Cooperation between Cancer Cells and Regulatory T Cells to Promote Immune-escape through Integrin αvβ8-Mediated TGF-β Activation

Among the strategies allowing cancer cells to escape the immune system, the presence of TGF-b in the tumor micro-environment is one of the most potent. However, TGF-b is secreted in an inactive form and mechanisms responsible for its activation within the tumor remain unknown. Here, we demonstrate that regulatory T cells (Tregs) compose the main cells expressing the b8 chain of avb8 integrin (Itgb8) in the tumors and that the Itgb8 pos Treg population activates TGF-b produced by the cancer cells and stored in the tumor micro-environment. Itgb8 ablation in Tregs impaired TGF-b signaling in T lymphocytes present in the tumor but not in the tumor draining lymph nodes. The cytotoxic function of CD8 pos T lymphocytes inltrating the tumors was subsequently exacerbated leading to an ecient control of the tumor growth. Similar observations were made in patient tumors after anti-Itgb8 antibody treatment. Thus, this study reveals that Tregs work in concert with cancer cells to produce bioactive-TGF-b and create a powerful-immunosuppressive micro-environment.


Introduction
The tenet of tumor immunotherapy is based on the ability of the immune system to survey for malignant transformation and be e cient at eliminating cancer cells. However, solid tumors can escape from the immune system by orchestrating a micro-environment that limits an e cient anti-tumor immune response.
In the tumor micro-environment, Transforming Growth Factor beta (TGF-β) is regarded as a key cytokine promoting potent immunosuppression 1 . Among the three isoforms of TGF-β (TGF-β 1-3), TGF-β1 is prevalent within tumors 2 3 . This polypeptide cytokine, highly conserved in all mammals 4 , impairs numerous functions of effector T lymphocytes and promotes both development and stability of CD4 pos Foxp3 pos regulatory T cells (Tregs) 5,6 . Subsequently, the selective targeting of TGF-β signaling in T lymphocytes leads to an e cient elimination of cancer cells by effector T lymphocytes 7 repressing their cytotoxic functions 8 . Hence, neutralization of TGF-β-immunoregulatory effects has been thought of as a promising anti-cancer therapy. However, major safety issue were raised, one of which being the risk of unleashing massive autoimmunity, given the key role of TGF-b signaling in the repression of T lymphocytes activation 5, 6 9 10 .
Importantly, TGF-β is one of the few cytokines secreted in an inactive form. This small latency complex is composed of the mature cytokine encircled by the latency-associated peptide (LAP), which are noncovalently associated. LAP covers all the contact sites of the mature cytokine that must interact with TGF-β receptor complexes (TGFβRI and TGFβRII) to induce TGF-β signaling, including the phosphorylation of SMAD2/3 11 . Within solid tumors, the latent TGF-β complex can be secreted by several cell types, including cancer cells, and Tregs 1 . Nevertheless, unlike the TGF-β produced by Tregs, TGF-β secreted by cancer cells seems essential for the repression of the anti-tumor immune response 12,13 . As long as LAP maintains close contacts with the mature cytokine, the secreted latent TGF-β can be stored in the tumor micro-environment, attached to the extracellular matrix, without any immune regulatory functionality 14 . Hence, activation of the secreted TGF-β latent complex, which involves exposure of the receptor-binding domain of the mature cytokine, is therefore indispensable for TGF-βmediated immune regulatory functions in tumors. Thus, deciphering the mechanisms by which the activation of TGF-β present in the tumor micro-environment occurs is essential to our comprehension of solid tumors escape the immune system and will highlight potential new effective anti-cancer therapies that speci cally target TGF-β activation within the tumor micro-environment and thus limiting autoimmune side effects associated to the privation of TGF-b activation In this study, we demonstrate that the expression of the integrin αvβ8 in Tregs is essential to e ciently activate TGF-β produced by cancer cells and promote tumor immune escape. In the absence of expression of the β8 integrin chain (Itgβ8) in Tregs, TGF-β signaling is impaired in tumor in ltrating effector T cells and their cytotoxic functions are unleashed leading to the e cient control of tumor growth. In patient tumors, treatment with a neutralizing anti-Itgβ8 antibody, as well as single-cell gene expression analysis on tumor in ltrating T cells, con rmed the relevance of our ndings in mice to human pathology. Overall, our results reveal an unexpected collaboration between cancer cells and Tregs to create an e cient TGF-β-mediated immunosuppressive tumor micro-environment (TME), highlighting that the targeting of Itgβ8 could constitute an e cient novel immunotherapies.

