Biomedical subjects

Daniel C Douek

Publications and source records attributed to Daniel C Douek.

At least 37 records · Page 2Linked to original sources

PubMed2006

Systemic vaccination prevents the total destruction of mucosal CD4 T cells during acute SIV challenge.

BACKGROUND: Acute human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infections are accompanied by a systemic loss of memory CD4 T cells, with mucosal sites serving as a major site for viral replication, dissemination and CD4 T cell depletion. Protecting the mucosal CD4 T cell compartment thus is critical to contain HIV, and preserve the integrity of the mucosal immune system. The primary objective of this study was to determine if systemic vaccination with DNA/rAd-5 encoding SIV-mac239-env, gag and pol could prevent the destruction of CD4 T cells in mucosal tissues. METHODS: Rhesus macaques were immunized with DNA/r-Ad-5 encoding SIV genes and compared with those immunized with sham vectors following high dose intravenous challenge with SIVmac251. SIV specific CD4 and CD8 T cell responses, cell associated viral loads and mucosal CD4 T cell dynamics were evaluated. RESULTS: Strong SIV specific immune responses were induced in mucosal tissues of vaccinated animals as compared with sham controls. These responses expanded rapidly following challenge suggesting a strong anamnestic response. Immune responses were associated with a decrease in cell associated viral loads, and a loss of fewer mucosal CD4 T cells. Approximately 25% of mucosal CD4 T cells were preserved in vaccinated animals as compared with <5% in sham controls. These results demonstrate that systemic immunization strategies can induce immune responses in mucosal tissues that can protect mucosal CD4 T cells from complete destruction following challenge. CONCLUSIONS: Preservation of mucosal CD4 T cells can contribute to maintaining immune competence in mucosal tissues and provide a substantial immune benefit to the vaccinees.

Animals↗

PubMed2006

Lymphatic tissue fibrosis is associated with reduced numbers of naive CD4+ T cells in human immunodeficiency virus type 1 infection.

The organized structure of lymphatic tissues (LTs) constitutes a microenvironment referred to as a niche that plays a critical role in immune system homeostasis by promoting cellular interactions and providing access to cytokines and growth factors on which cells are dependent for survival, proliferation, and differentiation. In chronic human immunodeficiency virus type 1 (HIV-1) infection, immune activation and inflammation result in collagen deposition and disruption of this LT niche. We have previously shown that these fibrotic changes correlate with a reduction in the size of the total population of CD4+ T cells. We now show that this reduction is most substantial within the naïve CD4+ T-cell population and is in proportion to the extent of LT collagen deposition in HIV-1 infection. Thus, the previously documented depletion of naïve CD4+ T cells in LTs in HIV-1 infection may be a consequence not only of a decreased supply of thymic emigrants or chronic immune activation but also of the decreased ability of those cells to survive in a scarred LT niche. We speculate that LT collagen deposition might therefore limit repopulation of naïve CD4+ T cells with highly active antiretroviral therapy, and thus, additional treatments directed to limiting or reversing inflammatory damage to the LT niche could potentially improve immune reconstitution.

Adult↗

PubMed2006

Preferential infection shortens the life span of human immunodeficiency virus-specific CD4+ T cells in vivo.

CD4(+) T-cell help is essential for effective immune responses to viruses. In human immunodeficiency virus (HIV) infection, CD4(+) T cells specific for HIV are infected by the virus at higher frequencies than other memory CD4(+) T cells. Here, we demonstrate that HIV-specific CD4(+) T cells are barely detectable in most infected individuals and that the corresponding CD4(+) T cells exhibit an immature phenotype compared to both cytomegalovirus (CMV)-specific CD4(+) T cells and other memory CD4(+) T cells. However, in two individuals, we observed a rare and diametrically opposed pattern in which HIV-specific CD4(+) T-cell populations of large magnitude exhibited a terminally differentiated immunophenotype; these cells were not preferentially infected in vivo. Clonotypic analysis revealed that the HIV-specific CD4(+) T cells from these individuals were cross-reactive with CMV. Thus, preferential infection can be circumvented in the presence of cross-reactive CD4(+) T cells driven to maturity by coinfecting viral antigens, and this physical proximity rather than activation status per se is an important determinant of preferential infection based on antigen specificity. These data demonstrate that preferential infection reduces the life span of HIV-specific CD4(+) T cells in vivo and thereby compromises the generation of effective immune responses to the virus itself; further, this central feature in the pathophysiology of HIV infection can be influenced by the cross-reactivity of responding CD4(+) T cells.

