Coronavirus induced disease 2019 (COVID-19) can be complicated by severe organ damage leading to dysfunction of the lungs and other organs. The processes that trigger organ damage in COVID-19 are ...incompletely understood.
Samples were donated from hospitalized patients. Sera, plasma, and autopsy-derived tissue sections were examined employing flow cytometry, enzyme-linked immunosorbent assays, and immunohistochemistry.
Here, we show that severe COVID-19 is characterized by a highly pronounced formation of neutrophil extracellular traps (NETs) inside the micro-vessels. Intravascular aggregation of NETs leads to rapid occlusion of the affected vessels, disturbed microcirculation, and organ damage. In severe COVID-19, neutrophil granulocytes are strongly activated and adopt a so-called low-density phenotype, prone to spontaneously form NETs. In accordance, markers indicating NET turnover are consistently increased in COVID-19 and linked to disease severity. Histopathology of the lungs and other organs from COVID-19 patients showed congestions of numerous micro-vessels by aggregated NETs associated with endothelial damage.
These data suggest that organ dysfunction in severe COVID-19 is associated with excessive NET formation and vascular damage.
Deutsche Forschungsgemeinschaft (DFG), EU, Volkswagen-Stiftung
As substantial constituents of the multiple myeloma (MM) microenvironment, pro-inflammatory macrophages have emerged as key promoters of disease progression, bone destruction, and immune impairment. ...We identify beta-2-microglobulin (β2m) as a driver in initiating inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed β2m promotes β2m amyloid aggregation in MAMs, resulting in lysosomal rupture and ultimately production of active interleukin-1β (IL-1β) and IL-18. This process depends on activation of the NLRP3 inflammasome after β2m accumulation, as macrophages from NLRP3-deficient mice lack efficient β2m-induced IL-1β production. Moreover, depletion or silencing of β2m in MM cells abrogates inflammasome activation in a murine MM model. Finally, we demonstrate that disruption of NLRP3 or IL-18 diminishes tumor growth and osteolytic bone destruction normally promoted by β2m-induced inflammasome signaling. Our results provide mechanistic evidence for β2m’s role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 represents a potential therapeutic approach in MM.
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•β2m that accumulates in multiple myeloma (MM) is ingested by MM-associated macrophages•β2m uptake leads to β-fibril aggregation, lysosomal rupture, and inflammasome activation•β2m contributes to inflammation in MM via IL-1β and IL-18 secretion•Inhibition of NLRP3 delays onset and reduces severity of MM in preclinical models
MM is characterized by β2m abundance. The role of β2m remains unknown. Hofbauer et al. show that MM-associated macrophages take up β2m. Phagocytosed β2m forms β-fibrils and leads to lysosomal rupture and inflammasome activation. They reveal β2m as a promoter of inflammation, which plays a role in MM initiation and progression.
Acute myeloid leukemia (AML) represents the most common acute leukemia among adults. Despite recent progress in diagnosis and treatment, long-term outcome remains unsatisfactory. The success of ...allogeneic stem cell transplantation underscores the immunoresponsive nature of AML, creating the basis for further exploiting immunotherapies. However, emerging evidence suggests that AML, similar to other malignant entities, employs a variety of mechanisms to evade immunosurveillance. In light of this, T-cell inhibitory myeloid-derived suppressor cells (MDSC) are gaining interest as key facilitators of immunoescape. Accumulation of CD14
HLA-DR
monocytic MDSCs has been described in newly diagnosed AML patients, and deciphering the underlying mechanisms could help to improve anti-AML immunity. Here, we report that conventional monocytes readily take-up AML-derived extracellular vesicles (EV) and subsequently undergo MDSC differentiation. They acquired an CD14
HLA-DR
phenotype, expressed the immunomodulatory indoleamine-2,3-dioxygenase, and upregulated expression of genes characteristic for MDSCs, such as
and
. The Akt/mTOR pathway played a critical role in the AML-EV-induced phenotypical and functional transition of monocytes. Generated MDSCs displayed a glycolytic switch, which rendered them more susceptible toward glycolytic inhibitors. Furthermore, palmitoylated proteins on the AML-EV surface activated Toll-like receptor 2 as the initiating event of Akt/mTOR-dependent induction of MDSC. Therefore, targeting protein palmitoylation in AML blasts could block MDSC accumulation to improve immune responses. SIGNIFICANCE: These findings indicate that targeting protein palmitoylation in AML could interfere with the leukemogenic potential and block MDSC accumulation to improve immunity.
