Call of project -  2020

Role of lactic acid in the tumor escape - Partners : Aurélie Moreau - CRTI U1064, Team 1(coordinator),  Yves Delneste & Pascale Jeannin - CRCINA U1232, Team 7, Fabienne Haspot - Humanized Rodent Facility, LabEx IGO &  Jordi Ochando - ISCIII, Madrid

Nutrients are key components of tumor development. Indeed, tumor cells efficiently capture glucose and rely mainly on the glycolytic pathway to produce ATP allowing rapid cell proliferation. This glycolytic pathway leads to an accumulation of lactic acid in the tumor microenvironment that favors angiogenesis, metastasis as well as tumor escape by functional modifications of immune cells. Indeed, as shown previously in mice, we recently demonstrated that lactic acid induces the differentiation of human monocytes into macrophages with a pro-tumor and inflammatory phenotype. Although it is generally accepted that the tumor has a major role in the production of lactic acid, recent studies suggest that lactic acid produced by myeloid cells may also contribute to dampen immune responses. Supporting this observation, we recently discovered that in vitro-generated human tolerogenic mononuclear phagocytes are highly glycolytic and therefore secrete lactic acid which is metabolized by T cells and contributes to the inhibition of their proliferation and cytokine production.
Based on these results, the overall objective of this project is to determine the mechanism by which either exogenous (produced by tumor cells) or endogenously produced lactic acid impacts the functional polarization of mononuclear phagocytes in the tumor microenvironment and to evaluate whether targeting lactic acid metabolism in these cells may represent a novel strategy to dampen tumor-associated immune suppression.

Next generation IL-34 and application in diseases (NEXT-IL34) - Partners : Carole Guillonneau & Ignacio Anegon - CRTI U1064, Team 2 (coordinators), Erwan Mortier - CRCINA U1232, Team 1, Luis Barbeito - Pasteur Institut, Uruguay,  Christophe Blanquart - CRCINA U1232, Team 4, Andrew Dick - University of Bristol, UK, Stephanie Gras - Monash University, Australia & François-XavierHubert - AbolerIS Pharma

The use of immunosuppressors in transplantation and autoimmune diseases has allowed remarkable success in the short and medium term, but unwanted side effects still lead to high morbidity and mortality. Therefore, new treatments are needed that will be more specific for anti-donor immune responses or with less side effects and that would allow at least to decrease the use of immunosuppressors. Cytokines are powerful tools for controlling immune responses. The discovery in 2008 of IL-34 as a new ligand of CSF-1R opened new perspectives and we described recently the immunoregulatory properties of IL-34 in rat and human. The general objective of this project is to generate and test improved formats of IL-34 for more efficient and/or more targeted in vivo use and to better define the actions of IL-34 on monocytes, DCs, microglia and Tregs.

B cell functional heterogeneity in both protective and pathogenic immune responses :Towards a better understanding of regulatory B cell subsets ontogeny and dynamics (BECOME) - Partners : Sophie Hillion - LBAI U1227, Sophie Brouard - CRTI U1064 & Céline Delaloy - MICMAC U1236

B-cell with functional duality have been described in diverse pathophysiological situations exhibiting either pro-inflammatory or suppressive function on the immune response. These suppressive B cells act mainly through the production of IL-10. However, this is not exclusive and other mechanisms exist and even coexist with IL-10 production like for example the production of Granzyme B. Bregs have a high degree of phenotype heterogeneity according to the immune contexts, species and tissue localization. And for now, the existence of a stable marker that unambiguously define Breg subsets has not been found. Consensual framework actually proposes that B cells could acquire regulatory functions under a specific environment leading to the convergent existence of immuno-regulatory progenitors prone to differentiate into regulatory plasmablasts- plasma cells. However open questions remain regarding the ontogeny and dynamics of those different cells in human. In this collaborative effort gathering three groups from the Labex IGO (U1227, U1236, U1264), we aim to decipher the dynamics of Breg subsets at the single cell level using single-cell RNA and ATAC sequencing as well as mass cytometry to catch the transient states leading to the acquisition of regulatory functions. This will enable to explore the molecular mechanisms underlying this process. Finally, we plan to explore the distribution of the Breg subsets in patients using high multi-parameter mass cytometry in blood but also using imaging mass cytometry in tissues (Hyperion Platform). This part will benefit from the access of our three groups to well characterized patient cohorts including kidney transplanted patients (U1064), patients with Sjögren syndrome (U1227), multiple sclerosis and rheumatoid arthritis (U1236).

