Dmp1 (Dentin matrix protein 1), encoding a non-collagenous extracellular matrix protein, plays an important role in dentin and bone mineralization. However, the transcriptional regulation of lncRNA on Dmp1 has not been reported. In this study, we identified a novel lncRNA named lnc-DMP1, which is near the Dmp1 gene region and undergoes remarkable changes during mandible development. lnc-DMP1 is co-localized and significantly expressed correlation with Dmp1 in embryonic and postnatal mouse mandibles. In MC3T3-E1 cells, lnc-DMP1 positively regulates DMP1 expression and skeletal mineralization. Furthermore, lnc-DMP1 induces the promoter activity of Dmp1 by modulating H3K27Ac enrichment in the Dmp1 promoter. In conclusion, our results indicate that lnc-DMP1 is a novel lncRNA near the Dmp1 gene region and regulates Dmp1 expression by modulating the H3K27 acetylation level of Dmp1 promoter.Several long non-coding RNAs (lncRNAs) have been reported regulate the expression of neighbor protein-coding genes at post-transcriptional, transcriptional and epigenetic levels.  Xist in X-chromosome inactivation was first described  are defined as a subgroup of non-coding RNA molecules that consist of at least 200 nucleotides and exhibit no or limited protein-coding capability . Since tescribed , studiesescribed , antivirescribed  and diffescribed . Studieset genes . Other lspeckles . Some cyspeckles .Dmp1 leads to severe defects in the odontogenesis of the dentin and cartilage formation of bones in mice , a highly phosphorylated acidic non-collagenous phosphoprotein, is initially identified by cDNA cloning in rat odontoblasts and highly expressed in bone tissues . DMP1 pl in mice , 2005. In humans .Dmp1 by c-Fos and c-Jun, two AP-1 transcriptional factor family members, which play important roles in early osteoblast differentiation. They also demonstrated that JunB can interact with p300 and dramatically modulate the promoter activity of Dmp1 during osteoblast mineralization  to investigate whether lnc-DMP1 was significantly correlated with Dmp1. Subsequently, whether lnc-DMP1 could participate both mRNA and protein expression of DMP1 and mineralization were tested in osteoblast cells MC3T3-E1. Finally, luciferase assay and chromatin immunoprecipitation (ChIP) methods were performed to investigate whether lnc-DMP1 could transcriptionally regulate the Dmp1 expression in MC3T3-E1 cells by modulating the enrichment of H3K27Ac in Dmp1 promoter.Since the transcription factor JunB regulates the expression of nsferase . Meanwhinsferase . So, we Embryonic  and postnatal (2 and 4 weeks old) C57 mice were purchased from the Animal Experiment Center of Sun Yat-sen University . The body weight of the mice was 10–15 g, and the mice were fed at room temperature (20–22°C) (License code: SYKX: 2012-0081). The mice were anesthetized by intraperitoneally injecting 5% chloral hydrate (0.1 mL/10 g) and then fixed on a dry ice anatomy bench. The mandibles were extracted under a microscope, frozen in liquid nitrogen and stored at −80°C. After sampling was completed, the mice were sacrificed by dislocating their necks and then treated in accordance with the death treatment method for experimental animals. All animal experiments were conducted following the Ministry of Health national guidelines for housing and care of laboratory animals and performed in accordance with institutional regulations after review and approval by the Institutional Animal Care and Use Committee at the Sun Yat-sen University (IACUC-DB-13-132).2 at 37°C.The MC3T3-E1 cell line  was cultured in α-MEM  and pretreated with 10% fetal bovine serum. The cells were cultured in an incubator with humidified atmosphere and 5% COTotal RNA was isolated from the mandibles of embryonic (18 days old) and postnatal (2 weeks old) C57 mice with TRIzol reagent  in accordance with the protocol. RNA quantity and quality were evaluated with Agilent Bioanalyzer 2100. The qualified RNA samples were purified and synthesized to cDNA. After cluster amplification was conducted through PCR, a sequencing library was sequenced with Illumina HiSeqTM2000. Raw sequences were filtered to remove the joint, undetected sequences and low-quality sequences. Transcriptional transcripts were constructed from the remaining sequence, and known non-coding RNAs and protein-encoding fragments were discarded. The new non-coded sequence and the known non-coding sequence were compared and statistically analyzed to screen the differentially expressed lncRNAs in the two-time-point tissue samples.P-value by controlling the false discovery rate (FDR) to determine the P-value of the domain. LncRNAs with | log2(E18D/P2W) | >4 and FDR < 0.01 were considered differentially expressed in E18D and P2W samples.On the basis of the Poisson distribution model, we simultaneously calculated the differential expression multiples [log2(E18D/P2W)] of lncRNA in E18D and P2W samples in accordance with the gene expression  and examined the differences in Dmp1 probes overnight at 37°C. Afterward, the slides were washed twice with 2 × saline sodium citrate (SSC) and three times with deionized water formamide: 2 × SSC (50:50) for 20 min. DAPI solution was used to stain the cell nuclei. Fluorescence images were acquired through fluorescence microscopy.The mandible samples of embryonic  and postnatal (2 and 4 weeks old) C57 mice were collected and fixed in 4% paraformaldehyde for 48 h and then decalcified in EDTA. The decalcified specimens were paraffin embedded, and 5 mm serial sections were prepared for the experiments. The slides were hybridized with lnc-DMP1 and For lnc-DMP1 overexpression, the full length of lnc-DMP1 was cloned into the lentivirus vector pCDH-CMV-MCS-EF1-copGFP . The shRNA sequence targeting lnc-DMP1 was cloned into the lentivirus vector psi-LVRU6MH (Gene) for the interference of the lnc-DMP1 expression. All of the vectors were transfected into MC3T3-E1 cells in accordance with the manufacturer’s instructions. The efficiency of transfection was confirmed through qRT-PCR. The cells were subjected to RNA extraction, Western blot assay and functional assays.GAPDH was used as reference genes, and the relative gene expression level was calculated by using the comparative threshold (Ct) cycle method (2Ct–ΔΔ).The total RNA of mandible samples and cells was extracted with TRIzol reagent in accordance with the manufacturer’s instructions (Thermo Fisher Scientific) and reverse transcribed with a high-capcity cDNA reverse transcription kit . SYBR Green PCR master Mix (Vazyme) and primers  were useThe cultured cells were extracted by RIPA (Radio Immunoprecipitation Assay) ordinary type reagent (Vazyme), which is a traditional cell tissue lysis buffer to gain proteins for western and IP experiments. Then, 30 μg of each protein was loaded to each lane, fractionated by 10% SDS-PAGE, transferred onto PVDF membranes and probed with the primary antibody of DMP1 and DSPP  as a target and GAPDH  as an internal control at 4°C overnight. The secondary antibodies were added for 2 h. The blots were visualized through enhanced chemiluminescence and autoradiography.The lnc-DMP1 overexpression and RNAi stable cell lines were induced by osteoinductive factors  for 28 days. After mineralization was induced, the medium was discarded. The cells were rinsed with PBS three times, fixed with 95% ethanol for 20 min and stained in alizarin red solution for 10 min. The calcification nodules formed in each group were compared.pDmp1 reporter vector that including both renilla luciferase with 35S promoter and firefly luciferase driven by the Dmp1 promoter (2000 bp before transcription start site). The plasmids of CMV empty vector, CMV-lnc-DMP1, U6-Sh empty vector and U6-Sh-lnc-DMP1 were co-transfected with pGL3-pDmp1 plasmid into MC3T3-E1 cell. Lipofectamine 2000 was used. After 48 h of transfection, the cells were collected, and luciferase activity was measured by using a dual luciferase reporter assay kit  in accordance with the manufacturer’s protocol. The firefly luciferase activities of the Dmp1 promoter co-transfected with CMV vectors or U6-Sh vectors were firstly normalized to renilla luciferase activity of itself, then showed as a relative LUC activity to the empty vector control CMV-lnc-DMP1 or U6-Sh empty vector.The dual-luciferase reporter vector pGL3-Basic vector, which contains renilla luciferase with 35S promoter and firefly luciferase without driven promoter, was used to construct the pGL3-ChIP was conducted by using a ChIP kit  in accordance with the manufacturer’s instructions. In brief, the cells were incubated with formaldehyde, and the link between DNA and protein was built. Then, the cross-linked chromatin was sonicated into fragments. Anti-H3K27ac antibodies (Abcam) were used for the immunoprecipitation of the chromatin fragments, and IgG was used as the negative control. Subsequently, the precipitated chromatin DNA was measured through qRT-PCR.In vitro, part of purified PCR product pDmp1 DNAs, which was 378 bp length of Dmp1 promoter before the transcriptional start site (TSS), were biotin-labeled with a Biotin DNA labeling mix . The Biotin-labeled and unbiotin-labeled pDmp1 DNAs were mixed with MC3T3-E1 cell protein extract in 500 μL incubation buffer  with 0.2% TritonX 100 and protease inhibitor) and then incubated at 4°C overnight. Afterward, 50 μL of washed streptavidin agarose beads (Thermo Fisher Scientific) was added to each binding reaction and further incubated at RT for 1 h. The beads were washed briefly with wash buffer [RIP buffer  with 0.1% TritonX 100] five times and boiled in SDS buffer. The retrieved proteins were detected by Western blot.The primer sequences are shown in t-test. Differences with a P < 0.05 were considered