Anti-Tumor Necrosis Factor α Therapeutics Differentially Affect Leishmania Infection of Human Macrophages

Tumor necrosis factor α (TNFα) drives the pathophysiology of human autoimmune diseases and consequently, neutralizing antibodies (Abs) or Ab-derived molecules directed against TNFα are essential therapeutics. As treatment with several TNFα blockers has been reported to entail a higher risk of infectious diseases such as leishmaniasis, we established an in vitro model based on Leishmania-infected human macrophages, co-cultured with autologous T-cells, for the analysis and comparison of anti-TNFα therapeutics. We demonstrate that neutralization of soluble TNFα (sTNFα) by the anti-TNFα Abs Humira®, Remicade®, and its biosimilar Remsima® negatively affects infection as treatment with these agents significantly reduces Leishmania-induced T-cell proliferation and increases the number of infected macrophages. By contrast, we show that blockade of sTNFα by Cimzia® does not affect T-cell proliferation and infection rates. Moreover, compared to Remicade®, treatment with Cimzia® does not impair the expression of cytolytic effector proteins in proliferating T-cells. Our data demonstrate that Cimzia® supports parasite control through its conjugated polyethylene glycol (PEG) moiety as PEGylation of Remicade® improves the clearance of intracellular Leishmania. This effect can be linked to complement activation, with levels of complement component C5a being increased upon treatment with Cimzia® or a PEGylated form of Remicade®. Taken together, we provide an in vitro model of human leishmaniasis that allows direct comparison of different anti-TNFα agents. Our results enhance the understanding of the efficacy and adverse effects of TNFα blockers and they contribute to evaluate anti-TNFα therapy for patients living in countries with a high prevalence of leishmaniasis

