Shaving Is an Epiphenomenon of Type I and II Anti-CD20-Mediated Phagocytosis, whereas Antigenic Modulation Limits Type I Monoclonal Antibody Efficacy.

Rituximab is an anti-CD20 mAb used in the treatment of B cell malignancies. Loss of surface CD20 Ag from the surface of target cells is thought to be one mechanism governing resistance to rituximab, but how this occurs is not completely understood. Two explanations for this have been proposed: antigenic modulation whereby mAb:CD20 complexes are internalized in a B cell intrinsic process and shaving, in which mAb:CD20 complexes undergo trogocytic removal by effector cells, such as macrophages. However, there is conflicting evidence as to which predominates in clinical scenarios and hence the best strategies to overcome resistance. In this study, we investigated the relative importance of modulation and shaving in the downregulation of surface mAb:CD20. We used both murine and human systems and treated ex vivo macrophages with varying concentrations of non-FcγR-interacting beads to achieve differential macrophage saturation states, hence controllably suppressing further phagocytosis of target cells. We then monitored the level and localization of mAb:CD20 using a quenching assay. Suppression of phagocytosis with bead treatment decreased shaving and increased modulation, suggesting that the two compete for surface rituximab:CD20. Under all conditions tested, modulation predominated in rituximab loss, whereas shaving represented an epiphenomenon to phagocytosis. We also demonstrate that the nonmodulating, glycoengineered, type II mAb obinutuzumab caused a modest but significant increase in shaving compared with type II BHH2 human IgG1 wild-type mAb. Therefore, shaving may represent an important mechanism of resistance when modulation is curtailed, and glycoengineering mAb to increase affinity for FcγR may enhance resistance because of shaving.

Escherichia coli K1 utilizes host macropinocytic pathways for invasion of brain microvascular endothelial cells.

Eukaryotic cells utilize multiple endocytic pathways for specific uptake of ligands or molecules, and these pathways are commonly hijacked by pathogens to enable host cell invasion. Escherichia coli K1, a pathogenic bacterium that causes neonatal meningitis, invades the endothelium of the blood-brain barrier, but the entry route remains unclear. Here, we demonstrate that the bacteria trigger an actin-mediated uptake route, stimulating fluid phase uptake, membrane ruffling and macropinocytosis. The route of uptake requires intact lipid rafts as shown by cholesterol depletion. Using a variety of perturbants we demonstrate that small Rho GTPases and their downstream effectors have a significant effect on bacterial invasion. Furthermore, clathrin-mediated endocytosis appears to play an indirect role in E. coli K1 uptake. The data suggest that the bacteria effect a complex interplay between the Rho GTPases to increase their chances of uptake by macropinocytosis into human brain microvascular endothelial cells.

A Stromal Cell Niche for Human and Mouse Type 3 Innate Lymphoid Cells.

Adaptive immunity critically depends on the functional compartmentalization of secondary lymphoid organs. Mesenchymal stromal cells create and maintain specialized niches that support survival, activation, and expansion of T and B cells, and integrated analysis of lymphocytes and their niche has been instrumental in understanding adaptive immunity. Lymphoid organs are also home to type 3 innate lymphoid cells (ILC3), innate effector cells essential for barrier immunity. However, a specialized stromal niche for ILC3 has not been identified. A novel lineage-tracing approach now identifies a subset of murine fetal lymphoid tissue organizer cells that gives rise exclusively to adult marginal reticular cells. Moreover, both cell types are conserved from mice to humans and colocalize with ILC3 in secondary lymphoid tissues throughout life. In sum, we provide evidence that fetal stromal organizers give rise to adult marginal reticular cells and form a dedicated stromal niche for innate ILC3 in adaptive lymphoid organs.

Oxidative stress-induced mitochondrial dysfunction drives inflammation and airway smooth muscle remodeling in patients with chronic obstructive pulmonary disease.

