Lupus nephritis is a major cause of morbidity in patients with systemic lupus erythematosus. Among the different types of lupus nephritis, intracapillary immune complex (IC) deposition and accumulation of monocytes are hallmarks of lupus nephritis class III and IV. The relevance of intracapillary ICs in terms of monocyte recruitment and activation, as well as the nature and function of these monocytes are not well understood. For the early focal form of lupus nephritis (class III) we demonstrate a selective accumulation of the proinflammatory population of 6-sulfo LacNAc+ (slan) monocytes (slanMo), which locally expressed TNF-α. Immobilized ICs induced a direct recruitment of slanMo from the microcirculation via interaction with Fc γ receptor IIIA (CD16). Interestingly, intravenous immunoglobulins blocked CD16 and prevented cell recruitment. Engagement of immobilized ICs by slanMo induced the production of neutrophil-attracting chemokine CXCL2 as well as TNF-α, which in a forward feedback loop stimulated endothelial cells to produce the slanMo-recruiting chemokine CX3CL1 (fractalkine). In conclusion, we observed that expression of CD16 equips slanMo with a unique capacity to orchestrate early IC-induced inflammatory responses in glomeruli and identified slanMo as a pathogenic proinflammatory cell type in lupus nephritis.
Florina Olaru, Thomas Döbel, Anke S. Lonsdorf, Stephanie Oehrl, Michael Maas, Alexander H. Enk, Marc Schmitz, Elisabeth F. Gröne, Hermann-J. Gröne, Knut Schäkel
Foxp3-positive regulatory T cells (Tregs) are crucial for the maintenance of immune homeostasis and keep immune responses in check. Upon activation, Tregs are transferred into an effector state expressing transcripts essential for their suppressive activity, migration, and survival. However, it is not completely understood how different intrinsic and environmental factors control differentiation. Here, we present for the first time to our knowledge data suggesting that Treg-intrinsic expression of CD83 is essential for Treg differentiation upon activation. Interestingly, mice with Treg-intrinsic CD83 deficiency are characterized by a proinflammatory phenotype. Furthermore, the loss of CD83 expression by Tregs leads to the downregulation of Treg-specific differentiation markers and the induction of an inflammatory profile. In addition, Treg-specific conditional knockout mice showed aggravated autoimmunity and an impaired resolution of inflammation. Altogether, our results show that CD83 expression in Tregs is an essential factor for the development and function of effector Tregs upon activation. Since Tregs play a crucial role in the maintenance of immune tolerance and thus prevention of autoimmune disorders, our findings are also clinically relevant.
Marina Doebbeler, Christina Koenig, Lena Krzyzak, Christine Seitz, Andreas Wild, Thomas Ulas, Kevin Baßler, Dmitry Kopelyanskiy, Alina Butterhof, Christine Kuhnt, Simon Kreiser, Lena Stich, Elisabeth Zinser, Ilka Knippertz, Stefan Wirtz, Christin Riegel, Petra Hoffmann, Matthias Edinger, Lars Nitschke, Thomas Winkler, Joachim L. Schultze, Alexander Steinkasserer, Matthias Lechmann
Multiple modes of immunosuppression restrain immune function within tumors. We previously reported that phosphoinositide 3-kinase δ (PI3Kδ) inactivation in mice confers resistance to a range of tumor models by disrupting immunosuppression mediated by regulatory T cells (Tregs). The PI3Kδ inhibitor idelalisib has proven highly effective in the clinical treatment of chronic lymphocytic leukemia and the potential to extend the use of PI3Kδ inhibitors to nonhematological cancers is being evaluated. In this work, we demonstrate that the antitumor effect of PI3Kδ inactivation is primarily mediated through the disruption of Treg function, and correlates with tumor dependence on Treg immunosuppression. Compared with Treg-specific PI3Kδ deletion, systemic PI3Kδ inactivation is less effective at conferring resistance to tumors. We show that PI3Kδ deficiency impairs the maturation and reduces the capacity of CD8+ cytotoxic T lymphocytes (CTLs) to kill tumor cells in vitro, and to respond to tumor antigen–specific immunization in vivo. PI3Kδ inactivation antagonized the antitumor effects of tumor vaccines and checkpoint blockade therapies intended to boost the CD8+ T cell response. These findings provide insights into mechanisms by which PI3Kδ inhibition promotes antitumor immunity and demonstrate that the mechanism is distinct from that mediated by immune checkpoint blockade.
