CD19-CAR T-cell therapy induces deep tissue depletion of B cells.

OBJECTIVES: CD19-targeting chimeric antigen receptor (CAR) T-cell therapy can induce long-term drug-free remission in patients with autoimmune diseases (AIDs). The efficacy of CD19-CAR T-cell therapy is presumably based on deep tissue depletion of B cells; however, such effect has not been proven in humans in vivo. METHODS: Sequential ultrasound-guided inguinal lymph node biopsies were performed at baseline and after CD19-CAR T-cell therapy in patients with AIDs. Results were compared with lymph node biopsies from rituximab (RTX)-treated AID patients with absence of peripheral B cells. Conventional and immunohistochemistry staining were performed on lymph node tissue to assess architecture as well the number of B cells, follicular dendritic cells (FDCs), plasma cells, T cells and macrophages. RESULTS: Sequential lymph node biopsies were analysed from five patients with AID before and after CD19-CAR T-cell therapy and from five patients with AID after RTX treatment. In addition, non-lymphoid organ biopsies (colon, kidney and gallbladder) from three additional patients with AID after CD19-CAR T-cell therapy were analysed. CD19+ and CD20+ B cells were completely depleted in the lymph nodes after CD19-CAR T-cell therapy, but not after RTX treatment. Plasma cells, T cells and macrophages in the lymph nodes remained unchanged. Follicular structures were disrupted and FDCs were depleted in the lymph nodes after CD19-CAR T-cell therapy, but not after RTX. Non-lymphoid organs were completely depleted of B cells. DISCUSSION: This study demonstrates complete B-cell depletion in secondary lymphoid tissues of patients with AIDs following CD19-CAR T-cell therapy combined with standard lymphodepleting therapy.

The STAT4/MLL1 Epigenetic Axis Regulates the Antimicrobial Functions of Murine Macrophages.

Macrophages are critical immune cells for the clearance of microbial pathogens and cellular debris from peripheral tissues. Macrophage inflammatory responses are governed by gene expression patterns, and these patterns are often subject to epigenetic control. Chromatin modifications, such as histone methylation, regulate gene accessibility in macrophages, and macrophage polarization is governed in part by the expression and function of chromatin-modifying enzymes. The histone methyltransferase mixed-lineage leukemia 1 (MLL1) preferentially modifies lysine residue 4 on the unstructured protein tail of histone H3. MLL1 expression and function have been shown to be governed by signal transduction pathways that are activated by inflammatory stimuli, such as NF-κB. Therefore, we sought to investigate the role of MLL1 in mediating macrophage inflammatory responses. Bone marrow-derived macrophages from mice with a targeted MLL1 gene knockout (Lys2-Cre+/- MLL1fx/fx) exhibited decreased proinflammatory gene expression with concurrent decreases in activating histone methylation. However, MLL1-deficient macrophages also exhibited increased phagocytic and bacterial killing activity in vitro. RNA profiling of MLL1-knockout macrophages identified numerous genes involved with inflammatory responses whose expression was altered in response to TLR ligands or proinflammatory cytokines, including STAT4. STAT4-dependent cytokines, such as type I IFNs were able to drive MLL1 expression in macrophages, and MLL1-knockout macrophages exhibited decreased activating histone methylation in the STAT4 promoter. These results implicate an important role for MLL1-dependent epigenetic regulation of macrophage antimicrobial functions.

Inhibition of phosphodiesterase 4 (PDE4) reduces dermal fibrosis by interfering with the release of interleukin-6 from M2 macrophages.

OBJECTIVES: To investigate the disease-modifying effects of phosphodiesterase 4 (PDE4) inhibition in preclinical models of systemic sclerosis (SSc). METHODS: We studied the effects of PDE4 inhibition in a prevention and a treatment model of bleomycin-induced skin fibrosis, in the topoisomerase mouse model as well as in a model of sclerodermatous chronic graft-versus-host disease. To better understand the mode of action of PDE4 blockade in preclinical models of SSc, we investigated fibrosis-relevant mediators in fibroblasts and macrophages from healthy individuals and patients suffering from diffuse-cutaneous SSc on blockade of PDE4. RESULTS: Specific inhibition of PDE4 by rolipram and apremilast had potent antifibrotic effects in bleomycin-induced skin fibrosis models, in the topoisomerase I mouse model and in murine sclerodermatous chronic graft-versus-host disease. Fibroblasts were not the direct targets of the antifibrotic effects of PDE4 blockade. Reduced leucocyte infiltration in lesional skin on PDE4 blockade suggested an immune-mediated mechanism. Further analysis revealed that PDE4 inhibition decreased the differentiation of M2 macrophages and the release of several profibrotic cytokines, resulting in reduced fibroblast activation and collagen release. Within these profibrotic mediators, interleukin-6 appeared to play a central role. CONCLUSIONS: PDE4 inhibition reduces inflammatory cell activity and the release of profibrotic cytokines from M2 macrophages, leading to decreased fibroblast activation and collagen release. Importantly, apremilast is already approved for the treatment of psoriasis and psoriatic arthritis. Therefore, PDE4 inhibitors might be further developed as potential antifibrotic therapies for patients with SSc. Our findings suggest that particularly patients with inflammation-driven fibrosis might benefit from PDE4 blockade.

