The balance between effector CD4+ T cells secreting IL-17 (Th17) and regulatory T cells (Treg) plays an important role in autoimmune disorders that include rheumatoid arthritis (RA) and Crohn's ...disease. Tumor necrosis factor (TNF)-α is a key pro-inflammatory cytokine that contributes to disease pathogenesis. We investigated the interplay between CD45RA+ Treg and TNF-α in the regulation of human Th17 differentiation. We found that CD45RA+ Treg promoted while TNF-α inhibited naive CD4+ T-cell differentiation into IL-17 and CCL20 co-expressing Th17 cells without influencing their IL-22 release. Unexpectedly, CD45RA+ Treg depletion abrogated TNF-α suppressive function. Finally, dendritic cell-derived TNF-α suppressed the development of IL-17+CCL20+ expressing Th17 cells. In conclusion, CD45RA+ Treg positively governs human Th17 development, which is impaired by TNF-α. We propose that TNF-α may represent a negative feedback mechanism to control IL-17/CCL20- but not IL-22-associated autoimmune pathologies.
Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia in Western countries, is a very heterogeneous disease characterized by a peripheral accumulation of abnormal CD5+ B lymphocytes ...in the immune system. Despite new therapeutic developments, there remains an unmet medical need for CLL. Here, we demonstrate that the use of N-methylated thrombospondin-1 (TSP-1)–derived peptides is an efficient way to kill the malignant CLL cells, including those from high-risk individuals with poor clinical prognosis, del11q, del17p, 2p gain, or complex karyotype. PKT16, our hit N-methylated peptide, triggers the elimination of the leukemic cells, sparing the nontumor cells, including the hematopoietic precursors, and reduces the in vivo tumor burden of a CLL-xenograft mice model. A complementary analysis underscores the improved cytotoxic efficiency of PKT16 compared with the previously described TSP-1–derived probes, such as PKHB1. PKT16 elicits an original caspase-independent programmed necrotic mode of cell death, different from necroptosis or ferroptosis, implicating an intracellular Ca2+ deregulation that provokes mitochondrial damage, cell cycle arrest, and the specific death of the malignant CLL cells. The activation of the Gαi proteins and the subsequent drop of cyclic adenosine monophosphate levels and protein kinase A activity regulate this cytotoxic cascade. Remarkably, PKT16 induces the molecular hallmarks of immunogenic cell death, as defined by the calreticulin plasma membrane exposure and the release of adenosine triphosphate and high-mobility group box 1 protein from the dying CLL cells. Thus, PKT16 appears to be able to stimulate an anticancer in vivo immune response. Collectively, our results pave the way toward the development of an efficient strategy against CLL.
•N-methylated thrombospondin-1 mimetic peptides selectively kill the leukemic CD5+ B cells, sparing mature and precursor nontumor cells.•CD47 peptide triggering eliminates drug-resistant CLL cells by inducing an original form of cell death regulated by G proteins.
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How do effector CD4 T cells escape cell death during the contraction of the immune response (IR) remain largely unknown. CD47, through interactions with thrombospondin-1 (TSP-1) and SIRP-α, is ...implicated in cell death and phagocytosis of malignant cells. Here, we reported a reduction in SIRP-α-Fc binding to effector memory T cells (T(EM)) and in vitro TCR-activated human CD4 T cells that was linked to TSP-1/CD47-induced cell death. The reduced SIRP-α-Fc binding (CD47(low) status) was not detected when CD4 T cells were stained with two anti-CD47 mAbs, which recognize distinct epitopes. In contrast, increased SIRP-α-Fc binding (CD47(high) status) marked central memory T cells (T(CM)) as well as activated CD4 T cells exposed to IL-2, and correlated with resistance to TSP-1/CD47-mediated killing. Auto-aggressive CD4 effectors, which accumulated in lymph nodes and at mucosal sites of patients with Crohn's disease, displayed a CD47(high) status despite a high level of TSP-1 release in colonic tissues. In mice, CD47 (CD47(low) status) was required on antigen (Ag)-specific CD4 effectors for the contraction of the IR in vivo, as significantly lower numbers of Ag-specific CD47(+/+)CD4 T cells were recovered when compared to Ag-specific CD47(-/-) CD4 T cells. In conclusion, we demonstrate that a transient change in the status of CD47, i.e. from CD47(high) to CD47(low), on CD4 effectors regulates the decision-making process that leads to CD47-mediated cell death and contraction of the IR while maintenance of a CD47(high) status on tissue-destructive CD4 effectors prevents the resolution of the inflammatory response.