CD4-Positive T-Lymphocytes↗

PubMed2005

T-cell responses directed against multiple HLA-A*0201-restricted epitopes derived from Wilms' tumor 1 protein in patients with leukemia and healthy donors: identification, quantification, and characterization.

PURPOSE: Antigens derived from the Wilms' tumor (WT1) protein, which is overexpressed in leukemias, are attractive targets for immunotherapy. Four HLA-A*0201-restricted WT1-derived epitopes have been identified: WT37, WT126, WT187, and WT235. We determined the natural immunogenecity of these antigens in patients with hematologic malignancies and healthy donor. EXPERIMENTAL DESIGN: To detect very low frequencies of WT1-specific CD8+ T cells, we used quantitative reverse transcription-PCR to measure IFN-gamma mRNA production by WT1 peptide-pulsed CD8+ T cells from 12 healthy donors, 8 patients with chronic myelogenous leukemia, 6 patients with acute myelogenous leukemia, and 8 patients with acute lymphoblastic leukemia. RESULTS: Responses were detected in 5 of 8 chronic myelogenous leukemia patients, 4 of 6 patients with acute myelogenous leukemia, and 7 of 12 healthy donors. No responses were detected in patients with acute lymphoblastic leukemia. The magnitude and extent of these CD8+ T-cell responses was greater in patients with myeloid leukemias than in healthy donors. Clonotypic analysis of WT1-specific CD8+ T cells directly ex vivo in one case showed that this naturally occurring population was oligoclonal. Using fluorescent peptide-MHC class I tetramers incorporating mutations in the alpha3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor, we were able to confirm the presence of high-avidity T-cell clones within the antigen-specific repertoire. CONCLUSION: The natural occurrence of high-avidity WT1-specific CD8+ T cells in the periphery could facilitate vaccination strategies to expand immune responses against myeloid leukemias.

Amino Acid Sequence↗

PubMed2005

Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses.

The forces that govern clonal selection during the genesis and maintenance of specific T cell responses are complex, but amenable to decryption by interrogation of constituent clonotypes within the antigen-experienced T cell pools. Here, we used point-mutated peptide-major histocompatibility complex class I (pMHCI) antigens, unbiased TCRB gene usage analysis, and polychromatic flow cytometry to probe directly ex vivo the clonal architecture of antigen-specific CD8(+) T cell populations under conditions of persistent exposure to structurally stable virus-derived epitopes. During chronic infection with cytomegalovirus and Epstein-Barr virus, CD8(+) T cell responses to immunodominant viral antigens were oligoclonal, highly skewed, and exhibited diverse clonotypic configurations; TCRB CDR3 sequence analysis indicated positive selection at the protein level. Dominant clonotypes demonstrated high intrinsic antigen avidity, defined strictly as a physical parameter, and were preferentially driven toward terminal differentiation in phenotypically heterogeneous populations. In contrast, subdominant clonotypes were characterized by lower intrinsic avidities and proportionately greater dependency on the pMHCI-CD8 interaction for antigen uptake and functional sensitivity. These findings provide evidence that interclonal competition for antigen operates in human T cell populations, while preferential CD8 coreceptor compensation mitigates this process to maintain clonotypic diversity. Vaccine strategies that reconstruct these biological processes could generate T cell populations that mediate optimal delivery of antiviral effector function.

Amino Acid Sequence↗

PubMed2005

Lymphopenia and interleukin-2 therapy alter homeostasis of CD4+CD25+ regulatory T cells.

