To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isola...

Although the immune system evolved to fight infections, it may also attack and destroy solid tumors. In most cases, tumor rejection is initiated by CD8(+) cytotoxic T lymphocytes (CTLs), which infiltrate solid tumors, recognize tumor antigens, and kill tumor cells. We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs. We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocitie...

IL-10 is a major regulator in inflammatory responses. Although various transcription factors were defined to enhance IL-10, the molecular mechanism for the initiation of Il-10 transcription, remains unknown. mRNA profiling of six distinct primary CD4+ T cell populations showed differential expression of the transcription factor GATA-3 correlated with levels of IL-10 expression. We showed that ectopic expression of GATA-3 in naive primary CD4+ T cells enhanced expression of IL-10 by these cells and uncovered a possible mechanism for this effect. We found that GATA...

Distinct dendritic cell (DC) subsets have been suggested to be preprogrammed to direct either T helper cell (Th) type 1 or Th2 development, although more recently different pathogen products or stimuli have been shown to render these DCs more flexible. It is still unclear how distinct mouse DC subsets cultured from bone marrow precursors, blood, or their lymphoid tissue counterparts direct Th differentiation. We show that mouse myeloid and plasmacytoid precursor DCs (pDCs) cultured from bone marrow precursors and ex vivo splenic DC subsets can induce the developm...

Dendritic cells (DCs) play a key role in the orchestration of the immune system by virtue of their capacity to control both immunity and tolerance induction. The functions of DCs depend on the subset as well as their location and activation state. A number of in vitro protocols, developed to recapitulate DC generation from hematopoietic precursors, have suggested the importance of microenvironment and cytokine milieu in driving the generation of DCs endowed with peculiar phenotypic and functional features. Recently, some important concepts on the development of D...

Dendritic cells (DCs) play a crucial role in bridging innate and acquired immune responses to pathogens. In human immunodeficiency virus type 1 (HIV-1) infection, immature DCs (iDCs) are also main targets for HIV-1 at the mucosal level. In this study, we evaluated the effects of HIV-1-DC interactions on the maturation and functional activity of these cells. Exposure of human monocyte-derived iDCs to either aldrithiol-2-inactivated HIV-1 or gp120 led to an upmodulation of activation markers indicative of functional maturation. Despite their phenotype, these cells ...

The molecular mechanisms that regulate expression of the immunosuppressive cytokine IL-10 remain poorly understood. In this study, by measuring sensitivity to DNase I digestion, we show that production of IL-10 by primary mouse bone marrow-derived macrophages stimulated through pattern recognition receptors was associated with chromatin remodeling of the IL-10 locus. We also demonstrate that the IL-10 locus is remodeled in primary Th2 cells and IL-10-producing regulatory T cells that have been differentiated in vitro. Strikingly, a novel DNase I-hypersensitive si...

Dendritic cells (DCs) are professional APCs of the immune system that play a key role in regulating T cell-based immunity. The capacity of DCs to activate T cells depends on their maturation state as well as their ability to migrate to the T cell areas of draining lymph nodes. In this study, we investigated the effects of DC maturation stimuli on the actin cytoskeleton and beta(1) integrin-dependent adhesion and migration. Podosomes, specialized adhesion structures found in immature monocyte-derived DCs as well as myeloid DCs, rapidly dissolve in response to matu...

The direct assessment of T helper (T(H))-cell responses specific for antigens is essential to evaluate pathogenic and protective immunity. Presently, analysis and isolation of antigen-specific T(H) cells is restricted to cells that produce cytokines, or can be performed only with a rare selection of specific peptide major histocompatibility complex class II (MHC II) multimers. Here we report a new method that enables the assessment and isolation of T(H) cells specific for a defined antigen according to CD154 expression induced after stimulation in vitro. We show ...

The beta2-integrin LFA-1 facilitates extravasation of monocytes (MOs) into the underlying tissues, where MOs can differentiate into dendritic cells (DCs). Although DCs express LFA-1, unlike MOs, they cannot bind to ICAM-1. We hypothesized that an altered integrin organization on the DC plasma membrane might cause this effect and investigated the relationship between membrane organization and function of LFA-1 on MOs and DCs. High-resolution mapping of LFA-1 surface distribution revealed that on MOs LFA-1 function is associated with a distribution in well-defined ...