Itgβ8 is mainly expressed in regulatory T cells in tumors
In vivo, the activation of TGF-β1 is largely dependent on integrins, including the αvβ8 integrin, whose expression is regulated by that of the β8 subunit (Itgβ8) 11 15 . In order to understand the mechanisms leading to the activation of the latent complex in the tumor, we rst analyzed Itgβ8 cellular expression in the TME.
To monitore Itgβ8 by ow cytometry, we took an unbiased approach by generating an Itgb8-td-Tomato reporter mice, in which we previously validated that td-tomato positive cells expressed Itgb8 protein in different cell types, including T lymphocytes 16 . Flow cytometry analysis of tumors (melanoma and breast cancer) revealed that among host cells composing the TME, Itgβ8 pos cells were mainly (85-95%) CD45 pos hematopoietic cells (Fig. 1A-B). T lymphocytes (CD3 pos ), and particularly the CD4 pos Foxp3 pos (Treg) subset, composed the main portion of hematopoietic cells expressing Itgβ8, with approximately 80% of Itgβ8 pos CD45 pos cells being CD4 pos Foxp3 pos irrelevant of the tumor type ( Fig. 1C-F). Moreover, within the Treg compartment, we found that about 40-45% of cells expressed Itgβ8 (Fig. 1G-H) and only Itgβ8 pos Tregs were endowed with the capacity to e ciently activate TGF-β1 ( Figure 1I) whereas both Itgβ8 pos Treg and Itgβ8 neg Treg populations expressed similar levels of this cytokine ( Figure 1J). Thus, this rst set of data reveals that Tregs constitute a large part of the Itgβ8-expressing host cells within the TME.
Itgβ8 expression in Tregs impairs anti-tumor response and promotes tumor-growth Next, in order to assess whether Itgβ8 expression by Tregs confers them abilities to control the anti-tumor  immune responses by providing a bioactive source of TGF-b, we rst selectively ablated Itgb8 in Tregs, using Foxp3-Cre Itgb8 / mice (Foxp3 ΔItgβ8 ). Importantly, in Foxp3 ΔItgβ8 mice Tregs retain their numbers,  localization, as well as their suppressive functions, including the ability to produce TGF-β1. Moreover, no autoimmunity signs, neither uncontrolled effector T cell activation have been observed in Foxp3 ΔItgβ8 animals 17,18 .
Strikingly, in contrast to their littermate controls (Foxp3 Ctrl ), Foxp3 ΔItgβ8 mice showed a profound impairment of tumor growth irrelevant of the tumor type ( Fig. 2A-F). Notably, we observed that 25-50% of the Foxp3 ΔItgβ8 animals exhibited a complete control of the tumor progression depending of the tumor type ( Figure G-H). Thus, Itgβ8 expression in Tregs promoted tumor growth, implying that the Itgβ8 pos Treg population could affect the anti-tumor function of the effector T cells.