statistically significant (∗P < 0.05 and ∗∗P < 0.01).Data were analyzed with SPSS 20.0 and presented as mean ± SD. Differences between the two groups were assessed by using two-tailed Student’s To examine the expression profiles of lncRNAs in the development of mouse mandibles, we performed lncRNA sequencing (lncRNA-seq) analysis. RNA was collected from the mouse mandibles of embryonic18-day-old (E18D) and postnatal 2-week-old (P2W) mice. A total of 38,566 lncRNAs were identified in the two samples . ThroughDmp1 in cis, we searched lncRNAs located within 100 kb of the Dmp1 gene region (Chromosome 5:104202613-104214102) from the lncRNA-seq data. We found that lnc19585 and lnc741 were located downstream of Dmp1, with distances of 16.7 and 45.6 kb, respectively, whereas lnc30475 was located upstream of Dmp1, with distance of 68.6 kb  and lnc-Dmp1 (red fluorescence) were highly co-localized (yellow fluorescence) in all of the developmental processes of mouse mandibles, and the co-localization was distributed in the region, including the nuclei (blue fluorescence) and the cytoplasm. We found that the expression levels of lnc-DMP1 and Dmp1 decreased in the postnatal mouse mandibles  was enriched in the promoter region of Dmp1 in human H1-hESC cells. Hence, we conducted a ChIP assay to detect the enrichment of H3K27Ac in the promoter of Dmp1 in MC3T3-E1 cells. The results showed that the acetylation of H3K27 in the promoter of Dmp1 was enriched (Dmp1 promoter to the H3K27Ac protein (lncRNA can bind adaptor protein and target transferase to drive histone modification and active gene transcription . Accordienriched . The DNA protein .Dmp1. To confirm this hypothesis, we performed ChIP assay to detect the enrichment of H3K27Ac in the promoter of Dmp1 in lnc-DMP1 OE and lnc-DMP1 RNAi stable lines. As shown in Dmp1 promoter, whereas the RNAi of lnc-DMP1 decreased the enrichment of H3K27Ac of the Dmp1 promoter. These results suggested that lnc-DMP1 induced the promoter activity by modulating the H3K27Ac enrichment of Dmp1 promoter.Given that the enhanced H3K27Ac histone modification is usually an important agonist for transcriptional regulation, we hypothesized that lnc-DMP1 participated in the acetylation of H3K27 in the promoter of Dmp1 is highly expressed in bone and dentin tissues and plays a key role in mineralization, but the mechanism of its regulation needs to further investigated (Dmp1.lncRNAs play important regulatory roles during multiple biological processes and in various diseases . Histonestigated . In thisDmp1 gene region and exhibited the most remarkable changes during mandible development. The RNA-FISH assay demonstrated that lnc-DMP1 and Dmp1 were expressed in the same areas in the mandibles and co-localized in the cytoplasm and the nuclei. Moreover, the significant correlation between the expression levels of lnc-DMP1 and Dmp1 at different stages of mandible development implied the presence of a regulatory mechanism between them. After constructing the stable lines of lnc-DMP1 overexpression and RNAi in osteoblast cell lineMC3T-E1, we found that overexpressing lnc-DMP1 promoted both mRNA and protein expression of DMP1 and skeletal mineralization in MC3T3-E1 cells. These results indicated that lnc-DMP1 positively regulated the expression of DMP1 to control the mineralization. The luciferase results implied that lnc-DMP1 induced the promoter activity of Dmp1. Given that the acetylation level of H3K27 was positively correlated with the transcriptional activity, the results of the ChIP assays confirmed our hypothesis that lnc-DMP1 enhanced Dmp1 expression by enriching H3K27Ac in Dmp1 promoter.lnc-DMP1, a novel lncRNA validated by the lncRNA sequence in our study, was near the Dmp1 during the development of mouse mandibles. The lnc-DMP1 regulated Dmp1 expression and mineralization by modulating the H3K27ac enrichment of Dmp1 promoter in osteoblast cells. In our future studies, we will further investigate chromatin-modifying complexes, which interact with lnc-DMP1 to modulate Dmp1 expression. Collectively, our findings will serve as a basis for exploring the mechanism of lncRNAs in the regulation of bone regeneration, including periodontal tissue reconstruction, and for tissue engineering and clinical practice.In summary, our study identified a novel lnc-DMP1 positively related to the PRJNA540617 and BioSample accessions are SAMN11549457 and SAMN11549458.The lnc-RNA sequencing data mentioned in our manuscript has been submitted to the SRA in NCBI, the SubmissionID is BioProject ID is XX, YR, BL, YY, XK, and PZ performed the experiments and/or analyzed the data. HW designed the research. YR participated the design of some experiments. XX and HW wrote the manuscript. All co-authors corrected the manuscript.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.


Zero-determinant (ZD) strategies, a recently found novel class of strategies in repeated games, has attracted much attention in evolutionary game theory. A ZD strategy unilaterally enforces a linear relation between average payoffs of players. Although existence and evolutional stability of ZD strategies have been studied in simple games, their mathematical properties have not been well-known yet. For example, what happens when more than one players employ ZD strategies have not been clarified. In this paper, we provide a general framework for investigating situations where more than one players employ ZD strategies in terms of linear algebra. First, we theoretically prove that a set of linear relations of average payoffs enforced by ZD strategies always has solutions, which implies that incompatible linear relations are impossible. Second, we prove that linear payoff relations are independent of each other under some conditions. These results hold for general games with public monitoring including perfect-monitoring games. Furthermore, we provide a simple example of a two-player game in which one player can simultaneously enforce two linear relations, that is, simultaneously control her and her opponent’s average payoffs. All of these results elucidate general mathematical properties of ZD strategies. Game theory is a powerful framework explaining rational behaviors of human beings  and evolRecently, in repeated games with perfect monitoring, a novel class of strategies, called zero-determinant (ZD) strategy, was discovered . SurprisThe contributions of this paper are four-fold. First, we extend ZD strategy for general multi-player multi-action repeated games with public monitoring, where players know the structure of games  but cannot observe actions of other players. A typical example of such situation is auction. In a sealed-bid auction, a player cannot know actions (bids) of other players, but only knows the result of the game (whether she is the winner or not). Second, we prove, in terms of a linear-algebraic argument, that linear payoff relations enforced by players with ZD strategies are consistent, that is, always have solutions. Third, we introduce the notion of independence of ZD strategies, and prove, again in terms of a linear-algebraic argument, that linear payoff relations enforced by players with ZD strategies are independent under a general condition. Fourth, as an application of linear algebraic formulation, we provide a simple example of a two-player game in which one player can simultaneously enforce two linear relations. This means that she can simultaneously control her and her opponent’s average payoffs, which has never been reported in the context of ZD strategies. All of these results develop deeper understanding of mathematical properties of ZD strategies in general games.δ ≤ 1 [δ = 1) was investigated [δ < 1 case [δ = 1.We remark on discounting. In standard repeated games, discounting of future payoffs is considered by introducing a discounting factor stigated . After t< 1 case , 25, 26.N-player multi-action repeated game, in which player n ∈ {1, ⋯, N} has Mn possible actions, where Mn is a positive integer. Let N players. Let n decides the next action stochastically according to her own previous action τ ∈ B with the conditional probability B is some set. We also define the conditional probability that common information τ arises when actions of players in the preceding round are σ′ by W(τ|σ′).  