%0 Journal Article
%~ PubMed
%A Mitchell, Andrew J
%A Yau, Belinda
%A McQuillan, James A
%A Ball, Helen J
%A Too, Lay Khoon
%A Abtin, Arby
%A Hertzog, Paul
%A Leib, Stephen L
%A Jones, Cheryl A
%A Gerega, Sebastien K
%A Weninger, Wolfgang
%A Hunt, Nicholas H
%T Inflammasome-Dependent IFN-γ Drives Pathogenesis in Streptococcus pneumoniae Meningitis.
%B Journal of Immunology
%D 2012
%C United States
%I American Association of Immunologists
%V 189
%N 10
%P 4970-4980
%@ 1550-6606
%X 
%Z FOR Codes: 110801


%0 Conference Proceedings
%A Keeble, J
%A Goh, C C
%A Wang, Y
%A Weninger, Wolfgang
%A Ng, L G
%T Intravital multiphoton imaging of immune cells
%B Singaporean-French Bioimaging Workshop
%D 2012
%C Singapore September 2011
%I Springer- Verlag
%V 120
%N 
%P 3-16
%@ 
%E Lom nie, Nicholas
%E Gouaillard, Alexandre
%E Racoceanu, Daniel
%X 
%Z FOR Codes: 29903


%0 Journal Article
%~ PubMed
%A Li, Jackson LiangYao
%A Goh, Chi Ching
%A Keeble, Jo L
%A Qin, Jim S
%A Roediger, Ben
%A Jain, Rohit
%A Wang, Yilin
%A Chew, Weng Keong
%A Weninger, Wolfgang
%A Ng, Lai Guan
%T Intravital multiphoton imaging of immune responses in the mouse ear skin.
%B Nature Protocols
%D 2012
%C United Kingdom
%I Nature Publishing Group
%V 7
%N 2
%P 221-234
%@ 1750-2799
%X Multiphoton (MP) microscopy enables the direct in vivo visualization, with high spatial and temporal resolution, of fluorescently tagged immune cells, extracellular matrix and vasculature in tissues. This approach, therefore, represents a powerful alternative to traditional methods of assessing immune cell function in the skin, which are mainly based on flow cytometry and histology. Here we provide a step-by-step protocol describing experimental procedures for intravital MP imaging of the mouse ear skin, which can be easily adapted to address many specific skin-related biological questions. We demonstrate the use of this procedure by characterizing the response of neutrophils during cutaneous inflammation, which can be used to perform in-depth analysis of neutrophil behavior in the context of the skin microanatomy, including the epidermis, dermis and blood vessels. Such experiments are typically completed within 1 d, but as the procedures are minimally invasive, it is possible to perform longitudinal studies through repeated imaging.
%Z FOR Codes: 110304


%0 Journal Article
%~ PubMed
%A Lucas, Keryn M
%A Mohana-Kumaran, Nethia
%A Lau, Diana T
%A Zhang, Xu Dong
%A Hersey, Peter
%A Huang, David
%A Weninger, Wolfgang
%A Haass, Nikolas K
%A Allen, John D
%T Modulation of NOXA and MCL-1 as a strategy for sensitizing melanoma cells to the BH3-mimetic ABT-737.
%B Clinical Cancer Research
%D 2012
%C United States
%I American Association for Cancer Research
%V 18
%N 3
%P 783-795
%@ 1078-0432
%X PURPOSE:  Drug resistance in melanoma is commonly attributed to ineffective apoptosis pathways. Inhibiting antiapoptotic BCL-2 and its relatives is an attractive strategy for sensitizing lymphoid malignancies to drugs but it has been largely unsuccessful for melanoma and other solid tumors. ABT-737, a small-molecule BH3-mimetic, selectively inhibits BCL-2, BCL-XL and BCL-w and shows promise for treating leukemia, lymphoma and small cell lung cancer. Melanoma cells are insensitive to ABT-737 but MCL-1 inhibition reportedly increases the sensitivity of other tumors to the compound. EXPERIMENTAL DESIGN:  The efficacy of MCL-1 and BFL-1 inhibition for sensitizing melanoma cells to ABT-737 was investigated by shRNA-mediated knockdown or overexpression of their antagonist NOXA in two-dimensional cell culture, a three-dimensional organotypic spheroid model, and an in vivo model.RESULTS:  MCL-1 downregulation or NOXA overexpression strongly sensitized melanoma cells to ABT-737 in vitro. NOXA-inducing cytotoxic drugs also strongly sensitized melanomas to ABT-737 although, surprisingly, not vice versa. The drugs most suitable are not necessarily those normally used to treat melanoma. Resistance to ABT-737 occurred quickly in 3D melanoma spheroids through reduced NOXA expression although experiments with both xenografts and 3D spheroids suggest that penetration of ABT-737 into tumor masses may be the principal limitation, which may be obviated through use of more diffusible BH3-mimetics.CONCLUSION:  Sensitization of tumors to BH3-mimetics by cytotoxic drugs that induce NOXA is a therapeutic strategy worth exploring for the treatment of melanoma and other solid cancers.
%Z FOR Codes: 111204 111201 111207