To con rm this scenario, we next analyzed the immune compartment of tumors and that of their draining lymph nodes (tdLN). Interestingly, the proportion of Natural Killers (NK) cells and T cells, including Tregs, were similar in both TME and tdLN between Foxp3 ΔItgβ8 mice and Foxp3 Ctrl animals ( Figure S1A). In line with this observation, the proliferative status of T cells and NK cells was similar between Foxp3 ΔItgβ8 mice control animals in both tdLN and TME ( Figure S1B). Moreover, the deprivation of Itgb8 on Tregs failed to affect the distribution of T lymphocytes within the TME (data not shown). Thus, we ruled-out a speci c role of the Itgβ8 pos Tregs in controlling proliferation, recruiting of effector immune T cells into the TME as well as T cell priming in tdLN. However, the inhibition of tumor-growth observed in Foxp3 ΔItgβ8 mice was completely lost when animals were depleted of their CD8 pos T lymphocytes ( Fig. 3A-B).
Thus, altogether these observations suggested that Itgβ8 pos Tregs exert their pro-tumoral effects by impairing the anti-tumor functions of CD8 pos T lymphocytes. In agreement with this assumption, we observed that CD8 pos T lymphocytes of the TME of Foxp3 ΔItgβ8 mice exhibited higher cytotoxic functions based on the production of granzyme B cytotoxic granules (GzB) in association with the surface expression of CD107 (Lamp1) compared to Foxp3 Ctrl animals (Fig. 3C). Production of IFN-γ was also exacerbated in tumor in ltrating in both CD4 Pos T cells and CD8 pos T cells from Foxp3 ΔItgβ8 mice compared to Foxp3 Ctrl animals ( Figure S2).
Supporting the exacerbated cytotoxic features of CD8 pos T lymphocytes in the TME of Foxp3 ΔItgβ8 mice, as well as the control of tumor growth in these animals, histology analysis showed higher numbers of apoptotic cells in tumors from Foxp3 ΔItgβ8 mice than control animals ( Fig. 3D-E). Importantly, in clear contrast to the TME, we failed to nd any exacerbation of the cytotoxic phenotype of CD8 pos T cells in the tdLN of Foxp3 ΔItgβ8 mice (Fig. 3F). This observation, combined with the absence of systemic T effector cell activation in secondary lymphoid organs of in Foxp3 ΔItgβ8 mice 17,18 , reveals a speci c role for Itgβ8 pos Tregs in the repression of the cytotoxic functions of CD8 pos T lymphocytes selectively in the TME.
Altogether, these data identify Itgβ8 as a key mediator of Treg induced suppression of the anti-tumor cytotoxic function of CD8 pos T cells present in the TME with direct consequences on tumor progression.
Itgβ8 expression on Tregs promotes TGF-β signaling controlling effector tumor T cells Given the role of αvβ8 in TGF-β activation 11 , and the unique ability of the Itgβ8 pos Treg subset to activate TGF-β1 compared to Itgβ8 neg Tregs ( gure 1Ι), we next assessed whether the repression of CD8 pos T cell cytotoxic functions in the TME of Foxp3 ΔItgβ8 mice was due to an increase of the TGF-β signaling in the effector cells in the tumor. This assumption was even more motivated by the fact that, we found that the percentage of T cells with high activation of TGF-β signaling pathway, monitored by the phosphorylation of SMAD2-3, was halved in the TME of Foxp3 ΔItgβ8 mice compared to Foxp3 Ctrl animals ( Fig. 4A).
Notably, in contrast to the TME, and in line with the absence of T cell over activation in tdLN of Foxp3 ΔItgβ8 mice the levels of phosphorylation of SMAD2-3 in T lymphocytes from tdLN were similar between Foxp3 ΔItgβ8 mice and Foxp3 Ctrl animals ( Fig. 4B). Hence, Itgβ8 pos Tregs are responsible of the increase of TGF-b signaling in T cells present in the TME.