Acute myeloid leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 30%. Emerging evidence suggest that immune alterations favor leukemogenesis ...and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. Here, we demonstrate increased frequencies of CD14
monocytic MDSCs in newly diagnosed AML that co-express CD33 but lack HLA-DR (HLA-DR
). AML-blasts induce HLA-DR
cells from healthy donor-derived monocytes in vitro that suppress T-cells and express indoleamine-2,3-dioxygenase (IDO). We investigated whether a CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with pre-clinical activity against AML-blasts by redirection of T-cells can eradicate CD33
MDSCs. In fact, T-cells eliminate IDO
CD33
MDSCs in the presence of AMG 330. Depletion of total CD14
cells (including MDSCs) in peripheral blood mononuclear cells from AML patients did not enhance AMG 330-triggered T-cell activation and expansion, but boosted AML-blast lysis. This finding was corroborated in experiments showing that adding MDSCs into co-cultures of T- and AML-cells reduced AML-blast killing, while IDO inhibition promotes AMG 330-mediated clearance of AML-blasts. Taken together, our results suggest that AMG 330 may achieve anti-leukemic efficacy not only through T-cell-mediated cytotoxicity against AML-blasts but also against CD33
MDSCs, suggesting that it is worth exploring the predictive role of MDSCs for responsiveness towards an AMG 330-based therapy.
Abstract
Previous studies indicated a role of the reconstituting immune system for disease outcome upon high-dose chemotherapy (HDCT) and autologous stem cell transplantation (auto-SCT) in multiple ...myeloma (MM) and lymphoma patients. Since immune cell metabolism and function are closely interconnected, we used flow-cytometry techniques to analyze key components and functions of the metabolic machinery in reconstituting immune cells upon HDCT/auto-SCT. We observed increased proliferative activity and an upregulation of the glycolytic and fatty acid oxidation (FAO) machinery in immune cells during engraftment. Metabolic activation was more pronounced in T-cells of advanced differentiation stages, in CD56
bright
NK-cells, and CD14
++
CD16
+
intermediate monocytes. Next, we investigated a potential correlation between the immune cells’ metabolic profile and early progression or relapse in lymphoma patients within the first twelve months following auto-SCT. Here, persistently increased metabolic parameters correlated with a rather poor disease course. Taken together, reconstituting immune cells display an upregulated bioenergetic machinery following auto-SCT. Interestingly, a persistently enhanced metabolic immune cell phenotype correlated with reduced PFS. However, it remains to be elucidated, if the clinical data can be confirmed within a larger set of patients and if residual malignant cells not detected by conventional means possibly caused the metabolic activation.
Immune cell activity is a major factor for disease progression in inflammatory bowel diseases (IBD). Classifying the type and functional state of immune cells is therefore crucial in clinical ...diagnostics of IBD. Label-free optical technologies exploiting NADH and FAD autofluorescence, such as multiphoton microscopy, have been used to describe tissue morphology in healthy and inflamed colon samples. Nevertheless, a strategy for the identification of single immune cell subtypes within the tissue is yet to be developed. This work aims to initiate an understanding of autofluorescence changes depending on immune cell type and activation state. For this, NADH and FAD autofluorescence signals of different murine immune cell subtypes under native conditions, as well as upon in vitro stimulation and cell death, have been evaluated. Autofluorescence was assessed using flow cytometry and multiphoton microscopy. Our results reveal significantly increased NADH and FAD signals in innate immune cells compared to adaptive immune cells. This allowed identification of relative amounts of neutrophils and CD4+ T cells in mixed cell suspensions, by using NADH signals as a differentiation marker. Furthermore, in vitro stimulation significantly increased NADH and FAD autofluorescence in adaptive immune cells and macrophages. Cell death induced a significant drop in NADH autofluorescence, while FAD signals were hardly affected. Taken together, these results demonstrate the value of autofluorescence as a tool to characterize immune cells in different functional states, paving the way to the label-free clinical classification of IBD in the future.
Autoimmune lymphoproliferative syndrome (ALPS) is a human disorder characterized by defective Fas signaling, resulting in chronic benign lymphoproliferation and accumulation of TCRαβ+ CD4− CD8− ...double-negative T (DNT) cells. Although their phenotype resembles that of terminally differentiated or exhausted T cells, lack of KLRG1, high eomesodermin, and marginal T-bet expression point instead to a long-lived memory state with potent proliferative capacity. Here we show that despite their terminally differentiated phenotype, human ALPS DNT cells exhibit substantial mitotic activity in vivo. Notably, hyperproliferation of ALPS DNT cells is associated with increased basal and activation-induced phosphorylation of serine-threonine kinases Akt and mechanistic target of rapamycin (mTOR). The mTOR inhibitor rapamycin abrogated survival and proliferation of ALPS DNT cells, but not of CD4+ or CD8+ T cells in vitro. In vivo, mTOR inhibition reduced proliferation and abnormal differentiation by DNT cells. Importantly, increased mitotic activity and hyperactive mTOR signaling was also observed in recently defined CD4+ or CD8+ precursor DNT cells, and mTOR inhibition specifically reduced these cells in vivo, indicating abnormal programming of Fas-deficient T cells before the DNT stage. Thus, our results identify the mTOR pathway as a major regulator of lymphoproliferation and aberrant differentiation in ALPS.
•ALPS DNT cells and their putative precursors reveal high proliferative activity in vivo, which is associated with hyperactive mTOR signaling.•Rapamycin therapy controls mitotic activity and abnormal differentiation of ALPS DNT cells and reduces CD4+ or CD8+ precursor DNT cells.