%0 Journal Article
%~ PubMed
%A Tan, Sioh-Yang
%A Cavanagh, Lois L
%A d'Advigor, William
%A Shackel, Nick
%A de St Groth, Barbara Fazekas
%A Weninger, Wolfgang
%T Phenotype and functions of conventional dendritic cells are not compromised in aged mice.
%B Immunology and Cell Biology
%D 2012
%C United Kingdom, Australia
%I Nature Publishing Group
%V 90
%N 7
%P 722-732
%@ 0818-9641
%X Aging has profound effects on the immune system, including thymic involution, reduced diversity of the T cell receptor repertoire, reduced effector T cell and B cell function and chronic increase of proinflammatory cytokine production by innate immune cells. The precise effects of aging on conventional dendritic cells (cDC), the main antigen presenting cells of the immune system, however, are not well understood. We found that in aged mice the number of cDC in the spleen and lymph nodes remained stable, whereas the number of cDC in the lungs increased with age. Whereas cDC in mice showed similar cycling kinetics in all organs tested, cDC reconstitution by aged bone marrow precursors was relatively higher than that of their young counterparts. With the exception of CD86, young and aged cDC did not differ in their expression of co-stimulatory molecules at steady state. Most toll-like receptor (TLR) ligands induced comparable upregulation of co-stimulatory molecules CD40, CD86 and B7H1 on young and aged cDC, whereas TLR2 and TLR5 stimulation resulted in reduced upregulation of CD80 and CD86 on aged cDC in vitro. In vivo, influenza infection-induced upregulation of CD86, but not other co-stimulatory molecules, was lower in aged DC. Young and aged DC were equally capable of direct and cross presentation of antigens in vitro. Transcriptome analysis did not reveal any significant difference between young and aged cDC. These data show that unlike T and B cells, the maintenance of cDC throughout the life of a healthy animal is relatively robust during the aging process.Immunology and Cell Biology advance online publication, 10 January 2012; doi:10.1038/icb.2011.104.
%Z FOR Codes: 110704


%0 Journal Article
%~ PubMed
%A Pai, Saparna
%A Danne, Karyn J
%A Qin, Jim
%A Cavanagh, Lois L
%A Smith, Adrian
%A Hickey, Michael J
%A Weninger, Wolfgang
%T Visualizing leukocyte trafficking in the living brain with 2-photon intravital microscopy.
%B Frontiers in Cellular Neuroscience
%D 2012
%C Switzerland
%I Frontiers Research Foundation
%V 6
%N 
%P 67
%@ 1662-5102
%X 
%Z FOR Codes: 1109


%0 Journal Article
%~ PubMed
%A Sumaria, Nital
%A Roediger, Ben
%A Ng, Lai Guan
%A Qin, Jim
%A Pinto, Rachel
%A Cavanagh, Lois L
%A Shklovskaya, Elena
%A Fazekas de St Groth, Barbara
%A Triccas, James A
%A Weninger, Wolfgang
%T Cutaneous immunosurveillance by self-renewing dermal gammadelta T cells.
%B The Journal of Experimental Medicine
%D 2011
%C United States
%I Rockefeller University Press
%V 208
%N 3
%P 505-518
%@ 1540-9538
%X The presence of ???? T cell receptor (TCR)-expressing cells in the epidermis of mice, termed dendritic epidermal T cells (DETCs), is well established. Because of their strict epidermal localization, it is likely that DETCs primarily respond to epithelial stress, such as infections or the presence of transformed cells, whereas they may not participate directly in dermal immune responses. In this study, we describe a prominent population of resident dermal ???? T cells, which differ from DETCs in TCR usage, phenotype, and migratory behavior. Dermal ???? T cells are radioresistant, cycle in situ, and are partially depend on interleukin (IL)-7, but not IL-15, for their development and survival. During mycobacterial infection, dermal ???? T cells are the predominant dermal cells that produce IL-17. Absence of dermal ???? T cells is associated with decreased expansion in skin draining lymph nodes of CD4(+) T cells specific for an immunodominant Mycobacterium tuberculosis epitope. Decreased CD4(+) T cell expansion is related to a reduction in neutrophil recruitment to the skin and decreased BCG shuttling to draining lymph nodes. Thus, dermal ???? T cells are an important part of the resident cutaneous immunosurveillance program. Our data demonstrate functional specialization of T cells in distinct microcompartments of the skin.
%Z FOR Codes: 110707


%0 Journal Article
%~ PubMed
%A Roediger, Ben
%A Weninger, Wolfgang
%T How nickel turns on innate immune cells.
%B Immunology and Cell Biology
%D 2011
%C United Kingdom, Australia
%I Nature Publishing Group
%V 89
%N 1
%P 1-2
%@ 0818-9641
%X 
%Z FOR Codes: 110701


%0 Journal Article
%~ PubMed
%A Shklovskaya, Elena
%A O'Sullivan, Brendan J
%A Ng, Lai Guan
%A Roediger, Ben
%A Thomas, Ranjeny
%A Weninger, Wolfgang
%A Fazekas de St Groth, Barbara
%T Langerhans cells are precommitted to immune tolerance induction.
%B Proceedings of the National Academy of Sciences of the United States of America
%D 2011
%C United States
%I National Academy of Sciences
%V 108
%N 44
%P 18049-18054
%@ 0027-8424
%X Antigen-dependent interactions between T lymphocytes and dendritic cells (DCs) can produce two distinct outcomes: tolerance and immunity. It is generally considered that all DC subsets are capable of supporting both tolerogenic and immunogenic responses, depending on their exposure to activating signals. Here, we tested whether epidermal Langerhans cells (LCs) can support immunogenic responses in vivo in the absence of antigen presentation by other DC subsets. CD4 T cells responding to antigen presentation by activated LCs initially proliferated but then failed to differentiate into effector/memory cells or to survive long term. The tolerogenic function of LCs was maintained after exposure to potent adjuvants and occurred despite up-regulation of the costimulatory molecules CD80, CD86, and IL-12, but was consistent with their failure to translocate the NF-??B family member RelB from the cytoplasm to the nucleus. Commitment of LCs to tolerogenic function may explain why commensal microorganisms expressing Toll-like receptor (TLR) ligands but confined to the skin epithelium are tolerated, whereas invading pathogens that breach the epithelial basement membrane and activate dermal DCs stimulate a strong immune response.
%Z FOR Codes: 110799