In order to con rm that the exacerbated cytotoxic features of CD8 pos T cells in the TME of Foxp3 ΔItgβ8 mice were directly linked to the increase TGF-β signaling in effector T cells by Itgβ8 pos Tregs, we developed genetic approaches allowing to sustain high levels of TGF-β signaling activation in effector T cells. The T cell compartment of CD3ε de cient mice was reconstituted with puri ed Tregs from either Foxp3 Ctrl mice or Foxp3 ΔItgβ8 mice and Foxp3 neg T cells expressing either a constitutively active (CA) form (TGFβRI CA ) or the unmodi ed form of TGFβRI (TGFβRI WT ) 19 (Fig. 4C). In TGFβRI CA -expressing T cells, the TGF-β signaling pathway remains activated even in the absence of bio-active source of TGF-β in their micro-environment as we previously described it 19,20 . Similarly to data illustrated in Fig. 3C, we observed that the absence of Itgβ8 expression in Tregs (Treg ΔItgβ8 ) increased the cytotoxic features of transferred wild type CD8 pos T cells. In contrast, the maintenance of TGF−β signaling in effector T cells was su cient to completely prevent the over-activation of their cytotoxic program as well as the repression of tumor growth we routinely observed in the absence of Itgβ8 expression on Tregs ( Fig. 4D-E).
Thus, within the TME, Itgβ8 expression on Tregs increases the levels of TGF-β signaling activation in effector T lymphocytes which is su cient to repress their cytotoxic functions.
Activation of cancer-cell-produced TGF-β1 by Itgβ8 pos Tregs leads to tumor CD8 T cell loss of function Our aforementioned data, combined with inability of Itgb8 expression to modulate Tgf-b1 expression in Tregs (Fig. 1I) and the minor role of TGF-β1-produced by Tregs in the control of the effector T cell functions in the TME 12 , strongly suggest that Itgb8 pos Tregs could contribute to the activation of TGF-b1 produced by other cells of the TME. As LAP re ects the inactive form of TGF-β, we evaluated the presence of LAP within the TME either in the presence or in the absence of Itgβ8 in Tregs. Strikingly, the classic brillar staining of the large latent complex was 2-3 times increased in the TME of Foxp3 ΔItgβ8 mice compared to Foxp3 Ctrl animals ( Fig. 5A-B). In order to address, the source of inactive TGF-b1 which accumulate in the TME of Foxp3 ΔItgβ8 mice, we selective ablated tgf-b1 in cancer cells regarded as high producer cells of TGF-b1 (TGF-β1 KO ) in the TME 23 ( Figure S3A). The accumulation of inactive form of TGF-β1 was lost in the TME of TGF-β1 KO cancer cells (Fig. 5C). Of note, the absence of TGF-b1 production by cancer cells strongly impaired the tumor growth in wild-type mice but not in T cell de cient animals (CD3 KO ) (Figure S3B C). Con rming the importance of TGF-b1 produced by cancer cells in the control of T cell anti-tumor immune response, the cytotoxic functions of CD8 T cells from the TME of TGF-β1 KO cancer cells were 2-3 times exacerbated TGF-β1 su cient cancer cells (Fig. 5D-E). Importantly the production of TGF-b1 by cancer cells had no signi cant impact Treg homeostasis ( Figure S3D) and T cell activation in the tdLN ( Figure S3E-F). Thus, Itgb8 expression by Tregs contributes to the activation TGF-b1 produced by cancer cells in the TME, with direct consequences on the repression the cytotoxic functions of CD8 pos T cells present in the TME and thus on tumor immune escape .

Itgβ8 expression on tumor in ltrating T cells is associated with poor patient survival and CD8 T cell activation
We next analyzed the relevance of our data in mice to the human pathology particularly in melanoma patients. First, we con rmed that human T cells expressed ITGb8 in the TME by analyzing single-cell mRNAseq, and reported that ITGB8 expression was prevalent in the Foxp3 pos compartment of the TME of various tumor types, with 65-70% of Itgβ8 pos T cells being Foxp3 pos T cells ( Figure S4). We then made use of publicly available sets of single cell-sequencing analysis data and obtained a speci c geneexpression signature of Itgβ8 pos T cells in ltrating the tumors, allowing us to perform multivariable survival analysis. We analyzed 358 patients bearing melanoma and revealed that high ITGB8 score in tumor in ltrating T cells was associated with poor survival (Fig. 6A). Of note the poor survival prognostic associated to presence of Itb8 Tregs was con rmed in other tumor types except in colorectal cancer ( Figure S5) The better survival prognostic observed in colorectal patients with high ITGB8 score in Tregs from the TME was in agreement with the ability of Itgβ8 pos Tregs to repress established chronic intestinal in ammation in mice 18 which was largely depicted to promote colorectal cancer progression 21 .
