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Daniel C Douek

Publications and source records attributed to Daniel C Douek.

At least 55 records · Page 3Linked to original sources
PubMed2004

CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract.

The mechanisms underlying CD4(+) T cell depletion in human immunodeficiency virus (HIV) infection are not well understood. Comparative studies of lymphoid tissues, where the vast majority of T cells reside, and peripheral blood can potentially illuminate the pathogenesis of HIV-associated disease. Here, we studied the effect of HIV infection on the activation and depletion of defined subsets of CD4(+) and CD8(+) T cells in the blood, gastrointestinal (GI) tract, and lymph node (LN). We also measured HIV-specific T cell frequencies in LNs and blood, and LN collagen deposition to define architectural changes associated with chronic inflammation. The major findings to emerge are the following: the GI tract has the most substantial CD4(+) T cell depletion at all stages of HIV disease; this depletion occurs preferentially within CCR5(+) CD4(+) T cells; HIV-associated immune activation results in abnormal accumulation of effector-type T cells within LNs; HIV-specific T cells in LNs do not account for all effector T cells; and T cell activation in LNs is associated with abnormal collagen deposition. Taken together, these findings define the nature and extent of CD4(+) T cell depletion in lymphoid tissue and point to mechanisms of profound depletion of specific T cell subsets related to elimination of CCR5(+) CD4(+) T cell targets and disruption of T cell homeostasis that accompanies chronic immune activation.

CD4-Positive T-Lymphocytes↗
PubMed2004

Unusual immunophenotype of CD8+ T cells in familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, fatal disorder of infancy. We report here a 17-day-old female infant who presented with high fever, hepatosplenomegaly, hypertriglyceridemia, hypofibrinogenemia, thrombocytopenia, and liver failure. Leukocytosis was detected with circulating "atypical" lymphoid cells. Flow cytometric studies revealed expanded subpopulations of CD8+ T cells with unusual immunophenotypic features, including a subset that lacked CD5 expression. A liver biopsy showed hemophagocytic lymphohistiocytosis with exuberant infiltrates of CD8+ T cells that lacked perforin. Mutational studies revealed a 666C-->A (H222Q) missense mutation in the perforin gene. T-cell receptor studies on flow-sorted T-cell subpopulations revealed no evidence of monoclonality. Analysis of T-cell receptor excision circle levels indicated long proliferative history in the aberrant CD8+ T-cell subsets. This case provides an instructive example of uncontrolled reactive proliferation of CD8+ T cells in FHL, resulting in atypical morphology and unusual immunophenotypic features that might suggest malignancy in other clinical settings.

Biopsy↗
PubMed2004

Adult human liver contains CD8pos T cells with naive phenotype, but is not a site for conventional alpha beta T cell development.

Normal adult human liver (AHL) contains populations of unconventional lymphocytes that have been shown in the mouse to mature locally. The presence of lymphoid progenitors together with IL-7, recombinase-activating gene, and pre-TCR-alpha expression in AHL suggests similar local T cell development activity in humans. Flow cytometry was used to characterize potentially naive hepatic alphabeta-T cells. We looked for evidence of TCR-alphabeta cell development in AHL by quantifying delta deletion TCR excision circles (TRECs) in CD3(pos) populations isolated from the liver and matched blood of eight individuals. Phenotypic analysis of hepatic T cells suggests the presence of Ag-inexperienced populations. TRECs were detected in all blood samples (mean, 164.10 TRECs/ micro g DNA), whereas only two hepatic samples were positive at low levels (59.40 and 1.92). The relatively high level of CD8(pos) T cells in these livers with a naive phenotype suggests that in addition to its role as a graveyard for Ag-specific activated CD8(pos) T cells, naive CD8(pos) T cells may enter the liver without prior activation. The almost complete absence of TRECs suggests that normal AHL is not a site for the development of conventional alphabeta T cells.

Adult↗
PubMed2004

The functional profile of primary human antiviral CD8+ T cell effector activity is dictated by cognate peptide concentration.

Antiviral CD8(+) T cells can elaborate at least two effector functions, cytokine production and cytotoxicity. Which effector function is elaborated can determine whether the CD8(+) T cell response is primarily inflammatory (cytokine producing) or antiviral (cytotoxic). In this study we demonstrate that cytotoxicity can be triggered at peptide concentrations 10- to 100-fold less than those required for cytokine production in primary HIV- and CMV-specific human CD8(+) T cells. Cytolytic granule exocytosis occurs at peptide concentrations insufficient to cause substantial TCR down-regulation, providing a mechanism by which a CD8(+) T cell could engage and lyse multiple target cells. TCR sequence analysis of virus-specific cells shows that individual T cell clones can degranulate or degranulate and produce cytokine depending on the Ag concentration, indicating that response heterogeneity exists within individual CD8(+) T cell clonotypes. Thus, antiviral CD8(+) T cell effector function is determined primarily by Ag concentration and is not an inherent characteristic of a virus-specific CD8(+) T cell clonotype or the virus to which the response is generated. The inherent ability of viruses to induce high or low Ag states may be the primary determinant of the cytokine vs cytolytic nature of the virus-specific CD8(+) T cell response.