%0 Journal Article
%~ PubMed
%A Kong, Carlyn U
%A Ng, Lai Guan
%A Nambiar, Jonathan K
%A Spratt, Joanne M
%A Weninger, Wolfgang
%A Triccas, James A
%T Targeted induction of antigen expression within dendritic cells modulates antigen-specific immunity afforded by recombinant BCG.
%B Vaccine
%D 2011
%C United Kingdom
%I Elsevier Ltd
%V 29
%N 7
%P 1374-1381
%@ 0264-410X
%X The targeted modulation of antigen expression by recombinant vaccine vehicles would significantly aid development of effective immunotherapeutic strategies. In this report we demonstrate that the Mycobacterium tuberculosis hspX promoter can be used to regulate in vivo induction of antigens expressed by recombinant Bacille Calmette Gu??rin (rBCG). HspX promoter induction occurred rapidly upon entry of rBCG into cultured dendritic cells (DCs), as evidenced by GFP levels in DCs when infected with BCG:P(hspX)-GFP, in which P(hspX) controlled GFP expression. Vaccination of mice with BCG:P(hspX)-GFP led to rapid in vivo induction of GFP associated with an influx of GFP(+) DCs at the infection site. P(hspX)-driven antigen expression resulted in an improved capacity of DCs to prime antigen-specific T cells, as infection of DCs with BCG:P(hspX)-85B, where the hspX promoter controls expression of M. tuberculosis Ag85B, led to enhanced proliferation of Ag85B-reactive CD4(+) T cells compared to BCG overexpressing Ag85B using the strong Mycobacterium bovis hsp60 promoter. This enhancement of rBCG-induced immunity was also evident in vivo; mice vaccinated with BCG:P(hspX)-85B displayed markedly improved generation of Ag85B-reactive IFN-??-secreting T cells compared to control BCG-vaccinated mice, which was most pronounced at extended times points post-vaccination. These data reveal a novel strategy to enhance the development and maintenance of vaccine-specific T cell responses.
%Z FOR Codes: 110801 110704


%0 Journal Article
%~ PubMed
%A Ng, Lai Guan
%A Qin, Jim S
%A Roediger, Ben
%A Wang, Yilin
%A Jain, Rohit
%A Cavanagh, Lois L
%A Smith, Adrian L
%A Jones, Cheryl A
%A de Veer, Michael
%A Grimbaldeston, Michele A
%A Meeusen, Els N
%A Weninger, Wolfgang
%T Visualizing the Neutrophil Response to Sterile Tissue Injury in Mouse Dermis Reveals a Three-Phase Cascade of Events.
%B Journal of Investigative Dermatology
%D 2011
%C United Kingdom, United States
%I Nature Publishing Group
%V 131
%N 10
%P 2058-2068
%@ 1523-1747
%X Neutrophil granulocytes traffic into sites of organ injury in which they may not only participate in tissue repair and pathogen clearance but may also contribute to collateral cell damage through the release of noxious mediators. The dynamics and mechanisms of neutrophil migration in the extravascular space toward loci of tissue damage are not well understood. Here, we have used intravital multi-photon microscopy to dissect the behavior of neutrophils in response to tissue injury in the dermis of mice. We found that, following confined physical injury, initially rare scouting neutrophils migrated in a directional manner toward the damage focus. This was followed by the attraction of waves of additional neutrophils, and finally stabilization of the neutrophil cluster around the injury. Although neutrophil migration in the steady state and during the scouting phase depended on pertussis toxin-sensitive signals, the amplification phase was sensitive to interference with the cyclic adenosine diphosphate ribose pathway. We finally demonstrated that neutrophil scouts also transit through the non-inflamed dermis, suggesting immunosurveillance function by these cells. Together, our data unravel a three-step cascade of events that mediates the specific accumulation of neutrophils at sites of sterile tissue injury in the interstitial space.
%Z FOR Codes: 110707 110304


%0 Journal Article
%~ Isi
%A John, B.
%A Weninger, W.
%A Hunter, C. A.
%T Advances in imaging the innate and adaptive immune response to Toxoplasma gondii
%B Future Microbiology
%D 2010
%C United Kingdom
%I Future Medicine Ltd.
%V 5
%N 9
%P 1321-1328
%@ 1746-0913
%X 
%Z FOR Codes: 110801


