophage content.

Sources of funding:

This work was supported by Institut National de Santé et de la Recherche Médicale (INSERM), research grants ANR-08-EBIO-003 (PL.T) from l'Agence Nationale de la Recherche (ANR), British Heart Foundation (Z.M).

Online Appendix-Methods

Cell culture and proliferation assays

Cells were cultured in RPMI 1640 supplemented with Glutamax, 10% FCS, 0.02 mM mercapto-ethanol and antibiotics. To analyze T cell proliferation, 1x10 5 CD4 + cells/well were cocultured with 10 4 CD11c + dendritic cells and soluble anti-CD3 (1g/ml) for 72 h. One Ci [ 3 H] thymidine was added to each well during the last 16 h. Cells were cultured at 37°C for 72 h and pulsed with 1 Ci [ 3 H] thymidine (Amersham) for the last 18 h. Thymidine incorporation was assessed using a TopCount NXT scintillation counter (Perkin Elmer). To analyze regulatory T cell functions, effectors T cells were cultured alone or co-cultured with purified regulatory T cells (at 1:8, 1:4, 1:2 or 1:1 ratio) in bottom-rounded 96-well microplates (0.5x10 5 cells/well; total volume 200 l/well). Cells were stimulated with purified soluble CD3-specific antibody (1 g/ml, Pharmingen) in the presence of antigen-presenting cells purified on CD11c-coated magnetic beads (Miltenyi Biotech). Cells were cultured at 37°C for 72 h and pulsed with 1 Ci [ 3 H] thymidine (Amersham) for the last 18 h. Thymidine incorporation was assessed using a TopCount NXT scintillation counter (Perkin Elmer).

To explore in vivo proliferation, Cd4Cre Egfr +/+ and Cd4Cre Egfr lox/lox purified CD4 + T cells from pooled spleens and lymph nodes were labeled with CFSE fluorescent dye (5uM, VybrantCFDASE; Invitrogen-Molecular Probes) according to the instructions of the manufacturer. Apoe -/-/Rag2 -/-mice received 20x10 6 CD4 + T cell-depleted splenocytes resupplemented with 8x10 6 purified CD4 + T cells from either Cd4Cre Egfr +/+ or Cd4Cre Egfr lox/lox mice. The proliferation of adoptively transferred cells was visualized by flow cytometric analysis of CFSE-labeled CD4 + T cells at day 7 after transfer.

Human lymphocytes were isolated from blood (negative selection, Miltenyi Biotech).

Blood samples from patients (with non-small lung cell cancer) and healthy volunteers was collected after informed consent, as part of the OncoHEGP clinical protocol at Georges Pompidou European Hospital, approved by the institutional ethics committee (CPP Ile de France II n°2012-08-09). Venous blood was centrifuged with a Histopaque TM gradient (density:

1.077 g/cm 3 , 30 min, 500g, 20 °C; Sigma-Aldrich, Saint Louis, US). Cells were washed twice with warm PBS and cell viability, as well as concentration, was determined using the trypan blue exclusion test. Cells were cultured in the same medium as that used for mouse cells. Specific T cell proliferation was induced using CD3/CD28-coated beads (Invitrogen™, Life Technologies, Paisley, UK) with different Bead/T cell ratio. EGFR engagement was blocked using the specific pharmacological inhibitor AG-1478 (Calbiochem™, Merck Millipore, Darmstadt, Germany).

We also used the specific EGFR mAb, Erbitux (Cetuximab®, Merk Sorono, 1μg/mL), which links the EGFR extracellular domain and prevents binding of ligands.

Cytosolic calcium recording

Intracellular Ca2+ activity was determined as previously described (1). Briefly, CD4+ T cells were loaded at 1x10 6 /mL with Fura-2-AM (5µM, with 0.015% Pluronic F127 in RPMI medium) (Molecular Probes, ThermoFisher Scientific) for 30 min at room temperature, washed and then resuspended in RPMI medium. Cells were attached to poly(L)-lysine-coated petri dish (Sigma-Aldrich) for 20 min at 37°C and Fura-2 dual excitation was accomplished using 340-380nm excitation filters at 5 seconds intervals. Cells were then perfused with the bath solution in the presence or absence of 2 mM extracellular Ca2+ and stimulated with 5µg/mL of anti-CD3

antibody (BD biosciences). Variation of fluorescence ratio reveal intracellular variation of free AUTHOR RESPONSIBILITIES: RELATIONSHIP WITH INDUSTRY AND FINANCIAL DISCLOSURE

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