Ee Lyn Lim, Fiorella M. Cugliandolo, Dalya R. Rosner, David Gyori, Rahul Roychoudhuri, Klaus Okkenhaug
Redundancy and compensation provide robustness to biological systems but may contribute to therapy resistance. Both tumor-associated macrophages (TAMs) and Foxp3+ regulatory T (Treg) cells promote tumor progression by limiting antitumor immunity. Here we show that genetic ablation of CSF1 in colorectal cancer cells reduces the influx of immunosuppressive CSF1R+ TAMs within tumors. This reduction in CSF1-dependent TAMs resulted in increased CD8+ T cell attack on tumors, but its effect on tumor growth was limited by a compensatory increase in Foxp3+ Treg cells. Similarly, disruption of Treg cell activity through their experimental ablation produced moderate effects on tumor growth and was associated with elevated numbers of CSF1R+ TAMs. Importantly, codepletion of CSF1R+ TAMs and Foxp3+ Treg cells resulted in an increased influx of CD8+ T cells, augmentation of their function, and a synergistic reduction in tumor growth. Further, inhibition of Treg cell activity either through systemic pharmacological blockade of PI3Kδ, or its genetic inactivation within Foxp3+ Treg cells, sensitized previously unresponsive solid tumors to CSF1R+ TAM depletion and enhanced the effect of CSF1R blockade. These findings identify CSF1R+ TAMs and PI3Kδ-driven Foxp3+ Treg cells as the dominant compensatory cellular components of the immunosuppressive tumor microenvironment, with implications for the design of combinatorial immunotherapies.
David Gyori, Ee Lyn Lim, Francis M. Grant, Dominik Spensberger, Rahul Roychoudhuri, Stephen J. Shuttleworth, Klaus Okkenhaug, Len R. Stephens, Phillip T. Hawkins
Allergic asthma is a chronic inflammatory disease dominated by a CD4+ T helper 2 (Th2) cell signature. The immune response amplifies in self-enforcing loops, promoting Th2-driven cellular immunity and leaving the host unable to terminate inflammation. Posttranscriptional mechanisms, including microRNAs (miRs), are pivotal in maintaining immune homeostasis. Since an altered expression of various miRs has been associated with T cell–driven diseases, including asthma, we hypothesized that miRs control mechanisms ensuring Th2 stability and maintenance in the lung. We isolated murine CD4+ Th2 cells from allergic inflamed lungs and profiled gene and miR expression. Instead of focusing on the magnitude of miR differential expression, here we addressed the secondary consequences for the set of molecular interactions in the cell, the interactome. We developed the Impact of Differential Expression Across Layers, a network-based algorithm to prioritize disease-relevant miRs based on the central role of their targets in the molecular interactome. This method identified 5 Th2-related miRs (mir27b, mir206, mir106b, mir203, and mir23b) whose antagonization led to a sharp reduction of the Th2 phenotype. Overall, a systems biology tool was developed and validated, highlighting the role of miRs in Th2-driven immune response. This result offers potentially novel approaches for therapeutic interventions.
Ayşe Kılıç, Marc Santolini, Taiji Nakano, Matthias Schiller, Mizue Teranishi, Pascal Gellert, Yuliya Ponomareva, Thomas Braun, Shizuka Uchida, Scott T. Weiss, Amitabh Sharma, Harald Renz
The excessive production of type I IFNs is a hallmark and a main pathogenic mechanism of many autoimmune diseases, including systemic lupus erythematosus (SLE). In these pathologies, the sustained secretion of type I IFNs is dependent on the improper activation of plasmacytoid DCs (pDCs) by self–nucleic acids. However, the nature and origin of pDC-activating self–nucleic acids is still incompletely characterized. Here, we report that exosomes isolated from the plasma of SLE patients can activate the secretion of IFN-α by human blood pDCs in vitro. This activation requires endosomal acidification and is recapitulated by microRNAs isolated from exosomes, suggesting that exosome-delivered microRNAs act as self-ligands of innate single-stranded endosomal RNA sensors. By using synthetic microRNAs, we identified an IFN induction motif that is responsible for the TLR7-dependent activation, maturation, and survival of human pDCs. These findings identify exosome-delivered microRNAs as potentially novel TLR7 endogenous ligands able to induce pDC activation in SLE patients. Therefore, microRNAs may represent novel pathogenic mediators in the onset of autoimmune reactions and potential therapeutic targets in the treatment of type I IFN–mediated diseases.
Valentina Salvi, Veronica Gianello, Sara Busatto, Paolo Bergese, Laura Andreoli, Ugo D’Oro, Alessandra Zingoni, Angela Tincani, Silvano Sozzani, Daniela Bosisio
Studies in human peripheral blood monocyte–derived macrophages in vitro have shown clear evidence that multiple macrophage polarization states exist. The extent to which different alveolar macrophage (AM) polarization states exist in homeostasis or in the setting of severe injury such as acute respiratory distress syndrome (ARDS) is largely unknown. We applied single-cell cytometry TOF (CyTOF) to simultaneously measure 36 cell-surface markers on CD45+ cells present in bronchoalveolar lavage from healthy volunteers, as well as mechanically ventilated subjects with and without ARDS. Visualization of the high-dimensional data with the t-distributed stochastic neighbor embedding algorithm demonstrated wide diversity of cell-surface marker profiles among CD33+CD71+CD163+ AMs. We then used a κ-nearest neighbor density estimation algorithm to statistically identify distinct alveolar myeloid subtypes, and we discerned 3 AM subtypes defined by CD169 and PD-L1 surface expression. The percentage of AMs that were classified into one of the 3 AM subtypes was significantly different between healthy and mechanically ventilated subjects. In an independent cohort of subjects with ARDS, PD-L1 gene expression and PD-L1/PD-1 pathway–associated gene sets were significantly decreased in AMs from patients who experienced prolonged mechanical ventilation or death. Unsupervised CyTOF analysis of alveolar leukocytes from human subjects has potential to identify expected and potentially novel myeloid populations that may be linked with clinical outcomes.
