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S. Figure 1. Involvement of Pxn in CD103-mediated adhesion and spreading of CD8+/CD103+ TRM cells. S. Figure 2. T-cell treatment with saracatinib or infection with shRNA targeting Pxn does not alter cell viability and cell surface protein expression levels. S. Figure 3. Transfection of the JTag T-cell clone with CD103-GFP constructs. S. Figure 4. The alphaE cytoplasmic tail is required for Pxn-dependent CD103 polarization and CD103-mediated early T-cell signaling.

The alphaE cytoplasmic tail is required for Pxn-dependent CD103 polarization and CD103-mediated early T-cell signaling

CD8+/CD103+ tissue-resident memory T cells (TRM cells) accumulate in several human solid tumors, where they have been associated with a favorable prognosis. However, the role of CD103, the α subunit of the integrin αEβ7 (also known as CD103), in the retention and functions of these TRM is undefined. In this report, we investigated the role of CD103 cytoplasmic domain and the focal adhesion-associated protein paxillin (Pxn) in downstream signaling and functional activities triggered through αE/CD103 chain. Binding to immobilized recombinant (r)E-cadherin-Fc of CD103 integrin expressed on tumor-specific CTL clones promotes phosphorylation of Pxn and Pyk2 and binding of Pxn to the αE/CD103 subunit tail. Inhibition of Pxn phosphorylation by the Src inhibitor saracatinib or its knockdown via shRNA dramatically altered adhesion and spreading of freshly isolated CD8+/CD103+ lung tumor–infiltrating lymphocytes and CD103+ tumor-specific CTL clones. Inhibition of Pxn phosphorylation with saracatinib in these CTL clones also severely compromised their functional activities toward autologous tumor cells. Using Jurkat T cells as a model to study CD103 integrin activation, we demonstrated a key role of serine residue S1163 of the αE chain intracellular domain in polarization of CD103 and recruitment of lysosomes and Pxn at the contact zone of T lymphocytes with rE-cadherin-Fc–coated beads. Overall, our results show how Pxn binding to the CD103 cytoplasmic tail triggers αEβ7 integrin outside-in signaling that promotes CD8+ T-cell migratory behavior and effector functions. These results also explain the more favorable prognosis associated with retention of TRM cells in the tumor microenvironment. Cancer Res; 77(24); 7072–82. ©2017 AACR.

Homing of CD8+ T lymphocytes to the tumor microenvironment is an important step for mounting a robust antitumor immune response. TGFβ is responsible for CD103 (αEβ7) integrin induction in activated intraepithelial CD8+ T lymphocytes. However, the interplay between TGFβ and CD103 and their contribution to T-cell infiltration and antitumor activity remain unknown. Here, we used viable human lung tumor slices and autologous tumor antigen-specific T-lymphocyte clones to provide evidence that CD103 is directly involved in T-lymphocyte recruitment within epithelial tumor islets and intratumoral early T-cell signaling. Moreover, TGFβ enhanced CD103-dependent T-cell adhesion and signaling, whereas it inhibited leukocyte function-associated antigen (LFA)-1 (αLβ2) integrin expression and LFA-1-mediated T-lymphocyte functions. Mechanistic investigations revealed that TGFβ bound to its receptors (TGFBR), which promoted the recruitment and phosphorylation of integrin-linked kinase (ILK) by TGFBR1. We further show that ILK interacted with the CD103 intracellular domain, resulting in protein kinase B (PKB)/AKT activation, thereby initiating integrin inside-out signaling. Collectively, our findings suggest that the abundance of TGFβ in the tumor microenvironment may in fact engage with integrin signaling pathways to promote T-lymphocyte antitumor functions, with potential implications for T-cell-based immunotherapies for cancer. Cancer Res; 76(7); 1757–69. ©2016 AACR.

Legend for Supplementary Figures S1-S5 and Supplementary Movies.

Statistical analyses of results shown in Figure 1A (S1); Statistical analyses of results shown in Supplementary Figure 1A (S2); Statistical analyses of results shown in Supplementary Figure 2B (S3).

CTL CD103+ on rE-cadh-Fc from TGFβ Signaling Intersects with CD103 Integrin Signaling to Promote T-Lymphocyte Accumulation and Antitumor Activity in the Lung Tumor Microenvironment

Supplemental video 1. 786-0 cells described in Figure 3B were recorded by time-lapse video microscopy

PDF file - 127K, Surface expression of E-cadherin (CD324) and ICAM-1 (CD54) on IGR-Heu tumor cells.