denotes the state distribution at time t. We assume that all players know the function W(τ|σ′) but cannot directly observe σ′. When B = Σ and W(τ|σ′) = δτ,σ′, the above formulation reduces to that of perfect monitoring games. Otherwise, it represents games with public monitoring, where players cannot directly observe actions of other players. The model treated here can therefore be regarded as an extension of repeated games with perfect monitoring to those with imperfect monitoring, and the extension includes the former as a special case.We consider an σ, a payoff of player n is defined as sn(σ). Let sn ≡ (sn(σ′))σ′∈Σ be the M-dimensional vector representing the payoffs of player n, which we call the payoff vector of player n. It should be noted that in the following analysis we do not assume the payoffs to be symmetric, unless otherwise stated.For each state δ is one, the payoffs of players are the average payoffs with respect to the stationary distribution of the Markov chain. Let Ps)((σ) denote the stationary distribution, which may depend on the initial condition when the Markov chain is not irreducible. It satisfiesσn− ≡ σ∖σn with an arbitrary n, we obtainn repeats the previous action with probability one, one can readily see that n and σn. We remark that all players are assumed to know the functional form of W(τ|σ′), and that Tn(σn|σ′) as well, are solely under control of player n. Because of the normalization condition Because a discounting factor n associated with action σn. .Let n vector .) A straρ ≡ (Ps)((σ))σ∈Σ be the vector representation of the stationary distribution Ps)((σ). When player n chooses a strategy v with respect to the stationary distribution ρ vanishes.Let Let strategy , 27 to mDefinition 1. A zero-determinant (ZD) strategy is defined as a strategyfor which dim Vn ≥ 1 holds.en equal to the expected payoff en ≡ 〈sn(σ)〉s of player n in the steady state. When player n employs a ZD strategy, it amounts to enforcing linear relations e with α satisfying To see that this is indeed an extended definition of the ZD strategy, note that any vector e1, e2) have a solution? Let N′ be the set of players who employ ZD strategies. The set N′. If E is empty, then it implies that the set of ZD strategies is inconsistent in the sense that there is no valid solution of the linear relations enforced by the players.A question naturally arises: When more than one of the players employ ZD strategies, are they “consistent”, that is, do linear payoff relations enforced by the players always have solutions? For example, in a two-player game, when player 1 enforces Definition 2. ZD strategies are said to be consistent when E is not empty.N′ as a variant of a ZD strategy alliance [N′ agree to coordinate on the linear relations to be enforced on the expected payoffs. The above question then amounts to asking whether it is possible for a player to serve as a counteracting agent who participates in the ZD strategy alliance with a hidden intention to invalidate it by adopting a ZD strategy that is inconsistent with others.In the multi-player setting, one may regard alliance , where tThe following proposition is the first main result of this paper.Proposition 1. Any set of ZD strategies is consistent.Proof. We first note that the following property holds for strategy vectors, whose proof is given in Methods.Lemma 1. LetThenVn)n∈N′ of ZD strategies, let K be the dimension of span(Vn)n∈N′, and let Vn)n∈N′. The expected payoff vector A, b, and For any set span(Vn)n∈N′ to be consistent (because A is augmented matrix). An equivalent expression of this condition is that there is no vector 1M, leading to contradiction.The Rouché-Capelli theorem  tells usProposition 1 states that it is impossible for any player to serve as a counteracting agent to invalidate ZD strategy alliances. This statement is quite general in that it applies to any instance of repeated games covered by our formulation.In Ref. , it was Mn − 1), depending on n with Mn ≥ 3 to adopt a ZD strategy for which dim Vn ≥ 2 holds. The dimension of Vn corresponds to the number of independent linear relations to be enforced on the expected payoffs of the players, so that it implies that one player may be able to enforce multiple independent linear relations. On the other hand, our result on the absence of inconsistent ZD strategies implies that for any set N′ of ZD players the dimension of span(Vn)n∈N′ should be at most N, the number of players, since any set of ZD strategies should contain at most N independent linear relations if it is consistent. This in turn implies that if the dimension of span(Vn)n∈N′ is equal to N for a subset N′ of players then players not in N′ cannot employ independent ZD strategy any more.Since the dimension of Another naturally-arising question would be regarding independence for a set of ZD strategies, which we define as follows:Definition 3. A setof ZD strategies is independent if any set {vn}n∈N′vof non-zero vectors nin Vnis linearly independent. Otherwise, is said to be dependent.If a set of ZD strategies is dependent, then there exists a ZD player whose ZD strategy adds no linear constraints other than those already imposed by other ZD players. One of the simplest example of a dependent set of ZD strategies is the case where two players enforce exactly the same linear relation to the expected payoffs. Our second main result is to show that any set of ZD strategies is independent under a general condition.Proposition 2. Let N′ be a subset of players. Assume thatdoes not have zero elements for any n ∈ N′ and any σn ∈ {1, …, Mn}. Then, any setof ZD strategies of players in N′ is independent.See Methods for the proof.s1 and s2 for players 1 and 2 be T ≠ S. If player 1 adopts the strategye1 = e2. This strategy is a well-known tit-for-tat strategy [It should be noted that when strategy . By symmMn of possible actions for player n is more than two, player n may be able to employ a ZD strategy with dim Vn ≥ 2 to simultaneously enforce more than one linear relations.  = 0 is an absorbing state regardless of the strategy of player 2 in this case.We consider the 3 × 3 symmetric gamee1 = e2 = C is allowed with some C. This is explained as follows: Assume that player 1 can simultaneously enforce e1 = C1 and e2 = C2 with C1 ≠ C2 by one ZD strategy. Because the game is symmetric, player 2 can also simultaneously enforce e1 = C2 and e2 = C1 independently by one ZD strategy. This contradicts the consistency of ZD strategies (Proposition 1). Therefore, the only possibility is e1 = e2 = C.In general, when one player simultaneously enforces two linear relations in two-player multi-action symmetric games, only The above argument can be extended straightforwardly to the multi-player case. For that purpose, we introduce some notions of symmetric multi-player games. The following definition of a symmetric multi-player game is due to von Neumann and Morgenstern [Definition 4. A game is symmetric with respect to a permutation πon {1, …, N} if Mn = Mπ(n)holds for any n ∈ {1, …, N} and if π preserves the payoff structure of the game, that is,σholds for any  ∈ Σ and for any n ∈ {1, …, N}, σwhere π ≡ (σπ(1), …, σπ(N)).The following definition is due to Ref. .Definition 5. A game is weakly symmetric if for any pair of players n andthere exists some permutation π on {1, …, N} satisfyingsuch that the game is symmetric with respect to π.N-player weakly symmetric game. Assume that one player simultaneously enforces N independent linear relations on the average payoffs {en}n ∈ {1, …, N} of N players via adopting an N-dimensional ZD strategy. (Note that for this to be possible the number Mn of actions should satisfy Mn ≥ N + 1). Then, the average payoffs {en}n∈{1,…,N} should be simultaneously controlled, but they should satisfy e1 = e2 = ⋯ = en due to the consistency of ZD strategies.Consider an N in weakly symmetric N-player games can be seen in the following two propositions, whose proofs are given in Methods.The difficulty of construction of a ZD strategy of one player with dimension Proposition 3. In a weakly symmetric N-player game, if the strategy vectors of one player contain no zero element, then a ZD strategy of the player with dimension N is impossible.Proposition 4. In a weakly symmetric N-player game, if payoffs sn(σ) σof player n are different from each other for all , then a ZD strategy with dimension N is impossible.In this paper, we have derived ZD strategies for general multi-player multi-action public-monitoring games, in which players cannot observe actions of other players. By formulating ZD strategy in terms of linear algebra, we have proved that linear payoff relations enforced by ZD players are consistent. Furthermore, we have proved that linear payoff relations enforced by players with ZD strategies are independent under a general condition. We emphasize that these results hold not only for imperfect-monitoring games but also for perfect-monitoring games. We have also provided a simple example in which one player can simultaneously enforce more than one linear constraints on the expected payoffs. These results elucidate constraints on ZD strategies in terms of linear algebra.N + 1 for zero-sum games, and construction of ZD strategies for zero-sum games is expected to be more difficult compared to non-zero-sum games. Consistency together with constraints on payoffs such as symmetry and linear dependence may be useful to specify the space of ZD strategies which can exist. Specifying a general criterion for the existence of ZD strategies is an important future problem.Although we have discussed mathematical properties of ZD strategies if exist, we do not know the criterion for whether ZD strategies exist or not when a game is given. For example, we can easily show that ZD strategy does not exist for the rock-paper-scissors game, which is the simplest two-player three-action symmetric zero-sum game. ≥0holds fo(σn|σ′)≥0holds foσ′ ∈ Σ satisfying n ∈ N′. The above argument has shown that the inequality  = 0 holds. Since σn ∈ {1, …, Mn}, they are all equal to zero. Since σn ∈ {1, …, Mn} and consequently α = 0N+1 holds, leading to contradiction.Let π, player n with π(n) ≠ n cannot enforce linear relations απ = α ≠ 0N+1. In other words, in a symmetric game no player to whom the game is symmetric can enforce a linear relation with the same symmetry as the game itself.It should be noted that Lemma 3 holds even if one takes Proposition 3 is a direct consequence of Lemma 3 in weakly symmetric multi-player games.k takes an N-dimensional ZD strategy determining the average payoffs en for n = 1, ⋯, N. Due to the above discussion, only e1 = ⋯ = eN = C is allowed. Letting n ∈ {1, …, N}, one can take N-dimensional ZD strategy. Let cn) = k such that the game is symmetric with respect to π. Noting that Without loss of generality, we assume that player n ∈ {1, …, N}, define σ* satisfying sn(σ*) = C. On the other hand, we also obtain for an arbitrary σ** satisfying sn(σ**) = C. Then, because we have assumed that all elements of the payoff vector sn are different from each other, we have arrived at a contradiction.For from Eqs  and 21)n ∈ {1, …S1 Text(PDF)Click here for additional data file.


