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CD14 + cells are able to differentiate into large and adherent cells if in contact with chronic lymphocytic leukemia (CLL) cells or healthy B lymphocytes. In CLL these cells, called CLL-nurse like cells (NLCs), express a very high amount of CD163 and CD68 and are able to rescue CLL cells through CCL4 production. Adherent cells derived from healthy donors, called HD-NLCs, express very little CD163 and CD68, do not produce CCL4 and are unable to rescue CCL cells. This study reveals that CLL-NLCs are the specific nurse cells in CLL, protecting CLL cells from death.

In the tumoral micro-environment (TME) of chronic lymphocytic leukemia (CLL), nurse-like cells (NLC) are tumor-associated macrophages which play a critical role in the survival and chemoresistance of tumoral cells. This pro-survival activity is known to involve soluble factors, but few data are available on the relative role of cells cross-talk. Here, we used a transcriptome-based approach to systematically investigate the expression of various receptor/ligand pairs at the surface of NLC/CLL cells. Their relative contribution to CLL survival was assessed both by fluorescent microscopy to identify cellular interactions and by the use of functional tests to measure the impact of uncoupling these pairs with blocking monoclonal antibodies. We found for the first time that lymphocyte function-associated antigen 3 (LFA-3), expressed in CLL at significantly higher levels than in healthy donor B-cells, and CD2 expressed on NLC, were both key for the specific pro-survival signals delivered by NLC. Moreover, we found that NLC/CLL interactions induced the shedding of soluble LFA-3. Importantly, in an exploratory cohort of 60 CLL patients receiving frontline immunochemotherapy, increased levels of soluble LFA-3 were found to correlate with shorter overall survival. Altogether, these data suggest that LFA-3/CD2 interactions promote the survival of CLL cells in the tumor microenvironment.

Phosphoantigens (PAgs) activate V9V2 T lymphocytes, inducing their potent and rapid response in vitro and in vivo. However, humans and nonhuman primates that receive repeated injections of PAgs progressively lose their V9V2 T cell response to them. To elucidate the molecular mechanisms of this in vivo desensitization, we analyzed the transcriptome of circulating V9V2 T cells from macaques injected with PAg. We showed that three PAg injections induced the activation of the PPAR pathway in V9V2 T cells. Thus, we analyzed the in vitro response of V9V2 T cells stimulated with a PPAR agonist. We demonstrated that in vitro PPAR pathway activation led to the inhibition of the BrHPP-induced activation and proliferation of human V9V2 T cells. Since the PPAR pathway is involved in the antigen-selective desensitization of human V9V2 T cells, the use of PPAR inhibitors could enhance cancer immunotherapy based on V9V2 T cells.

Chemoimmunotherapywith rituximab (R-chemo) or obinutuzumab (G-chemo) is standard of care for patients with previously untreated symptomatic or high-tumor-burden follicular lymphoma. Median progression-free survival (PFS) with R-chemo plus R maintenance exceeds 10 years, and G-chemo plus G maintenance improves PFS relative to the corresponding R-containing regimen. Despite these positive results, a sizable proportion of patients continue to progress during or shortly after initial treatment. While no single definition of early relapse has been established, progression of disease within 24 months of initial treatment (POD24) is now widely accepted as a critical adverse prognostic factor. Multiple studies have shown increased mortality risk in patients with POD24 versus those without POD24. Unfortunately, tools for the assessment of POD24 risk are suboptimal, and it is not currently possible in clinical practice to identify individual patients who are at increased risk for early relapse. Treatment strategies for patients with POD24 are not well defined. G-chemo regimens appear to reduce the risk of POD24 relative to R-chemo regimens, although the impact on survival outcomes remains unclear. Beyond standard therapy, autologous stem cell transplant and emerging treatment modalities, such as bispecific antibodies and chimeric antigen receptor T-cells, may have a role in future management. Until standard treatments are defined, mitigating the risk of early relapse with effective up-front treatment remains the priority.