BACKGROUND: Inflammation and oxidative stress play critical roles in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial oxidative stress might be involved in driving the oxidative stress-induced pathology. OBJECTIVE: We sought to determine the effects of oxidative stress on mitochondrial function in the pathophysiology of airway inflammation in ozone-exposed mice and human airway smooth muscle (ASM) cells. METHODS: Mice were exposed to ozone, and lung inflammation, airway hyperresponsiveness (AHR), and mitochondrial function were determined. Human ASM cells were isolated from bronchial biopsy specimens from healthy subjects, smokers, and patients with COPD. Inflammation and mitochondrial function in mice and human ASM cells were measured with and without the presence of the mitochondria-targeted antioxidant MitoQ. RESULTS: Mice exposed to ozone, a source of oxidative stress, had lung inflammation and AHR associated with mitochondrial dysfunction and reflected by decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial oxidative stress, and reduced mitochondrial complex I, III, and V expression. Reversal of mitochondrial dysfunction by the mitochondria-targeted antioxidant MitoQ reduced inflammation and AHR. ASM cells from patients with COPD have reduced ΔΨm, adenosine triphosphate content, complex expression, basal and maximum respiration levels, and respiratory reserve capacity compared with those from healthy control subjects, whereas mitochondrial reactive oxygen species (ROS) levels were increased. Healthy smokers were intermediate between healthy nonsmokers and patients with COPD. Hydrogen peroxide induced mitochondrial dysfunction in ASM cells from healthy subjects. MitoQ and Tiron inhibited TGF-ß-induced ASM cell proliferation and CXCL8 release. CONCLUSIONS: Mitochondrial dysfunction in patients with COPD is associated with excessive mitochondrial ROS levels, which contribute to enhanced inflammation and cell hyperproliferation. Targeting mitochondrial ROS represents a promising therapeutic approach in patients with COPD.

Blood flukes exploit Peyer's Patch lymphoid tissue to facilitate transmission from the mammalian host.

Schistosomes are blood-dwelling parasitic helminths which produce eggs in order to facilitate transmission. Intestinal schistosomes lay eggs in the mesenteries, however, it is unclear how their eggs escape the vasculature to exit the host. Using a murine model of infection, we reveal that Schistosoma mansoni exploits Peyer's Patches (PP) gut lymphoid tissue as a preferential route of egress for their eggs. Egg deposition is favoured within PP as a result of their more abundant vasculature. Moreover, the presence of eggs causes significant vascular remodeling leading to an expanded venule network. Egg deposition results in a decrease in stromal integrity and lymphoid cellularity, including secretory IgA producing lymphocytes, and the focal recruitment of macrophages. In mice lacking PP, egg excretion is significantly impaired, leading to greater numbers of ova being entrapped in tissues and consequently, exacerbated morbidity. Thus, we demonstrate how schistosomes directly facilitate transmission from the host by targeting lymphoid tissue. For the host, PP-dependency of egg egress represents a trade-off, as limiting potentially life-threatening morbidity is balanced by loss of PP structure and perturbed PP IgA production.

IL-7-producing stromal cells are critical for lymph node remodeling.

Nonhematopoietic stromal cells of secondary lymphoid organs form important scaffold and fluid transport structures, such as lymph node (LN) trabeculae, lymph vessels, and conduits. Furthermore, through the production of chemokines and cytokines, these cells generate a particular microenvironment that determines lymphocyte positioning and supports lymphocyte homeostasis. IL-7 is an important stromal cell-derived cytokine that has been considered to be derived mainly from T-cell zone fibroblastic reticular cells. We show here that lymphatic endothelial cells (LECs) are a prominent source of IL-7 both in human and murine LNs. Using bacterial artificial chromosome transgenic IL-7-Cre mice, we found that fibroblastic reticular cells and LECs strongly up-regulated IL-7 expression during LN remodeling after viral infection and LN reconstruction after avascular transplantation. Furthermore, IL-7-producing stromal cells contributed to de novo formation of LyveI-positive lymphatic structures connecting reconstructed LNs with the surrounding tissue. Importantly, diphtheria toxin-mediated depletion of IL-7-producing stromal cells completely abolished LN reconstruction. Taken together, this study identifies LN LECs as a major source of IL-7 and shows that IL-7-producing stromal cells are critical for reconstruction and remodeling of the distinct LN microenvironment.

Galectin-1-expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow.