%0 Journal Article
%~ PubMed
%A Vukovic, Jana
%A Blomster, Linda V
%A Chinnery, Holly R
%A Weninger, Wolfgang
%A Jung, Steffen
%A McMenamin, Paul G
%A Ruitenberg, Marc J
%T Bone marrow chimeric mice reveal a role for CX3CR1 in maintenance of the monocyte-derived cell population in the olfactory neuroepithelium.
%B Journal of leukocyte biology
%D 2010
%C United States
%I Federation of American Societies for Experimental
%V 88
%N 4
%P 645-54
%@ 1938-3673
%X Macrophages in the olfactory neuroepithelium are thought to play major roles in tissue homeostasis and repair. However, little information is available at present about possible heterogeneity of these monocyte-derived cells, their turnover rates, and the role of chemokine receptors in this process. To start addressing these issues, this study used Cx???cr1(gfp) mice, in which the gene sequence for eGFP was knocked into the CX???CR1 gene locus in the mutant allele. Using neuroepithelial whole-mounts from Cx???cr1(gfp/+) mice, we show that eGFP(+) cells of monocytic origin are distributed in a loose network throughout this tissue and can be subdivided further into two immunophenotypically distinct subsets based on MHC-II glycoprotein expression. BM chimeric mice were created using Cx???cr1(gfp/+) donors to investigate turnover of macrophages (and other monocyte-derived cells) in the olfactory neuroepithelium. Our data indicate that the monocyte-derived cell population in the olfactory neuroepithelium is actively replenished by circulating monocytes and under the experimental conditions, completely turned over within 6 months. Transplantation of Cx???cr1(gfp/gfp) (i.e., CX???CR1-deficient) BM partially impaired the replenishment process and resulted in an overall decline of the total monocyte-derived cell number in the olfactory epithelium. Interestingly, replenishment of the CD68(low)MHC-II(+) subset appeared minimally affected by CX???CR1 deficiency. Taken together, the established baseline data about heterogeneity of monocyte-derived cells, their replenishment rates, and the role of CX???CR1 provide a solid basis to further examine the importance of different monocyte subsets for neuroregeneration at this unique frontier with the external environment.
%Z FOR Codes: 110704


%0 Journal Article
%~ PubMed
%A Mrass, Paulus
%A Petravic, Janka
%A Davenport, Miles P
%A Weninger, Wolfgang
%T Cell-autonomous and environmental contributions to the interstitial migration of T cells.
%B Seminars in immunopathology
%D 2010
%C Germany
%I Springer
%V 32
%N 3
%P 257-74
%@ 1863-2300
%X A key to understanding the functioning of the immune system is to define the mechanisms that facilitate directed lymphocyte migration to and within tissues. The recent development of improved imaging technologies, most prominently multi-photon microscopy, has enabled the dynamic visualization of immune cells in real-time directly within intact tissues. Intravital imaging approaches have revealed high spontaneous migratory activity of T cells in secondary lymphoid organs and inflamed tissues. Experimental evidence points towards both environmental and cell-intrinsic cues involved in the regulation of lymphocyte motility in the interstitial space. Based on these data, several conceptually distinct models have been proposed in order to explain the coordination of lymphocyte migration both at the single cell and population level. These range from "stochastic" models, where chance is the major driving force, to "deterministic" models, where the architecture of the microenvironment dictates the migratory trajectory of cells. In this review, we focus on recent advances in understanding na??ve and effector T cell migration in vivo. In addition, we discuss some of the contradictory experimental findings in the context of theoretical models of migrating leukocytes.
%Z FOR Codes: 60106 110702


%0 Journal Article
%~ PubMed
%A Puttur, Franz K
%A Fernandez, Marian A
%A White, Rose
%A Roediger, Ben
%A Cunningham, Anthony L
%A Weninger, Wolfgang
%A Jones, Cheryl A
%T Herpes Simplex Virus Infects Skin {gamma}{delta} T Cells before Langerhans Cells and Impedes Migration of Infected Langerhans Cells by Inducing Apoptosis and Blocking E-Cadherin Downregulation.
%B Journal of immunology (Baltimore, Md. : 1950)
%D 2010
%C United States
%I American Association of Immunologists
%V 185
%N 1
%P 477-87
%@ 1550-6606
%X The role individual skin dendritic cell (DC) subsets play in the immune response to HSV remains unclear. We investigated the effect of HSV on DC virus uptake, viability, and migration after cutaneous infection in vitro and in vivo. HSV increased the emigration of skin DCs from whole skin explants over 3 d postinfection (p.i.) compared with mock controls, but the kinetics of emigration was influenced by the skin DC subset. Uninfected (bystander) Langerhans cells (LCs) were the major emigrant DC subset at 24 h p.i., but thereafter, large increases in infected CD103(+)langerin(+) dermal DC (dDC) and uninfected langerin(-) dDC emigration were also observed. LC infection was confirmed by the presence of HSV glycoprotein D (gD) and was associated with impaired migration from cultured skin. Langerin(+) dDC also expressed HSV gD, but infection did not impede migration. We then followed the virus in live MacGreen mice in which LCs express GFP using a fluorescent HSV-1 strain by time-lapse confocal microscopy. We observed a sequential infection of epidermal cells, first in keratinocytes and epidermal gammadelta T cells at 6 h p.i., followed by the occurrence of HSVgD(+) LCs at 24 h p.i. HSV induced CCR7 upregulation on all langerin(+) DC, including infected LCs, and increased production of skin TNF-alpha and IL-1beta. However, a large proportion of infected LCs that remained within the skin was apoptotic and failed to downregulate E-cadherin compared with bystander LCs or mock controls. Thus, HSV infection of LCs is preceded by infection of gammadelta T cells and delays migration.
%Z FOR Codes: 110804 110701 110304


%0 Journal Article
%~ PubMed
%A Wilson, Emma H
%A Weninger, Wolfgang
%A Hunter, Christopher A
%T Trafficking of immune cells in the central nervous system.
%B The Journal of clinical investigation
%D 2010
%C United States
%I American Society for Clinical Investigation
%V 120
%N 5
%P 1368-79
%@ 1558-8238
%X The CNS is an immune-privileged environment, yet the local control of multiple pathogens is dependent on the ability of immune cells to access and operate within this site. However, inflammation of the distinct anatomical sites (i.e., meninges, cerebrospinal fluid, and parenchyma) associated with the CNS can also be deleterious. Therefore, control of lymphocyte entry and migration within the brain is vital to regulate protective and pathological responses. In this review, several recent advances are highlighted that provide new insights into the processes that regulate leukocyte access to, and movement within, the brain.
%Z FOR Codes: 110903


