Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo−/Colo+) and colon (CX−/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo−/CX− derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis

Stress or heat shock proteins (HSPs) are the most conserved proteins present in both prokaryotes and eukaryotes. Their expression is induced in response to a wide variety of physiological and environmental insults. These proteins play an essential role as molecular chaperones by assisting the correct folding of nascent and sress-accumulated misfolded proteins, and preventing their aggregation. HSPs have a dual function depending on their intracellular or extracellular location. Intracellular HSPs have a protective function. They allow the cells to survive lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. Several HSPs have also been demonstrated to directly interact with various components of the tightly regulated programmed cell death machinery, upstream and downstream of the mitochondrial events. On the other hand, extracellular located or membrane-bound HSPs mediate immunological functions. They can elicit an immune response modulated either by the adaptive or innate immune system. This review will focus on HSP27, HSP70, and HSP90. We will discuss the dual role of these HSPs, protective vs. immunogenic properties, making a special emphasis in their utility as targets in cancer therapy.

Exosomes are nanometer-sized vesicles released from cells into the blood, where they can transmit signals throughout the body. Shown to act on the heart, exosomes' composition and the signaling pathways they activate have not been explored. We hypothesized that endogenous plasma exosomes can communicate signals to the heart and provide protection against ischemia and reperfusion injury. This study sought to isolate and characterize exosomes from rats and healthy volunteers, evaluate their cardioprotective actions, and identify the molecular mechanisms involved. The exosome-rich fraction was isolated from the blood of adult rats and human volunteers and was analyzed by protein marker expression, transmission electron microscopy, and nanoparticle tracking analysis. This was then used in ex vivo, in vivo, and in vitro settings of ischemia-reperfusion, with the protective signaling pathways activated on cardiomyocytes identified using Western blot analyses and chemical inhibitors. Exosomes exhibited the expected size and expressed marker proteins CD63, CD81, and heat shock protein (HSP) 70. The exosome-rich fraction was powerfully cardioprotective in all tested models of cardiac ischemia-reperfusion injury. We identified a pro-survival signaling pathway activated in cardiomyocytes involving toll-like receptor (TLR) 4 and various kinases, leading to activation of the cardioprotective HSP27. Cardioprotection was prevented by a neutralizing antibody against a conserved HSP70 epitope expressed on the exosome surface and by blocking TLR4 in cardiomyocytes, identifying the HSP70/TLR4 communication axis as a critical component in exosome-mediated cardioprotection. Exosomes deliver endogenous protective signals to the myocardium by a pathway involving TLR4 and classic cardioprotective HSPs.