AR mutations is a routine approach in AIS diagnosis. However, some AIS patients lack AR mutations, which complicates the diagnosis. Here, we describe a patient suffering from partial androgen insensitivity syndrome (PAIS) and lacking AR mutations. The whole exome sequencing of the patient and his family members identified a heterozygous FKBP4 gene mutation, c.956T>C (p.Leu319Pro), inherited from the mother. The gene encodes FKBP prolyl isomerase 4, a positive regulator of the AR signaling pathway. This is the first report describing a FKBP4 gene mutation in association with a human disorder of sexual development (DSD). Importantly, the dysfunction of a homologous gene was previously reported in mice, resulting in a phenotype corresponding to PAIS. Moreover, the Leu319Pro amino acid substitution occurred in a highly conserved position of the FKBP4 region, responsible for interaction with other proteins that are crucial for the AR functional heterocomplex formation and therefore the substitution is predicted to cause the disease. We proposed the FKBP4 gene as a candidate AIS gene and suggest screening that gene for the molecular diagnosis of AIS patients lacking AR gene mutations.Androgen insensitivity syndrome (AIS), manifesting incomplete virilization in 46,XY individuals, is caused mostly by androgen receptor (AR) gene mutations. Therefore, a search for  Androgens govern the development of reproductive and non-reproductive pathways of the male body during the successive life periods, resulting in human sexual dimorphism. Testosterone and dihydrotestosterone are the most crucial androgens inducing male development. In the male embryo, they are responsible for the development of the Wolffian duct into the internal male sex organs and virilization of external genitalia, respectively. At the puberty period, androgens govern the growth of male internal and external genitalia as well as the growth of skeletal muscle, development of larynx, and general growth spurt. In the adult, androgens regulate spermatogenesis, muscle mass, bone metabolism as well as behavior; for a review, see ,2,3. AndAR gene (located on the X chromosome) cause the dysregulation of the AR signaling pathway, resulting in androgen insensitivity syndrome (AIS) in 46,XY individuals. This syndrome is characterized by abnormalities of the internal and external genitalia virilization as well as secondary sex characteristics. This occurs despite the presence of hormonally active bilateral testes producing normal levels of testosterone and anti-Müllerian hormone (AMH), for a review, see [AR gene, in some AIS individuals no AR gene mutations have been found. Namely, around 10% of CAIS and 60–80% of PAIS patients do not carry such mutations; for a review, see [MAMLD1 gene [NR5A1 gene, encoding steroidogenic factor 1 [AR mutations are genetically unexplained. It was hypothesized that mutations in the AR co-regulator proteins could account for the AIS phenotype [AR gene mutation revealed reduced AR transactivation ability since the mRNA level of an AR target gene was significantly lowered. This data supports the existence of cellular components, besides the AR, affecting androgen signaling during sexual differentiation and suggests their involvement in the etiology of AIS when disrupted. That phenotypic AIS subgroup characterized by a lowered expression of AR target while lacking AR gene mutation is called AIS type II [Loss-of-function mutations in the iew, see . The speiew, see . Althougiew, see ,13. StudLD1 gene  or the Nfactor 1 . Neverthhenotype . Accordi type II . TherefoFKBP4 gene mutation in a patient manifesting PAIS with hypospadias.Here, we report for the first time a potentially causative 2). Later on, at the age of 13, urological examinations revealed the size of penis and testes still smaller compared to the average, namely penis length was 28 mm, the right testis size was 58 mm × 24 mm (7.28 mL), the left one was 60 mm × 21 mm (6.59 mL).The chromosomal analysis of the patient showed a 46,XY karyotype without evidence of mosaicism. At the age of 10, urological examinations revealed the size of penis and testes being smaller, compared to the average at his age: penis length was 25 mm, the right testis 9 mm × 7 mm (0.33 mL), the left one 12 mm × 9 mm (0.565 mL). Pelvic ultrasound investigation did not reveal any structure of Müllerian ducts. The patient was overweight , estradiol, luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), prolactin (PRL), dehydroepiandrosterone sulfate (DHEAS), cortisol, and progesterone within the proper range, in accordance with age . The humTaking into consideration (1) the characteristics of the disorder of sexual development (DSD) phenotype of the patient, (2) his male karyotype, (3) the results of the hCG stimulation tests excluding defects in testosterone biosynthesis, (4) the normal AMH level excluding gonadal dysgenesis, and (5) normal LH and FSH levels excluding hypogonadotropic hypogonadism, we concluded that the patient suffered from PAIS.AR gene mutations are significantly less frequent in PAIS than in CAIS patients [AR gene mutation, it uncovered a heterozygous mutation in the FKBP4 autosomal gene encoding an AR interactor [Since patients , we procteractor  being a teractor ,8,9,10. Importantly, the mutation was located within one of three tetratricopeptide repeats (TPR), responsible for interaction with several proteins crucial for AR activity. Moreover, the 319 position in which the Leu319Pro substitution occurred is highly conserved among vertebrate Fkbp4 homologues . The FKBFKBP4 gene mutation, six additional heterozygous variants in five genes  involved in sexual development were identified in the patient . However, three  out of the six variants were predicted as neutral using Meta-SNP , biosample accessions: SAMN16449689, SAMN16449690, SAMN16449691.AR gene mutation is significant, which makes their genetic diagnosis difficult. Here, we report a patient manifesting a PAIS phenotype, lacking any AR gene mutation and instead carrying a novel FKBP4 gene variant. The gene seems to be a strong candidate for PAIS, since the knockout of its homologue in the mouse caused a phenotype consistent with PAIS, including hypospadias [FKBP4 gene mutation in a group of patients with hypospadias was described but no variant associated with that condition was found [FKBP4 variant identified in our PAIS patient carries a missense p.Leu319Pro mutation. This mutation is considered causative for several reasons. Firstly, it is located within the TPR domain, which is crucial for FKBP4-Hsp90 interaction within the AR heterocomplex, as well as for FKBP4 interaction with S100A1 and S100A2 proteins, which regulate FKBP4-Hsp90 complex formation [Fkbp4 mutant males did not display hypospadias and were fertile, in contrast to homozygous Fkbp4 mutant animals displaying a phenotype corresponding to PAIS [SOX8 gene, encoding a transcriptional factor closely related to SRY , caused a 46,XY DSD phenotype [Sox8 knockout did not affect the sex determination process. Instead, it caused Sertoli cell function impairment in the adult, leading to infertility or decreased fertility in males [FKBP4 heterozygous variant and a lack of the abnormal phenotype in heterozygous knockout in mice does not rule out a causative effect of the FKBP4 variant in the patient.The number of AIS patients with no ospadias . Moreoveospadias  and its ospadias , leadingospadias ,8,9,10. as found . The FKBormation . Taking  to PAIS . Still, henotype . Meanwhiin males . TherefoAKR1C4, CYP17A1, FREM2, IL17RD, NKD2, involved in sexual development, do not seem to be causative for the patient’s phenotype. Namely, three among these variants were predicted to be neutral  via vaginal delivery without complications. At birth, the patient manifested a scrotal form of hypospadias, small phallus (18 mm) with ventral chordae, bilateral cryptorchidism . The fourth degree of virilization was assessed according to the Prader scale. At the age of 6, the patient underwent surgery due to hypospadias, accompanied by orchiopexy. At the age of 10, a comprehensive examination, such as karyotyping, urological examinations (including testicle and pelvic ultrasound investigation), and hormonal analysis (including testosterone synthesis stimulation test) were performed. At the age of 13, urological examinations and the testosterone synthesis stimulation test were repeated. The patient expressed male psycho-sexual identity. His parents consented to endocrine and genetic studies.At the age of 10, the patient was subjected to a complex hormonal examination. The levels of AMH, LH, FSH, TSH, PRL, DHEAS, cortisol, and progesterone were determined using the cobas e 411 instrument  and applying commercial kits . The efficiency of testosterone synthesis was examined upon 3-day testosterone stimulation with 1500 IU hCG  . The hCG®). Sequencing was performed on Illumina HiSeq 2000 device, and Fastq files were obtained using the Illumina CASAVA v1.8.1 software. The raw data were analyzed using our bioinformatic pipeline hosted on the Vital-IT Center of the Swiss Institute of Bioinformatics . Exome capture was performed on the proband and parents’ DNA samples by using the SureSelect Human All Exon v3 kit , as precs  , the 100FKBP4 NM_002014.3, AKR1C4 NM_001818, CYP17A1 NM_000102, FREM2 NM_207361, IL17RD NM_017563, NKD2 NM_001271082. Accession numbers of vertebrate FKBP4 proteins used are: Homo sapiens NP_002005.1, Mus musculus NP_034349, Anolis carolinensis XP_008109533, Gallus gallus NP_001006250, Xenopus laevis NP_001084593, Danio rerio XP_005173673.Accession numbers of Homo sapiens mRNAs used are:


Microsatellite or simple sequence repeat (SSR) instability within genes can induce genetic variation. The SSR signatures remain largely unknown in different clades within Euarchontoglires, one of the most successful mammalian radiations. Here, we conducted a genome-wide characterization of microsatellite distribution patterns at different taxonomic levels in 153 Euarchontoglires genomes. Our results showed that the abundance and density of the SSRs were significantly positively correlated with primate genome size, but no significant relationship with the genome size of rodents was found. Furthermore, a higher level of complexity for perfect SSR (P-SSR) attributes was observed in rodents than in primates. The most frequent type of P-SSR was the mononucleotide P-SSR in the genomes of primates, tree shrews, and colugos, while mononucleotide or dinucleotide motif types were dominant in the genomes of rodents and lagomorphs. Furthermore, (A)n was the most abundant motif in primate genomes, but (A)n, (AC)n, or (AG)n was the most abundant motif in rodent genomes which even varied within the same genus. The GC content and the repeat copy numbers of P-SSRs varied in different species when compared at different taxonomic levels, reflecting underlying differences in SSR mutation processes. Notably, the CDSs containing P-SSRs were categorized by functions and pathways using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotations, highlighting their roles in transcription regulation. Generally, this work will aid future studies of the functional roles of the taxonomic features of microsatellites during the evolution of mammals in Euarchontoglires. Microsatellites, or simple sequence repeats (SSRs) are tandem repetitions of relatively short DNA motifs present in perfect (P-SSR), compound (C-SSR), and imperfect (I-SSR) forms in nearly all known genomes . PolymorAside from the ubiquity and functional significance of SSRs, tremendous progress has been made in characterizing the distribution patterns of SSRs in diverse eukaryotic genomes . Indeed,Euarchontoglires is a superclade of placental mammals that includes primates, rodents, lagomorphs, tree shrews, and colugos. Dynamic evolutionary signatures of microsatellites in Euarchontoglires genomes may be present because many species in this group are characterized by their successful adaptive radiation to various ecological niches. Although over 150 Euarchontoglires genomes are now available in the GenBank database, the SSR distribution patterns have only been studied in a handful of species in this clade . Thus, a1 for microsatellite identification and analysis. Ambiguous nucleotides were removed from the genomes prior to analysis. Detailed taxonomic information of these organisms was gathered from the National Center of Biotechnology Information (NCBI) database. The features of these genomes are presented in Currently, the genomes of 153 species  within Euarchontoglires are publicly available . All curAccording to the methods described previously , SSRs i. FurtherIn this study, the prevalence of SSRs in the genome was assessed by SSR abundance (loci/Mb) and SSR density (bp/Mb) as described by The GC content of mono- to hexanucleotide P-SSRs was also calculated for the GC composition analysis in each organism using in-house Python scripts. Meanwhile, the preference analysis of the repeat copy numbers (RCNs) and analysis of the coefficient of variability (CV) of the P-SSRs were analyzed according to the methods described by 2; To characterize the functional roles of the CDSs containing P-SSRs, these sequences were aligned with the NCBI non-redundant database and the SWISS-PROT database using Diamond  with a c3, and R (version 3.5.1) with the “ggplot2” package.SPSS  was used for the calculation of the Pearson correlation coefficient and the significance test. Figures were produced using Microsoft Office Excel 2013, ImageGPDaubentonia madagascariensis) to 9.87% (Myocastor coypus) of the Euarchontoglires genomes. Of these, I-SSRs were the dominant SSR category (62.05–82.58%) of the SSRs recovered, followed by P-SSRs (16.77–34.64%), and C-SSRs (0.60–4.17%). r = 0.619, p < 0.01; SSR length: Pearson, r = 0.495, p < 0.01). Moreover, the total number and total length of the SSRs in rodents, primates, lagomorphs, and tree shrews were positively correlated with genome size . Although the abundance and density of SSRs in rodent genomes were not significantly correlated with genome size , the abundance and density of SSRs in primate genomes were significantly positively correlated with genome size . Furthermore, no significant relationship between genome size and SSR abundance or SSR density was found in lagomorphs and tree shrews. In the genic regions, the abundance of SSRs also followed the pattern I-SSRs > P-SSRs > C-SSRs , however, dinucleotide P-SSRs dominated in Microtus agrestis, Microtus ochrogaster, and Microtus oeconomus but not in Microtus arvalis, in which the proportion of mononucleotide P-SSRs (36.74%) was slightly higher than that of dinucleotide P-SSRs (35.69%). Next, the numbers of P-SSRs in the CDS, exon, and intron regions of the Euarchontoglires genomes were further analyzed . On the other hand, the results showed that the most frequent repeated motif in each P-SSR category (mono- to hexanucleotide P-SSRs) exhibited different levels of variation among the mammals studied. Moreover, such variation was greater in rodents than in primates  varied within Euarchontoglires, a few taxon-specific features were observed . For exaPeromyscus maniculatus. The specific repeat motifs in genic and intergenic regions of different clades were further analyzed (not shown), which revealed that the number of the specific repeat motif categories in rodents was higher than that in other clades of Euarchontoglires.As described by Peromyscus genus, for example, the CV of the RCN of the hexanucleotide P-SSR in genomes varied from 30.39% (Peromyscus nudipes) to 343.14% (Peromyscus eremicus). Furthermore, the trend line for the CV of the mono- to hexanucleotide P-SSRs among different species also differed considerably , while di- (12 species) or hexanucleotide P-SSRs (15 species) had the highest GC content in rodents . This is likely explained by the fact that genome nucleotide composition could shape the prevalence of certain repeat units . Nonetheless, comparable results in genomes or in genomic regions of the five clades in this study could be observed when we plotted our results by using the average value of CV, e.g., the CVs of the RCNs of P-SSRs in CDS regions (SSRs with variable-length repeating motifs cause many human diseases . As expe regions . Microsa regions . Therefo regions , heteroz regions  and vari regions .Recent studies have shown that the functional significance of SSRs in the modulation of gene expression and genome organization implies their functional conservation across species. An increasing number of SSRs have been developed from the transcriptomes of many organisms because of their importance as a source of functional markers . HoweverTaxon-specific microsatellite distribution patterns were observed among different clades within Euarchontoglires. The dominant P-SSR categories in primates, tree shrews, and colugos were identical, but varied in rodents and lagomorphs. The enrichment of the most prevalent repeat motifs in specific clades was detected at the order level. However, the GC content and CV of P-SSRs varied greatly among different species at all taxonomic levels, suggesting that SSR variation might contribute to genetic variation among these mammals. We showed that the CDSs containing P-SSRs across all Euarchontoglires genomes were enriched for functions related to transcription. However, more work is needed to clarify the precise evolution and functional roles of taxon-specific P-SSRs in Euarchontoglires.The original contributions presented in the study are included in the article/XS, CZ, and YW: conceptualization. XS and TY: writing-original draft. XS, TY, XZ, and YY: data curation. TY, XY, and JZ: formal analysis. All authors contributed to the article and approved the submitted version.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 reviewer XZ declared a past co-authorship with one of the authors XS to the handling editor.


We observed that the antigen repertoire displayed on MHC-I molecules at the surface of MCA205 fibrosarcoma is modified upon treatment with IP2. In particular, IP2 enhances the presentation of an exon-derived epitope from the tumor suppressor nischarin. The combination of IP2 with a peptide vaccine targeting the nischarin-derived epitope showed a synergistic antitumor effect in vivo. These findings identify the spliceosome as a druggable target for the development of epitope-based immunotherapies.The success of cancer immunotherapy relies on the induction of an immunoprotective response targeting tumor antigens (TAs) presented on MHC-I molecules. We demonstrated that the splicing inhibitor isoginkgetin and its water-soluble and non-toxic derivative IP2 act at the production stage of the pioneer translation products (PTPs). We showed that IP2 increases PTP-derived antigen presentation in cancer cells in vitro and impairs tumor growth in vivo. IP2 action is long-lasting and dependent on the CD8 + T cell activation and showed greater antitumour activity in vivo. These findings identify the spliceosome as a druggable target for the development of epitope-based immunotherapies.Darrigrand, Pierson et al. study the effect of mRNA splicing inhibitors on the generation of the MHC class I ligands. They developed a derivative of the spliceosome inhibitor isoginkgetin which is less toxic and more effective at enhancing antigen presentation and CD8 Initial clinical trials investigating vaccines targeting tumor antigens (TA) have not met expectations. The main failures have been associated with immunosuppressive mechanisms and with a suboptimal choice of antigens3. One of the important events that drives tumor escape from immunosurveillance and is correlated with a poor prognosis is the loss or the downregulation of MHC-I antigen presentation by tumor cells5. The latter take advantage of defects in components of the MHC-I presentation pathway to escape cytotoxic T lymphocyte (CTL) and natural killer (NK) cell recognition6. Moreover, along with the overall decrease in MHC-I antigen presentation, the very nature and the number of antigens presented at the cell surface, namely the MHC-I immunopeptidome, is of critical importance for immune recognition. For example, the loss of expression at the tumor cell surface of a specific TA identified and targeted with immunotherapy, such as Her/neu in breast cancer or CEA in colon cancer, can lead to immune evasion8. To counter this phenomenon, current strategies aim at enlarging the range of targetable tumor antigens and restoring MHC-I antigen presentation12.All nucleated cells of jawed vertebrates