Call of projets - 2018

Tracking auto-reactive CD4 T cells in autoimmune diseases and during immune related adverse events after immunotherapy targeting checkpoint inhibitor -  Amédée Renand (UMR1064)

Targeting PD-1 and other check point inhibitors during cancer immunotherapy is a “revolution” and leads to the control of cancer disease by the immune system. Unfortunately, these types of immunotherapy have been shown to induce side effects which look like autoimmune disorders, called Immune Related Adverse Events (IRAEs). Thus eliciting the link between autoimmunity, PD-1 and IRAEs is of major clinical, fundamental and therapeutic interest. In the laboratory we work on the characterization of auto-reactive CD4 T cells in patients with autoimmune hepatitis (AIH). We were able to detect auto-reactive Soluble Liver antigen (SLA)-specific CD4 T cells, which express high level of PD-1, only in the blood of patients with SLA auto-antibodies (the SLA self antigen has been described to be highly specific of the AIH). Objectives are to: 1) identify the immune signature of auto-reactive CD4 T cells in AIH; 2) develop a generic strategy to directly identify auto-reactive CD4 T cells ex vivo; and 3) determine the relevance of tracking auto-reactive-like CD4 T cells as potential biomarkers of IRAEs.

Functional role of peptides derived from meloe long non coding RNA in melanoma - Partners : Catherine Rabu (UMR1232) & Ignacio Anegon (UMR1064)

Meloe RNA is a polycistronic long non coding RNA transcribed specifically in the melanocytic lineage. We identified this RNA a few years ago when we were looking for antigens recognized by T lymphocytes from a metastatic melanoma patient who benefited from T cell therapy. MELOE-1 is highly immunogenic in both healthy subjects and patients and we identified multiple class II epitopes and a class I epitope in its sequence. The tumor specificity of this antigen associated with the existence of a broad T cell repertoire against it makes it an ideal target for immunotherapy strategies. In fact, MELOE-1 antigen is currently exploited as an immunotherapeutic target in a T cell immunotherapy clinical trial (NCT02424916). Since then, we have explored further the modes of translation of this lncRNA and the peptides produced and we found that meloe actually encodes at least 3 short peptides named MELOE-1, 2 and 3. MELOE-1 and 2 are translated by an alternative IRES-dependent (Internal Ribosome Entry Sequence) mechanism exclusively in melanoma cell lines while MELOE-3 is translated in a cap-dependent manner, both in melanocytes and in melanoma cell lines. Interestingly, MELOE-3 is poorly immunogenic as compared to MELOE-1 thus suggesting a correlation between immunogenicity, expression profile and IRES dependency. A putative role of MELOE-1 peptide has never been assessed and is the purpose of the present project. To achieve this goal, we will develop two strategies: i) Overexpression of MELOE-1 peptide in melanoma cells by transfection of MELOE-1 ORF under a strong promoter ; ii) Invalidation of MELOE-1 ORFs (or MELOE-3 ORF as control) in melanoma cells using the CRISPR/Cas9 technology.