%0 Journal Article
%~ PubMed
%A John, Beena
%A Harris, Tajie H
%A Tait, Elia D
%A Wilson, Emma H
%A Gregg, Beth
%A Ng, Lai Guan
%A Mrass, Paulus
%A Roos, David S
%A Dzierszinski, Florence
%A Weninger, Wolfgang
%A Hunter, Christopher A
%T Dynamic Imaging of CD8(+) T cells and dendritic cells during infection with Toxoplasma gondii.
%B PLoS Pathogens
%D 2009
%C United States
%I Public Library of Science
%V 5
%N 7
%P e1000505
%@ 1553-7374
%X To better understand the initiation of CD8(+) T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8(+) T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8(+) T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8(+) T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8(+) T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.
%Z FOR Codes: 110701


%0 Journal Article
%~ PubMed
%A Hoeller, Christoph
%A Richardson, Stephen K
%A Ng, Lai Guan
%A Valero, Teresa
%A Wysocka, Maria
%A Rook, Alain H
%A Weninger, Wolfgang
%T In vivo imaging of cutaneous T-cell lymphoma migration to the skin.
%B Cancer Research
%D 2009
%C United States
%I American Association for Cancer Research (A AC R)
%V 69
%N 7
%P 2704-2708
%@ 0008-5472
%X Cutaneous T-cell lymphoma (CTCL) is characterized by the accumulation of malignant CD4(+) T cells in the skin. Although the expression of adhesion molecules and chemokine receptors on CTCL cells has been studied extensively on ex vivo isolated cells, very little is known about the dynamics and mechanisms of CTCL trafficking in vivo. However, detailed knowledge of the molecular cues mediating CTCL migration may be used to interfere with their homing to the skin. We made use of real-time intravital epifluorescence video and two-photon microscopy to visualize malignant T cells from Sezary syndrome (SS), a leukemic variant of CTCL, in dermal microvessels in mouse ear skin. We found that SS cells rolled along dermal venules in a P-selectin- and E-selectin-dependent manner at ratios similar to CD4(+) memory T cells from normal donors. We furthermore show that the chemokine CCL17/TARC, but not CCL27/CTACK, was sufficient to induce the arrest of SS cells in the microvasculature. However, a combination of both chemokines was required to induce extravasation of SS cells. Together, our experiments delineate the molecular adhesion cascade operant in SS cell homing to the skin in vivo.
%Z FOR Codes: 111299


%0 Journal Article
%~ PubMed
%A Levental, Kandice R
%A Yu, Hongmei
%A Kass, Laura
%A Lakins, Johnathon N
%A Egeblad, Mikala
%A Erler, Janine T
%A Fong, Sheri F T
%A Csiszar, Katalin
%A Giaccia, Amato
%A Weninger, Wolfgang
%A Yamauchi, Mitsuo
%A Gasser, David L
%A Weaver, Valerie M
%T Matrix crosslinking forces tumor progression by enhancing integrin signaling.
%B Cell
%D 2009
%C United States
%I Cell Press
%V 139
%N 5
%P 891-906
%@ 1097-4172
%X Tumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM remodeling to cancer is appreciated; the relevance of stiffening is less clear. We found that breast tumorigenesis is accompanied by collagen crosslinking, ECM stiffening, and increased focal adhesions. Induction of collagen crosslinking stiffened the ECM, promoted focal adhesions, enhanced PI3 kinase (PI3K) activity, and induced the invasion of an oncogene-initiated epithelium. Inhibition of integrin signaling repressed the invasion of a premalignant epithelium into a stiffened, crosslinked ECM and forced integrin clustering promoted focal adhesions, enhanced PI3K signaling, and induced the invasion of a premalignant epithelium. Consistently, reduction of lysyl oxidase-mediated collagen crosslinking prevented MMTV-Neu-induced fibrosis, decreased focal adhesions and PI3K activity, impeded malignancy, and lowered tumor incidence. These data show how collagen crosslinking can modulate tissue fibrosis and stiffness to force focal adhesions, growth factor signaling and breast malignancy.
%Z FOR Codes: 60111 111201


%0 Journal Article
%~ PubMed
%A Wolf, Amaya I
%A Buehler, Darya
%A Hensley, Scott E
%A Cavanagh, Lois L
%A Wherry, E John
%A Kastner, Philippe
%A Chan, Susan
%A Weninger, Wolfgang
%T Plasmacytoid dendritic cells are dispensable during primary influenza virus infection.
%B Journal of Immunology
%D 2009
%C United States
%I American Association of Immunologists
%V 182
%N 2
%P 871-879
%@ 1550-6606
%X Plasmacytoid dendritic cells (pDC) are thought to be pivotal in the first line of defense against viral infections. Although previous studies have suggested that pDC regulate the immune response against respiratory syncytial virus, their role in pulmonary infection with influenza virus has remained unclear. Using mice with GFP-tagged pDC, we observed a marked increase in pDC numbers in the lung airways 3 days after intranasal infection with influenza virus A/PR/8/34. To further investigate their potential involvement in the disease, we made use of pDC-deficient IkarosL/L mice. In the absence of pDC, the recruitment of T cells to the bronchoalveolar space was delayed, which could be reversed by the adoptive transfer of pDC before infection. Surprisingly, however, when compared with wild-type animals, IkarosL/L mice revealed a similar course of disease, as determined by weight loss, viral titers, levels of neutralizing Ab, and lung pathology. Moreover, the activation and differentiation of influenza-specific CD8+ effector T cells was unaltered in the absence of pDC, as was the generation of CD8+ memory T cells. Taken together, our study suggests that pDC regulate the accumulation of T cells in the bronchoalveolar space during early influenza virus infection, but are dispensable for the control of this disease.
%Z FOR Codes: 110701