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Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western Countries. This pathology is characterized by an accumulation of monoclonal, non-functional and mature CD5+ CD19+ leukemic B-cells (CLL cells) in lymph nodes, peripheral blood and bone marrow. Despite a high resistance to the in vivo apoptosis, CLL cells die spontaneously in vitro due to a lack in ex vivo conditions of sustaining cells and factors from their microenvironment such as stroma cells (Lagneaux L et al, Blood. 1998; 91:2387-2396), follicular dendritic cells or Nurse-Like-Cells (NLC) (Burger JA et al, Blood. 2000; 96:2655-2663). NLC are derived from CD14+ cells in contact with CLL cells in vitro (Tsukada N et al, Blood. 2002; 99:1030-1037) and were found in lymph nodes of CLL patients (Ysebaert L et al, Leuk Lymphoma. 2011; 52:1404-1406). NLC were shown to have a Tumour Associated Macrophages phenotype and gene expression profile. These cells have been first described to be essential for in vitro CLL cells survival partially through the production of soluble factors such as CXCL12 (Burger JA et al, Blood. 2000; 96:2655-2663), CCL3 and CCL4 (Burger JA et al, Blood. 2009; 113:3050-3058). Thus, other mechanisms are required for CLL cells survival. Indeed, we showed that contact of CLL cells with NLC was necessary to protect CLL cells from the in vitro apoptosis. We then investigated the mechanism of these interactions at a molecular level. We also determined their influences on the in vitro CLL cells survival and on the NLC-induced chemoresistance. We observed close and strong interactions evaluated by the measurement of trogocytosis from NLC to CLL cells. Trogocytosis is an active phenomenon with transfer of membrane fragments from one cell to another. We showed that NLC/CLL cells trogocytosis is dependant to actin polymerization and SRC phosphorylation. To find possible couples of molecules involved in this contact, we compared different transcriptomic data from NLC, monocyte, CLL cells and B lymphocytes. We highlighted potential couples of molecules and confirmed their expression on CLL cells and NLC by flow cytometry analysis. Finally, we obtained 3 couples probably implicated: Lymphocyte Function-Associated Antigen 3 (LFA-3)/CD2, Platelet/Endothelial Cell Adhesion Molecule 1 (PECAM1)/CD38 and Intercellular Adhesion Molecule 1 (ICAM-1)/LFA-1. Antibody blocking strategies revealed that LFA-3, which is up-regulated in CLL cells compared to healthy donors B lymphocytes, was necessary for the interaction between CLL cells/NLC when PECAM1, ICAM-1 and their co-partners were not essential (figure 1). Furthermore, this contact, through LFA-3, induced Akt phosphorylation but not ERK1/2 phosphorylation in CLL cells. Finally, we showed that LFA-3 and its receptor CD2 are necessary to the rescue of CLL cells by NLC (figure 2). To go further, we tested the chemoprotective effect of NLC on CLL cells. We showed that NLC slightly protect CLL cells against bendamustin but not against rituximab, dasatinib or ibrutinib. We hypothesized that the contact through LFA-3 could be involved in this chemoresistance. However, we did not observed a significant effect of the combination of bendamustin and LFA-3-blocking compared to bendamustin alone suggesting that this chemoprotection of CLL cells by NLC involved another pathway. Altogether, our results indicate that overexpression of LFA-3 by CLL cells and its critical implication in the interaction with NLC might be a new therapeutic target in CLL to disturb the interaction of CLL cells with their microenvironment. Figure 1: LFA-3 blocking but not ICAM-1 and PECAM1 decrease trogocytosis from NLC to CLL cells. a) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-LFA-3. b) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-ICAM-1. c) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-PECAM1. Figure 1:. LFA-3 blocking but not ICAM-1 and PECAM1 decrease trogocytosis from NLC to CLL cells. a) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-LFA-3. b) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-ICAM-1. c) Representative overlay of an experiment of trogocytosis from NLC to CLL cells treated or not by a blocking antibody anti-PECAM1. Figure 2: LFA-3 is critical for the survival of CLL cells in contact with NLC. Figure 2:. LFA-3 is critical for the survival of CLL cells in contact with NLC. Disclosures No relevant conflicts of interest to declare.