present antigenic peptides (APs) at their surface through the major histocompatibility complex class I (MHC-I) presentation pathway. MHC-I peptides (MIPs) are 8- to 12-amino acid-long and reflect the inherent metabolic cellular activity16, we showed that one of the most important sources of APs is a pioneer translation event that occurs on pre-mRNAs, before introns are spliced out, and independently of the translation of the corresponding full length proteins. The pioneer translation products (PTPs) produced through this non-canonical translation event can therefore derive from intronic sequences, 3’ or 5’ untranslated regions (UTR) as well as alternative reading frames18. The discovery of PTPs emphasizes the existence of a nuclear translation mechanism of precursor mRNAs that participates to the production of MHC-I peptides (MIPs). Moreover, PTPs play a role in the dynamic of cancer development. When inoculated into mice, it has been shown that cancer cells presenting PTP-derived antigens at their surface can be recognized by specific T cells leading to tumor growth reduction19. Besides, purified PTPs encompassing a model immunodominant epitope efficiently promote anticancer immune response when injected as a peptide vaccine into mice.In order to understand the dynamics of the MHC-I immunopeptidome, we focused on the source of APs for the MHC-I presentation pathway. Exploring the concept of defective ribosomal products (DRIPs)21. However, the efficiency of those monoclonal antibodies is limited to a fraction of patients and is often associated with deleterious side effects22. Attention is being paid to small molecules with immunomodulatory properties, which offer a cost-effective production, a reversible action and reduced systemic side effects due to their short half-lives23.Cancer therapies have been constantly evolving with the hope of finding the most effective agents to eradicate tumors with the least toxic effects on healthy tissues. Unlike conventional cancer treatments that focus on tumor cells, immunotherapies target the immune system to strengthen and stimulate the patient’s own defenses against cancer. Breakthrough of the last decade, the immune checkpoint inhibitors against cytotoxic T lymphocyte associated protein 4 (CTLA-4), programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) have shown unprecedented antitumor effect in particular against advanced melanoma or non-small cell lung cancerGinkgo biloba tree. Multiple properties of the molecule have been reported and associated with its antitumor activity. Isoginkgetin was first shown to inhibit tumor cell invasion by inhibiting the production of the matrix metalloproteinase 9 (MMP-9)24. Indeed, isoginkgetin-induced downregulation of the NF-κB pathway leads to the upregulation of the MMP-9 inhibitor (TIMP-1) in human fibrosarcoma. More recently, it has been demonstrated that isoginkgetin inhibits 20 S proteasome activity and induces a toxic accumulation of polyubiquitinated proteins25. Eventually, isoginkgetin was described as a general inhibitor of pre-mRNA splicing, which stalls spliceosome assembly at the prespliceosomal A complex26. Pre-mRNA splicing is catalyzed in the nucleus by the spliceosome, a conserved and dynamic multi-RNA/protein complex composed of five small nuclear RNAs (snRNAs) in interaction with over 180 proteins27. A growing number of studies report that the deregulation of the spliceosome complex entails aberrant splicing patterns in many cancers contributing to abnormal tumor cell proliferation and progression31. In a recent study, we observed that splicing inhibition positively modulates the presentation of a PTP-derived model antigen in HEK-293T cells treated with isoginkgetin18.The biflavonoid isoginkgetin is a natural compound extracted from the leaves of the Here we show that the biflavonoid isoginkgetin and its water-soluble derivative IP2 enhance the presentation of PTP-derived antigens at the surface of cancer cells in vitro. In addition, IP2 induces a long-lasting anticancer immune response in vivo. Finally, IP2 was shown to reshape the MHC-I immunopeptidome of MCA205 fibrosarcoma. Our findings shed light on a new immunomodulatory agent whose antitumor activity relies on the induction of immunogenic epitopes that can be targeted in the context of epitope-based immunotherapies.50 determined by MTT assay  were treated with increasing doses of isoginkgetin up to the limit of IC+ T-cell response32. Therefore, we assessed the effect of isoginkgetin treatment on the antitumor response against SL8-expressing tumors in vivo. MCA205 fibrosarcoma and B16F10 melanoma stably expressing the globin-SL8-intron construct were inoculated subcutaneously into C57BL/6 mice. At days 5, 10, and 15 post-tumor inoculation, the mice were injected intraperitoneally with 18 mg/kg of isoginkgetin and the tumor growth was monitored. We observed a 60% decrease in the growth of MCA205-globin-SL8-intron at day 27  and two predicted binders of H-2Db molecules (affinity below 500 nM) were found to be enriched at the surface of MCA205 fibrosarcoma upon treatment with IP2. To refine our selection, we used the IEDB Analysis Resource36 to compute the immunogenicity score of the predicted strong binders, which accounts for peptide preferences in T-cell recognition. Eventually, two H-2Kb epitopes with an affinity for MHC-I molecules lower than 500 nM and a predicted immunogenicity score above 0.1 were shown to be enriched in at least two out of three IP2-treated samples, the SGYEFIHKL 9mer from the tyrosine–tRNA ligase (UniProtKB - E9PX65) and the TNQDFIQRL 9mer (TL9) from the nischarin (UniProtKB–B7ZN33), which has been described as a tumor suppressor in breast and ovarian cancer  encompassing the 9-amino acid-long sequence in central position was produced and its immunogenicity was assessed ex vivo. An irrelevant 39-amino acid-long SLP was also randomly generated and synthesized; both sequences are shared in Supplementary Fig. The TNQDFIQRL epitope shows a good computed immunogenicity score and the TL9 SLP was shown to be immunogenic in two different immunoassays therefore the antitumor potential of the combination between IP2 and the TL9 SLP was assessed in vivo. In a prophylactic setting, mice were vaccinated twice with 100 µg of TL9 SLP, then challenged subcutaneously with MCA205 fibrosarcoma and finally treated intraperitoneally with 24 mg/kg of IP2 Fig. . VaccinaOverall, we show here in the mouse MCA205 fibrosarcoma model that IP2 induces the presentation of immunogenic epitopes that can be targeted in cancer vaccines. This suggests the use of IP2 to build new epitope-based cancer immunotherapies.39. Tumors bearing spliceosome alterations are key targets for treatments with splicing inhibitors some of which are currently under development in acute myeloid leukemia40. Here we observed that treatment with the splicing inhibitor isoginkgetin or its derivative IP2 reduces the growth of MCA205 fibrosarcoma and B16F10 melanoma bearing the PTP-derived SL8 epitope and significantly extends mice survival 1 have the capacity to modify quantitatively and qualitatively the repertoire of epitopes presented at the surface of treated cells. We show here that interfering with the spliceosome using the IP2 compound leads to a remodeling of the MHC-I immunopeptidome of MCA205 fibrosarcoma  and tumor-specific (TSAs) antigens from different regions of the genome in the context of epitope-based immunotherapies.Importantly, IP2 treatment slows down the growth of WT fibrosarcoma and melanoma, which suggests that an immunodominant epitope is not required to observe an impact on tumor growth but however necessary to observe complete tumor regression Fig. . While t56 related to the secretion of exosomes from breast cancer cells57. The nischarin-derived TL9 epitope has been identified throughout multiple proteomic studies referenced in the Immune Epitope Database (IEDB) but has never been further exploited so far. Importantly, the TL9 epitope was found to be expressed in healthy tissues58 however we observed neither an antitumor effect nor any deleterious auto-immune response in mice injected with the TL9 SLP alone. This suggests that the basal level of expression of the antigen at the surface of MCA205 fibrosarcoma is not sufficient to allow CTL recognition, which is no more the case after IP2 treatment. The 39-amino-acid-long TL9 SLP was generated by translating the corresponding sequence identified in the RNA-seq analysis of MCA205 fibrosarcoma. Interestingly, we identified a point mutation in the cancer cell transcriptome, which translates into an amino acid switch, from Alanine (A) to Aspartic acid (D), at the position preceding the epitope. The flanking regions of the epitopes in synthetic long peptides can be optimized to promote epitope generation and presentation59, we hypothesize that this mutation found in the nischarin gene in MCA205 cells could potentially play a role in the increase presentation of the epitope compared with other healthy tissues. Future work will be needed to explore the effect of IP2 treatment on the presentation of the TL9 epitope with and without this mutation within the flanking regions, as well as other IP2-induced MHC ligands in cancerous and noncancerous cells.The exon-derived TL9 epitope from the tumor suppressor nischarin was shown to be immunogenic and to synergize with IP2 in cancer vaccines to inhibit the growth of MCA205 fibrosarcoma. Recent studies highlighted the role of nischarin in the regulation of breast tumorigenesis60. We reaffirm here that we did not observe any toxicity related to the use of the IP2 compound in vivo. While a deeper understanding of isoginkgetin and IP2 effects on splicing is needed, their positive impact on the immune response opens a large field of study on the link between splicing regulation and MHC-I immunopeptidome regulation. This could lead to the development of innovative anticancer immunotherapies targeting the spliceosome.Eventually, this work suggests a role for splicing regulation in the generation of MHC-I peptide for cancer immunosurveillance. The frequent mutations affecting the spliceosome in cancer cells may promote their evasion from T cells and NK cells. This raises the potential of the spliceosome as a druggable target in cancer