CTL CD103+ on rICAM-Fc from TGFβ Signaling Intersects with CD103 Integrin Signaling to Promote T-Lymphocyte Accumulation and Antitumor Activity in the Lung Tumor Microenvironment

Supplementary Figures S1-S5. Figure S1. Differential susceptibility of 786-0, PRC3, and WT7 cell lines to NK-mediated lysis. Figure S2. Role of ANGPTL4 and ADM in RCC cell susceptibility to NK-mediated lysis. Figure S3. VHL and NK ligand expression in RCC cells. Figure S4. Silencing of HIF-2α and NK ligand expression in 786-0 cells. Figure S5. VHL status, targeting of HIF-2α or silencing of ITPR1 does not regulate autophagy in 786-0 cells.

PDF file - 120K, Adhesion of T-cell clones to recombinant adhesion molecules under shear stress.

Supplemental video 2. 786-0 cells described in Figure 3B were recorded by time-lapse video microscopy

PDF file - 9K

CTL CD103+ treated TGF-β on rICAM-Fc from TGFβ Signaling Intersects with CD103 Integrin Signaling to Promote T-Lymphocyte Accumulation and Antitumor Activity in the Lung Tumor Microenvironment

Understanding how specific cancer mutations modulate the tumor microenvironment is essential for developing new precision cancer therapies. The histone methyltransferase MLL3 is a tumor suppressor that is frequently mutated together with p53 and PI3-kinase in breast cancer. Herein, using a mouse mammary-stem-cell-based tumor model and CRISPR gene editing, we recapitulated this genetic setting and revealed that Mll3 loss promotes tumorigenesis through enhancing tumor-intrinsic aggressiveness and establishing an immunosuppressive microenvironment. Specifically, HIF1α was upregulated in Mll3-mutant tumors and controlled proliferation and stem cell activity of the tumor cells. HIF1α also transcriptionally upregulated expression of the chemokine CCL2 by tumor cells. CCL2 further contributed to rapid CCR2-dependent accumulation of activated, proliferative KLRG1+Foxp3+ regulatory T (Treg) cells at very early stages of tumor initiation, promoting faster tumor onset. These results link MLL3 loss to HIF signaling, CCL2 and Treg cells, and the establishment of an early immune-suppressive microenvironment, a finding that may open new therapeutic opportunities for MLL3-mutant cancers.One Sentence SummaryMLL3 loss promotes tumorigenesis by enhancing recruitment of regulatory T cells.

While cognate antigen drives clonal expansion of memory CD8

While cognate antigen drives clonal expansion of memory CD8+ T (CD8+ TM) cells to achieve sterilizing immunity in immunized hosts, not much is known on how cognate antigen contributes to early prot...

SummaryWhile cognate antigen drives clonal expansion of memory CD8+ T cells to achieve sterilizing immunity in immunized hosts, not much is known on how cognate antigen contributes to early mechanisms of protection before clonal expansion occurs. Herein, using distinct models of immunization, we establish that cognate antigen recognition by CD8+ TM cells on dendritic cells initiates their rapid and coordinated production of a burst of CCL3, CCL4 and XCL1 chemokines under the transcriptional control of IRF4. Using intravital microscopy imaging and in vivo monoclonal antibody labelling, we reveal that memory CD8+ T cells undergo antigen-mediated arrest in splenic red pulp clusters of CCR2+ monocytes where they locally deliver both IFNγ- and chemokine-potentiating microbicidal activities to achieve early protection. Thus, rapid and effective memory CD8+ T cell responses require a complex series of spatially and temporally coordinated stepwise molecular and cellular events that quickly restrict microbial pathogen growth and optimize the local delivery of effector molecules before clonal expansion occurs.

The lytic function of CTL relies on the polarized release of cytotoxic granules (CG) at the immune synapse (IS) with target cells. CTL also contain CCL5 in cytoplasmic storage vesicles (CCL5V) distinct from CG, the role of which, in regulating T cell effector functions, is not understood. Using human CD8+ T cells specific to a lung tumor–associated Ag, we show in this article that CTL release both secretory compartments into the immune synapse with autologous tumor cells. Moreover, we demonstrate that disorganization of the T cell microtubule cytoskeleton and defects in hMunc13-4 or Rab27a abrogate CG exocytosis and synaptic secretion of the chemokine. Mechanistically, synaptic release of CCL5 cytoplasmic storage vesicles likely occurs upon their coalescence with the Rab27a-hMunc13-4 compartment and results in autocrine, CCR5-dependent induction of CXCR4 cell surface expression, thereby promoting T cell migration in response to CXCL12. We propose that CCL5 polarized delivery represents a mechanism by which CTL control immune synapse duration.