Dying cells, apoptotic or necrotic, are swiftly eliminated by professional phagocytes. We previously reported that CD47 engagement by CD47 mAb or thrombospondin induced caspase-independent cell death ...of chronic lymphocytic leukemic B cells (B-CLL). Here we show that human immature dendritic cells (iDCs) phagocytosed the CD47 mAb–killed leukemic cells in the absence of caspases 3, 7, 8, and 9 activation in the malignant lymphocytes. Yet the dead cells displayed the cytoplasmic features of apoptosis, including cell shrinkage, phosphatidylserine exposure, and decreased mitochondrial transmembrane potential (ΔΨm). CD47 mAb–induced cell death also occurred in normal resting and activated lymphocytes, with B-CLL cells demonstrating the highest susceptibility. Importantly, iDCs and CD34+ progenitors were resistant. Structure-function studies in cell lines transfected with various CD47 chimeras demonstrated that killing exclusively required the extracellular and transmembrane domains of the CD47 molecule. Cytochalasin D, an inhibitor of actin polymerization, and antimycin A, an inhibitor of mitochondrial electron transfer, completely suppressed CD47-induced phosphatidylserine exposure. Interestingly, CD47 ligation failed to induce cell death in mononuclear cells isolated from Wiskott-Aldrich syndrome (WAS) patients, suggesting the involvement of Cdc42/WAS protein (WASP) signaling pathway. We propose that CD47-induced caspase-independent cell death be mediated by cytoskeleton reorganization. This form of cell death may be relevant to maintenance of homeostasis and as such might be explored for the development of future therapeutic approaches in lymphoid malignancies.
Type 4 phosphodiesterase (PDE4) inhibitors reportedly induce apoptosis in chronic lymphocytic leukemia (CLL) cells. Following clinical improvement of one previously untreated CLL patient with ...sildenafil therapy, we evaluated the in vitro induction of apoptosis in CLL cells by 4 PDE5/6 inhibitors, including sildenafil, vardenafil, zaprinast, and methoxyquinazoline (MQZ). After 24 hours of culture, the various PDE inhibitors differed in their ability to induce apoptosis, with zaprinast displaying no killing effect. Normal B cells isolated from control donors were totally resistant to PDE-induced apoptosis. Vardenafil was 3 and 30 times more potent an inducer of apoptosis than sildenafil and MQZ, respectively. Both vardenafil and sildenafil failed to elevate adenosine 3′5′ cyclic monophosphate (cAMP) levels, largely excluding an inhibitory effect on cAMP-PDE3, -PDE4, and -PDE7. Vardenafil- or sildenafil-treated B-CLL cells displayed up to 30% intracellular active caspase 3. Drug-induced apoptosis was inhibited by the caspase inhibitor z-VAD.fmk, prevented by interleukin-4 (IL-4), and significantly reduced by stromal-derived factor1-α (SDF-1α). We conclude that vardenafil and sildenafil induce caspase-dependent apoptosis of B-CLL cells in vitro and thus might be considered in the treatment of CLL patients. However, further in vivo investigations should be warranted.
Monocyte maturation program into macrophages (MΦ) is well defined in murine gut under homeostatic or inflammatory conditions. Obviously, in vivo tracking of monocytes in inflamed tissues remains ...difficult in humans. Furthermore, in vitro models fall short in generating the surrogates of transient extravasated tissue inflammatory monocytes. Here, we aimed to unravel environmental cues that replicated the human monocyte “waterfall” process in vitro by first, generating tissue‐like inflammatory monocytes, which were then shifted toward MΦ. Purified CD14+CD16− monocytes, cultured with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), IFN‐γ and IL23, differentiated into CD14+CD163− cells that displayed a monocyte‐like morphology. In vitro generated inflammatory CD14+CD163− (inflammatory monocyte‐like cells) cells promoted IL‐1β‐dependent memory Th17 and Th17/Th1 responses, like the CD14+CD163− mo‐like cells that accumulate in inflamed colon of Crohn's disease patients. Next, in vitro generated inflammatory monocyte‐like cells converted to functional CD163+ MΦ following exposure to TGF‐β and IL10. Gene set enrichment analysis further revealed a shared molecular signature between converted CD163+ MΦ and MΦ detected in various inflamed nonlymphoid and lymphoid diseased tissues. Our findings propose a two‐step in vitro culture that recapitulates human monocyte maturation cascade in inflamed tissue. Manipulation of this process might open therapeutic avenues for chronic inflammatory disorders.
We replicated in vitro human monocyte “waterfall” process that might occur in intestinal tissue. Monocytes were differentiated into pro‐inflammatory monocyte‐like cells that were shifted toward macrophages. The latter shared transcriptional profile with tissue macrophages endowed with repair function. Understanding this process sheds light on therapeutic avenues for chronic inflammatory disorders.