Antigens↗
PubMed2004

Where does HIV live?

Explore the source record for details and available documents.

Acquired Immunodeficiency Syndrome↗
PubMed2004

High prevalence of autoreactive, neuroantigen-specific CD8+ T cells in multiple sclerosis revealed by novel flow cytometric assay.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) with features suggestive of T-cell-mediated pathology. Most prior reports have focused on CD4(+) T cells with the underlying assumption that MS is predominantly a CD4(+) T helper 1 (Th1)-mediated disease. In this report, we used a novel flow cytometric approach to evaluate autoreactive T-cell responses against a large variety of neuroantigenic targets. We found that both CD4(+) and CD8(+) T cells targeted against several CNS autoantigens were widely prevalent in patients with MS and healthy individuals. Whereas the distribution of CD4(+) responses was similar in different groups, patients with relapsing-remitting MS showed a higher proportion of CNS-specific CD8(+) responses. Autoreactive CD4(+) T cells from patients with MS exhibited a more differentiated Th1 phenotype compared with healthy subjects. Similarly, CNS-specific CD8(+) T-cell responses from patients with MS were functionally distinct from those in healthy individuals. Collectively, these studies reveal the high prevalence of class I-restricted autoreactive CD8(+) T-cell responses in MS that has been underappreciated thus far. The results emphasize the need to evaluate both CD4(+) and CD8(+) T-cell responses in MS and to make both subsets a consideration in the development of novel therapeutic strategies.

Adult↗
PubMed2004

HIV-infected Langerhans cells preferentially transmit virus to proliferating autologous CD4+ memory T cells located within Langerhans cell-T cell clusters.

Langerhans cells (LC) are likely initial targets for HIV following sexual exposure to virus and provide an efficient means for HIV to gain access to lymph node T cells. The purpose of this study was to examine the nature of the CD4(+) T cell that becomes infected by HIV-infected LC. We infected human LC within tissue explants ex vivo and then, 3 days later, cocultured HIV-infected LC with different subsets of autologous CD4(+) T cells. Using multicolor flow cytometric analyses of LC-CD4(+) T cell cocultures, we documented that HIV-infected LC preferentially infected memory (as compared with naive) CD4(+) T cells. Proliferating and HIV-infected CD4(+) memory T cells were more frequently detected in conjugates of LC and autologous CD4(+) T cells, suggesting that T cells become activated and preferentially get infected through cluster formation with infected LC, rather than getting infected with free virus produced by single HIV-infected LC or T cells. p24(+) Memory CD4(+) T cells proliferated well in the absence of superantigen; by contrast, p24(+) T cells did not divide or divided only once in the presence of staphylococcal enterotoxin B, suggesting that virus production was rapid and induced apoptosis in these cells before significant proliferation could occur. These results highlight that close interactions between dendritic cells, in this case epidermal LC, and T cells are important for optimal HIV replication within specific subsets of CD4(+) T cells. Disrupting cluster formation between LC and memory CD4(+) T cells may be a novel strategy to interfere with sexual transmission of HIV.

Antigens, Bacterial↗
PubMed2004

HLA class II polymorphisms determine responses to bacterial superantigens.

The excessive immunological response triggered by microbial superantigens has been implicated in the etiology of a wide range of human diseases but has been most clearly defined for the staphylococcal and streptococcal toxic shock syndromes. Because MHC class II presentation of superantigens to T cells is not MHC-restricted, the possibility that HLA polymorphisms could influence superantigenicity, and thus clinical susceptibility to the toxicity of individual superantigens, has received little attention. In this study, we demonstrate that binding of streptococcal and staphylococcal superantigens to HLA class II is influenced by allelic differences in class II. For the superantigen streptococcal pyrogenic exotoxin A, class II binding is dependent on DQ alpha-chain polymorphisms such that HLA-DQA1*01 alpha-chains show greater binding than DQA1*03/05 alpha-chains. The functional implications of differential binding on T cell activation were investigated in various experimental systems using human T cells and murine Vbeta8.2 transgenic cells as responders. These studies showed quantitative and qualitative differences resulting from differential HLA-DQ binding. We observed changes in T cell proliferation and cytokine production, and in the Vbeta specific changes in T cell repertoire that have hitherto been regarded as a defining feature of an individual superantigen. Our observations reveal a mechanism for the different outcomes seen following infection by toxigenic bacteria.