CD8

CD8+/CD103+ tissue-resident memory T cells (TRM cells) accumulate in several human solid tumors, where they have been associated with a favorable prognosis. However, the role of CD103, the α subunit of the integrin αEβ7 (also known as CD103), in the retention and functions of these TRM is undefined. In this report, we investigated the role of CD103 cytoplasmic domain and the focal adhesion-associated protein paxillin (Pxn) in downstream signaling and functional activities triggered through αE/CD103 chain. Binding to immobilized recombinant (r)E-cadherin-Fc of CD103 integrin expressed on tumor-specific CTL clones promotes phosphorylation of Pxn and Pyk2 and binding of Pxn to the αE/CD103 subunit tail. Inhibition of Pxn phosphorylation by the Src inhibitor saracatinib or its knockdown via shRNA dramatically altered adhesion and spreading of freshly isolated CD8+/CD103+ lung tumor–infiltrating lymphocytes and CD103+ tumor-specific CTL clones. Inhibition of Pxn phosphorylation with saracatinib in these CTL clones also severely compromised their functional activities toward autologous tumor cells. Using Jurkat T cells as a model to study CD103 integrin activation, we demonstrated a key role of serine residue S1163 of the αE chain intracellular domain in polarization of CD103 and recruitment of lysosomes and Pxn at the contact zone of T lymphocytes with rE-cadherin-Fc–coated beads. Overall, our results show how Pxn binding to the CD103 cytoplasmic tail triggers αEβ7 integrin outside-in signaling that promotes CD8+ T-cell migratory behavior and effector functions. These results also explain the more favorable prognosis associated with retention of TRM cells in the tumor microenvironment. Cancer Res; 77(24); 7072–82. ©2017 AACR.

Recent findings have revealed roles for systemic and mucosa-resident memory CD8(+) T cells in the orchestration of innate immune responses critical to host defense upon microbial infection. Here we integrate these findings into the current understanding of the molecular and cellular signals controlling memory CD8(+) T cell reactivation and the mechanisms by which these cells mediate effective protection in vivo. The picture that emerges presents memory CD8(+) T cells as early sensors of danger signals, mediating protective immunity both through licensing of cellular effectors of the innate immune system and via the canonical functions associated with memory T cells. We discuss implications for the development of T cell vaccines and therapies and highlight important areas in need of further investigation.

Homing of CD8+ T lymphocytes to the tumor microenvironment is an important step for mounting a robust antitumor immune response. TGFβ is responsible for CD103 (αEβ7) integrin induction in activated intraepithelial CD8+ T lymphocytes. However, the interplay between TGFβ and CD103 and their contribution to T-cell infiltration and antitumor activity remain unknown. Here, we used viable human lung tumor slices and autologous tumor antigen-specific T-lymphocyte clones to provide evidence that CD103 is directly involved in T-lymphocyte recruitment within epithelial tumor islets and intratumoral early T-cell signaling. Moreover, TGFβ enhanced CD103-dependent T-cell adhesion and signaling, whereas it inhibited leukocyte function-associated antigen (LFA)-1 (αLβ2) integrin expression and LFA-1-mediated T-lymphocyte functions. Mechanistic investigations revealed that TGFβ bound to its receptors (TGFBR), which promoted the recruitment and phosphorylation of integrin-linked kinase (ILK) by TGFBR1. We further show that ILK interacted with the CD103 intracellular domain, resulting in protein kinase B (PKB)/AKT activation, thereby initiating integrin inside-out signaling. Collectively, our findings suggest that the abundance of TGFβ in the tumor microenvironment may in fact engage with integrin signaling pathways to promote T-lymphocyte antitumor functions, with potential implications for T-cell-based immunotherapies for cancer. Cancer Res; 76(7); 1757–69. ©2016 AACR.

