WP1. New immune markers and therapeutic targets for immunomodulating or depleting therapies
This WP will cover three projects that aim at characterizing new immune mechanisms regulating antitumor immunity and allograft rejection (project P1), at accelerating obtention of preclinical and clinical POC of new approaches, through set up of new animal models (project P2) and improvement of immune monitoring (project P3).
Within Project P1, the five IGO partners will share their complementary expertise to screen in vitro and ex vivo the immunomodulatory function of several human cell subsets (T cells, CD4 and CD8 T effectors and Tregs, Bregs, type II macrophages and neutrophils, plasmacytoid DC, MDSCs, mesenchymal stromal cells…) and candidate molecules (eg IL15, IL34, TORID, CLEC-1, IL22BP, HO-1, IDO…) in a broad array of preclinical models. Project P2 will aim at setting up dedicated or existing humanized mouse and rat models to identify common pathways shared by the immune system to fight tumors and reject transplants and to evaluate the therapeutic potential of strategies (eg using anti-CD28, Hepcidin, Tranilast, tolerogenic DCs…) already designed by IGO members. The main objectives of project P3 will be to (i) implement a new technology combining flow cytometry and mass spectrometry for very high content single cell analysis, (ii) develop new monitoring approaches for in depth phenotypic and functional analyses of human innate and adaptive immune cell subsets and (iii) implement a centralized data base for sharing standardized immune monitoring protocols.
Task 1 – Immunobiology
Task 1.1 “Suppressor cells” – Partners: EA2216, INSERM ESPRI, ERI29 (Brest), INSERM U1064 (Nantes), IJMR 917 (Rennes)
B cells play a complex role in the development of autoimmune diseases, tumors or in organ transplants. They have long been considered pathogenic especially because they produce antibodies in response to stimulation. It appears now that they have regulatory effects independent of antibody production based on the control of the dendritic cells that initiate immune responses, and the control of T cells that are effectors of immune responses. Thus, B cells contribute to hindering the development of inflammatory responses, having a beneficial effect against the severity of autoimmune diseases. Conversely, slowing immunosuppressive responses, they promote the development of tumors that escape immune controls, whilst they allow the acceptance of transplanted organs by preventing immune responses against grafts.
The main objective of this project is to identify and phenotypically and functionally characterize subpopulations of regulatory human B cells using different types of co-cultures developed in the three laboratories. Secondarily, identification and characterization of these subpopulations will extend their monitoring in various autoimmune diseases (Brest), in cancer (Brest, Rennes) or in transplanted patients (Nantes).
State of progress / results:
Operationally tolerant patient (TOL) recipients show a higher frequency of CD20+ CD24high CD38 high transitional and CD20+ CD38low CD24low naïve B cells compared to patients with stable graft function, associated with a decreased frequency of CD20- CD38+ CD138+ differentiated plasma cells, suggestive of abnormal B cell differentiation. B cells from TOL proliferate normally but produce more IL-10. In addition, B cells from tolerant recipients exhibit a defective expression of factors of the end step of differentiation into plasma cells and show a higher propensity for cell death apoptosis compared to patients with stable graft function. This in vitro profile is consistent with down-regulation of B cell differentiation genes and anti-apoptotic B cell genes observed in these patients in vivo. These data suggest that a balance between B cells producing IL-10 and a deficiency in plasma cells may encourage an environment favorable to the tolerance maintenance (Chesneau et al. Am J Transplant, 2014).
Chronic antibody-mediated rejection (cAbMR) now represents a major complication in transplantation and is a challenge in current therapeutics. We showed that patients with cAbMR display a unique B-cell phenotype with a reduced ratio of activated to memory B cells associated with an impaired immunosuppressive activity. The regulatory functions of the B cells depended on their maturation status. Furthermore, we have established that “activated B cells” were able to induce the expansion of regulatory T cells and to mediate durable suppressive function on autologous T cell proliferation through a TGF/IDO axis, that was deficient in B cells from cABMR patients. Thus, phenotypic and functional analyses of the B-cell compartment may be indicated for appropriate follow-up after transplantation and drive therapy in the establishment of transplant tolerance processes (Nouel et al. Kidney Int, 2013 and Ann Rheum Dis, 2014).
In the phenotypical approach (Quentin Simon PhD), we have now chosen to focus on the CD24high CD38high transitional B cell population in humans. Indeed, transitional B cells are currently ascribe to regulatory B-cells. We want to better understand the precise nature of this subset related to its regulatory function. This project has been organized in three phases: (1) Identification and characterization of the different subpopulations contained within the CD24high CD38high subset using compilation of phenotypic analyses of cytometry data with bioinformatic tools (Figure1) (2) A functional characterization of the identified B cell subsets (3) The study of their potential regulatory activities on T-cell immune response using in vitro models
The precise identification of regulatory B cells will not only open a way in the monitoring of different treatments, but also in the development of new pharmacological tools to increase or to eliminate their effects. For example, the promotion of regulatory B lymphocytes in vivo may provide new therapeutic approaches to induce tolerance in autoimmunity and transplantation. Conversely, immunosuppression led by tumor cells could be reduced after depletion of regulatory B cells, thereby increasing the therapeutic effects of many cancer treatments. In this context, B chronic lymphocytic leukemia (CLL) cells shares common phenotypic characteristics with regulatory B cells (Figure 2). Then, the aim of this specific part of the project is to determine whether B CLL cells share also functional characteristics with regulatory B cells. If yes, could they be responsible for immunosuppression observed in this disease? The particular abnormal ratio between CLL T cells and CLL B cells in vivo as well as the environment may also influence the properties of the regulatory B cells. Further investigations will be lead (Audrey Mohr PhD).