Amino Acid Sequence↗
PubMed2004

T cell receptor recognition motifs govern immune escape patterns in acute SIV infection.

Escape from adaptive T cell immunity through transmutation of viral antigenic structure is a cardinal feature in the pathogenesis of SIV/HIV infection and a major obstacle to antiretroviral vaccine development. However, the molecular determinants of this phenomenon at the T cell receptor (TCR)-antigen interface are unknown. Here, we show that mutational escape is intimately linked to the structural configuration of constituent TCR clonotypes within virus-specific CD8(+) T cell populations. Analysis of 3416 SIV-specific TCR sequences revealed that polyclonal T cell populations characterized by highly conserved TCRB CDR3 motifs were rendered ineffectual by single residue mutations in the cognate viral epitope. Conversely, diverse clonotypic repertoires without discernible motifs were not associated with viral escape. Thus, fundamental differences in the mode of antigen engagement direct the pattern of adaptive viral evolution. These findings have profound implications for the development of vaccines that elicit T cell immunity to combat pathogens with unstable genomes.

Acute Disease↗
PubMed2004

T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis.

Identification of T-cell subsets that are infected in vivo is essential to understanding the pathogenesis of human immunodeficiency virus (HIV) disease; however, this goal has been beset with technical challenges. Here, we used polychromatic flow cytometry to sort multiple T-cell subsets to 99.8% purity, followed by quantitative PCR to quantify HIV gag DNA directly ex vivo. We show that resting memory CD4(+) T cells are the predominantly infected cells but that terminally differentiated memory CD4(+) T cells contain 10-fold fewer copies of HIV DNA. Memory CD8(+) T cells can also be infected upon upregulation of CD4; however, this is infrequent and HIV-specific CD8(+) T cells are not infected preferentially. Naïve CD4(+) T-cell infection is rare and principally confined to those peripheral T cells that have proliferated. Furthermore, the virus is essentially absent from naïve CD8(+) T cells, suggesting that the thymus is not a major source of HIV-infected T cells in the periphery. These data illuminate the underlying mechanisms that distort T-cell homeostasis in HIV infection.

Adult↗
PubMed2003

Identification and in vitro expansion of CD4+ and CD8+ T cells specific for human neutrophil elastase.

Human neutrophil elastase (HNE) and proteinase 3 (PRO3) are myeloid tissue-restricted serine proteases, aberrantly expressed by myeloid leukemia cells. PRO3 and HNE share the PR1 peptide sequence that induces HLA-A*0201-restricted cytotoxic T cells (CTLs) with antileukemia reactivity. We studied the entire HNE protein for its ability to induce CTLs. In an 18-hour culture, HNE-loaded monocytes stimulated significant intracellular interferon gamma (IFN-gamma) production by CD4+ and CD8+ T cells in 12 of 20 and 8 of 20 healthy individuals, respectively. Lymphocytes from 2 HNE responders were pulsed weekly for 4 weeks to generate HNE-specific CTLs. One of 2 HLA-A*0201-negative individuals inhibited the colony formation of HLA-identical chronic myelogenous leukemia progenitor cells (73% inhibition at 50:1 effector-target [E/T] ratio), indicating that peptides other than PR1 can induce leukemia-reactive CTLs. Repetitive stimulations with HNE in 2 of 5 HLA-A*0201+ individuals increased PR1 tetramer-positive CD8+ T-cell frequencies from 0.1% to 0.29% and 0.02% to 0.55%, respectively. These CTLs recognized PR1 peptide or killed HNE-loaded targets. These results indicate that exogenously processed HNE is a source of PR1 peptide as well as other peptide sequences capable of inducing leukemia-specific CD8+ and CD4+ T cells. HNE could, therefore, be used in an HLA-unrestricted manner to induce leukemia-reactive CTLs for adoptive immunotherapy.

Amino Acid Sequence↗
PubMed2003

Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation.