immunotherapy. Potential side effects inherent to the alteration of such essential cellular mechanisms must be considered in the light of the benefice/risk balance. The use of proteasome inhibitors for myeloma or lymphoma treatment is one pertinent example2 in RPMI 1640 medium (Life Technologies) in the presence of 1% glutamine, 1% sodium pyruvate, 1% nonessential amino-acids, 1% penicillin/streptomycin and 10% FBS (Life Technologies). B16F10 mouse melanoma cell line was cultured at 37 °C under 5% CO2 in DMEM medium (Life Technologies) containing 1% glutamine, 1% penicillin/streptomycin and 10% FBS. The SL8/H-2Kb-specific B3Z CD8+ T-cell hybridoma61 were cultured at 37 °C under 5% CO2 in RPMI 1640 medium supplemented with 1% glutamine, 0.1% 2-mercaptoethanol, 1% penicillin/streptomycin, and 10% FBS. Once a month, mycoplasma contamination in cell cultures was assessed using the Venor®GeM OneStep mycoplasma detection kit (Minerva biolabs). All cells were used within four weeks after thawing (≈10 passages). MCA205 cells were transfected with the reagent jetPRIME (Ozyme) according to the manufacturer protocol. B16F10 cells were transfected with the reagent GeneJuice (Millipore) according to the manufacturer protocol. The plasmids YFP-Globin-SL8-intron, YFP-Globin-SL8-exon and PCDNA3 have been described previously17. The pre-mRNA splicing inhibitor isoginkgetin (Merck Millipore) was resuspended in DMSO. The IP2 and M2P2 derivatives were synthesized from the isoginkgetin powder and resuspended in PBS. Synthetic long peptides (39AA) were synthesized by GL Biochem, resuspended at 5 mg/mL in PBS at delivery and stored at −20 °C before use.MCA205 mouse fibrosarcoma cell line was cultured at 37 °C under 5% CO1H NMR, 31P NMR, and 13C NMR spectra were recorded in either CDCl3, D2O, or DMSO-d6 on Bruker Avance 300 spectrometers, or 200 spectrometers. The chemical shifts of 1H and 13C are reported in ppm relative to the solvent residual peaks. IR spectra were measured on a Bruker Vector 22 spectrophotometer. Melting points were recorded on a Büchi B-450 apparatus and are uncorrected. High resolution mass spectra (HRMS) were recorded on a MicroMass LCT Premier TOF ESI Spectrometer. For the synthesis of compound 1  :100:0:0 to 99:1:0.1) to yield 635 mg of compound 1 as a yellow powder . 1H NMR  δ: 13.00 , 12.79 , 8.02 , 7.97 , 7.46 , 7.18 , 6.99 , 6.95 , 6.84 , 6.65 , 4.32–4.18 , 4.11–3.95 , 3.81 , 1.37 , 1.22 . 13C NMR  δ: 183.0 (CO), 182.7 (CO), 164.4 (Ar), 162.9 (Ar), 162.3 (Ar), 161.9 (Ar), 161.1 (Ar), 157.1 (Ar), 156.1 (Ar), 156.0 (Ar), 154.4 (Ar), 153.6 (Ar), 153.5 (Ar), 131.2 (ArH), 128.6 (ArH), 127.9 (2ArH), 123.1 (Ar), 123.1 (Ar), 121.5 (Ar), 114.7 (2ArH), 111.3 (ArH), 108.3 (Ar), 104.9 (ArH), 104.3 (ArH), 103.9 (ArH), 103.9 (ArH), 103.7 (ArH), 99.1 (ArH), 99.0 (ArH), 65.2 , 64.9 , 56.1 (OCH3), 55.6 (OCH3), 16.3 (CH3), 16.2 (CH3), 16.1 (CH3). 31 P NMR  δ: −7.30, −7.50. IR (neat): 2989 (CH), 1653 and 1604 (CO), 1582 cm−1. HRMS (ESI-TOF) m/z; [M + H] + Calcd. for C40H41O16P2 839.1870; found 839.1865. Melting point = 155 °C. For the synthesis of IP2  δ: 7.64 , 7.51 , 7.24 , 7.00 , 6.81 , 6.58–6.41 , 6.33 , 3.83 , 3.63 . 13 C NMR  δ: 182.5 (CO), 164.5 (Ar), 161.8 (Ar), 160.9 (Ar), 160.8 (Ar), 160.4 (Ar), 159.4 (Ar), 159.3 (Ar), 158.6 (Ar), 156.9 (Ar), 153.7 (Ar), 130.9 (ArH), 128.3 (ArH), 127.3 (2ArH), 122.5 (Ar), 122.1 (Ar), 121.5 (Ar), 114.0 (2ArH), 111.8 (ArH), 107.2 (Ar), 107.1 (Ar), 105.5 (Ar), 104.9 (Ar), 103.6 (ArH), 103.3 (ArH), 102.6 (ArH), 99.8 (ArH), 99.7 (ArH), 56.1 (OCH3), 55.4 (OCH3). 31P NMR  δ: −0.49, −1.59. IR (neat): 3423 (broad), 2989 (CH), 1650 and 1600 (CO), 1563 cm−1. HRMS (ESI-TOF) m/z; [M-H]- Calcd. for C32H19O16P2 725.0467; found 725.0466. Melting point >350 °C. HPLC purity could not be determined due to the hydrolysis of phosphate during analysis. For the synthesis of compound 2, K2CO3  and iodomethane  were successively added to a solution under argon of compound 1  in anhydrous DMF (1.5 mL). The mixture was stirred at room temperature for 20 h and filtered. The solid was washed with ethyl acetate and the filtrate was washed with brine (3 × 30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to yield 110 mg of compound 2 as a yellow powder .1H NMR  δ: 7.99 , 7.93 , 7.38 , 7.16 , 7.11 , 7.04 , 6.80 , 6.67 , 4.31 – 4.18 , 4.16–3.90 , 3.78 , 1.36 , 1.22 . 13 C NMR  δ: 177.9 (CO), 177.4 (CO), 162.3 (Ar), 161.2 (Ar), 161.0 (Ar), 160.9 (Ar), 160.5 (Ar), 160.4 (Ar), 158.9 (Ar), 156.4 (Ar), 154.8 (Ar), 154.8 (Ar), 152.3 (Ar), 152.2 (Ar), 130.7 (ArH), 128.1 (ArH), 127.5 (2ArH), 123.4 (Ar), 123.3 (Ar), 121.6 (Ar), 114.5 (2ArH), 112.0 (Ar), 111.1 (ArH), 108.0 (ArH), 107.3 (ArH), 101.1 (ArH), 101.1 (ArH), 99.9 (ArH), 99.8 (ArH), 99.5 (ArH), 65.2 , 65.1 , 64.9 , 56.9 (OCH3), 56.8 (OCH3), 56.0 (OCH3), 55.5 (OCH3), 16.3 (CH3), 16.2 (CH3), 16.1 (CH3), 16.0 (CH3). 31P NMR  δ: −7.30, −7.50. IR (neat): 3492, 2984–2934 (CH), 1647 and 1605 (CO), 1573 cm−1. HRMS (ESI-TOF) m/z; [M + H] + Calcd. for C42H45O16P2 867.2177; found 867.2177. Melting point = 142 °C. For the synthesis of M2P2, TMSI  was added to a solution under argon of compound 2  in anhydrous dichloromethane (8 mL) and the reaction mixture was stirred at room temperature for 16 h. After evaporation of the solvent under reduced pressure, the yellow solid was dissolved in anhydrous methanol (4 mL) and NaOH  was added. The solution was stirred at room temperature for 30 min and the solvent was evaporated under reduced pressure to yield M2P2  as a yellow solid. 1H NMR  δ: 7.81 , 7.71 , 7.41 , 7.23–6.93 , 6.81 , 6.54 , 6.33 , 4.05 , 3.94 , 3.70 , 3.54 . 13C NMR  δ: 179.9 (CO), 162.4 (Ar), 161.5 (Ar), 160.8 (Ar), 160.4 (Ar), 160.3 (Ar), 160.2 (Ar), 160.1 (Ar), 159.9 (Ar), 159.7 (Ar), 159.4 (Ar), 158.9 (Ar), 157.9 (Ar), 155.6 (Ar), 130.9 (ArH), 128.1 (ArH), 127.1 (2ArH), 126.7 (ArH), 122.8 (Ar), 122.1 (Ar), 113.8 (2ArH), 111.6 (ArH), 108.2 (Ar), 107.6 (Ar), 105.9 (ArH), 104.9 (ArH), 100.9 (ArH), 99.6 (ArH), 56.3 (OCH3), 56.2 (OCH3), 56.2 (OCH3), 56.0 (OCH3). 31P NMR  δ: −0.16, −0.80. IR (neat): 3393 (broad), 2984 (CH), 1631 and 1593 (CO), 1565 cm−1. HRMS could not be determined due to the hydrolysis of phosphate during analysis Melting point >350 °C. HPLC purity could not be determined due to the hydrolysis of phosphate during analysis.Solvents and reagents are obtained from commercial suppliers and were used without further purification. Analytical TLC was performed using Merck silica gel F254 (230–400 mesh) plates and analyzed by UV light. For silica gel chromatography, the flash chromatography technique was used, with Merck silica gel 60 (230–400 mesh) and p.a. grade solvents unless otherwise noted. The 4 cells were co-cultured with 1 × 105 SL8/H-2Kb-specific B3Z CD8+ T-cell hybridoma for 18 h at 37 °C with 5% CO2 overnight. Cells were centrifuged at 1200 rpm for 5 min, washed twice with PBS 1× and lysed for 5 min at 4 °C under shaking. Lysis buffer was composed of 0.2% TritonX-100, 10 mM DTT, 90 mM K2HPO4, 8,5 mM KH2PO4. The lysates were centrifuged at 3000 rpm for 10 min to pellet cell debris and the supernatants were transferred to a 96-well optiplate . A revelation buffer containing 10 mM MgCl2, 11,2 mM β-mercaptoethanol, 0,0015% IGEPAL® CA-630 et 40 µM 4-Methylumbelliferyl β-D-Galactopyranoside (MUG) was added and the plate was incubated at room temperature for 3 h. Finally, β-galactosidase activity was measured using the FLUOstar OPTIMA .MCA205 fibrosarcoma and B16F10 melanoma were transfected with the plasmid YFP-Globin-SL8-intron or with the PCDNA3 empty plasmid as described in the previous section. Twenty-four hours after transfection, cells were treated with different doses of isoginkgetin (Merk Millipore), IP2 or M2P2 for 18 h. Cell were then washed three times with PBS 1X and 5 × 106 cells were incubated with mouse FcR blocking reagent for 10 min at 4 °C according to the manufacturer protocol (Miltenyi Biotech). Cells were stained with anti-mouse MHC class I (H-2Kb)-PerCP-Cy5.5  or with the corresponding PerCp-Cy5.5-conjugated mouse (BALB/c) IgG2a, κ isotype for 30 min at 4 °C. Cells were stained with DAPI prior to acquisition for dead cell exclusion. To study the kinetics of endogenous H-2Kb recovery at the cell surface, cells were treated for 18 h with the different drugs. The day after, cells were washed and treated with ice-cold citric acid buffer  at 1 × 107 cells per milliliter for 120 s, washed three times with PBS, and resuspended in culture medium. At the indicated time point, an aliquot of cells  was collected and stained with anti-mouse H-2Kb PE or anti-mouse SIINFEKL-peptide bound to H-2Kb-PE. All flow cytometry experiments were conducted using the BD LSRII flow cytometer (BD Biosciences) and data are analyzed with the FlowJow software (V10).Mouse cells were treated with drugs for 18 h. Cells were harvested and 1 × 10Mouse cells were treated with drugs for 18 h. Floating and adherent cells were collected, washed in PBS and resuspended in 1× Annexin V Binding Buffer with APC Annexin V (BD Pharmingen) according to the manufacturer protocol. Cells were gently vortexed and incubated 15 min at RT in the dark. Cells were stained with DAPI prior to flow cytometry analysis. Cells were analyzed using the BD LSRII flow cytometer (BD Biosciences) and data were analyzed with the FlowJow software (V10).4 cells per well for 24 h. Cells were then treated with increasing doses of drugs overnight. After removing the medium, the MTT  (Sigma–Aldrich) powder was resuspended in PBS, filtered and added into the plate wells at 2.5 mg/ml. The plate was incubated for 2 h at 37 °C, with 5% CO2. It was then centrifuged, and the supernatant was carefully removed in order not to disturb the precipitated formazan. Crystals were dissolved in DMSO at 200 µL/well and the plate was shaken for 10 min. Absorbance was measured at 544 nm using the FLUOstar OPTIMA.Cells were plated in a 96-well plate at 1 × 10Cells were transfected with the YFP-Globin-SL8-intron as described previously and then treated with drugs for 48 h. Cells were then harvested and total cellular RNA was extracted and purified using the RNeasy Mini kit (Qiagen) according to the manufacturer protocol. The reverse transcription was carried out with 0.5 μg of RNA with the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer protocol. The StepOne