Both cancer and organ transplantation face very significant obstacles with respect to the impact of immunosuppression mechanisms on the efficacy of therapy. Thus, both fields share the need of a better understanding of the biology of immune regulation mechanisms that hold promises for better treatments. This project aims to assess the role of CD8 regulatory T cells (Treg) and their downstream mechanisms in suppressing immune responses and therefore promoting graft and tumor survival or, on the contrary, in increasing immune responses thus favoring graft and tumor rejection. Together, the results of this project will provide new insights into human CD8+ Treg in human transplantation and in cancer. A better understanding of the tolerogenic potential of CD8 Treg in human could enable new strategies for inducing tolerance in transplantation and increased immunity in cancer.
Subproject 2: IL4I1 specific CD8 Treg in cancer (UMR 1102)
This subproject will address the hypothesis that IL4I1, an L-phenylalanine oxidase that plays an immunosuppressive role in cancer, is recognized by human CD8 Treg from cancer patients.
Specifically, the objectives of this study will be to:
– Define peptides binding to MHC class I molecules derived from the signal sequence of IL4I1 (IL4I1SS).
– Assess the repertoire of CD8 T cells specific for IL4I1SS in healthy donors.
– Assess the presence/prevalence of IL4I1SS specific CD8 Treg in cancer patients.
Task 1.1.3. Regulatory myeloid cells
The functional heterogeneity of myeloid cells depends, at least in part, on the local microenvironment and on their differentiation process. An inappropriate differentiation/polarization may have profound consequences on the immune response. Cancer and transplantation are two faces of immune-associated pathologies in which myeloid cells play a pivotal role. It is now largely documented that established tumors propagate conditions that favor their immune escape. An important mechanism is the acumulation of infiltrating suppressive myeloid cells such as myeloid-derived suppressor cells (MDSCs). At the opposite, in the field of transplantation, graft-specific tolerance needs to be induced. Fine manipulation of immune tolerance in the context of autoimmunity has been developed during the last two decades in animal models as well as in the clinic. These approaches aim at avoiding or minimizing toxic immunosuppressive treatments as well as preserving intact immune surveillance againt pathogens. One of the emerging strategies to efficiently modulate the immune system is the adoptive transfer of immunoregulatory cells.
MDSCs are a clinically applicable source of suppressive cells for their potential use in cell therapy. A translational view implicates the development of a clinically acceptable method for the production of these cells as well as a better understanding of their development, stability and function. The objective of this project is to combine the expertise of two research teams in cancer and transplantation immunology to decipher the mechanisms of generation/polarization of immunosuppressive myeloid cells and, ultimately, to propose innovative therapeutical strategies.
This work has led to a recent publication describing the immunosuppressive potential of MDSCs in a mouse model of allogenic transplantation :
Drujont L, Carretero-Iglesia L, Bouchet-Delbos L, Beriou G, Merieau E, Hill M, Delneste Y, Cuturi MC, Louvet C. Evaluation of the therapeutic potential of bone marrow-derived myeloid suppressor cell (MDSC) adoptive transfer in mouse models of autoimmunity and allograft rejection. PLoS One. 2014 Jun 13;9(6):e100013.
We are currently assessing improved conditions of MDSC generation/selection/stability as well as analyzing their migration ability into the graft. In parallel, we will study the role of two homolog genes in the biology of MDSCs through the analysis of a genetically deficient mouse that we have successfully generated. Indeed, a better understanding of MDSC-mediated suppression could lead to innovative therapeutical strategies to induce tolerance in the context of graft rejection but also to promote tumor regression.
Task 1.2 “HLA-E-restricted T cell populations and immunoregulatory activity of soluble HLA-E in transplantation and in melanoma”
IGO participants suggest a key role of the non-classical MHC molecule HLA-E in modulating immune responses in the context of cancer and transplantation. Hence, the general aim of this project is to re-evaluate the role of cell-bound and soluble HLA-E in both natural and acquired immune responses through interacting with invariable CD94/NKG2 or hypervariable T cell receptors.
One part of this project is to assess the frequency and the possible adverse impact of HLA-E-restricted immune response to CMV on renal graft outcome and to develop predictive test for patient monitoring and treatment. This project also aims to investigate the existence of anti-tumor HLA-E-restricted T cells in melanoma.
Moreover, another objective is to investigate the properties of the release of soluble HLA-E by vascular endothelial cells and tumor cells in the context of transplantation, viral (CMV) infection and cancer (melanoma), including the potential predictive and prognostic value of sHLA-E levels in the blood of patients.
Task 1.3 – Immunosuppressive factors
This work package will fund the generation or acquisition of reagents (antibodies, fusion molecules, cytokines, tetramers, etc…) that will be needed to evaluate the role of critical molecules or cellular functions by the other work packages of the Labex IGO. Priority will be given to the generation of new reagents. The acquisition of reagents will need to be justified and be necessary for the proposed projects in the most specific manner and not just as a general screening strategy; as an example, the acquisition of commercialized antibodies for extensive phenotyping will not be funded.