%0 Journal Article
%A Herbert, O C
%A Barnetson, R
%A Weninger, Wolfgang
%A Kramer, U
%A Behrendt, H
%A Ring, J
%T Western lifestyle and increased prevalence of atopic diseases: a example from a small Papua New Guinean island
%B World Allergy Organization Journal
%D 2009
%C United States
%I Lippincott Williams & Wilkins
%V 2
%N 7
%P 130-137
%@ 1939-4551
%X 
%Z FOR Codes: 110399


%0 Journal Article
%~ PubMed
%A Mrass, Paulus
%A Kinjyo, Ichiko
%A Ng, Lai Guan
%A Reiner, Steven L
%A Puré, Ellen
%A Weninger, Wolfgang
%T CD44 mediates successful interstitial navigation by killer T cells and enables efficient antitumor immunity.
%B Immunity
%D 2008
%C United States
%I Cell Press
%V 29
%N 6
%P 971-85
%@ 1074-7613
%X Although T lymphocytes are constitutively nonadherent cells, they undergo facultative polarity during migration and upon interaction with cells presenting cognate antigen, suggesting that cell polarity might be critical for target cell destruction. Using two-photon imaging of tumor-infiltrating T lymphocytes, we found that CD44, a receptor for extracellular matrix proteins and glycosaminoglycans, was crucial for interstitial T cell navigation and, consequently, efficient tumor cell screening. CD44 functioned as a critical regulator of intratumoral movement by stabilizing cell polarity in migrating T cells, but not during target cell interactions. Stable anterior-posterior asymmetry was maintained by CD44 independently of its extracellular domain. Instead, migratory polarity depended on the recruitment of ezrin, radixin, moesin (ERM) proteins by the intracellular domain of CD44 to the posterior cellular protrusion. Our results formally demonstrate that CD44-dependent T lymphocyte locomotion within target sites represents an essential immunologic checkpoint that determines the potency of T cell effector functions.
%Z FOR Codes: 110304


%0 Journal Article
%~ PubMed
%A Cavanagh, Lois L
%A Weninger, Wolfgang
%T Dendritic cell behaviour in vivo: lessons learned from intravital two-photon microscopy.
%B Immunology and cell biology
%D 2008
%C United Kingdom
%I Nature Publishing Group
%V 86
%N 5
%P 428-38
%@ 1440-1711
%X Dendritic cells (DC) are central regulators of immune responses. Their functional characterization has, thus far, mainly relied on the analysis of ex vivo isolated cells or immunohistology, which provides information in a static manner. While these approaches have enabled an excellent understanding of the role of DC in antigen uptake, processing and presentation, there has been a clear need to investigate the behaviour of DC in the context of intact tissues in real time. This demand has recently been met by the availability of intravital two-photon microscopy, which allows for the visualization of single cells deep within intact organs over time. Thus, during the past few years, exciting new data have been generated as to how DC behave within secondary lymphoid and peripheral tissues both under homoeostatic and inflammatory conditions. Here, we will review what two-photon microscopy studies have taught us about the migration of DC in the interstitial space as well as their interactions with adaptive immune cells.
%Z FOR Codes: 110704


%0 Journal Article
%~ PubMed
%A Acharya, Pinak S
%A Majumdar, Sonali
%A Jacob, Michele
%A Hayden, James
%A Mrass, Paul
%A Weninger, Wolfgang
%A Assoian, Richard K
%A Puré, Ellen
%T Fibroblast migration is mediated by CD44-dependent TGF beta activation.
%B Journal of cell science
%D 2008
%C United Kingdom
%I The Company of Biologists Ltd
%V 121
%N Pt 9
%P 1393-1402
%@ 0021-9533
%X CD44 contributes to inflammation and fibrosis in response to injury. As fibroblast recruitment is critical to wound healing, we compared cytoskeletal architecture and migration of wild-type (CD44WT) and CD44-deficient (CD44KO) fibroblasts. CD44KO fibroblasts exhibited fewer stress fibers and focal adhesion complexes, and their migration was characterized by increased velocity but loss of directionality, compared with CD44WT fibroblasts. Mechanistically, we demonstrate that CD44WT cells generated more active TGFbeta than CD44KO cells and that CD44 promotes the activation of TGFbeta via an MMP-dependent mechanism. Reconstitution of CD44 expression completely rescued the phenotype of CD44KO cells whereas exposure of CD44KO cells to exogenous active TGFbeta rescued the defect in stress fibers and migrational velocity, but was not sufficient to restore directionality of migration. These results resolve the TGFbeta-mediated and TGFbeta-independent effects of CD44 on fibroblast migration and suggest that CD44 may be critical for the recruitment of fibroblasts to sites of injury and the function of fibroblasts in tissue remodeling and fibrosis.
%Z FOR Codes: 60199