CD4(+)CD25(+) regulatory T (T(reg)) cells have a crucial role in maintaining immune tolerance. Mice and humans born lacking T(reg) cells develop severe autoimmune disease, and depletion of T(reg) cells in lymphopenic mice induces autoimmunity. Interleukin (IL)-2 signaling is required for thymic development, peripheral expansion and suppressive activity of T(reg) cells. Animals lacking IL-2 die of autoimmunity, which is prevented by administration of IL-2-responsive T(reg) cells. In light of the emerging evidence that one of the primary physiologic roles of IL-2 is to generate and maintain T(reg) cells, the question arises as to the effects of IL-2 therapy on them. We monitored T(reg) cells during immune reconstitution in individuals with cancer who did or did not receive IL-2 therapy. CD4(+)CD25(hi) cells underwent homeostatic peripheral expansion during immune reconstitution, and in lymphopenic individuals receiving IL-2, the T(reg) cell compartment was markedly increased. Mouse studies showed that IL-2 therapy induced expansion of existent T(reg) cells in normal hosts, and IL-2-induced T(reg) cell expansion was further augmented by lymphopenia. On a per-cell basis, T(reg) cells generated by IL-2 therapy expressed similar levels of FOXP3 and had similar potency for suppression compared to T(reg) cells present in normal hosts. These studies suggest that IL-2 and lymphopenia are primary modulators of CD4(+)CD25(+) T(reg) cell homeostasis.

Adolescent↗

PubMed2005

Thymic expression of peripheral tissue antigens in humans: a remarkable variability among individuals.

The majority of maturing T lymphocytes that recognize self-antigens is eliminated in the thymus upon exposure to their target antigens. This physiological process of negative selection requires that tissue-specific antigens be expressed by thymic cells, a phenomenon that has been well studied in experimental animals. Here, we have examined the expression in human thymi of four retinal antigens, that are capable of inducing autoimmune ocular disease retinal S-antigen (S-Ag), recoverin, RPE65 and inter-photoreceptor retinoid-binding protein (IRBP)], as well as four melanocyte-specific antigens, two of which are used as targets for melanoma immunotherapy [gp100, melanoma antigen recognized by T cells 1, tyrosinase-related protein (TRP)-1 and TRP-2]. Using reverse transcription (RT)-PCR, we found that all thymic samples from the 18 donors expressed mRNA transcripts of most or all the eight tested tissue antigens. Yet, the expression of the transcripts varied remarkably among the individual thymic samples. In addition, S-Ag, RPE65 and IRBP were detected by immunostaining in rare cells in sections of human thymi by antibodies against these proteins. Quantitative real-time RT-PCR analysis revealed that the retinal antigen transcripts in the human thymus are present at trace levels, that are lower by approximately five orders of magnitude than those in the retina. Our observations thus support the notions that thymic expression is a common feature for all tissue-specific antigens and that the levels of expression play a role in determining the susceptibility to autoimmunity against these molecules.

Antigens, Neoplasm↗

PubMed2005

In vitro induction of myeloid leukemia-specific CD4 and CD8 T cells by CD40 ligand-activated B cells gene modified to express primary granule proteins.

The primary granule proteins (PGP) of myeloid cells are a source of multiple antigens with immunotherapeutic potential for myeloid leukemias. Therefore, we developed a method to induce T-cell responses to PGP protein sequences. We found that gene-transfected antigen-presenting cells efficiently expand functionally competent PGP-specific CD4 and CD8 T cells. The system was optimized using T-cell responses to autologous CD40-activated B cells (CD40-B) transfected with a cytomegalovirus pp65-encoding expression vector. To generate leukemia-specific T cells, expression vectors encoding the PGP proteinase 3 (PR3), human neutrophil elastase, and cathepsin-G were transfected into CD40-B cells to stimulate post-allogeneic stem cell transplantation T cells from five patients with myeloid and three with lymphoid leukemias. T-cell responses to PGP proteinase 3 and human neutrophil elastase were observed in CD8+ and CD4+ T cells only in patients with myeloid leukemias. T-cell responses against cathepsin-G occurred in both myeloid and lymphoblastic leukemias. T cells from a patient with chronic myelogenous leukemia (CML) and from a posttransplant CML patient, expanded against PGP, produced IFN-gamma or were cytotoxic to the patient's CML cells, demonstrating specific antileukemic efficacy. This study emphasizes the clinical potential of PGP for expansion and adoptive transfer of polyclonal leukemia antigen-specific T cells to treat leukemia.

Antigen-Presenting Cells↗

PubMed2005

Antigen-specific T-cell memory is preserved in children treated for acute lymphoblastic leukemia.