Flow cytometric detection of antigen-specific CD8+ T cells has previously been limited to MHC-class I tetramer staining or intracellular cytokine production, neither of which measure the cytolytic potential of these cells. Here we present a novel technique to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced degranulation, a necessary precursor of cytolysis. This assay measures the exposure of CD107a and b, present in the membrane of cytotoxic granules, onto the cell surface as a result of degranulation. Acquisition of cell surface CD107a and b is associated with loss of intracellular perforin and is inhibited by colchicine, indicating that exposure of CD107a and b to the cell surface is dependent on degranulation. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide, concordant with production of intracellular IFNgamma. Finally, CD107-expressing CD8+ T cells are shown to mediate cytolytic activity in an antigen-specific manner. Measurement of CD107a and b expression can also be combined with MHC-class I tetramer labeling and intracellular cytokine staining to provide a more complete assessment of the functionality of CD8+T cells expressing cognate T cell receptors (TCR).

Antigens, CD↗
PubMed2003

Functional leukemia-associated antigen-specific memory CD8+ T cells exist in healthy individuals and in patients with chronic myelogenous leukemia before and after stem cell transplantation.

Antigens implicated in the graft-versus-leukemia (GVL) effect in chronic myeloid leukemia (CML) include WT1, PR1, and BCR-ABL. To detect very low frequencies of these antigen-specific CD8+ T cells, we used quantitative polymerase chain reaction (qPCR) to measure interferon-gamma (IFN-gamma) mRNA production by peptide-pulsed CD8+ T cells from HLA-A*0201+ healthy volunteers and from patients with CML before and after allogeneic stem cell transplantation (SCT). Parallel assays using cytomegalovirus (CMV) pp65 tetramers demonstrated the IFN-gamma copy number to be linearly related to the frequency of tetramer-binding T cells, sensitive to frequencies of 1 responding CD8+ T cell/100 000 CD8+ T cells. Responses to WT1 and PR1 but not BCR-ABL were detected in 10 of 18 healthy donors. Responses to WT1, PR1, or BCR-ABL were observed in 9 of 14 patients with CML before SCT and 5 of 6 after SCT, often to multiple epitopes. Responses were higher in patients with CML compared with healthy donors and highest after SCT. These antigen-specific CD8+ T cells comprised central memory (CD45RO+CD27+CD57-) and effector memory (CD45RO-CD27-CD57+) T cells. In conclusion, leukemia-reactive CD8+ T cells derive from memory T cells and occur at low frequencies in healthy individuals and at higher frequencies in patients with CML. The increased response in patients after SCT suggests a quantitative explanation for the greater effect of allogeneic SCT.

CD3 Complex↗
PubMed2003

T cells containing T cell receptor excision circles are inversely related to HIV replication and are selectively and rapidly released into circulation with antiretroviral treatment.

OBJECTIVE: To examine baseline predictors of T-cell receptor rearrangement excision circle (TREC) levels and their changes during treatment with combined antiretroviral therapy. METHODS: Peripheral blood and lymph node lymphocytes were examined for the presence of TREC by real-time polymerase chain reaction and circulating lymphocyte phenotypes were examined by flow cytometry. Correlates for CD4 and CD8 cell TREC levels at baseline were identified among CD4 and CD8 immunophenotypes, viral load and patient demographics; the significance of TREC changes after initiation of antiretroviral therapy was assessed. RESULTS: Circulating TREC levels correlated inversely with age, with HIV RNA levels, with activation markers on circulating T cells and with naive CD4 but not CD8 cell frequencies. With initiation of antiretroviral therapy, TREC and naive T cell frequencies increased in peripheral blood during the first 2 weeks of treatment and these changes correlated negatively with TREC frequencies in lymph node aspirates, particularly among CD8 T cells. CONCLUSIONS: These findings suggest that recent thymic emigrants are sequestered in lymphoid tissue during uncontrolled HIV replication and are selectively released into circulation rapidly after initiation of antiretroviral therapies.

Adult↗
PubMed2003

Identification and monitoring of graft-versus-host specific T-cell clone in stem cell transplantation.

Allogeneic haemopoietic stem cell transplantation can be complicated by acute graft-versus-host disease (GVHD). If the alloreactive T-cell clones responsible for this disorder could be identified before stem cell transplantation, they could be quantitatively monitored afterwards to allow initiation of immunosuppression before GVHD becomes apparent. We identified an alloreactive CD4+ T-cell clone from a donor before stem cell transplantation, and then monitored its frequency in the recipient's peripheral blood after transplantation by use of clonotypic quantitative real-time PCR. Greatly increased concentrations of this clone (nearly 100% of circulating CD4+ T cells) preceded and closely correlated with the onset of clinical GVHD, suggesting that a sole alloreactive T-cell clone was responsible for initiation of this disease in vivo. This simple identification and monitoring of potentially GVHD-causing clones can be undertaken before stem cell transplantation with no previous knowledge of the alloantigen responsible, and could allow earlier diagnosis and intervention in GVHD.

Adult↗