Eric D. Morrell, Alice Wiedeman, S. Alice Long, Sina A. Gharib, T. Eoin West, Shawn J. Skerrett, Mark M. Wurfel, Carmen Mikacenic
Myeloid leukocytes are essentially involved in both tumor progression and control. We show that neo-adjuvant treatment of mice with an inhibitor of CSF1 receptor (CSF1R), a drug that is used to deplete tumor-associated macrophages, unexpectedly promoted metastasis. CSF1R blockade indirectly diminished the number of NK cells due to a paucity of myeloid cells that provide the survival factor IL-15 to NK cells. Reduction of the number of NK cells resulted in increased seeding of metastatic tumor cells to the lungs but did not impact on progression of established metastases. Supplementation of mice treated with CSF1R-inhibitor with IL-15 restored numbers of NK cells and diminished metastasis. Our data suggest that CSF1R blockade should be combined with administration of IL-15 to reduce the risk of metastasis.
Michal Beffinger, Paulino Tallón de Lara, Sònia Tugues, Marijne Vermeer, Yannick Montagnolo, Isabel Ohs, Virginia Cecconi, Giulia Lucchiari, Aron Gagliardi, Nikola Misljencevic, James Sutton, Roman Spörri, Burkhard Becher, Anurag Gupta, Maries van den Broek
Allogeneic stem cell transplantation (allo-SCT) can cure some patients with hematopoietic malignancy, but this relies on the development of a donor T cell alloreactive immune response. T cell activity in the first 2 weeks after allo-SCT is crucial in determining outcome, despite the clinical effects of the early alloreactive immune response often not appearing until later. However, the effect of the allogeneic environment on T cells is difficult to study at this time point due to the effects of profound lymphopenia. We approached this problem by comparing T cells at week 2 after allograft to T cells from autograft patients. Allograft T cells were present in small numbers but displayed intense proliferation with spontaneous cytokine production. Oligoclonal expansions at week 2 came to represent a substantial fraction of the established T cell pool and were recruited into tissues affected by graft-versus-host disease. Transcriptional analysis uncovered a range of potential targets for immune manipulation, including OX40L, TWEAK, and CD70. These findings reveal that recognition of alloantigen drives naive T cells toward a unique phenotype. Moreover, they demonstrate that early clonal T cell responses are recruited to sites of subsequent tissue damage and provide a range of targets for potential therapeutic immunomodulation.
Charlotte F. Inman, Suzy A. Eldershaw, Joanne E. Croudace, Nathaniel J. Davies, Archana Sharma-Oates, Tanuja Rai, Hayden Pearce, Mirjana Sirovica, Y.L. Tracey Chan, Kriti Verma, Jianmin Zuo, Sandeep Nagra, Francesca Kinsella, Jane Nunnick, Rasoul Amel-Kashipaz, Charles Craddock, Ram Malladi, Paul Moss
Chimeric antigen receptor–modified (CAR-modified) T cells have shown promising therapeutic effects for hematological malignancies, yet limited and inconsistent efficacy against solid tumors. The refinement of CAR therapy requires an understanding of the optimal characteristics of the cellular products, including the appropriate composition of CD4+ and CD8+ subsets. Here, we investigated the differential antitumor effect of CD4+ and CD8+ CAR T cells targeting glioblastoma-associated (GBM-associated) antigen IL-13 receptor α2 (IL13Rα2). Upon stimulation with IL13Rα2+ GBM cells, the CD8+ CAR T cells exhibited robust short-term effector function but became rapidly exhausted. By comparison, the CD4+ CAR T cells persisted after tumor challenge and sustained their effector potency. Mixing with CD4+ CAR T cells failed to ameliorate the effector dysfunction of CD8+ CAR T cells, while surprisingly, CD4+ CAR T cell effector potency was impaired when coapplied with CD8+ T cells. In orthotopic GBM models, CD4+ outperformed CD8+ CAR T cells, especially for long-term antitumor response. Further, maintenance of the CD4+ subset was positively correlated with the recursive killing ability of CAR T cell products derived from GBM patients. These findings identify CD4+ CAR T cells as a highly potent and clinically important T cell subset for effective CAR therapy.
Dongrui Wang, Brenda Aguilar, Renate Starr, Darya Alizadeh, Alfonso Brito, Aniee Sarkissian, Julie R. Ostberg, Stephen J. Forman, Christine E. Brown
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