Interestingly, our analysis con rmed that FOXP3 expression alone in the T cells of TME was not su cient to predict patient prognosis in any tumor types as previously showed 22 ( Figure S5). Of note, given that ITGB8 expression was reported to be increased on activated human Tregs 18 , we also removed the gene signature of activated Tregs in the ITGB8 Treg signature and obtained similar survival prognostics as with the ITGB8 Treg total gene-signature for all the tumor types we analyzed ( Figure S5). In line with poor survival associated with the presence of Itgb8 Tregs in the TME of patients, we observed that the expression of ITGB8 Treg signature in the TME was inversely corelated with the activation of CD8 T cells present in the same TME (Fig. 6B). Thus, these data suggest that ITGB8 expression in Tregs present in the TME might be useful as predictor of poor patient survival and activation of CD8 T cells in tumors.
Moreover combined with our analysis in mice, the aforementioned observations suggest that neutralizing Itgb8 ability to activate TGF-b in patient tumors could be associated with stronger CD8 T cells activation in the TME.
Neutralization of Itgβ8 exacerbates cytotoxic T cell function in TME of patients Finally, we assessed whether neutralizing Itgb8 ability to activate TGF-b in patient tumors could affect effector t cells ability to respond to TGF-b and develop e cient anti-tumor response in the TME. To this end, we used an ex-vivo culture approach in which two serial sections of live tumor were cultured either in the presence or in the absence of neutralizing anti-Itgβ8 antibody (Fig. 7A). This technique allowed us to address the effects of the anti-Itgb8 antibody on same TME of given same patient in which the immune system compartment and its interactions with the tumor tissues were conserved. After treatment, CD8 pos T cells from the tumors were analyzed by ow cytometry (Fig. 7B). We rst monitored the effects of the anti-Itgb8 antibody treatment on TGF-b signaling in patient melanoma. In response to anti-Itgβ8 antibody, we observed 30-50% of reduction in phosphorylation of SMAD2/3 in CD8 pos T cells from TME demonstrating that neutralizing Itgβ8 in the human tumors affects the levels of TGF-β signaling in CD8 pos T cells in ltrating the TME (Fig. 7C-D). Strikingly, we also observed a 2-5 fold-increase of cytotoxic features of CD8 pos T cells present in the TME in the majority of the melanoma after anti-Itgβ8 antibody treatment compared to untreated condition (Fig. 7E-F). Of note, similar observations were made in breast cancers in response to neutralizing anti-Itgβ8 antibody treatment ( Figure S6). Thus, neutralizing Itgβ8 is su cient to impair TGF-β signaling in CD8 pos T lymphocytes in ltrating human tumors and boost their cytotoxic functions, opening the path towards clinical applications based on Itgb8 targeting in cancer.

Discussion
The presence of Tregs in the TME is usually associated with a weakness of the effector T cell responses and poor prognosis in patients. Though Tregs do not need to produce their own TGF-β1 to repress the effector T cell functions in the TME 12 , this study reveals that Tregs, and particularly the Itgβ8 pos population, are essential to increase the levels of activated TGF-β produced by cancer cells responsible for an e cient repression of T cell cytotoxic functions within the TME. Thus, this collaborative work between cancer cells and Itgβ8 pos Tregs increases the ability of the cancer cells to escape the immune system and fosters cancer progression.
In certain cancers, Itgb8 expression is observed in tumor cells express and the forced expression of Itgb8 in cancer cell lines was associated with TGF-b1 activation in vitro as well as the impairment of metastasis growth and vascularization modi cations of the tumors after their implantation in mice 23 .
