Tumor stiffening reversion through collagen crosslinking inhibition improves T cell migration and anti-PD-1 treatment

Only a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing five preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase, was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy

Transdifferentiation of Human Circulating Monocytes Into Neuronal-Like Cells in 20 Days and Without Reprograming

Despite progress, our understanding of psychiatric and neurological illnesses remains poor, at least in part due to the inability to access neurons directly from patients. Currently, there are in vitro models available but significant work remains, including the search for a less invasive, inexpensive and rapid method to obtain neuronal-like cells with the capacity to deliver reproducible results. Here, we present a new protocol to transdifferentiate human circulating monocytes into neuronal-like cells in 20 days and without the need for viral insertion or reprograming. We have thoroughly characterized these monocyte-derived-neuronal-like cells (MDNCs) through various approaches including immunofluorescence (IF), flow cytometry, qRT-PCR, single cell mRNA sequencing, electrophysiology and pharmacological techniques. These MDNCs resembled human neurons early in development, expressed a variety of neuroprogenitor and neuronal genes as well as several neuroprogenitor and neuronal proteins and also presented electrical activity. In addition, when these neuronal-like cells were exposed to either dopamine or colchicine, they responded similarly to neurons by retracting their neuronal arborizations. More importantly, MDNCs exhibited reproducible differentiation rates, arborizations and expression of dopamine 1 receptors (DR1) on separate sequential samples from the same individual. Differentiation efficiency measured by cell morphology was on average 11.9 ± 1.4% (mean, SEM, n = 38,819 cells from 15 donors). To provide context and help researchers decide which in vitro model of neuronal development is best suited to address their scientific question,we compared our results with those of other in vitro models currently available and exposed advantages and disadvantages of each paradigm

Dendritic Cells Crosspresent Antigens from Live B16 Cells More Efficiently than from Apoptotic Cells and Protect from Melanoma in a Therapeutic Model

Dendritic cells (DC) are able to elicit anti-tumoral CD8+ T cell responses by cross-presenting exogenous antigens in association with major histocompatibility complex (MHC) class I molecules. Therefore they are crucial actors in cell-based cancer immunotherapy. Although apoptotic cells are usually considered to be the best source of antigens, live cells are also able to provide antigens for cross-presentation by DC. We have recently shown that prophylactic immunotherapy by DC after capture of antigens from live B16 melanoma cells induced strong CD8+ T-cell responses and protection against a lethal tumor challenge in vivo in C57Bl/6 mice. Here, we showed that DC cross-presenting antigens from live B16 cells can also inhibit melanoma lung dissemination in a therapeutic protocol in mice. DC were first incubated with live tumor cells for antigen uptake and processing, then purified and irradiated for safety prior to injection. This treatment induced stronger tumor-specific CD8+ T-cell responses than treatment by DC cross-presenting antigens from apoptotic cells. Apoptotic B16 cells induced more IL-10 secretion by DC than live B16 cells. They underwent strong native antigen degradation and led to the expression of fewer MHC class I/epitope complexes on the surface of DC than live cells. Therefore, the possibility to use live cells as sources of tumor antigens must be taken into account to improve the efficiency of cancer immunotherapy

Local IFNα enhances the anti-tumoral efficacy of systemic anti-PD1 to prevent tumor relapse

International audienceBackground Tumor relapse constitutes a major challenge for anti-tumoral treatments, including immunotherapies. Indeed, most cancer-related deaths occur during the tumor relapse phase. Methods We designed a mouse model of tumor relapse in which mice transplanted with E7 + TC1 tumor cells received a single therapeutic vaccination of STxB-E7+IFNα. Unlike the complete regression observed after two vaccinations, such a treatment induced a transient shrinkage of the tumor mass, followed by a rapid tumor outgrowth. To prevent this relapse, we tested the efficacy of a local administration of IFNα together with a systemic therapy with anti-PD1 Ab. The immune response was analyzed during both the tumor regression and relapse phases. Results We show that, during the regression phase, tumors of mice treated with a single vaccination of STxB-E7 + IFNα harbor fewer activated CD8 T cells and monocytes than tumors doomed to fully regress after two vaccinations. In contrast, the systemic injection of an anti-PD1 Ab combined with the peri-tumoral injection of IFNα in this time frame promotes infiltration of activated CD8 T cells and myeloid cells, which, together, exert a high cytotoxicity in vitro against TC1 cells. Moreover, the IFNα and anti-PD1 Ab combination was found to be more efficient than IFNα or anti-PD1 used alone in preventing tumor relapse and was better able to prolong mice survival. Conclusions Together, these results indicate that the local increase of IFNα in combination with an anti-PD1 therapy is an effective way to promote efficient and durable innate and adaptive immune responses preventing tumor relapse

Nurselike cells sequester B cells in disorganized lymph nodes in chronic lymphocytic leukemia via alternative production of CCL21

International audienceAbstract Tumor microenvironment exerts a critical role in sustaining homing, retention, and survival of chronic lymphocytic leukemia (CLL) cells in secondary lymphoid organs. Such conditions foster immune surveillance escape and resistance to therapies. The physiological microenvironment is rendered tumor permissive by an interplay of chemokines, chemokine receptors, and adhesion molecules as well as by direct interactions between malignant lymphocytes and stromal cells, T cells, and specialized macrophages referred to as nurselike cells (NLCs). To characterize this complex interplay, we investigated the altered architecture on CLL lymph nodes biopsies and observed a dramatic loss of tissue subcompartments and stromal cell networks as compared with nonmalignant lymph nodes. A supplemental high density of CD68+ cells expressing the homeostatic chemokine CCL21 was randomly distributed. Using an imaging flow cytometry approach, CCL21 mRNA and the corresponding protein were observed in single CD68+ NLCs differentiated in vitro from CLL peripheral blood mononuclear cells. The chemokine was sequestered at the NLC membrane, helping capture of CCR7-high-expressing CLL B cells. Inhibiting the CCL21/CCR7 interaction by blocking antibodies or using therapeutic ibrutinib altered the adhesion of leukemic cells. Our results indicate NLCs as providers of an alternative source of CCL21, taking over the physiological task of follicular reticular cells, whose network is deeply altered in CLL lymph nodes. By retaining malignant B cells, CCL21 provides a protective environment for their niching and survival, thus allowing tumor evasion and resistance to treatment. These findings argue for a specific targeting or reeducation of NLCs as a new immunotherapy strategy for this disease