Divergent effects of Tlr9 deletion in experimental late venous thrombosis resolution and vein wall injury.

Deep-vein thrombosis (DVT) resolves via a sterile inflammatory response. Defining the inflammatory response of DVT may allow for new therapies that do not involve anticoagulation. Previously, we have shown that Toll-like receptor 9 (Tlr9) gene deleted mice had impaired venous thrombosis (VT) resolution. Here, we further characterise the role of Tlr9 signalling and sterile inflammation in chronic VT and vein wall responses. First, we found a human precedent exists with Tlr9+ cells present in chronic post thrombotic intraluminal tissue. Second, in a stasis VT mouse model, endogenous danger signal mediators of uric acid, HMGB-1, and neutrophil extracellular traps marker of citrullinated histone-3 (and extracellular DNA) were greater in Tlr9-/- thrombi as compared with wild-type (WT), corresponding with larger VT at 8 and 21 days. Fewer M1 type (CCR2+) monocyte/macrophages (MØ) were present in Tlr9-/- thrombi than WT controls at 8 days, suggesting an impaired inflammatory cell influx. Using bone marrow-derived monocyte (BMMØ) cell culture, we found decreased fibrinolytic gene expression with exposure to several endogenous danger signals. Next, adoptive transfer of cultured Tlr9+/+ BMMØ to Tlr9-/- mice normalised VT resolution at 8 days. Lastly, although the VT size was larger at 21 days in Tlr9-/- mice and correlated with decreased endothelial antigen markers, no difference in fibrosis was found. These data suggest that Tlr9 signalling in MØ is critical for later VT resolution, is associated with necrosis clearance, but does not affect later vein wall fibrosis. These findings provide insight into the Tlr9 MØ mechanisms of sterile inflammation in this disease process.

Epigenetic regulation of IL-12-dependent T cell proliferation.

It is well established that the cytokine IL-12 and the transcription factor STAT4, an essential part of the IL-12 signaling pathway, are critical components of the Th1 differentiation process in T cells. In response to pathogenic stimuli, this process causes T cells to proliferate rapidly and secrete high amounts of the cytokine IFN-γ, leading to the Th1 proinflammatory phenotype. However, there are still unknown components of this differentiation pathway. We here demonstrated that the expression of the histone methyltransferase Mll1 is driven by IL-12 signaling through STAT4 in humans and mice and is critical for the proper differentiation of a naïve T cell to a Th1 cell. Once MLL1 is up-regulated by IL-12, it regulates the proliferation of Th1 cells. As evidence of this, we show that Th1 cells from Mll1(+/-) mice are unable to proliferate rapidly in a Th1 environment in vitro and in vivo. Additionally, upon restimulation with cognate antigen Mll1(+/-), T cells do not convert to a Th1 phenotype, as characterized by IFN-γ output. Furthermore, we observed a reduction in IFN-γ production and proliferation in human peripheral blood stimulated with tetanus toxoid by use of a specific inhibitor of the MLL1/menin complex. Together, our results demonstrate that the MLL1 gene plays a previously unrecognized but essential role in Th1 cell biology and furthermore, describes a novel pathway through which Mll1 expression is regulated.

Preventive azithromycin treatment reduces noninfectious lung injury and acute graft-versus-host disease in a murine model of allogeneic hematopoietic cell transplantation.