Our results do not exclude other cellular actors than Itgβ8 pos Tregs participate to TGF-b activation in the TME. Indeed, we observed that TGF-b signaling in T cells in ltrating the tumors is not fully abolished in Foxp3 ΔItgβ8 mice. However, no role of Itgb8 pos cancer cells have been assigned to the regulation of the CD8 T cell cytotoxic functions in the TME 23 . Hence, we propose that once Itgb8 pos Tregs colonize the tumor, they help enforce the activation of latent TGF-b1 produced by cancer cells so far ensured by others cells of the TME, including cancer cells themselves in the tumors where they express αvβ8 integrin. This help from the Tregs allows the TME to reach the optimal activation of TGF-β1 which block the cytotoxic functions of CD8 T cells and promote tumor immune escape. The control of TGF-b signaling in CD8 T cells by activating TGF-b1 is likely facilitated by the unique ability of Tregs to be in the close vicinity of CD8 T cells in the TME 24 . The ability of Itgβ8 pos Tregs to activate TGF-β1 produced by cancer cells is in agreement with recent biochemical investigations on αvβ8-mediated TGF-β1 activation, made outside the Treg context, suggesting that the latent complex released by a given cell can be activated by αvβ8 integrin expressed by others 25 . Moreover, Tregs have been shown to be capable of acquiring at their surface latent complex produced by other cells 26 .
Interestingly, the capacity of Itgβ8 pos Tregs to increase the levels of bioactive TGF-β1, to ensure a repression of the cytotoxic functions of T cells appears particularly of importance in the TME. Indeed, in clear contrast to the TME, the absence of Itgβ8 expression in Tregs failed to alter TGF-β signaling in effector T cells in the tdLN, implying that either other cells expressing Itgβ8 or other mechanisms, independent of the Itgβ8, play a key role in the activation of TGF-β in the secondary lymphoid organs. In line with this, while a modi cation on LAP of the RGD sequence recognized by avb8 integrin recapitulates the autoimmune syndromes observed in the absence of TGF-β1 14 , no signs of autoimmunity nor immune disorders were described in Foxp3 ΔItgβ8 mice, 17,18 . Moreover, depending of the tissue, the predominant role of certain cells have been depicted in the activation of TGF-b. In the gut, Itgβ8 pos dendritic cells appear as key activators of TGF-β, whereas in the skin this function seems to be more dependent on keratinocytes 27, 28 29 . In addition, the in ammatory context favors the role of Itgβ8 pos Tregs in activating latent TGF-b 18 . Whether some in ammatory factors present in the TME reduce the expression of Itgβ8 by other cells than Tregs or repress alternative mechanisms of TGF-β activation could be considered as suggested in the gut 30 .
Secreted latent complex can be stored in the micro-environment of the secreting cells and thus be accessible to integrins 31 . Our data reveal that cancer cells as a major source of latent TGF-β complex stored in the TME which is activated by Itgβ8 pos Tregs. However, we do not exclude that Itgβ8 pos Tregs can also activate TGF-β once secreted. Indeed, one of the features of Tregs is to express at their surface high amounts of the protein GARP, which can bind latent complex, then present it to αvβ8 integrin and thus contribute to the activation of secreted latent TGF-β 26 , 32 . However, in contrast to the absence of Itgb8, the deletion in Tregs of lrrc32, which encodes for GARP, is not su cient to affect tumor growth 33 . While GARP expression on Tregs contributes to the activation of secreted latent complexes, that of Itgβ8 contributes to the activation of latent complexes stored in the TME. Since much of the secreted latent complex of TGF-b is stored in the tissue 31 , this could explain why the absence of Itgβ8 expression in Tregs, and not that of GARP, is su cient to in uence TGF-b signal given to effector T cells and repress tumor-growth.