%0 Journal Article
%~ PubMed
%A Ng, Lai Guan
%A Hsu, Alice
%A Mandell, Michael A
%A Roediger, Ben
%A Hoeller, Christoph
%A Mrass, Paulus
%A Iparraguirre, Amaya
%A Cavanagh, Lois L
%A Triccas, James A
%A Beverley, Stephen M
%A Scott, Phillip
%A Weninger, Wolfgang
%T Migratory dermal dendritic cells act as rapid sensors of protozoan parasites.
%B PLoS pathogens
%D 2008
%C United States
%I Public Library of Science
%V 4
%N 11
%P e1000222
%@ 1553-7374
%X Dendritic cells (DC), including those of the skin, act as sentinels for intruding microorganisms. In the epidermis, DC (termed Langerhans cells, LC) are sessile and screen their microenvironment through occasional movements of their dendrites. The spatio-temporal orchestration of antigen encounter by dermal DC (DDC) is not known. Since these cells are thought to be instrumental in the initiation of immune responses during infection, we investigated their behavior directly within their natural microenvironment using intravital two-photon microscopy. Surprisingly, we found that, under homeostatic conditions, DDC were highly motile, continuously crawling through the interstitial space in a Galpha(i) protein-coupled receptor-dependent manner. However, within minutes after intradermal delivery of the protozoan parasite Leishmania major, DDC became immobile and incorporated multiple parasites into cytosolic vacuoles. Parasite uptake occurred through the extension of long, highly dynamic pseudopods capable of tracking and engulfing parasites. This was then followed by rapid dendrite retraction towards the cell body. DDC were proficient at discriminating between parasites and inert particles, and parasite uptake was independent of the presence of neutrophils. Together, our study has visualized the dynamics and microenvironmental context of parasite encounter by an innate immune cell subset during the initiation of the immune response. Our results uncover a unique migratory tissue surveillance program of DDC that ensures the rapid detection of pathogens.
%Z FOR Codes: 110304


%0 Journal Article
%~ PubMed
%A Wallace, Africa
%A Kapoor, Veena
%A Sun, Jing
%A Mrass, Paul
%A Weninger, Wolfgang
%A Heitjan, Daniel F
%A June, Carl
%A Kaiser, Larry R
%A Ling, Leona E
%A Albelda, Steven M
%T Transforming growth factor-beta receptor blockade augments the effectiveness of adoptive T-cell therapy of established solid cancers.
%B Clinical cancer research : an official journal of the American Association for Cancer Research
%D 2008
%C US
%I American Association for Cancer Research
%V 14
%N 12
%P 3966-3974
%@ 1078-0432
%X PURPOSE: Adoptive cellular immunotherapy is a promising approach to eradicate established tumors. However, a significant hurdle in the success of cellular immunotherapy involves recently identified mechanisms of immune suppression on cytotoxic T cells at the effector phase. Transforming growth factor-beta (TGF-beta) is one of the most important of these immunosuppressive factors because it affects both T-cell and macrophage functions. We thus hypothesized that systemic blockade of TGF-beta signaling combined with adoptive T-cell transfer would enhance the effectiveness of the therapy. EXPERIMENTAL DESIGN: Flank tumors were generated in mice using the chicken ovalbumin-expressing thymoma cell line, EG7. Splenocytes from transgenic OT-1 mice (whose CD8 T cells recognize an immunodominant peptide in chicken ovalbumin) were activated in vitro and adoptively transferred into mice bearing large tumors in the presence or absence of an orally available TGF-beta receptor-I kinase blocker (SM16). RESULTS: We observed markedly smaller tumors in the group receiving the combination of SM16 chow and adoptive transfer. Additional investigation revealed that TGF-beta receptor blockade increased the persistence of adoptively transferred T cells in the spleen and lymph nodes, increased numbers of adoptively transferred T cells within tumors, increased activation of these infiltrating T cells, and altered the tumor microenvironment with a significant increase in tumor necrosis factor-alpha and decrease in arginase mRNA expression. CONCLUSIONS: We found that systemic blockade of TGF-beta receptor activity augmented the antitumor activity of adoptively transferred T cells and may thus be a useful adjunct in future clinical trials.
%Z FOR Codes: 111201


%0 Journal Article
%~ PubMed
%A Ng, Lai Guan
%A Mrass, Paulus
%A Kinjyo, Ichiko
%A Reiner, Steven L
%A Weninger, Wolfgang
%T Two-photon imaging of effector T-cell behavior: lessons from a tumor model.
%B Immunological reviews
%D 2008
%C Denmark
%I Wiley-Blackwell Munksgaard
%V 221
%N 0
%P 147-162
%@ 1600-065X
%X Recent advances in two-photon microscopy have provided a new way of visualizing the behavior of fluorescently tagged cells within their natural microenvironment. This technology has allowed for generating a detailed picture of the cellular interaction dynamics operant in the activation of T cells and B cells during primary immune responses within secondary lymphoid organs. In contrast, relatively little is known about the migratory and interactive behavior of effector T cells within peripheral organs. We have recently developed a two-photon microscopy model that enables tracking of cytotoxic T cells within tumors. We have demonstrated that tumor-infiltrating T lymphocytes (TILs) follow random migratory paths and that their migratory properties depend on signals from the T-cell receptor. We further showed that TILs underwent short- and long-term interactions with tumor cells as well as macrophages. Recently, we succeeded in dynamic imaging of the distribution of fluorescently tagged molecules within TILs at subcellular resolution, which will be instrumental for defining the composition of the lytic synapse as well as the targeted release of cytotoxic granules by these cells. The purpose of this review is to put our findings into the context of the current literature and to point out the molecular cues mediating effector T-cell function as candidates for future investigation.
%Z FOR Codes: 110701