Noninfectious lung injury and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) are associated with significant morbidity and mortality. Azithromycin is widely used in allogeneic HCT recipients for pulmonary chronic GVHD, although current data appear controversial. We induced GVHD and noninfectious lung injury in lethally irradiated B6D2F1 mice by transplanting bone marrow and splenic T cells from allogeneic C57BL/6 mice. Experimental groups were treated with oral azithromycin starting on day 14 until the end of week 6 or week 14 after transplantation. Azithromycin treatment resulted in improved survival and decreased lung injury; the latter characterized by improved pulmonary function, reduced peribronchial and perivascular inflammatory cell infiltrates along with diminished collagen deposition, and a decrease in lung cytokine and chemokine expression. Azithromycin also improved intestinal GVHD but did not affect liver GVHD at week 6 early after transplantation. At week 14, azithromycin decreased liver GVHD but had no effect on intestinal GVHD. In vitro, allogeneic antigen-presenting cell (APC)- dependent T cell proliferation and cytokine production were suppressed by azithromycin and inversely correlated with relative regulatory T cell (Treg) expansion, whereas no effect was seen when T cell proliferation occurred APC independently through CD3/CD28-stimulation. Further, azithromycin reduced alloreactive T cell expansion but increased Treg expansion in vivo with corresponding downregulation of MHC II on CD11c(+) dendritic cells. These results demonstrate that preventive administration of azithromycin can reduce the severity of acute GVHD and noninfectious lung injury after allo-HCT, supporting further investigation in clinical trials.

Role of growth arrest-specific gene 6 in the development of fungal allergic airway disease in mice.

Growth arrest-specific gene (Gas)6 is a secreted vitamin K-dependent protein with pleiotropic effects via activation of receptor tyrosine kinase Tyro3, Axl, and Mertk receptors, but little is known about its role in allergic airway disease. We investigated the role of Gas6 in the development of fungal allergic airway disease in mice. The immune response was evaluated in Gas6-deficient (Gas6-/-) and wild-type (WT) mice and in recombinant Gas6-treated WT mice during Aspergillus fumigatus-induced allergic airway disease. Gas6 plasma levels were significantly elevated in adult clinical asthma of all severities compared with subjects without asthma. In a murine model of fungal allergic airway disease, increased protein expression of Axl and Mertk were observed in the lung. Airway hyperresponsiveness (AHR), whole lung Th2 cytokine levels, goblet cell metaplasia, and peribronchial fibrosis were ameliorated in Gas6-/- mice compared with WT mice with fungal allergic airway disease. Intranasal Gas6 administration into WT mice had a divergent effect on airway inflammation and AHR. Specifically, a total dose of 2 µg of exogenous Gas6 (i.e., low dose) significantly increased whole lung Th2 cytokine levels and subsequent AHR, whereas a total dose of 7 µg of exogenous Gas6 (i.e., high dose) significantly suppressed Th1 and Th2 cytokines and AHR compared with appropriate control groups. Mechanistically, Gas6 promoted Th2 activation via its highest affinity receptor Axl expressed by myeloid DCs. Intranasal administration of Gas6 consistently exacerbated airway remodeling compared with control WT groups. These results demonstrate that Gas6 enhances several features of fungal allergic airway disease.

Regression of eosinophil counts after diagnosis of chronic graft-versus-host disease as a potential marker for improved clinical outcome.

Eosinophilia after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been associated with the development of acute and chronic graft-versus-host disease (cGVHD). However, a limited number of studies have investigated the course of eosinophil counts in relation to the onset of cGVHD. In this study, the course of relative eosinophil counts (RECs) was retrospectively analyzed in 64 patients who developed cGVHD following allogeneic HSCT in relation to overall survival (OS), relapse rate and clinical course of cGVHD. At onset of cGVHD, eosinophilia was observed in 45% of the patients and developed one week prior to cGVHD diagnosis. Furthermore, a trend towards improved OS in patients with eosinophilia was observed. Beneficial effects were most evident in patients who exhibited decreasing eosinophil counts one week after diagnosis of cGVHD. By contrast, an increase in or stable eosinophil counts one week after diagnosis were associated with significantly impaired OS and a significantly higher rate of later aggravation of cGVHD. Findings of this study suggested that the course of eosinophil counts may provide a useful parameter in the assessment of cGVHD development and activity allowing the potential identification of patient subpopulations with a good outcome and reduced cGVHD-related mortality.

Autoantibody-mediated bone loss.

In rheumatoid arthritis (RA), the presence of autoantibodies such as the rheumatoid factor and antibodies against citrullinated proteins is highly correlated with the severity of disease and bone loss. For many years, the involvement of autoantibodies in bone resorption has merely been attributed to enhanced tissue infiltration and the production of inflammatory cytokines that promote osteoclastogenesis. However, recent research provides evidence for a direct activation of osteoclasts and their precursors by autoantibodies, which is independent of inflammation. The depletion of B-cells with rituximab that substantially reduces autoantibody levels seems to be as effective as the well-established treatment with tumor necrosis factor-antagonists in RA patients that do not respond to methotrexate, highlighting the significance of autoantibodies for RA and bone loss.