CD47 is a ubiquitously expressed molecule which has been attributed a role in many cellular processes. Its role in preventing cellular phagocytosis has defined CD47 as an obligatory self-molecule ...providing a ‘don't-eat-me-signal’. Additionally, CD47–CD172a interactions are important for cellular trafficking. Yet, the contribution of CD47 to T cell stimulation remains controversial, acting sometimes as a co-stimulator and sometimes as an inhibitor of TCR signalling or peripheral T cell responses. Most of the experiments leading to this controversy have been carried in in vitro systems. Moreover, the role of CD47 on thymocyte differentiation, which precisely relies on TCR signal strength, has not been evaluated. Here, we examine the in vivo role of CD47 in T cell differentiation using CD47-deficient mice. We find that, in the absence of CD47, thymocyte positive and negative selection processes are not altered. Indeed, our data demonstrate that the absence of CD47 does not influence the strength of TCR signalling in thymocytes. Furthermore, in agreement with a role for CD47–CD172a interactions in CD172a+ dendritic cell migration, we report a reduced proportion of thymic dendritic cells expressing CD172a in CD47-deficient mice. As the total proportion of dendritic cells is maintained, this creates an imbalance in the proportion of CD172a+ and CD172alow dendritic cells in the thymus. Together, these data indicate that the altered proportion of thymic dendritic cell subsets does not have a primordial influence on thymic selection processes.
1 INSERM, U872, Mort Cellulaire Programmée et Physiopathologie des Cellules Tumorales, Equipe 19, Centre de Recherche des Cordeliers, Paris, France
2 Université Pierre et Marie Curie-Paris 6, UMRS ...872, Paris, France
3 Université Paris Descartes, UMRS 872, Paris, France
4 Institut Pasteur, Paris, France
5 AP-HP, Groupe Hospitalier Pitie-Salpêtrière, Service d'Hématologie Biologique, Paris, France
6 Centre de Recherche du CHUM, Hôpital Notre-Dame, Laboratoire dImmunorégulation, Montréal, Canada
7 Cell Neurobiology Unit, Departament de Medicina Experimental, Universitat de Lleida, IRBLLEIDA, Lleida, Spain
Correspondence: Santos A. Susin. Centre de Recherche des Cordeliers. 15, rue de lEcole de Médecine, 75006, Paris, France. E-mail: santos.susin{at}crc.jussieu.fr
Background: Programmed cell death has been traditionally related with caspase activation. However, it is now accepted that caspase-independent forms of programmed cell death also regulate cell death. In chronic lymphocytic leukemia, CD47 ligation induces one of these alternative forms of cell death: type III programmed cell death. This poorly understood process is characterized by cytoplasmic hallmarks, such as mitochondrial damage. To gain insights into the molecular pathways regulating type III programmed cell death in chronic lymphocytic leukemia, we performed extensive biochemical and cell biology assessments.
Design and Methods: After CD47 triggering, purified B-cells from 20 patients with chronic lymphocytic leukemia were studied by flow cytometry, immunofluorescence and three-dimensional imaging, immunoblotting, electron microscopy, and fibrillar/globular actin measurements. Finally, we subjected CD47-treated chronic lymphocytic leukemia cells to a phagocytosis assay.
Results: We first confirmed that induction of type III programmed cell death is an efficient means of triggering cell death in chronic lymphocytic leukemia. Further, we demonstrated that the signaling events induced by CD47 ligation provoked a reduction in cell size. This alteration is related to F-actin disruption, as the two other cytoskeleton networks, microtubules and intermediate filaments, remain undisturbed in type III programmed cell death. Strikingly, we revealed that the pharmacological modulation of F-actin dynamics regulated this type of death. Finally, our data delineated a new programmed cell death pathway in chronic lymphocytic leukemia initiated by CD47 triggering, and followed by serine protease activation, F-actin rearrangement, mitochondrial damage, phosphatidylserine exposure, and cell clearance.
Conclusions: Our work reveals a key molecular tool in the modulation of cell death in chronic lymphocytic leukemia: F-actin. By assessing the regulation of F-actin and type III programmed cell death, this analysis provides new options for destroying chronic lymphocytic leukemia cells, such as a combination of therapies based on apoptosis regulators (e.g., caspases, Bcl-2, Bax) along with alternative therapies based on type III death effectors (e.g., F-actin).
Key words: actin, apoptosis, caspase-independent cell death, CD47, chronic lymphocytic leukemia, mitochondria.
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