In the bone marrow (BM), stromal cells constitute a supportive tissue indispensable for the generation of pro-B/pre-BI, pre-BII, and immature B lymphocytes. IL-7-producing stromal cells constitute a cellular niche for pro-B/pre-BI cells, but no specific stromal cell microenvironment was identified for pre-BII cells expressing a functional pre-B cell receptor (pre-BCR). However expression of the pre-BCR represents a crucial checkpoint during B-cell development. We recently demonstrated that the stromal cell derived-galectin1 (GAL1) is a ligand for the pre-BCR, involved in the proliferation and differentiation of normal mouse pre-BII cells. Here we show that nonhematopoietic osteoblasts and reticular cells in the BM express GAL1. We observed that pre-BII cells, unlike the other B-cell subsets, were specifically localized in close contact with GAL1(+) reticular cells. We also determined that IL-7(+) and GAL1(+) cells represent 2 distinct mesenchymal populations with different BM localization. These results demonstrate the existence of a pre-BII specific stromal cell niche and indicate that early B cells move from IL-7(+) to GAL1(+) supportive BM niches during their development.

Developing a 3D B Cell Lymphoma Culture System to Model Antibody Therapy.

Diffuse large cell B cell lymphoma (DLBCL) accounts for approximately 30%-40% of all non-Hodgkin lymphoma (NHL) cases. Current first line DLBCL treatment results in long-term remission in more than 60% of cases. However, those patients with primary refractory disease or early relapse exhibit poor prognosis, highlighting a requirement for alternative therapies. Our aim was to develop a novel model of DLBCL that facilitates in vitro testing of current and novel therapies by replicating key components of the tumor microenvironment (TME) in a three-dimensional (3D) culture system that would enable primary DLBCL cell survival and study ex vivo. The TME is a complex ecosystem, comprising malignant and non-malignant cells, including cancer-associated fibroblasts (CAF) and tumor-associated macrophages (TAM) whose reciprocal crosstalk drives tumor initiation and growth while fostering an immunosuppressive milieu enabling its persistence. The requirement to recapitulate, at least to some degree, this complex, interactive network is exemplified by the rapid cell death of primary DLBCL cells removed from their TME and cultured alone in vitro. Building on previously described methodologies to generate lymphoid-like fibroblasts from adipocyte derived stem cells (ADSC), we confirmed lymphocytes, specifically B cells, interacted with this ADSC-derived stroma, in the presence or absence of monocyte-derived macrophages (MDM), in both two-dimensional (2D) cultures and a 3D collagen-based spheroid system. Furthermore, we demonstrated that DLBCL cells cultured in this system interact with its constituent components, resulting in their improved viability as compared to ex-vivo 2D monocultures. We then assessed the utility of this system as a platform to study therapeutics in the context of antibody-directed phagocytosis, using rituximab as a model immunotherapeutic antibody. Overall, we describe a novel 3D spheroid co-culture system comprising key components of the DLBCL TME with the potential to serve as a testbed for novel therapeutics, targeting key cellular constituents of the TME, such as CAF and/or TAM.

IL7-hCD25 and IL7-Cre BAC transgenic mouse lines: new tools for analysis of IL-7 expressing cells.

IL-7 is a cytokine that is required for T-cell development and homeostasis as well as for lymph node organogenesis. Despite the importance of IL-7 in the immune system and its potential therapeutic relevance, questions remain regarding the sites of IL-7 synthesis, specific cell types involved and molecular mechanisms regulating IL-7 expression. To address these issues, we generated two bacterial artificial chromosome (BAC) transgenic mouse lines in which IL-7 regulatory elements drive expression of either Cre recombinase or a human CD25 (hCD25) cell surface reporter molecule. Expression of the IL-7.hCD25 BAC transgene, detected by reactivity with anti-hCD25 antibody, mimicked endogenous IL-7 expression. Fetal and adult tissues from crosses between IL-7.Cre transgenic mice and Rosa26R or R26-EYFP reporters demonstrated X-gal or YFP staining in tissues known to express endogenous IL-7 at some stage during development. These transgenic lines provide novel genetic tools to identify IL-7 producing cells in various tissues and to manipulate gene expression selectively in IL-7 expressing cells.

Environmental dimensionality controls the interaction of phagocytes with the pathogenic fungi Aspergillus fumigatus and Candida albicans.