STAT5-induced lunatic fringe during Th2 development alters delta-like 4-mediated Th2 cytokine production in respiratory syncytial virus-exacerbated airway allergic disease.

Notch activation plays an important role in T cell development and mature T cell differentiation. In this study, we investigated the role of Notch activation in a mouse model of respiratory syncytial virus (RSV)-exacerbated allergic airway disease. During RSV exacerbation, in vivo neutralization of a specific Notch ligand, Delta-like ligand (Dll)-4, significantly decreased airway hyperreactivity, mucus production, and Th2 cytokines. Lunatic Fringe (Lfng), a glycosyltransferase that enhances Notch activation by Dll4, was increased during RSV exacerbation. Lfng loss of function in Th2-skewed cells inhibited Dll4-Notch activation and subsequent IL-4 production. Further knockdown of Lfng in T cells in CD4Cre(+)Lfng(fl/fl) mice showed reduced Th2 response and disease pathology during RSV exacerbation. Finally, we identified STAT5-binding cis-acting regulatory element activation as a critical driver of Lfng transcriptional activation. These data demonstrate that STAT5-dependent amplification of Notch-modifying Lfng augments Th2 response via Dll4 and is critical for amplifying viral exacerbation during allergic airway disease.

Cytokine induced phenotypic and epigenetic signatures are key to establishing specific macrophage phenotypes.

Macrophages (MΦ) play an essential role in innate immune responses and can either display a pro-inflammatory, classically activated phenotype (M1) or undergo an alternative activation program (M2) promoting immune regulation. M-CSF is used to differentiate monocytes into MΦ and IFN-γ or IL-4+IL-13 to further polarize these cells towards M1 or M2, respectively. Recently, differentiation using only GM-CSF or M-CSF has been described to induce a M1- or M2-like phenotype, respectively. In this study, we combined both approaches by differentiating human MΦ in GM-CSF or M-CSF followed by polarization with either IFN-γ or IL-4+IL-13. We describe the phenotypic differences between CD14(hi) CD163(hi) CD206(int) FOLR2-expressing M-CSF MΦ and CD14(lo) CD163(lo) CD206(hi) GM-CSF MΦ but show that both macrophage populations reacted similarly to further polarization with IFN-γ or IL-4+IL-13 with up- and down-regulation of common M1 and M2 marker genes. We also show that high expression of the mannose receptor (CD206), a marker of alternative activation, is a distinct feature of GM-CSF MΦ. Changes of the chromatin structure carried out by chromatin modification enzymes (CME) have been shown to regulate myeloid differentiation. We analyzed the expression patterns of CME during MΦ polarization and show that M1 up-regulate the histone methyltransferase MLL and demethylase KDM6B, while resting and M2 MΦ were characterized by DNA methyltransferases and histone deacetylases. We demonstrate that MLL regulates CXCL10 expression and that this effect could be abrogated using a MLL-Menin inhibitor. Taken together we describe the distinct phenotypic differences of GM-CSF or M-CSF MΦ and demonstrate that MΦ polarization is regulated by specific epigenetic mechanisms. In addition, we describe a novel role for MLL as marker for classical activation. Our findings provide new insights into MΦ polarization that could be helpful to distinguish MΦ activation states.

Murine cytomegalovirus immediate-early 1 gene expression correlates with increased GVHD after allogeneic hematopoietic cell transplantation in recipients reactivating from latent infection.

The success of allogeneic (allo) hematopoietic cell transplantation (HCT) is limited by its treatment related complications, mostly graft versus host disease (GVHD) and fungal and viral infections. CMV reactivation after HCT has been associated with increased morbidity and mortality, and a causal relation between GVHD, immunosuppressive therapy and vice versa has been postulated. Using a low GVHD severity murine HCT model, we assessed the role of MCMV reactivation and GVHD development. BALB/c mice were infected with either murine CMV (MCMV) or mock and monitored for 25 weeks to establish latency, followed by sublethal irradiation conditioning and infusion of bone marrow plus splenocytes from either syngeneic (syn) BALB/c or allo B10.D2 donors. Engraftment of allo donor cells was confirmed by PCR for D2Mit265 gene product size. Day+100 mortality and overall GVHD severity in allo MCMV pre-infected recipients was higher than in allo mock controls. Pathologic changes of lung and liver GVHD in immediate-early gene 1 (IE1) positive recipients were significantly increased compared to mock controls, and were only slightly increased in IE1 negative. No significant gut injury was seen in any group. Aggravated lung injury in IE1 positive recipients correlated with higher BAL cell counts both for total cells and for CD4+ T cells when compared with mock controls, and also with protein expression of lung IFN-gamma and liver TNF. No evidence for CMV specific morphologic changes was seen on histopathology in any organ of IE1 positive recipients, suggesting that CMV reactivation is related to increased GVHD severity but does not require active CMV disease, strengthening the concept of a reciprocal relationship between CMV and GVHD.

CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells.

Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p<0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.

The chemokine system: a possible therapeutic target in acute graft versus host disease.

Allogeneic hematopoetic stem cell transplantation often presents the only chance for cure in a number of malignant and nonmalignant hematologic diseases. However, its beneficial effects are counterweighed by the development of potentially lethal complications, most importantly the development of acute and chronic graft-vs.-host disease (GVHD). Alloantigen-reactive immune responses mediate injury and destruction of GVHD target organs, including the gastrointestinal tract, the liver, the skin, and the lung. Donor leukocyte infiltration into the respective tissues is orchestrated by interactions between chemokines and chemokine receptors, which will be reviewed using a basic science - clinical comparative approach.

Co-ordinated regulation of plasmacytoid dendritic cell surface receptors upon stimulation with herpes simplex virus type 1.

Human plasmacytoid dendritic cells (PDC) are crucial for innate and adaptive immune responses against viral infections, mainly through production of type I interferons. Evidence is accumulating that PDC surface receptors play an important role in this process. To investigate the PDC phenotype in more detail, a chip-based expression analysis of surface receptors was combined with respective flow cytometry data obtained from fresh PDC, PDC exposed to interleukin-3 (IL-3) and/or herpes simplex virus type 1 (HSV-1). CD156b, CD229, CD305 and CD319 were newly identified on the surface of PDC, and CD180 was identified as a new intracellular antigen. After correction for multiple comparisons, a total of 33 receptors were found to be significantly regulated upon exposure to IL-3, HSV-1 or IL-3 and HSV-1. These were receptors involved in chemotaxis, antigen uptake, activation and maturation, migration, apoptosis, cytotoxicity and costimulation. Infectious and ultraviolet-inactivated HSV-1 did not differentially affect surface receptor regulation, consistent with the lack of productive virus infection in PDC, which was confirmed by HSV-1 real-time polymerase chain reaction and experiments involving autofluorescing HSV-1 particles. Viral entry was mediated at least in part by endocytosis. Time-course experiments provided evidence of a co-ordinated regulation of PDC surface markers, which play a specific role in different aspects of PDC function such as attraction to inflamed tissue, antigen recognition and subsequent migration to secondary lymphatic tissue. This knowledge can be used to investigate PDC surface receptor functions in interactions with other cells of the innate and adaptive immune system, particularly natural killer cells and cytotoxic T lymphocytes.

Impaired plasmacytoid dendritic cell innate immune responses in patients with herpes virus-associated acute retinal necrosis.

Plasmacytoid dendritic cells (PDC), the main producers of type I IFNs in the blood, are important for the recognition and control of viral and bacterial infections. Because several viruses induce IFN-alpha production, severe courses of herpes virus infections in nonimmunocompromised patients may be related to numerical or functional PDC deficits. To evaluate this hypothesis, PBMC and PDC were repeatedly isolated from nine patients with acute retinal necrosis (ARN), caused by herpes simplex or varicella zoster virus. The patients experienced meningitis/encephalitis and frequent infections in childhood (n = 2), recurrent herpes virus infections at unusual localizations (n = 2), ocular surgery (n = 1), infections (n = 4), and stress around ARN (n = 6). The median percentage of isolated PDC was significantly lower in patients compared with 18 age-matched healthy controls (p < 0.001), confirmed by FACS analysis using peripheral blood, and was extremely low during acute disease. PDC counts dropped in five controls suffering from respiratory infections or diarrhea. IFN-alpha production in PDC and PBMC exposed to different stimuli was significantly lower in patients than in controls (p < 0.05). Anergy to these stimuli was observed on four occasions, in particular during acute disease. PDC of patients showed up-regulated IFN regulatory factor-7 mRNA levels and evidence of in vivo activation (CD80) and maturation (CD83) (p < 0.05). CD8+ cell responses were significantly lower in patients vs controls (p = 0.04). These data support a risk factor model in which numerical and functional deficits in PDC-mediated innate immune responses contribute to an impaired control of latent herpes virus infections and subsequent development of ARN.