TCR-independent CD137 (4-1BB) signaling promotes CD8+-exhausted T cell proliferation and terminal differentiation.

CD137 (4-1BB)-activating receptor represents a promising cancer immunotherapeutic target. Yet, the cellular program driven by CD137 and its role in cancer immune surveillance remain unresolved. Using T cell-specific deletion and agonist antibodies, we found that CD137 modulates tumor infiltration of CD8+-exhausted T (Tex) cells expressing PD1, Lag-3, and Tim-3 inhibitory receptors. T cell-intrinsic, TCR-independent CD137 signaling stimulated the proliferation and the terminal differentiation of Tex precursor cells through a mechanism involving the RelA and cRel canonical NF-κB subunits and Tox-dependent chromatin remodeling. While Tex cell accumulation induced by prophylactic CD137 agonists favored tumor growth, anti-PD1 efficacy was improved with subsequent CD137 stimulation in pre-clinical mouse models. Better understanding of T cell exhaustion has crucial implications for the treatment of cancer and infectious diseases. Our results identify CD137 as a critical regulator of Tex cell expansion and differentiation that holds potential for broad therapeutic applications.

Eomes-Dependent Loss of the Co-activating Receptor CD226 Restrains CD8+ T Cell Anti-tumor Functions and Limits the Efficacy of Cancer Immunotherapy.

CD8+ T cells within the tumor microenvironment (TME) are exposed to various signals that ultimately determine functional outcomes. Here, we examined the role of the co-activating receptor CD226 (DNAM-1) in CD8+ T cell function. The absence of CD226 expression identified a subset of dysfunctional CD8+ T cells present in peripheral blood of healthy individuals. These cells exhibited reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program upon stimulation. CD226neg CD8+ T cells accumulated in human and mouse tumors of diverse origin through an antigen-specific mechanism involving the transcriptional regulator Eomesodermin (Eomes). Despite similar expression of co-inhibitory receptors, CD8+ tumor-infiltrating lymphocyte failed to respond to anti-PD-1 in the absence of CD226. Immune checkpoint blockade efficacy was hampered in Cd226-/- mice. Anti-CD137 (4-1BB) agonists also stimulated Eomes-dependent CD226 loss that limited the anti-tumor efficacy of this treatment. Thus, CD226 loss restrains CD8+ T cell function and limits the efficacy of cancer immunotherapy.

NK cells with adhesion defects and reduced cytotoxic functions are associated with a poor prognosis in multiple myeloma.

ABSTRACT: The promising results obtained with immunotherapeutic approaches for multiple myeloma (MM) call for a better stratification of patients based on immune components. The most pressing being cytotoxic lymphocytes such as natural killer (NK) cells that are mandatory for MM surveillance and therapy. Here, we performed a single-cell RNA sequencing analysis of NK cells from 10 patients with MM and 10 age/sex-matched healthy donors that revealed important transcriptomic changes in the NK cell landscape affecting both the bone marrow (BM) and peripheral blood compartment. The frequency of mature cytotoxic CD56dim NK cell subsets was reduced in patients with MM at the advantage of late-stage NK cell subsets expressing NF-κB and interferon-I inflammatory signatures. These NK cell subsets accumulating in patients with MM were characterized by low CD16 and CD226 expression and poor cytotoxic functions. MM CD16/CD226Lo NK cells also had adhesion defects with reduced lymphocyte function-associated antigen 1 (LFA-1) integrin activation and actin polymerization that may account for their limited effector functions in vitro. Finally, analysis of BM-infiltrating NK cells in a retrospective cohort of 177 patients with MM from the Intergroupe Francophone du Myélome (IFM) 2009 trial demonstrated that a high frequency of NK cells and their low CD16 and CD226 expression were associated with a shorter overall survival. Thus, CD16/CD226Lo NK cells with reduced effector functions accumulate along MM development and negatively affect patients' clinical outcomes. Given the growing interest in harnessing NK cells to treat myeloma, this improved knowledge around MM-associated NK cell dysfunction will stimulate the development of more efficient immunotherapeutic drugs against MM.

Imprinting of Mesenchymal Stromal Cell Transcriptome Persists even after Treatment in Patients with Multiple Myeloma.

INTRODUCTION: Multiple myeloma (MM) is a B-cell neoplasm characterized by clonal expansion of malignant plasma cells (MM cells) in the bone-marrow (BM) compartment. BM mesenchymal stromal cells (MSC) from newly diagnosed MM patients were shown to be involved in MM pathogenesis and chemoresistance. The patients displayed a distinct transcriptome and were functionally different from healthy donors' (HD) MSC. Our aim was to determine whether MM-MSC also contributed to relapse. METHODS: We obtained and characterized patients' MSC samples at diagnosis, two years after intensive treatment, without relapse and at relapse. RESULTS: Transcriptomic analysis revealed differences in gene expression between HD and MM-MSC, whatever the stage of the disease. An easier differentiation towards adipogenesis at the expense of osteoblatogeneis was observed, even in patients displaying a complete response to treatment. Although their transcriptome was similar, we found that MSC from relapsed patients had an increased immunosuppressive ability, compared to those from patients in remission. CONCLUSION: We demonstrated that imprinting of MSC transcriptome demonstrated at diagnosis of MM, persisted even after the apparent disappearance of MM cells induced by treatment, suggesting the maintenance of a local context favorable to relapse.