The fungal pathogens Aspergillus fumigatus and Candida albicans are major health threats for immune-compromised patients. Normally, macrophages and neutrophil granulocytes phagocytose inhaled Aspergillus conidia in the two-dimensional (2-D) environment of the alveolar lumen or Candida growing in tissue microabscesses, which are composed of a three-dimensional (3-D) extracellular matrix. However, neither the cellular dynamics, the per-cell efficiency, the outcome of this interaction, nor the environmental impact on this process are known. Live imaging shows that the interaction of phagocytes with Aspergillus or Candida in 2-D liquid cultures or 3-D collagen environments is a dynamic process that includes phagocytosis, dragging, or the mere touching of fungal elements. Neutrophils and alveolar macrophages efficiently phagocytosed or dragged Aspergillus conidia in 2-D, while in 3-D their function was severely impaired. The reverse was found for phagocytosis of Candida. The phagocytosis rate was very low in 2-D, while in 3-D most neutrophils internalized multiple yeasts. In competitive assays, neutrophils primarily incorporated Aspergillus conidia in 2-D and Candida yeasts in 3-D despite frequent touching of the other pathogen. Thus, phagocytes show activity best in the environment where a pathogen is naturally encountered. This could explain why "delocalized" Aspergillus infections such as hematogeneous spread are almost uncontrollable diseases, even in immunocompetent individuals.

Epidermal Notch1 recruits RORγ(+) group 3 innate lymphoid cells to orchestrate normal skin repair.

Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ(+) ILC3s into wounded dermis; RORγ(+) ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ(+) ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair.

Serodiagnosis of dengue during an outbreak at a tertiary care hospital in Delhi.

Dengue, an important human arboviral infection, is endemic in many parts of India. Outbreaks are now reported quite frequently from different parts of the country. We report here findings of serological investigation of serum samples received during a recent outbreak of dengue infection in Delhi from September to November 2003. Acute phase serum samples from suspected cases of dengue infection (including dengue fever, dengue haemorrhagic fever and dengue shock syndrome) of duration 5 days, were tested for dengue specific IgM antibodies by enzyme linked immuno sorbant assay (ELISA). Of the 874 serum samples tested, 456 (52.3% ) were positive for dengue specific IgM antibodies. The maximum number of positive cases was reported in October, peaking in second and third week. Patients in the age group of 21-30 yr accounted for 34.2 per cent of positive cases. This outbreak was less severe as compared to the previous 1996 outbreak and showed a shift towards an older age group.

Fate mapping reveals origin and dynamics of lymph node follicular dendritic cells.

Follicular dendritic cells (FDCs) regulate B cell function and development of high affinity antibody responses but little is known about their biology. FDCs associate in intricate cellular networks within secondary lymphoid organs. In vitro and ex vivo methods, therefore, allow only limited understanding of the genuine immunobiology of FDCs in their native habitat. Herein, we used various multicolor fate mapping systems to investigate the ontogeny and dynamics of lymph node (LN) FDCs in situ. We show that LN FDC networks arise from the clonal expansion and differentiation of marginal reticular cells (MRCs), a population of lymphoid stromal cells lining the LN subcapsular sinus. We further demonstrate that during an immune response, FDCs accumulate in germinal centers and that neither the recruitment of circulating progenitors nor the division of local mature FDCs significantly contributes to this accumulation. Rather, we provide evidence that newly generated FDCs also arise from the proliferation and differentiation of MRCs, thus unraveling a critical function of this poorly defined stromal cell population.

Inhibition of receptor tyrosine kinases restores immunocompetence and improves immune-dependent chemotherapy against experimental leishmaniasis in mice.

Receptor tyrosine kinases are involved in multiple cellular processes, and drugs that inhibit their action are used in the clinic to treat several types of cancer. However, the value of receptor tyrosine kinase inhibitors (RTKIs) for treating infectious disease has yet to be explored. Here, we have shown in mice that administration of the broad-spectrum RTKI sunitinib maleate (Sm) blocked the vascular remodeling and progressive splenomegaly associated with experimental visceral leishmaniasis. Furthermore, Sm treatment restored the integrity of the splenic microarchitecture. Although restoration of splenic architecture was accompanied by an increase in the frequency of IFN-gamma+CD4+ T cells, Sm treatment alone was insufficient to cause a reduction in tissue parasite burden. However, preconditioning by short-term Sm treatment proved to be successful as an adjunct therapy, increasing the frequency of IFN-gamma+ and IFN-gamma+TNF+CD4+ T cells, enhancing NO production by splenic macrophages, and providing dose-sparing effects when combined with a first-line immune-dependent anti-leishmanial drug. We propose, therefore, that RTKIs may prove clinically useful as agents to restore immune competence before the administration of chemo- or immunotherapeutic drugs in the treatment of visceral leishmaniasis or other diseases involving lymphoid tissue remodeling, including cancer.