real-time PCR system (Applied BioSystems) was used for qPCR and the reaction was performed with the Power SYBR green PCR master mix (Applied BioSystems). Results were analyzed using the StepOne software. Mouse gene specific primers were designed as follow: 18 S mouse forward primer, 5′-GCCGCTAGAGGTGAAATTCTTG-3′; 18 S mouse reverse primer, 5′-CATTCTTGGCAAATGCTTTCG-3′; Glob-SL8-exon forward primer, 5′-AGAAGTCTGCCGTTACTGCC-3′; Glob-SL8-exon reverse primer, 5′-AGGCCATCACTAAAGGCACC-3′; Glob-SL8-intron forward primer, 5′-GTATCAAGGTTACAAGACAG-3′; Glob-SL8-intron reverse primer, 5′-GGGAAAATAGACCAATAGGC-3′.1. Libraries were sequenced on the Illumina HiSeq 4000 sequencer using 50–base pair paired-end read method. All RNA-seq data were aligned to the mouse genome assembly GRCm38 (mm10) with the TopHat2 splice-junction mapper for RNA-Seq reads (version 2.1.1) with default parameters. For the differential gene expression analysis, the Cuffdiff program (version 2.2.1)62 was run using the following parameters: -library-type=fr-first-strand -compatible-hits-norm -library-norm-method geometric -min-reps-for-js-test 2 -dispersion-method per-condition -u -b. In all such analyses, the difference in expression of a gene was considered significant if the q value was less than 0.05 (the Cuffdiff default) and the fold change was superior to 2. Scatter plots were generated using ggplot2 in R environment. For the differential splicing analysis, the SUPPA2 program was run with the default settings64.MCA205 and B16F10 cells were treated with 15 μM of isoginkgetin or 35 μM of IP2 for 18 h. RNA was extracted using QIAGEN RNeasy columns. RNA-seq libraries were prepared from total RNA by poly(A) selection with oligo(dT) beads according to the manufacturer’s instructions (Life Technologies). The resulting RNA samples were then used as input for first-strand stranded library preparation using a custom protocol based on dUTP method as described previously6 MCA205 or B16F10 cells were lysed in 3 mL lysis buffer containing PBS, 0.25% Sodium Deoxycholate and protease inhibitor  during 1 h at 4 °C under agitation. Lysate was sonicated and then cleared by centrifugation for 1 h at 4000 × g at 4 °C. Supernatant was filtered through 0.40 µm filter and applied on affinity columns overnight . Flow was maintained at 10 rpm overnight by a peristaltic pomp. IP resins were prepared as follows: (i) 40 mg CNBr-activated Sepharose were activated in 1 mM HCl for 30 min at RT, (ii) 1 mg of Y-3 or B22-249 antibodies (kindly provided by Prof. Dr. Stefan Stevanović from University of Tübingen) was added in H2O, 0.5 M NaCl, 0.1 M NaHCO3 for 2 h at RT, (iii) resin was blocked in PBS, 0.2 M glycin for 1 h at RT, and finally (iv) resin was washed twice in PBS and resuspended at 1 mg antibody/mL in PBS. Centrifugation at 300 rpm for 4 min (no brake) and removal of the supernatant were performed between each step. On the second day, the peptides were eluted in 100 µL 0.2% trifluoroacetic acid (TFA) for 25 min at 4 °C under agitation. Elution was repeated three times. The four eluates were pooled, cleared by centrifugation for 5 min at 15,000 × g and filtered through Amicon 3 kDa MWCO . Prior to filtration, equilibration of the Omega membrane was performed by successive rinse/centrifugation cycles with MS-grade water, 0.1 N NaOH, MS-grade water and finally 0.2% TFA. Eventually, membrane was rinsed with 32.5% ACN, 0.2% TFA and the sample was concentrated using a Speed Vac Concentrator (Thermo Scientific). Samples were stored at −80 °C prior LC-MS/MS analysis.400 × 1018 pipette tips (Pierce Thermo Scientific), eluted in 40 µL acetonitrile 70% (Fisher Scientific)/0.1% formic acid, vacuum centrifuged and resuspended in 12 µL of 0.1% formic acid. All C18 zip-tipped peptides extracts were analyzed using an Orbitrap Fusion Tribrid equipped with an EASY-Spray Nano electrospray ion source and coupled to an Easy Nano-LC Proxeon 1200 system . Chromatographic separation of peptides was performed with the following parameters: Acclaim PepMap100 C18 pre-column , Pepmap-RSLC Proxeon C18 column , 300 nl/min flow, gradient rising from 95% solvent A  to 28% solvent B  in 105 min and then up to 40%B in 15 min followed by column regeneration for 50 min. Peptides were analyzed in the orbitrap in full ion scan mode at a resolution of 120,000 and with a mass range of m/z 400–650 using quadrupole isolation and an AGC target of 1.5 × 105. Fragments were obtained by Collision Induced Dissociation (CID) activation with a collisional energy of 35%. MS/MS data were acquired in the Orbitrap at a resolution of 30,000 in a top-speed mode, with a total cycle of 3 s with an AGC target of 7 × 104. The maximum ion accumulation times were set to 100 ms for MS acquisition and 150 ms for MS/MS acquisition in parallelization mode.The peptides were desalted using ZipTip C65 using a PWIZ-mzDB (https://github.com/mzdb/pwiz-mzdb) version 0.9.10, and then to MGF peaklists using a mzdb-access (https://github.com/mzdb/mzdb-access) version 0.7.0. Produced MGF files were submitted to Mascot database searches  against a mouse Uniprot protein sequence database released in July 2020 and downloaded from SwissProt website. The database contains 87,954 protein entries. Spectra were searched with a mass tolerance of 10 ppm in MS mode and 20 mmu in MS/MS mode. No missed cleavage was allowed (no enzyme search). Carbamidomethylation of cysteine residues and oxidation of methionine residues were set as variable modifications. For peptide validation, identification results were imported into the Proline software66 version 2.0 (http://proline.profiproteomics.fr) for target/decoy validation and label-free quantification. Peptide Spectrum Matches (PSM) with pretty rank equal to one and a Mascot score above 20 were retained. False Discovery Rate was then optimized to be below 1% at PSM level using an adjusted variant of the Mascot E-value67. For label-free quantification, peptide abundances were extracted by Proline using an m/z tolerance of 5 ppm. Alignment of the LC-MS runs was performed. Cross-assignment (a.k.a. match between runs) of peptide ion abundances was performed between the raw files using an m/z tolerance of 5 ppm and a retention time tolerance of 60 s. Precursor ions abundances were normalized using the quotient normalization method68 implemented in Proline. Fold change was calculated as the ratio of the mean normalized abundances in the IP2-treated and untreated biological replicates.Raw files were converted to mzDB files+ T cells were isolated from spleens of vaccinated mice using magnetic selection with CD8a (Ly-2) MicroBeads (Miltenyi Biotec). CD8+ T cells were co-cultured with BMDCs pulsed with 30 mg/mL of synthetic long peptides for 18 h in ELISpot plates coated with anti-IFNϒ capture antibody (Cell Sciences). Spots were revealed according to manufacturer instructions. IL-2 concentration was measured in the supernatants of the co-culture between CD8+ T cells and peptide-pulsed BMDCs using the Mouse IL-2 ELISA MAX Standard kit (BioLegend).C57BL/6 mice were vaccinated subcutaneously at the tail base with 100 µg of synthetic long peptide mixed with 30 µG ODN 1668. One week after, CD84 MCA205 wild-type (WT) or 7.5 × 104 MCA205-globin-SL8-intron cells, or with 5 × 104 B16F10 wild-type (WT) or B16F10-Globin-SL8-intron cells along with matrigel (VWR). Five days after challenge, mice were treated intraperitonealy with different dose of isoginkgetin, IP2 or M2P2. Intraperitoneal injection was repeated at day 10 and 15. Area of the tumor was recorded every 3–4 days until ethical limit points were reached. Mice which experienced complete tumor regression following treatment were kept for 100 days after tumor challenge and were then rechallenged subcutaneously on the right flank with 7.5 × 104 MCA205-Globin-SL8-intron cells and on the left flank with 5 × 104 B16F10 WT cells.Female wild-type C57BL/6 mice at the age of 6 weeks were obtained from Harlan France. NU/NU nude mice were obtained from Charles River France. Animal experiments were conducted in compliance with the EU Directive 63/2010, and protocols 2016_064_5677 and were approved by the Ethical Committee of the Gustave Roussy Campus Cancer . Mice were challenged subcutaneously in the right flank with 5 × 105 MCA205-globin-SL8-intron cells and thereafter treated with 24 mg/kg of IP2 at days 8 and 11. At day 12, OT-1 CD8+ T cells were isolated from spleens of OT-1 mice using magnetic selection with CD8a (Ly-2) MicroBeads (Miltenyi Biotec) and then intravenously injected into tumor-bearing mice at 3.0 × 106 cells/mouse. Three days after transfer, proliferation of OT-1 cells was measured in spleens by flow cytometry after staining with anti-CD8-FITC and APC-conjugated SL8/H-2Kb Dextramers (Immudex).C57BL/6 mice were challenged subcutaneously with 3.0 × 105 MCA205 WT were inoculated subcutaneously into the right flank. Mice were then treated twice a week with 24 mg/kg of IP2 for 2 weeks. In the therapeutic setting, C57BL/6 mice were injected subcutaneously in the right flank with 1.5 × 105 MCA205 WT and thereafter vaccinated once a week with 100 µg of synthetic long peptide mixed with 30 µG ODN 1668 and treated twice a week with 24 mg/kg of IP2 for 2 weeks.In the prophylactic setting, C57BL/6 mice were vaccinated twice subcutaneously at the tail base with 100 µg of synthetic long peptide mixed with 30 µG ODN 1668, two weeks apart (prime-boost). One week after the booster injection, 1.5 × 10t-test. A one-way analysis of variants (ANOVA) with Tukey’s post-hoc test was run when multiple conditions were tested. All data were processed on the GraphPad Prism (version 5) software.All data from T-cell activation assays were analyzed from at least three biological replicates, each one including three technical replicates. Data were presented as the means ± SEM of biological replicates. Significant changes were analyzed by a one-way analysis of variants (ANOVA) with Dunnett’s post-hoc test. All in vivo experiments were repeated at least twice with a minimum of six mice per group. Significant changes between two conditions were analyzed by a Student’s Further information on research design is available in the Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Description of Additional Supplementary FilesReporting SummaryPeer Review File