Toll-like receptor 4 selective inhibition in medullar microenvironment alters multiple myeloma cell growth.

Bone marrow (BM) mesenchymal stromal cells (MSCs) are abnormal in multiple myeloma (MM) and play a critical role by promoting growth, survival, and drug resistance of MM cells. We observed higher Toll-like receptor 4 (TLR4) gene expression in MM MSCs than in MSCs from healthy donors. At the clinical level, we highlighted that TLR4 expression in MM MSCs evolves in parallel with the disease stage. Thus, we reasoned that the TLR4 axis is pivotal in MM by increasing the protumor activity of MSCs. Challenging primary MSCs with TLR4 agonists increased the expression of CD54 and interleukin-6 (IL-6), 2 factors directly implicated in MM MSC-MM cell crosstalk. Then, we evaluated the therapeutic efficacy of a TLR4 antagonist combined or not with conventional treatment in vitro with MSC-MM cell coculture and in vivo with the Vk*MYC mouse model. Selective inhibition of TLR4 specifically reduced the MM MSC ability to support the growth of MM cells in an IL-6-dependent manner and delayed the development of MM in the Vk*MYC mouse model by altering the early disease phase in vivo. For the first time, we demonstrate that specific targeting of the pathological BM microenvironment via TLR4 signaling could be an innovative approach to alter MM pathology development.

Tumor cells educate mesenchymal stromal cells to release chemoprotective and immunomodulatory factors.

Factors released by surrounding cells such as cancer-associated mesenchymal stromal cells (CA-MSCs) are involved in tumor progression and chemoresistance. In this study, we characterize the mechanisms by which naïve mesenchymal stromal cells (MSCs) can acquire a CA-MSCs phenotype. Ovarian tumor cells trigger the transformation of MSCs to CA-MSCs by expressing pro-tumoral genes implicated in the chemoresistance of cancer cells, resulting in the secretion of high levels of CXC chemokine receptors 1 and 2 (CXCR1/2) ligands such as chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, and interleukin 8 (IL-8). CXCR1/2 ligands can also inhibit the immune response against ovarian tumor cells. Indeed, through their released factors, CA-MSCs promote the differentiation of monocytes towards M2 macrophages, which favors tumor progression. When CXCR1/2 receptors are inhibited, these CA-MSC-activated macrophages lose their M2 properties and acquire an anti-tumoral phenotype. Both ex vivo and in vivo, we used a CXCR1/2 inhibitor to sensitize ovarian tumor cells to carboplatin and circumvent the pro-tumoral effects of CA-MSCs. Since high concentrations of CXCR1/2 ligands in patients' blood are associated with chemoresistance, CXCR1/2 inhibition could be a potential therapeutic strategy to revert carboplatin resistance.

Functional Comparison between Healthy and Multiple Myeloma Adipose Stromal Cells.

Multiple myeloma (MM) is an incurable B cell neoplasia characterized by the accumulation of tumor plasma cells within the bone marrow (BM). As a consequence, bone osteolytic lesions develop in 80% of patients and remain even after complete disease remission. We and others had demonstrated that BM-derived mesenchymal stromal cells (MSCs) are abnormal in MM and thus cannot be used for autologous treatment to repair bone damage. Adipose stromal cells (ASCs) represent an interesting alternative to MSCs for cellular therapy. Thus, in this study, we wondered whether they could be a good candidate in repairing MM bone lesions. For the first time, we present a transcriptomic, phenotypic, and functional comparison of ASCs from MM patients and healthy donors (HDs) relying on their autologous MSC counterparts. In contrast to MM MSCs, MM ASCs did not exhibit major abnormalities. However, the changes observed in MM ASCs and the supportive property of ASCs on MM cells question their putative and safety uses at an autologous or allogenic level.

The translocation t(4;14) can be present only in minor subclones in multiple myeloma.

PURPOSE: Although the translocation t(4;14) is supposed to be a primary event in multiple myeloma, we have been surprised to observe that in large relapse series of patients, the t(4;14) can be observed only in subpopulations of plasma cells, in contrast to what is seen at diagnosis. This observation raised the question of possible subclones harboring the translocation that would be observable only at the time of relapse. EXPERIMENTAL DESIGN: To address this issue, we analyzed by FISH a cohort of 306 patients for whom we had at least two samples obtained at different disease phases. RESULTS: We observed a "gain" of the t(4;14) in 14 patients, and conversely, a "loss" of the translocation in 11 patients. Two hypotheses were raised: either an acquisition of the translocation during evolution or the existence of small t(4;14)-positive subclones at the time of diagnosis. To address this question, we had the opportunity to analyze two patients at the time of diagnosis by RT-PCR (reverse transcription-polymerase chain reaction) to look for the chimeric Eµ-MMSET transcript, and one patient positive at diagnosis, but negative at relapse. The samples were positive, supporting the second hypothesis. Furthermore, the IGH sequences of two patients who "lose" the t(4;14) were identical at diagnosis and relapse, confirming the existence of a common ancestral clone. CONCLUSION: Thus, the conclusion of this study is that the t(4;14) is not a primary event in multiple myeloma and that it can be present in silent subclones at diagnosis, but also at relapse.