Proactive vis a vis des risques engendres par les crues sur les barrages qu'elle exploite, EDF a entrepris depuis 25 ans, d'une part la re-evaluation du debit des crues extremes par la methode du Gradex, puis plus recemment de son evolution le Schadex et, d'autre part, des actions d'amelioration de la fiabilite ou/ et d'augmentation de la capacite des ouvrages d'evacuation des crues. L'etude de l'impact des ecarts entre la debitance des ouvrages d'evacuation et les debits des crues estimes, sur la cote de retenue maximale atteinte, induit une reponse parmi deux grandes familles de solutions, en tenant compte des particularites de chaque barrage et du deficit de debit constate. La premiere famille de solutions permet de maintenir la debitance des ouvrages grâce a la prise en compte de differents facteurs (laminage de la retenue, creux preventif ou baisse conditionnelle de plan d'eau) associes eventuellement a l'augmentation des cotes atteintes lors du passage de la crue de projet. Ce type de solution necessite prealablement de verifier la stabilite du barrage sous les nouvelles conditions et le cas echeant d'envisager la realisation de travaux de confortement. La seconde famille consiste a accroitre la debitance des ouvrages. Ces solutions ont ete couramment mises en œuvre dans le passe, generalement sur les ouvrages les plus anciens. Un certain nombre de projets plus recents ont egalement vu le jour ou sont en cours de developpement. Qu'il s'agisse de la modification d'ouvrages existants ou de la creation d'ouvrages complementaires, ces solutions concernent davantage les evacuateurs de surface meme si quelques projets impactent les vannes de fond. Parfois ces solutions repondent egalement a d'autres besoins que le strict passage d'un debit de crue (amelioration de l'exploitation des ouvrages, vidange de la retenue...).

Elimination of cancer cells by the immune system requires the induction of a strong and durable antitumor cytotoxic T lymphocyte (CTL) response. Immunotherapy approaches aim at generating tumor-specific CTL capable of migrating to the tumor site and at optimizing their functional activities toward target cells. Unfortunately, clinical trials indicate that despite an increase in the frequency and reactivity of antitumor CD8+ T lymphocytes, the efficacy of current immunotherapeutic strategies remains limited and rarely resulted in the eradication of malignant cells. Integrins and their ligands play critical roles in regulating T-cell effector functions, including adhesion to antigen presenting cells (APC), costimulation, migration to lymphoid organs and inflammatory sites, and extravasation. Although some of those are known to promote tumor cell proliferation and dissemination, others are required for T-lymphocyte homing and retention within the tumor microenvironment and for CTL activation and triggering of cytotoxic activity within a hostile ecosystem. In this chapter, we will briefly summarize findings involving integrins, in particular CD103 (αE/β7) and LFA-1 (αL/β2), and their respective ligands, E-cadherin and intercellular adhesion molecule 1 (ICAM-1), in regulating the effector phase of the antitumor T-cell response and we provide insights into the potential implication of their altered expression in tumor resistance to CTL-mediated cancer immunotherapy. The characterization of integrin-dependent pathways involved in the potentiation of antitumor CTL functions may lead to enhanced immune protection and improved cancer immunotherapy.

Clear cell renal cell carcinomas (RCC) frequently display inactivation of von Hippel-Lindau (VHL) gene leading to increased level of hypoxia-inducible factors (HIF). In this study, we investigated the potential role of HIF2α in regulating RCC susceptibility to natural killer (NK) cell–mediated killing. We demonstrated that the RCC cell line 786-0 with mutated VHL was resistant to NK-mediated lysis as compared with the VHL-corrected cell line (WT7). This resistance was found to require HIF2α stabilization. On the basis of global gene expression profiling and chromatin immunoprecipitation assay, we found ITPR1 (inositol 1,4,5-trisphosphate receptor, type 1) as a direct novel target of HIF2α and that targeting ITPR1 significantly increased susceptibility of 786-0 cells to NK-mediated lysis. Mechanistically, HIF2α in 786-0 cells lead to overexpression of ITPR1, which subsequently regulated the NK-mediated killing through the activation of autophagy in target cells by NK-derived signal. Interestingly, both ITPR1 and Beclin-1 silencing in 786-0 cells inhibited NK-induced autophagy and subsequently increased granzyme B activity in target cells. Finally, in vivo ITPR1 targeting significantly enhanced the NK-mediated tumor regression. Our data provide insight into the link between HIF2α, the ITPR1-related pathway, and natural immunity and strongly suggest a role for the HIF2α/ITPR1 axis in regulating RCC cell survival. Cancer Res; 74(23); 6820–32. ©2014 AACR.

(Abstract) This method allows evaluating the relative adhesion strength between T lymphocytes and specific adherent target cells using a shear force in flow chambers. It is based on the measure of the resistance of conjugates formed between T cells and adherent tumor cells to shear stress in a microfluidic system. For this purpose, T cells, stained with a CellTracker probe, are added into flow channels containing a monolayer of adherent target cells and their progressive detachment under a constant shear stress is then recorded using a fluorescent microscope.