PD-1 checkpoint inhibition in Leishmania infection of primary human cells

In this study we investigated the impact of Programmed Death-1/Programmed Death-1 ligand (PD-1/PD-L) axis in an in vitro Leishmania major (Lm) infection model consisting of primary human myeloid and lymphoid cells. Two different PD-1 checkpoint inhibitors (IgG1 and IgG4) were used to modulate the PD-1/PD-L interactions in different Lm-specific T-cell assay setups. As read-outs, Lm-induced T-cell proliferation and Lm infection rate in host cells was assessed. First, PD-1 ligand expression was demonstrated on three different Lm host cells, namely pro-inflammatory and anti-inflammatory human monocyte-derived macrophages (hMDM1 and hMDM2) and dendritic cells (hMDDC). PD-1 checkpoint blockade had no significant impact on Lm-induced T-cell proliferation or Lm infection rate in a co-culture of Lm-infected host cells together with autologous peripheral blood lymphocytes (PBLs). Using an approach with pre-cultured leucocytes (RESTORE-Assay by Römer et al. 2011) which mimics tissue-like conditions and renders T-cells more responsive to their cognate antigen, revealed a similar picture. Although PD-1 and its ligands were detectable in this assay, no significant differences in T-cell proliferation due to PD-1 checkpoint blockade were observed. Because PD-1/PD-L interactions are highly prominent during chronic inflammation and antigen stimulation, we investigated an approach using phytohemagglutinin-pre-stimulated PBLs in a co-culture with infected hMDM1, hMDM2 or hMDDC. In this approach, two different PD-1 checkpoint inhibitors (IgG1 and IgG4) increased T-cell effector functions in a similar manner. As a consequence Lm infection decreased, being the most pronounced in hMDDC, compared to hMDM1 and hMDM2. Focusing on hMDDC, effects mediated by PD-1 blockade were shown to be partially TNFα dependent. Moreover, treatment with the therapeutic PD-1 checkpoint inhibitor nivolumab specifically enhanced proliferation of CD4+ T-cells, increased expression of the TH1-specific transcription factor Tbet, T-cell activation markers and cytolytic effector proteins, which at large might be implicated in enhanced parasite killing. In all, our data describe an important role for the PD-1/PD-L axis upon Lm infection using a human primary cell system. These data contribute to a better understanding of the PD-1/PD-L-induced T-cell impairment during infectious disease and its influence on immune effector mechanisms to combat Lm infection.Ziel dieser Studie war die Untersuchung des Programmed Death-1/Programmed Death-1 ligand (PD-1/PD-L) Signalwegs innerhalb eines in vitro Leishmania major (Lm) Infektionsmodells bestehend aus humanen myeloiden und lymphoiden Zellen. Zwei verschiedene Subklassen von PD-1 Checkpoint Inhibitoren (IgG1 und IgG4) wurden zur Modulation des PD-1/PD-L Signalwegs in verschiedenen Versuchsaufbauten getestet, wobei der Einfluss auf Lm-induzierte T-Zellproliferation und Infektionsrate bestimmt wurde. Zunächst wurde gezeigt, dass die PD-1 Liganden auf drei verschiedenen Lm Wirtszellen exprimiert werden, und zwar auf pro- und anti-inflammatorischen humanen Monozyten-abgeleiteten Makrophagen (hMDM1 und hMDM2) sowie auf humanen Monozyten-abgeleiteten dendritischen Zellen (hMDDC). PD-1 Checkpoint Blockade hatte keinen Einfluss auf die Lm-induzierte T-Zellproliferation oder die Infektionsrate in einer Kokultur bestehend aus Lm Wirtszellen und peripheren Blutlymphozyten (PBLs). Ähnliche Ergebnisse wurden mit einem Versuchsaufbau bestehend aus vorkultivierten Leukozyten erzielt (RESTORE-Assay von Römer et al. 2011). Dabei wurden in-vitro gewebeartige Bedingungen geschaffen, die T-Zellen stärker auf Antigenstimulation reagieren lassen. Obwohl PD-1 sowie die zugehörigen Liganden messbar waren, hatte PD-1 Checkpoint Blockade keinen signifikanten Effekt auf die T-Zellproliferation. Da der PD-1/PD-L Signalweg insbesondere bei chronischer Entzündung und Antigenstimulation eine inhibierende Rolle spielt, entwickelten wir einen Versuchsansatz mit Phytohemagglutinin-vorstimulierten PBLs, welche mit infizierten hMDM1, hMDM2 oder hMDDC kokultiviert wurden. In diesem Versuchsansatz verstärkten die oben genannten PD-1 Checkpoint Inhibitoren (IgG1 und IgG4) T-Zell Effektor-Funktionen in ähnlicher Weise. Dadurch verringerte sich die Lm Infektion am stärksten in hMDDC im Vergleich zu hMDM1 und hMDM2. In der hMDDC Kokultur wurde gezeigt, dass die Effekte der PD-1-Blockade teilweise TNFα abhängig sind. Des Weiteren verstärkte der therapeutische IgG4 PD-1 Checkpoint Inhibitor Nivolumab spezifisch die CD4+ T-Zellproliferation, die Expression des TH1-spezifischen Transkriptionsfaktors Tbet, T-Zell Aktivierungsmarker sowie zytolytische Effektor-Proteine, welche womöglich in der Gesamtheit zur Parasitenabtötung beitragen. Insgesamt zeigen unsere Ergebnisse, dass der PD-1/PD-L Signalweg bei Lm Infektion von primären humanen Zellen eine wichtige Rolle spielt. Diese Daten können zu einem besseren Verständnis der durch PD-1/PD-L-induzierten T-Zellbeeinträchtigung während der humanen Leishmaniose beitragen sowie für die Entwicklung von Immuntherapieansätzen genutzt werden

RCF1-dependent respiratory supercomplexes are integral for lifespan-maintenance in a fungal ageing model

Mitochondrial respiratory supercomplexes (mtRSCs) are stoichiometric assemblies of electron transport chain (ETC) complexes in the inner mitochondrial membrane. They are hypothesized to regulate electron flow, the generation of reactive oxygen species (ROS) and to stabilize ETC complexes. Using the fungal ageing model Podospora anserina, we investigated the impact of homologues of the Saccharomyces cerevisiae respiratory supercomplex factors 1 and 2 (termed PaRCF1 and PaRCF2) on mtRSC formation, fitness and lifespan. Whereas PaRCF2’s role seems negligible, ablation of PaRCF1 alters size of monomeric complex IV, reduces the abundance of complex IV-containing supercomplexes, negatively affects vital functions and shortens lifespan. PaRcf1 overexpression slightly prolongs lifespan, though without appreciably influencing ETC organization. Overall, our results identify PaRCF1 as necessary yet not sufficient for mtRSC formation and demonstrate that PaRCF1-dependent stability of complex IV and associated supercomplexes is highly relevant for maintenance of the healthy lifespan in a eukaryotic model organism

