Theses supported by Labex IGO

B-cell differentiation leads to the generation of antibody-secreting cells (plasmablasts (PB) and plasma cells) through various developmental pathways. This thesis project aimed to study the influence of the cytokine microenvironment on B-cell fate and functional responses.
In a first study, we tried to define the molecular signature of PB through a transcriptional meta-analysis. As results, we underlined the robustness of the transcription factors IRF4 and IRF8 in the identification of PB. Based on these results, we followed activation and differentiation of various B-cell subsets discriminated on the expression of CD27 and IgD, in presence or in absence of IFNα or IL21.
We uncovered an IgD⁺ CD27^- B-cell population able to differentiate into PB, similarly to memory B cells. This subset distinguishes itself from the other IgD⁺ CD27^- naive B cells by the expression of CD45RB. This marker was recently used to identify memory B-cell subsets. However, contrary to memory B-cell subsets, the differentiation of IgD⁺ CD27^- CD45RB⁺ B cells into PB is inhibited in presence of IL21.Thus, these results suggest these cells are precursors of IgD⁺ CD27⁺ unswitched memory B cells (related to marginal zone B cells : MZB).
Patients with primary Sjögren’s syndrome had lower frequencies of peripheral blood unswitched memory B cells and their potential precursors. This suggests a defect of MZB pathway could be implicated in the pathogenesis of this autoimmune disease.

Melanoma is at the forefront of the development of innovative therapies. Recently the immune checkpoint inhibitors, in particular anti-PD-1 antibodies, have greatly improved patient survival. However, a significant fraction fails to respond to these therapies which justifies the development of new therapeutic strategies. Targeted radiotherapy (TRT) uses radiolabeled vectors such as antibodies (mAb) which target Ag expressed by the cancer cells. TRT delivers ionizing radiation in situ to eradicate the tumor with limited toxicity to the surrounding healthy tissues. We developed a RIV approach using a human anti-PD-L1 mAb radiolabeled with bismuth-213, an a-particle emitter, to investigate in a preclinical xenograft model of human melanoma, the therapeutic effects of targeted alpha radiotherapy (TAT). We demonstrated that treatment with the radiolabeled anti-PD-L1 mAb induced a significant delay in tumor growth leading to improved survival without major toxicity, in immunodeficient mice grafted with a human melanoma cell line expressing PD-L1. This preclinical study highlights the feasibility and therapeutic potency of TAT in the treatment of solid tumors such as melanoma. Based on these results and to improve anti-tumor efficacy, combination of TAT with an immunotherapy based on adoptive transfer of tumor specific modified T-cells has been initiated. ln parallel, the anti-PD-L1 mAb has been also used to develop a phenotypic imaging modality using positron emission tomography (Immune-PET) to analyze in vivo treatment efficacy according to PD-L1 expression on the tumor.

One of the challenges in transplantation is to identify early biomarkers predicting the occurrence of chronic rejection and to improve understanding of the causes leading to late loss of the graft in order to develop new therapeutic strategies. Of the factors associated with transplant failure and chronic rejection, memory T-cells are now considered to be one of the major barriers to successful transplantation, and our team's work has demonstrated an association between the frequency of CD8 Effector Memory (EM) re-expressing CD45RA (TEMRA) and the risk of graft loss. My Ph.D. work shows that the immunological functions of CD8 TEMRA are very similar to those of CD8 EM after joint stimulation of TCR and IL-15, including the ability to induce endothelial activation resulting in the creation of a pro-inflammatory environment and an adaptation of their metabolism to inflammatory stimuli. We also show that CD8 TEMRA in transplant patients have higher migratory properties than CD8 EM, including strong adhesion to activated endothelium and increased ability to transmigrate in response to chemokine CXCL12. We demonstrate that CXCL12 acts as a costimulatory molecule by activating the effector functions of TEMRA and CD8 EM. Finally, we have shown that IL-15 increases the migration of TEMRA and EM by promoting the generation of functional PSGL-1 by increasing the sialylation of this molecule.

Cytokines are powerful tools for regulating immune responses. IL-34 is a cytokine that bonds to CSF-1R, PTP and CD138 and is implicated in survival, proliferation and differentiation of monocytes/macrophages. We recently described the expression of IL-34 by induced FOXP3+CD4+and CD8+Tregs in healthy individuals in human and in a model of tolerance in transplantation in rat. Here we report that the absence of expression of IL-34 in Il34-/-rats impact the T CD8+compartment but not myeloid cells. Moreover, the deficiency lead to an unstable phenotype exacerbated under inflammatory conditions. In vivo, we revealed the inability of Il34-/-CD4 Tregs to protect from the wasting disease compared to Il34+/+ cells. We also showed the regulatory role of IL-34 to delay GVHD development and allograft rejection in immune humanized mice models. In addition, using two different transplanted human cohorts, we showed that the IL-34 serum level can’t be used as a prediction marker of kidney rejection. But a high expression of IL-34 by Tregs CD8+CD45RC low/-is a marker of good prognosis for a reduced GVHD incidence. Altogether, our data demonstrate the crucial role of IL-34 in the CD4+ Treg suppressive function as well as its therapeutical potential in transplantation.

Terminal B cell differentiation gives rise to antibody secreting plasma cells and memory B cells. It's a highly controlled process regulated by external inputs which guide B cell fate in the transition from naive B cells through to plasma cell. Here, we identify that IL-2 signaling triggers the plasma cell differentiation programme via the ERK transduction pathway instigating BACH2 repression. Results obtained using expression profiles of individual cells to bypass heterogeneous and asynchronous B cell responses confirm the crucial role of BACH2 regulating human B cell fate decision in a well-defined in vitro model of primary human naive B cell differentiation into plasma cell. My focus was then to study the temporal resolution of transcriptional dynamics following times series single-cell transcriptome analysis that should pinpoints key regulatory events that govern normal plasma cell differentiation in one hand and, on the other hand, the mechanisms that govern the differentiation arrest of plasma cell differentiation responsible for Waldenström disease, a rare incurable neoplasm. Altogether the data confirm the technological feasibility of the experimental strategies and should contribute to a better understanding of the complex mechanisms that control generation of plasma cell. This projec may ultimately help to achieve greater knowledge of the pathological process in Waldeström disease.