Decipher the role of the inhibitory myeloid C-type lectin-like receptor CLEC-1 in Foxp3+ Treg induction and anti-tumor response - Elise Chiffoleau (UMR1064) 

Emerging literature in these past few years demonstrates that tumors use physiological processes of C-type lectin receptors particularly those involved with wound healing to suppress myeloid cell activation and promote immune evasion. We previously identified the C-type receptor-like 1, CLEC-1 as highly expressed in rat tolerated allografts. We showed in both rodent and Human that CLEC-1 is expressed by myeloid cells such as macrophages, monocytes, dendritic cells (DCs) and neutrophils and that its expression is decreased by pro-inflammatory stimuli but enhanced by TGF?. Importantly, using CLEC-1 deficient rats, we showed that disruption of CLEC-1 signaling led to an enhanced Il12p40 expression in DCs and to an exacerbation of downstream in vitro and in vivo CD4+ Th1 and Th17 responses. As Th17 cells bear a reciprocal developmental relationship to inducible Tregs, we propose in this Labex project to finely evaluate the role of the myeloid receptor CLEC-1 in the generation and plasticity of inducible Tregs in the context of cancer. For that, we plan to use CLEC-1 KO mice and OVA antigen-specific mice models that will allow to specifically follow Tregs fate in in vitro assays and in in vivo cancer models. This will help to reinforce the hypothesis of CLEC-1 targeting as an original checkpoint inhibitor with innovative therapeutic application in oncology.

Tracking B cell differentiation in multiple sclerosis: from the blood to the CNS - Partners : Laure Michel (UMR 1236) & David Laplaud (UMR1064)

Multiple Sclerosis (MS) is an autoimmune chronic disease of the CNS affecting more than 2 Million people worldwide. Clinical trials on the efficacy of B-cell depleting therapies in relapsing MS, have suggested that B cells may contribute to MS pathogenesis, potentially through antibody independent mechanisms. The overarching hypothesis is that abnormalities in B cell differentiation contribute to MS disease processes, within distinct disease compartments, and throughout the clinical spectrum of MS. In this project, we plan to develop 4 different aims: (1) characterize the differentiation abilities of B cells in MS patients, (2) characterize the phenotype, cytotoxicity of plasma cells, (3) analyze the migration capacities of plasma cells inside the CNS of MS patients, and (4) analyze the phenotype and function of follicular helper T cells (TFH) in MS patients. Such knowledge about B cell differentiation will help to develop new tools to selectively deplete pathogenic plasma cells and spare the regulatory ones, but also to act upstream by targeting proinflammatory TFH cells.

Call of projets - 2016

CD8+CD28neg T cells, a threat to transplantation? - Partner 1 : N. Degauque (UMR1064) - Partner 2 : F. Haspot (UMR1064) - Partner 3 : C. Pecqueur (UMR1232)

Kidney transplantation is the treatment of choice for End-Stage Renal dysfunction from patient survival perspective as well as from an economical point-of-view. Preventing the occurrence of acute rejection is not anymore an issue. However, minimal success had been obtained to prevent late chronic rejection and current immunosuppressive drugs are associated with increased infections, viral reactivations and malignancies. It is thus critical to design innovative strategies to control the pathogenic cells involved in chronic rejection. We have shown that kidney transplant recipients with a high frequency of effector memory CD8 T cells re-expressing CD45RA (EMRA) exhibit a 2-fold higher risk of kidney dysfunction. Our preliminary results highlight that pre-existing or neo-formed pathogenic CD8+CD28- T cells are a barrier for long-term graft acceptance that cannot be control by actual IS drugs nor by the newly developed costimulation blockade drugs. The aim of the project is to characterize the development of pathogeneic CD8+CD28- T cells after kidney transplantation and to evaluate in humanized mouse model the ability to control their immune function by metabolic interferences. The project is divided into 3 tasks
Task #1. To investigate the adaptation of the metabolism of CD8 T cell subsets after kidney transplantation
Task #2. To develop humanized mouse models to study CD8+CD28- T cells pathogenicity
Task #3. To test the ability of metabolic interferences to control the immune response of CD8+CD28- T cells in preclinical models.