Tumor-Associated Macrophages (TAM) represent the major inflammatory component of the stroma of many tumors, able to affect different aspects of the neoplastic tissue. Many observations indicate that TAM express several M2-associated protumoral functions, including promotion of angiogenesis, matrix remodelling and suppression of adaptive immunity. The protumoral role of TAM in cancer is further supported by clinical studies that found a correlation between the high macrophage content of tumors and poor patient prognosis and by evidence showing that long-term use o...

As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 x 10(6) cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1-3 x 10(8) immature and mature DC per mouse at 90-95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss...

Tumors require a constant influx of myelomonocytic cells to support the angiogenesis and stroma remodeling needed for their growth. This is mediated by tumor-derived factors, which cause sustained myelopoiesis and the accumulation and functional differentiation of myelomonocytic cells, most of which are macrophages, at the tumor site. An important side effect of the accumulation and functional differentiation of these cells is that they can induce lymphocyte dysfunction. A complete understanding of the complex interplay between neoplastic and myelomonocytic cells...

Tumour-associated macrophages (TAM) represent the major inflammatory component of the stroma of many tumours, and can affect different aspects of the neoplastic tissue. Many observations indicate that TAM express several M2-associated pro-tumoural functions, including promotion of angiogenesis, matrix remodelling and suppression of adaptive immunity. The pro-tumoural role of TAM in cancer is further supported by clinical studies that found a correlation between the high macrophage content of tumours and poor patient prognosis. Evidence is presented here supportin...

Tumor-associated macrophages (TAM) are a major inflammatory infiltrate in tumors and a major component of the protumor function of inflammation. TAM in established tumors generally have an M2 phenotype with defective production of interleukin-12 (IL-12) and high IL-10. Here, we report that defective responsiveness of TAM from a murine fibrosarcoma and human ovarian carcinoma to M1 activation signals was associated with a massive nuclear localization of the p50 nuclear factor-kappaB (NF-kappaB) inhibitory homodimer. p50 overexpression inhibited IL-12 expression in...

Mass spectrometry (MS)-based proteomics has become a formidable tool for the investigation of posttranslational modifications to proteins, protein interactions, and organelles. Is it now ready to tackle comprehensive protein expression analysis?

For the last four decades, the treatment of cancer has relied on four treatment modalities, namely surgery, radiotherapy, cytotoxic chemotherapy, and hormonotherapy. Most of these therapies are believed to directly attack and eradicate tumor cells. The emerging concept that cancer is not just a disease of a tissue or an organ but also a host disease relies on evidence of tumor-induced immunosuppression and polymorphisms in genes involved in host protection against tumors. This theory is now gaining new impetus, based on our recent data showing that optimal therape...

If the large collection of microarray-specific statistical tools was applicable to the analysis of quantitative shotgun proteomics datasets, it would certainly foster an important advancement of proteomics research. Here we analyze two large multidimensional protein identification technology datasets, one containing eight replicates of the soluble fraction of a yeast whole-cell lysate and one containing nine replicates of a human immunoprecipitate, to test whether normalized spectral abundance factor (NSAF) values share substantially similar statistical propertie...

A cornucopia of physiological and pathological circumstances including anticancer chemotherapy and radiotherapy can induce cell death. However, the immunological consequences of tumor cell demise have remained largely elusive. The paradigm opposing 'apoptosis versus necrosis' as to their respective immunogenicity does not currently hold to predict long-term immunity. Moreover, the notion that tumor cells may be 'stressed' before death to be recognized by immune cells deserves to be underlined. 'Eat-me', 'danger' and 'killing' signals released by stressed tumor und...

Apoptotic cell death is initiated by a morphologically homogenous entity that was considered to be non-immunogenic and non-inflammatory in nature. However, recent advances suggest that apoptosis, under certain circumstances, can be immunogenic. In particular, some characteristics of the plasma membrane, acquired at preapoptotic stage, can cause immune effectors to recognize and attack preapoptotic tumor cells. The signals that mediate the immunogenicity of tumor cells involve elements of the DNA damage response (such as ataxia telangiectasia mutated and p53 activ...