Glioblastoma multiform (GBM) is the most frequent and aggressive primary brain tumor in adults, with a dismal prognosis and few therapeutic advances made over the last decade. Cellular immunotherapies are currently being explored to eliminate highly invasive/resistant GBM cells likely involved in tumor relapse. Nonalloreactive human Vγ9Vδ2 T lymphocytes are able to kill a wide range of human tumor cells and display effector functions setting them up as promising cell candidates for efficient immunotherapies. The work described in this thesis aimed at characterizing primary GBM (pGBM) cultures, through phenotypic, metabolic and transcriptomic analyses and at investigating their recognition and killing by Vγ9Vδ2 T cells both in vitro and in vivo. We showed that immunodeficient NSG mice carrying orthotopic human pGBM xenografts recapitulate tumor development in patients. Furthermore, we demonstrated that Vγ9Vδ2 T cells can survive and patrol within the brain following adoptive transfer and successfully eliminate infiltrative pGBM cells orthotopic zoledronate treatment. In order to bypass this sensitization, several human Vγ9Vδ2 T cells have been isolated and amplified from PBMCs of healthy donors and screened for their ability to naturally and specifically react against pGBM cells. These results evidence that allogeneic Vγ9Vδ2 T lymphocytes efficiently and preferentially eliminate mesenchymal pGBM cells through !"-TCR- and NKG2D-dependent pathways. Taken together, these results provide an important proof-of-concept for optimized targeted immunotherapies of brain tumors and identify pathways that need to be explored before considering human V!9V"2 T cells in clinical approaches.

The human cytomegalovirus (HCMV) primary infection is usually asymptomatic but leads to latent infection of blood progenitor cells. Immunocompromised patients are at high risks of HCMV reactivation, which is associated with severe end organ diseases and increased mortality in transplant patients. Standard anti-viral treatments based on nucleotide analogues decreased the occurrence of HCMV reactivation and diseases, but induce side effects and drug-resistant viral strains. In this thesis, we introduced new anti-viral approaches based on the CRISPR/Cas9 gene editing tool. Two strategies are designed to target the UL122/123 gene of HCMV encoding the immediate early proteins, essential for lytic viral replication and reactivation from latency. We validated that the disruption of the UL122/123 gene by the CRISPR/Cas9 system to abrogate viral replication. The multiplex CRISPR/Cas9 system (three gRNA) was much more efficient than the singleplex approach targeting the same gene. Target gene expression, concomitant genome replication and virion release were significantly impaired by the multiplex strategy. A further anti-HCMV CRISPR/Cas9 system was developed specifically to target the HCMV genome during latency. Two gRNAs target the viral genome at three target sites: LUNA, essential for reactivation, and the two homolog TR regions. We verified this duplex strategy on the lytic replicating virus and detected mutations at the target site as well as the reduction of viral genome copy number. In conclusion, the anti-HCMV strategies based on two or three gRNAs efficiently blocked viral replication. This provides the basis for the development of an anti-HCMV CRISPR/Cas9 therapy

Chronic lymphocytic leukemia (CLL) is characterized by expansion of CD5+ B cells associated with disruption of immune responses, contributing to the immunodeficiency and the disease progression. Regulatory B (Breg) cells may control the anti-tumor responses favoring tumor escape. Intriguingly, CLL B cells share phenotypical characteristics with these cells.

The main focus of this project is to evaluate the regulatory function of CLL B cells, aiming to estimate their influence on the lack of anti-tumor responses mediated by T cells. In vitro models of co-cultures between T and B cells are used to appraise the regulatory capacity of CLL B cells on T cell proliferation. We determined a defective spontaneous regulatory function for CLL B cells. Two groups of patients have been identified following CpG-ODN stimulation. The first group presents defective regulatory B cell functions compared with control B cells. In the second group, no inhibitory activity is detected. TLR-9 gene expression analysis highlighted differential gene expression between controls and the two groups of CLL patients. Moreover,ours observations indicate that patients with low Breg activity have more aggressive disease. These results suggest alteration of the TLR-9 pathway in CLL B cells. To go further, it will be of interest to identify the molecular mechanisms da maging the TLR-9 pathway. These results would contribute to clarify the lack of anti -tumor immune response found in the CLL patients.

While having considerably modified melanoma-patients’ management, a large fraction of patients remains refractory to the immunotherapies targeting PD-1 axis. The understanding of immunological mechanisms involved in clinical efficacy or tumor resistance is crucial to further improve therapeutic efficiency. PD-1 has been largely documented as a major regulator of anti-tumor T cell responses, but it also identifies melanoma-reactive T lymphocytes. We have demonstrated that PD-1 positive melanoma-specific T cell clones exhibit higher functional avidity than T cell clones unable to induce PD-1 through epigenetic mechanisms. Furthermore, the in vitro PD-1 blockade during the generation of melanoma-specific T cells for adoptive cell transfer allows the production of T effectors with optimized functions. The immune follow-up of anti-PD-1 treated melanoma patients demonstrated substantial changes, in all patients, within the Melan-A specific T cell repertoire. We observed the emergence of high functional avidity clonotypes’ exhibiting PD-1 and TIGIT co-expression in responding patients.