Exploring the immunomodulatory role of virtual memory CD8+ T cells: Role of IFN gamma in tumor growth control

International audienceVirtual memory CD8 + T cells (T VM ) have been described as cells with a memory-like phenotype but without previous antigen (Ag) exposure. T VM cells have the ability to respond better to innate stimuli rather than by TCR engagement, producing large amounts of interferon gamma (IFNγ) after stimulation with interleukin (IL)-12 plus IL-18. As a result of the phenotypic similarity, T VM cells have been erroneously included in the central memory T cell subset for many years. However, they can now be discriminated via the CD49d receptor, which is up-regulated only on conventional memory T cells (T MEM ) and effector T cells (T EFF ) after specific cognate Ag recognition by a TCR. In this work we show that systemic expression of IL-12 plus IL-18 induced an alteration in the normal T VM vs T MEM /T EFF distribution in secondary lymphoid organs and a preferential enrichment of T VM cells in the melanoma (B16) and the pancreatic ductal adenocarcinoma (KPC) tumor models. Using our KPC bearing OT-I mouse model, we observed a significant increase in CD8 + T cell infiltrating the tumor islets after IL-12+IL-18 stimulation with a lower average speed when compared to those from control mice. This finding indicates a stronger interaction of T cells with tumor cells after cytokine stimulation. These results correlate with a significant reduction in tumor size in both tumor models in IL-12+IL-18-treated OT-I mice compared to control OT-I mice. Interestingly, the absence of IFNγ completely abolished the high antitumor capacity induced by IL-12+IL-18 expression, indicating an important role for these cytokines in early tumor growth control. Thus, our studies provide significant new information that indicates an important role of T VM cells in the immune response against cancer

Blockade of β-Adrenergic Receptors Improves CD8 + T-cell Priming and Cancer Vaccine Efficacy

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IL-23 and IL-12p70 production by monocytes and dendritic cells in primary HIV-1 infection.

International audienceIL-12 enhances protective responses against HIV replication. Its production after in vitro stimulation is defective in chronic HIV infection, but higher responses can be found. IL-23 shares the p40 chain and some properties with IL-12 and enhances Th17 responses, but its role in HIV infection is unknown. The production of IL-12 and IL-23 and the respective contribution of monocytes and myeloid conventional DC (cDCs) during primary HIV infection were determined. Sixteen patients included in the French PRIMO-ANRS Cohort without antiretroviral treatment were followed prospectively and compared with uninfected donors. Intracellular p40 expression by monocytes and cDCs, analyzed by flow cytometry, was transiently increased in monocytes and cDCs in response to LPS and more consistently, in monocytes in response to LPS + IFN-gamma. IL-23 production, measured by ELISA after PBMC stimulation, was induced by LPS in strong correlation with VLs. IL-12p70 production required the addition of IFN-gamma and was transiently increased in patients compared with controls in correlation with VLs, whereas IL-23 was increased sustainedly. Therefore, an apparent domination of IL-23 over IL-12 responses occurred throughout primary HIV infection, and a potential restoration of IL-12 responses might be expected from a treatment mimicking activated T cell signals

TGFβ blocks IFNα/β release and tumor rejection in spontaneous mammary tumors

International audienceType I interferons (IFN) are being rediscovered as potent anti-tumoral agents. Activation of the STimulator of INterferon Genes (STING) by DMXAA (5,6-dimethylxanthenone-4-acetic acid) can induce strong production of IFNα/β and rejection of transplanted primary tumors. In the present study, we address whether targeting STING with DMXAA also leads to the regression of spontaneous MMTV-PyMT mammary tumors. We show that these tumors are refractory to DMXAA-induced regression. This is due to a blockade in the phosphorylation of IRF3 and the ensuing IFNα/β production. Mechanistically, we identify TGFβ, which is abundant in spontaneous tumors, as a key molecule limiting this IFN-induced tumor regression by DMXAA. Finally, blocking TGFβ restores the production of IFNα by activated MHCII + tumorassociated macrophages, and enables tumor regression induced by STING activation. On the basis of these findings, we propose that type I IFN-dependent cancer therapies could be greatly improved by combinations including the blockade of TGFβ

DC cytokine profile after culture with live or apoptotic B16 cells.

<p>DC were cultured alone (DC) or with live (zVAD treated) or apoptotic (γ-irradiated) B16 cells for 24 h and stimulated or not with LPS and IFNγ. As controls, B16 zVAD and B16γ cells were also cultured alone. DC were then assessed for their ability to produce IL-12p70 (A) or IL-10 (B) by ELISA. In the upper panels, data from one out of three independent experiments are shown. In the bottom panels, for LPS and IFNγ stimulation, relative IL-12p70 and IL-10 productions are expressed as a percentage of the cytokine production obtained for DC alone. Mean percentage values±SEM are shown. The significance of differences between series of results was assessed using a paired t test (n = 3, 3 independent experiments).</p