Genetic alterations in tumor cells provide promising targets for antitumor therapy. Recently, loss of methylthioadenosine phosphorylase (MTAP), a deletion frequently occurring in cancer, has been ...shown to create vulnerability to the inhibition of the protein arginine methyltransferase 5 (PRMT5). MTAP deficiency leads to accumulation of methylthioadenosine (MTA), which reduces PRMT5 activity, and thus, sensitizes the tumor cells to selective PRMT5 inhibitors (PRMT5i). PRMT5i are investigated as a new strategy to selectively kill MTAP-deficient tumor cells by blocking residual PRMT5 activity, but also to treat PRMT5-overexpressing tumors. Although many studies investigated the role of PRMT5 in cancer, only little data exist about the effect of PRMT5 inhibition on immune cells. As we could show that the tumor metabolite MTA suppresses T cells, we asked whether selective PRMT5 inhibition is detrimental for T-cell immune responses. Therefore, we examined the effect of the synthetic PRMT5 inhibitor EPZ015666 on human CD8
T cells in direct comparison with the naturally occurring PRMT5-inhibiting molecule MTA. Both compounds reduced T-cell proliferation, viability, and functionality. In addition, T-cell metabolism was impaired upon PRMT5 inhibition. These effects coincided with the induction of p53 expression and reduced AKT/mTOR signaling. Our data clearly demonstrate that PRMT5 activity is involved in various cellular processes of human CD8
T cells associated with essential T-cell functions. Therefore, not only tumor cells, but also antitumor immune responses, are compromised by PRMT5 inhibitors. This emphasizes the importance of considering side effects on the immune system when developing new strategies to specifically target not only MTAP-deficient tumors.
Macrophages are one of the key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD19 antibody tafasitamab, approved in combination with lenalidomide for the ...treatment of relapsed or refractory (r/r) diffuse large B cell lymphoma (DLBCL). However, antibody-dependent cellular phagocytosis (ADCP) in the tumor microenvironment can be counteracted by increased expression of the inhibitory receptor SIRPα on macrophages and its ligand, the immune checkpoint molecule CD47 on tumor cells. The aim of this study was to investigate the impact of the CD47-SIRPα axis on tafasitamabmediated phagocytosis and explore the potential of anti-CD47 blockade to enhance its antitumor activity. Elevated expression of both SIRPα and CD47 was observed in DLBCL patient-derived lymph node biopsies compared to healthy controls. CRISPR-mediated CD47 overexpression impacted tafasitamab-mediated ADCP in vitro and increased expression of SIRPα on macrophages correlated with decreased ADCP activity of tafasitamab against DLBCL cell lines. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Importantly, tafasitamab-mediated phagocytosis was elevated in combination with CD47 blockade using primary DLBCL cells and patient-derived lymphoma-associated macrophages (LAMs) in an autologous setting. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination treatment. Finally, combined treatment of tafasitamab and an anti-CD47 antibody resulted in enhanced tumor volume reduction and survival benefit in lymphoma xenograft mouse models. These findings provide evidence that CD47 blockade can enhance the phagocytic potential of tumor targeting immunotherapies such as tafasitamab and suggest there is value in exploring the combination in the clinic.
The recently discovered population of TCRαβ+ CD4-/CD8- (double-negative, DN) T-cells are highly potent suppressor cells in mice and humans. In preclinical transplantation models, adoptive transfer of ...DN T-cells specifically inhibits alloreactive T-cells and prevents transplant rejection or graft-vs.-host disease (GvHD). Interestingly, clinical studies in patients who underwent allogeneic stem cell transplantation reveal an inverse correlation between the frequency of circulating DN T-cells and the severity of GvHD, suggesting a therapeutic potential of human DN T-cells. However, their exact mode of action has not been elucidated yet. Investigating the impact of DN T-cells on conventional T-cells, we found that human DN T-cells selectively inhibit mTOR signaling in CD4 T-cells. Given that mTOR is a critical regulator of cellular metabolism, we further determined the impact of DN T-cells on the metabolic framework of T-cells. Intriguingly, DN T-cells diminished expression of glucose transporters and glucose uptake, whereas fatty acid uptake was not modified, indicating that DN T-cells prevent metabolic adaptation of CD4 T-cells upon activation (i.e., glycolytic switch) thereby contributing to their suppression. Further analyses demonstrated that CD4 T-cells also do not upregulate homing receptors associated with inflammatory processes. In contrast, expression of central memory-cell associated cell surface markers and transcription factors were increased by DN T-cells. Moreover, CD4 T-cells failed to produce inflammatory cytokines after co-culture with DN T-cells, whereas IL-2 secretion was enhanced. Taken together DN T-cells impair metabolic reprogramming of conventional CD4 T-cells by abrogating mTOR signaling, thereby modulating CD4 T-cell functionality. These results uncover a new mechanism of DN T-cell-mediated suppression, pointing out that DN T-cells could serve as cell-based therapy to limit alloreactive immune response.
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