Engineered CAR-Tregs - licensed to specific control of immune responses in transplantation - Partner 1 : C. Guillonneau (UMR1064) - Partner 2 : X. Saulquin (UMR1232)  

The establishment of tolerance for organ transplants is a major goal in the field of transplantation. Indeed, transplanted patients take immunosuppressive drugs suppressing all the immune system for the rest of their life, and although these drugs have allowed remarkable success, unwanted side effects still lead to high morbidity and mortality, even when avoiding excessive immunosuppression. We previously reported the suppressive properties of rat CD8+CD45RClow T cells and demonstrated their potential for cell therapy in transplantation. We showed that antigen-specific CD8+ Tregs had superior potential compared to polyclonal Tregs. This project aim to confer antigen-specificity using chimeric antigen receptors (CARs) directed to donor antigens and evaluate the potential of such strategy in transplantation.

 Boosting anti-tumor response by conferring metabolic autonomy to T cells - Partners : C. Louvet (UMR1064) & B. Vanhove (UMR1064)

Blockade of immune activation checkpoints and gene-engineering of T cells with anti tumor T cell receptors (TCR) or chimeric antigen receptors (CAR) have recently produced impressive results in the clinic for a variety of cancers. However, additional therapeutic weapons will likely be needed to reach tumor clearance, especially in solid tumors. The tumor microenvironment can exert strong suppressive effects on T cells, often considered as counter-regulatory mechanisms triggered by the T cells themselves. Beyond immune checkpoint targeting, we believe that conferring functional advantages to T cells by genetic engineering could be decisive to tip the balance in favor of immunity. Such strategies could be implemented through adoptive cell transfer (ACT) therapies but still remain poorly explored notably because most of the attention is currently focused on TCR/CAR. In this project, we explore the therapeutic potential of engineering critical enzyme-encoding genes with the aim to boost selective metabolic pathways in preclinical models of solid cancers.

Combining adoptive T cell transfer of engineered PD-1 deficient specific T cells with a-radioimmunotherapy for melanoma treatment - Partner 1: N. Labarriere Team 3 (UMR1232)  - Partner 2: E. Scotet Team 1 (UMR1232) - Partner 3: TH. Nguyen Team 2 (UMR1064) - Partner 4: J. Gaschet Team 13 (UMR1232)

The overall objective of this proposal is to assess, through a set of complementary in vitro and in vivo experimental approaches (eg, NSG mice engrafted with PDL-1 expressing human melanoma tumors), the anti-tumor efficacy of an innovative treatment combining ACT, with high avidity PD-1ko melanoma specific T lymphocytes, and α-RIT. This will be achieved through the implementation and the completion of the following milestones:
1/ Silencing of PDCD1 gene in high-avidity human melanoma-specific CD8+ αβ T lymphocytes. This step will be achieved by transfection or transduction of T lymphocytes with PDCD1-specific gRNA and Cas9. The design and validation of these tools will rely on the expertise of the local GenoCell Edit facility (Partner ≠3), specialized in this technology. The selection, amplification and in vitro validation of PD-1 inactivated melanoma-specific CD8+ αβ T cells will be performed by Partner ≠1.

2/ Anti-tumor efficiency of engineered CD8+ αβ T cells in vivo. The efficiency of engineered PD-1neg melanoma specific T cells to control the growth of human PDL-1 expressing melanoma tumors will be measured in NSG mice, and compared to that obtained with wild-type counterpart T cells (Partners ≠2 and ≠1).
3/ Development of radiolabeled anti-PDL-1 mAb. A commercially available anti-human PDL-1 specific mAb will be conjugated and labeled with Astatine-211 (for therapy) or Zirconium-89 (for molecular imaging) (Partner ≠4).