Immune system–mediated eradication of neoplastic cells requires induction of a strong long-lasting antitumor T-cell response. However, generation of tumor-specific effector T cells does not necessarily result in tumor clearance. CTL must first be able to migrate to the tumor site, infiltrate the tumor tissue, and interact with the target to finally trigger effector functions indispensable for tumor destruction. Chemokines are involved in circulation, homing, retention, and activation of immunocompetent cells. Although some of them are known to contribute to tumor growth and metastasis, others are responsible for changes in the tumor microenvironment that lead to extensive infiltration of lymphocytes, resulting in tumor eradication. Given their chemoattractive and activating properties, a role for chemokines in the development of the effector phase of the antitumor immune response has been suggested. Here, we emphasize the role of the chemokine–chemokine receptor network at multiple levels of the T-cell–mediated antitumor immune response. The identification of chemokine-dependent molecular mechanisms implicated in tumor-specific CTL trafficking, retention, and regulation of their in situ effector functions may offer new perspectives for development of innovative immunotherapeutic approaches to cancer treatment. Cancer Res; 72(24); 6325–32. ©2012 AACR.

Cancer immunotherapies specifically target tumors by optimizing the capacity of host cytotoxic T lymphocytes (CTL) to recognize and kill malignant cells. To destroy their target, CTL must migrate to the tumor, infiltrate the stroma and then interact with cancer cells. Recruitment of T lymphocytes within the tumor involves several sequential steps regulated by molecular interactions with a variety of stromal cell types and coordinated by chemotactic agents, extracellular matrix proteins and cell surface adhesion molecules. Integrins are one of the major families of adhesion molecules that contribute to lymphocyte development, migration, extravasation and activation. CD103 integrin and its ligand, the epithelial cell marker E-cadherin, play a crucial role in promoting both tumor cell cytotoxicity and cytokine secretion. Accumulating evidence also indicates that CD103 on CD8+ tumor-infiltrating lymphocytes (TIL) is associated with increased intraepithelial T-cell infiltration and that an enhanced CD103+ TIL subset correlates with improved patient survival in several cancers. However, the contribution of CD103 to promoting T-cell recruitment within epithelial tumor regions, and the influence of TGF-beta, abundant within the tumor microenvironment, on integrin functions and signaling remain poorly understood. In the present study we provide evidence that CD103 is directly involved in T-cell recruitment within epithelial tumor islets and in local early T-cell signaling. Moreover, TGF-beta triggers integrin-mediated functional activities by promoting phosphorylation of integrin-linked kinase (ILK) and its subsequent binding to CD103, thus initiating an “inside-out” signaling leading to activation and clustering of the integrin and thereby initiating an “outside-in” signal that contributes to T-cell stimulation, spreading and cytotoxicity. We conclude that TGF-beta, while known for its protumoral effects, promotes an antitumor CTL response by activating CD103 integrin. Citation Format: Marie Boutet, Jr., Fathia Mami-Chouaib, Sr. CD103 integrin promotes T-cell recruitment and activation within epithelial tumors. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B075.

Cytotoxic T lymphocytes (CTL) exert their lytic activity through the polarized release of cytotoxic granules at the immune synapse (IS) formed with specific target cells. Integrins and their cognate ligands on target cells play a major role in T-cell receptor (TCR)-mediated functions. We show here that cytotoxicity towards epithelial cancer cells requires, along with TCR engagement, the interaction of either CD103 with E-cadherin or LFA-1 with ICAM-1 thereby enhancing the strength of the T-cell/target cell adhesion. Integrin-dependent mature cytotoxic IS (mcIS) displays a cohesive ultrastructure characterized by polarized degranulation and chemokine release associated with target cell killing and chemokine receptor induction on effector cells. Our results provide new insights into molecular mechanisms regulating antitumor CTL responses and the role of integrins and chemokine receptors on the formation and duration of the cIS.

We have previously reported that interaction of integrin αE(CD103)β7, often expressed by tumor-infiltrating lymphocytes (TIL), with E-cadherin on epithelial tumor cells is required for polarized exocytosis of cytotoxic granules resulting in target cell lysis. Our results also indicated that binding of CD103 on tumor-specific cytotoxic T lymphocytes (CTL) to a recombinant E-cadherin-Fc molecule is sufficient to induce polarization of cytolytic granules in a phospholipase Cγ-dependent pathway, whereas degranulation also requires co-engagement of the T-cell receptor (TCR). In the present study, we compared the ultrastructure of the immune synapses formed between CD103+ or CD103- CTL and specific lung cancer cells, and analyzed the involvement of adhesion/costimulatory molecules in the activation of cytotoxicity versus stimulatory functions. Our results indicated that stimulatory and lytic synapses display differential activation threshold and intercellular molecular requirements. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-150. doi:10.1158/1538-7445.AM2011-LB-150