TGF-b signaling is known to directly affect the CD8 T cell cytotoxic function 8 . This study reveals that the activation of the latent complex by Itgβ8 pos Tregs directly in uences the levels of TGF-β signaling delivered to intra-tumor effector CD8 T cells and thus their cytotoxic function. The restoration of TGF-β signaling in effector T cells fully prevents their cytotoxic functions associated with the deletion of Itgβ8 on Tregs and it con rms that the modulation of TGF-b signaling in effector cells by Itgβ8 pos Tregs as the main mechanisms of action this regulatory subset in the TME. Based on several observations made in vitro and in the gut TGF-β has been proposed to promote the conversion Foxp3 neg T cells towards Foxp3 pos cells in the tumor 34 . Interestingly, in the absence of Itgβ8 pos Tregs, the proportion and the numbers of Tregs remained unchanged within the TME. Moreover, the lack of Tgfβ1 in cancer cells, which leads to the activate the cytotoxic function of CD8 T cells in the TME, failed to affect Treg homeostasis in the tumor. Hence, further investigations should con rm the ability of the activated TGF-b1 present in the TME to in uence T effector cell conversion into Tregs.
Targeting TGF-b effects on the immune cells in the TME is a important eld of investigation for numerous companies. Our data strongly suggest that targeting Itgb8 in patient could lead to potent activation the T cell cytotoxic program in the TME and control of the tumor progression. This idea is comforted by our exvivo experiments, revealing that anti-Itgb8 antibody treatment impairs TGF-b signaling in effector T cells and is su cient to boost their cytotoxic functions in the TME of patients. Though the best way to e ciently target Itgb8 effects in patients need to de ne, the exacerbation of the cytotoxic functions of T lymphocytes selectively in the TME suggests that targeting Itgβ8 pos Tregs may represent a promising immunotherapy avoiding the risk of unleashing massive auto-immunity following a systemic neutralization of TGF-b effects 10 .
In sum, this study reveals an unsuspected collaborative mechanism between cancer cells and Tregs with direct consequences on the repression of the anti-tumor function of effector T cells in tumors. Moreover, it provides evidence that targeting Itgβ8 could constitute a promising future anti-cancer immunotherapy in patients.

Experimental Procedures
Mice Itgb8-td-Tomato mice were generated as described 16 . Generated animals were cross on FOXP3-IRES-GFP background 35 to follow Tregs. FOXP3-Cre eYFP ;Itgβ8 -/-(Foxp3 Ctrl ) mice FOXP3-Cre eYFP ;Itgβ8 / (Foxp3 ΔItgβ8 ) mice 18 , CD4-Cre;Stop / ;tgfbr1 CA ;Foxp3 GFP mice 19 were used.. C57BL/6 mice and CD57BL/6 CD3ε KO (CD3 KO ) mice were purchased (Charles Rivers, France). Importantly, though Itgβ8 is mainly expressed in Tregs, we validated any leakiness of Foxp3-CRE construct in Foxp3 ΔItgβ8 mice, by breading animals on Rosa26 reported background 36 . All animals were between 2-6 months of age, all on a C57BL/6 background. Mice were maintained in AniCan SPF mouse facility Lyon, France. Patient tumors and anti-Itgβ8 antibody treatment Primary breast adenocarcinoma tumors and primary melanoma were obtained by the Biological Resource Center of Centre Léon Bérard and Hospital Lyon Sud respectively. Primary melanoma, at non-invasive stages, were obtained after surgery in different regions of the body. Primary breast tumors, irrelevant of their hormonal status, were analyzed. No gender (melanoma) and age (breast cancer and melanoma) selection was performed to establish the patient cohort. Importantly, patients never received anti-cancer treatments prior surgery. Fresh tumors were treated by the Ex-vivo facility of the Centre Léon Bérard Lyon France. They were imbedded in the Ex-vivo facility speci c matrix gel© and cut at 250 µm with microtome (Seica). Tumor slides were then cultured on Uvac 1264 in RPMI-completed medium, 1% FCS, 1% HEPES, 1% penicillin/streptomycin, 1% MEM-NEAA (LifeTechnologies), 1% NaPyruvate