Nivolumab Enhances In Vitro Effector Functions of PD-1+ T-Lymphocytes and Leishmania-Infected Human Myeloid Cells in a Host Cell-Dependent Manner

Functional impairment of T-cells and a concomitant augmented expression of programmed death-1 (PD-1) have been observed in visceral leishmaniasis patients, as well as in experimental models for visceral and cutaneous leishmaniasis. The PD-1/PD-1-ligand (PD-1/PD-L) interaction negatively regulates T-cell effector functions, which are required for parasite control during leishmaniasis. The aim of this study was to elucidate the impact of the PD-1/PD-L axis in a human primary in vitro infection model of Leishmania major (Lm). Blocking the PD-1/PD-L interaction with nivolumab increased T-cell proliferation and release of the proinflammatory cytokines TNFα and IFNγ during the cocultivation of Lm-infected human monocyte-derived macrophages (hMDMs) or dendritic cells (hMDDC) with autologous PD-1+-lymphocytes. As a consequence Lm infection decreased, being the most pronounced in hMDDC, compared to proinflammatory hMDM1 and anti-inflammatory hMDM2. Focusing on hMDDC, we could partially reverse effects mediated by PD-1 blockade by neutralizing TNFα but not by neutralizing IFNγ. Furthermore, PD-1 blockade increased intracellular expression of perforin, granulysin, and granzymes in proliferating CD4+-T-cells, which might be implicated in reduction of Lm-infected cells. In all, our data describe an important role for the PD-1/PD-L axis upon Lm infection using a human primary cell system. These data contribute to a better understanding of the PD-1-induced T-cell impairment during disease and its influence on immune effector mechanisms to combat Lm infection

Data_Sheet_1_Anti-Tumor Necrosis Factor α Therapeutics Differentially Affect Leishmania Infection of Human Macrophages.PDF

<p>Tumor necrosis factor α (TNFα) drives the pathophysiology of human autoimmune diseases and consequently, neutralizing antibodies (Abs) or Ab-derived molecules directed against TNFα are essential therapeutics. As treatment with several TNFα blockers has been reported to entail a higher risk of infectious diseases such as leishmaniasis, we established an in vitro model based on Leishmania-infected human macrophages, co-cultured with autologous T-cells, for the analysis and comparison of anti-TNFα therapeutics. We demonstrate that neutralization of soluble TNFα (sTNFα) by the anti-TNFα Abs Humira^®, Remicade^®, and its biosimilar Remsima^® negatively affects infection as treatment with these agents significantly reduces Leishmania-induced T-cell proliferation and increases the number of infected macrophages. By contrast, we show that blockade of sTNFα by Cimzia^® does not affect T-cell proliferation and infection rates. Moreover, compared to Remicade^®, treatment with Cimzia^® does not impair the expression of cytolytic effector proteins in proliferating T-cells. Our data demonstrate that Cimzia^® supports parasite control through its conjugated polyethylene glycol (PEG) moiety as PEGylation of Remicade^® improves the clearance of intracellular Leishmania. This effect can be linked to complement activation, with levels of complement component C5a being increased upon treatment with Cimzia^® or a PEGylated form of Remicade^®. Taken together, we provide an in vitro model of human leishmaniasis that allows direct comparison of different anti-TNFα agents. Our results enhance the understanding of the efficacy and adverse effects of TNFα blockers and they contribute to evaluate anti-TNFα therapy for patients living in countries with a high prevalence of leishmaniasis.</p

DARPin-targeting of measles virus: unique bispecificity, effective oncolysis, and enhanced safety

Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancers. Many naturally occurring viruses have a preferential, although nonexclusive, tropism for tumors and tumor cells. In addition, specific targeting of cancer cells can be achieved at the virus entry level. We optimized retargeting of cell entry by elongating the measles virus attachment protein with designed ankyrin repeat proteins (DARPins), while simultaneously ablating entry through the natural receptors. DARPin-targeted viruses were strongly attenuated in off-target tissue, thereby enhancing safety, but completely eliminated tumor xenografts. Taking advantage of the unique properties of DARPins of being fused without generating folding problems, we generated a virus simultaneous targeting two different tumor markers. The bispecific virus retained the original oncolytic efficacy, while providing proof of concept for a strategy to counteract issues of resistance development. Thus, DARPin-targeting opens new prospects for the development of personalized, targeted therapeutics