4/ Anti-tumor efficiency of PDL-1-specific α-RIT in vivo. The objective is to document in NSG mice engrafted with human PDL-1 expressing melanoma tumors the efficiency of α-RIT targeting PDL-1 on tumor growth and to characterize the expression of PDL-1 on remaining or resistant melanoma cells (Partners #2 and #4).
5/ Anti-tumor efficiency of combined ACT and α-RIT strategies in vivo. The impact of α-RIT immunotherapy targeting PDL-1 molecules expressed on melanoma cells, prior infusion of PD-1ko specific T cells will be assessed in NSG mice carrying human melanoma tumor grafts (Partners 1, 2 and 4).

Immune tolerance by IL-34: actions on macrophages and Tregs (TOL34) - Partners : I. Anegon (UMR1064) &  MC Cuturi

Call of projets - 2014

A new anti-CMV strategy to prevent primary infection during HSCT - Partners : F. Haspot and I. Anegon (UMR 1064)

Immunocompromised patients are at high risks of HCMV reactivation, which might be associated with severe end organ diseases and increased mortality in transplant patients. Thus, we used CRISPR/Cas9 to specifically target the UL122/123 HCMV’s essential. The multiplex CRISPR/Cas9 system (three gRNA) was much more efficient than the singleplex (one gRNA) approach targeting the same gene. Target gene expression, concomitant genome replication and virion release were significantly impaired by the multiplex strategy. Our anti-HCMV strategy based on three gRNAs efficiently blocked viral replication. A further anti-HCMV CRISPR/Cas9 system is under development to be able to target latently infected cells. This provides the basis for the development of an anti-HCMV CRISPR/Cas9 therapy.

Functional Contribution of MICA Polymorphic variants to Immune Responses in Organ Transplantation and in Cancer - Partners : B. Charreau (UMR 1064) & N. Gervois (UMR1232)

This project explores the emergent concept that some MICA gene polymorphisms encode for non-conventional MICA proteins which may affect MICA expression, regulation and functions. Our study focus on the genetic variant MICA A5.1 that associates with MICA*008, the most common allele, and on two MICA alleles encoding novel MICA isoforms that we recently identified (Charreau B. et al., EP13305955.0 5/7/2013, patented). Overall these 3 MICA alleles account for around 70% of the general population and of our transplant donors. The principal aim of this project is to determine how some frequent MICA gene polymorphisms encoding for non-conventional and/or truncated MICA proteins will affect MICA expression (membrane-bound and circulating proteins) and regulation on endothelial cells and melanoma cells, NKG2D-dependent NK cell activation and CD8 T-cell costimulation, and to decipher their contribution to the control of tumor progression and MICA alloimmunization in kidney transplantation.

Adoptive cell therapy for glioblastoma – characterization of tumor cell targets and analysis of their recognition by human t lymphocytes in vitro - Partners : E. Scotet (Team 1) and C. Pecqueur (Team 9) (UMR1232)

This project had several objectives centered on the study of human glioblastoma (GBM) biology and immunotherapy in novel physiological models in vitro and in vivo (eg., humanized xenografts in NSG mice). Promising results have been obtained with both in vitro and in vivo activities. Transcriptomic and functional studies have identified relevant GBM NKR-related molecules that are targeted by human Vg9Vd2 T cells. The funding of the project byt the Labex IGO has fostered the establishment of new local, national and international collaborations and the submission of additional national and EU network projects. A better characterization of the self-ligands recognized by Vg9Vd2 T cells on GBM cells is currently performed as well as the definition of some biomarkers (that may allow the pre-selection of the patients affected by the GBM sub-type that seem to optimally respond to the Vg9Vd2 ACT, in order to maximize the number of patients that may benefit from the treatment.

Tracking of tumor-specific T cells - Partners : Y. Guilloux and  E. Mortier (CRCINA U1232, Team 1)

Melanoma vaccination : coupling optimized long peptides to a viral protein that targets dendritic cells and favors cross-presentation - Partners : F. Lang and  P. Jeannin

HO-1 and tolerance (HOT) - Partners : I. Anegon (UMR 1064)  and  JF. Fonteneau