%0 Journal Article
%~ PubMed
%A Roediger, Ben
%A Ng, Lai
%A Smith, Adrian
%A de St Groth, Barbara
%A Weninger, Wolfgang
%T Visualizing dendritic cell migration within the skin.
%B Histochemistry and cell biology
%D 2008
%C Germany
%I Springer
%V 130
%N 6
%P 1131-46
%@ 0948-6143
%X Dendritic cells (DCs) within the skin are a heterogeneous population of cells, including Langerhans cells of the epidermis and at least three subsets of dermal DCs. Collectively, these DCs play important roles in the initiation of adaptive immune responses following antigen challenge of the skin as well as being mediators of tolerance to self-antigen. A key functional aspect of cutaneous DCs is their migration both within the skin and into lymphatic vessels, resulting in their emigration to draining lymph nodes. Here, we discuss our current understanding of the requirements for successful DC migration in and from the skin, and introduce some of the microscopic techniques developed in our laboratory to facilitate a better understanding of this process. In particular, we detail our current use of multi-photon excitation (MPE) microscopy of murine skin to dissect the migratory behavior of DCs in vivo.
%Z FOR Codes: 110304 1108


%0 Journal Article
%~ PubMed
%A Whitmore, Mark M
%A Iparraguirre, Amaya
%A Kubelka, Lindsey
%A Weninger, Wolfgang
%A Hai, Tsonwin
%A Williams, Bryan R G
%T Negative regulation of TLR-signaling pathways by activating transcription factor-3.
%B Journal of immunology
%D 2007
%C United States
%I American Association of Immunologists
%V 179
%N 6
%P 3622-3630
%@ 0022-1767
%X Activating transcription factor-3 (ATF3) is rapidly induced by LPS in mouse macrophages and regulates TLR4 responses. We show that ATF3 is rapidly induced by various TLRs in mouse macrophages and plasmacytoid dendritic cells (DCs), as well as plasmacytoid and myeloid subsets of human DCs. In primary macrophages from mice with a targeted deletion of the atf3 gene (ATF3-knockout (KO)), TLR-stimulated levels of IL-12 and IL-6 were elevated relative to responses in wild-type macrophages. Similarly, targeted deletion of atf3 correlated with enhanced responsiveness of myeloid DCs to TLR activation as measured by IL-12 secretion. Ectopic expression of ATF3 antagonized TLR-stimulated IL-12p40 activation in a reporter assay. In vivo, CpG-oligodeoxynucleotide, a TLR9 agonist, given i.p. to ATF3-KO mice resulted in enhanced cytokine production from splenocytes. Furthermore, while ATF3-KO mice challenged with a sublethal dose of PR8 influenza virus were delayed in body weight recovery in comparison to wild type, the ATF3-KO mice showed higher titers of serum neutralizing Ab against PR8 5 mo postinfection. Thus, ATF3 behaves as a negative regulatory transcription factor in TLR pathways and, accordingly, deficiency in atf3 alters responses to immunological challenges in vivo. ATF3 dysregulation merits further exploration in diseases such as type I diabetes and cancer, where altered innate immunity has been implicated in their pathogenesis.
%Z FOR Codes: 110704


%0 Journal Article
%~ PubMed
%A Fukunaga-Kalabis, Mizuho
%A Martinez, Gabriela
%A Liu, Zhao-Jun
%A Kalabis, Jiri
%A Mrass, Paul
%A Weninger, Wolfgang
%A Firth, Sue M
%A Planque, Nathalie
%A Perbal, Bernard
%A Herlyn, Meenhard
%T CCN3 controls 3D spatial localization of melanocytes in the human skin through DDR1.
%B The Journal of cell biology
%D 2006
%C USA
%I Rockefeller University Press
%V 175
%N 4
%P 563-9
%@ 0021-9525
%X Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis.
%Z FOR Codes: 111601 111201


%0 Journal Article
%~ PubMed
%A Mrass, Paulus
%A Takano, Hajime
%A Ng, Lai Guan
%A Daxini, Sachin
%A Lasaro, Marcio O
%A Iparraguirre, Amaya
%A Cavanagh, Lois L
%A von Andrian, Ulrich H
%A Ertl, Hildegund C J
%A Haydon, Philip G
%A Weninger, Wolfgang
%T Random migration precedes stable target cell interactions of tumor-infiltrating T cells.
%B The Journal of experimental medicine
%D 2006
%C USA
%I Rockefeller University Press
%V 203
%N 12
%P 2749-2761
%@ 0022-1007
%X The tumor microenvironment is composed of an intricate mixture of tumor and host-derived cells that engage in a continuous interplay. T cells are particularly important in this context as they may recognize tumor-associated antigens and induce tumor regression. However, the precise identity of cells targeted by tumor-infiltrating T lymphocytes (TILs) as well as the kinetics and anatomy of TIL-target cell interactions within tumors are incompletely understood. Furthermore, the spatiotemporal conditions of TIL locomotion through the tumor stroma, as a prerequisite for establishing contact with target cells, have not been analyzed. These shortcomings limit the rational design of immunotherapeutic strategies that aim to overcome tumor-immune evasion. We have used two-photon microscopy to determine, in a dynamic manner, the requirements leading to tumor regression by TILs. Key observations were that TILs migrated randomly throughout the tumor microenvironment and that, in the absence of cognate antigen, they were incapable of sustaining active migration. Furthermore, TILs in regressing tumors formed long-lasting (>or=30 min), cognate antigen-dependent contacts with tumor cells. Finally, TILs physically interacted with macrophages, suggesting tumor antigen cross-presentation by these cells. Our results demonstrate that recognition of cognate antigen within tumors is a critical determinant of optimal TIL migration and target cell interactions, and argue against TIL guidance by long-range chemokine gradients.
%Z FOR Codes: 1108