Despite profound T-cell immunodeficiency, most patients treated with chemotherapy do not succumb to infection. The basis for residual protective immunity in lymphopenic patients is not known. We prospectively measured T-cell numbers, thymopoiesis, and T-cell memory in 73 children undergoing a 2-year chemotherapy regimen for acute lymphoblastic leukemia (ALL) and compared them to an age-matched cohort of 805 healthy children. Most patients had profound defects in CD4 and CD8 T-cell numbers at diagnosis that did not recover during the 2 years of therapy. Thymic output and the fraction of naive T cells were significantly lower than in healthy controls. However, the remaining T-cell compartment was enriched for antigen-experienced, memory T cells defined both by phenotype and by function. This relative sparing of T-cell memory may, in part, account for the maintenance of protective immunity in lymphopenic patients treated for ALL. Moreover, because the memory T-cell compartment is least affected by ALL and its treatment, strategies to induce immunity to pathogens or tumor antigens in cancer patients may be most successful if they seek to expand pre-existing memory T cells.

Adolescent↗

PubMed2005

Characterization of subsets of CD4+ memory T cells reveals early branched pathways of T cell differentiation in humans.

The pathways for differentiation of human CD4(+) T cells into functionally distinct subsets of memory cells in vivo are unknown. The identification of these subsets and pathways has clear implications for the design of vaccines and immune-targeted therapies. Here, we show that populations of apparently naive CD4(+) T cells express the chemokine receptors CXCR3 or CCR4 and demonstrate patterns of gene expression and functional responses characteristic of memory cells. The proliferation history and T cell receptor repertoire of these chemokine-receptor(+) cells suggest that they are very early memory CD4(+) T cells that have "rested down" before acquiring the phenotypes described for "central" or "effector" memory T cells. In addition, the chemokine-receptor(+) "naive" populations contain Th1 and Th2 cells, respectively, demonstrating that Th1/Th2 differentiation can occur very early in vivo in the absence of markers conventionally associated with memory cells. We localized ligands for CXCR3 and CCR4 to separate foci in T cell zones of tonsil, suggesting that the chemokine-receptor(+) subsets may be recruited and contribute to segregated, polarized microenvironments within lymphoid organs. Importantly, our data suggest that CD4(+) T cells do not differentiate according to a simple schema from naive --> CD45RO(+) noneffector/central memory --> effector/effector memory cells. Rather, developmental pathways branch early on to yield effector/memory populations that are highly heterogeneous and multifunctional and have the potential to become stable resting cells.

CD4-Positive T-Lymphocytes↗

PubMed2005

Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection.

It has recently been established that both acute human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are accompanied by a dramatic and selective loss of memory CD4+ T cells predominantly from the mucosal surfaces. The mechanism underlying this depletion of memory CD4+ T cells (that is, T-helper cells specific to previously encountered pathogens) has not been defined. Using highly sensitive, quantitative polymerase chain reaction together with precise sorting of different subsets of CD4+ T cells in various tissues, we show that this loss is explained by a massive infection of memory CD4+ T cells by the virus. Specifically, 30-60% of CD4+ memory T cells throughout the body are infected by SIV at the peak of infection, and most of these infected cells disappear within four days. Furthermore, our data demonstrate that the depletion of memory CD4+ T cells occurs to a similar extent in all tissues. As a consequence, over one-half of all memory CD4+ T cells in SIV-infected macaques are destroyed directly by viral infection during the acute phase-an insult that certainly heralds subsequent immunodeficiency. Our findings point to the importance of reducing the cell-associated viral load during acute infection through therapeutic or vaccination strategies.

Acute Disease↗

PubMed2005

Interaction between the CD8 coreceptor and major histocompatibility complex class I stabilizes T cell receptor-antigen complexes at the cell surface.

The off-rate (k(off)) of the T cell receptor (TCR)/peptide-major histocompatibility complex class I (pMHCI) interaction, and hence its half-life, is the principal kinetic feature that determines the biological outcome of TCR ligation. However, it is unclear whether the CD8 coreceptor, which binds pMHCI at a distinct site, influences this parameter. Although biophysical studies with soluble proteins show that TCR and CD8 do not bind cooperatively to pMHCI, accumulating evidence suggests that TCR associates with CD8 on the T cell surface. Here, we titrated and quantified the contribution of CD8 to TCR/pMHCI dissociation in membrane-constrained interactions using a panel of engineered pMHCI mutants that retain faithful TCR interactions but exhibit a spectrum of affinities for CD8 of >1,000-fold. Data modeling generates a "stabilization factor" that preferentially increases the predicted TCR triggering rate for low affinity pMHCI ligands, thereby suggesting an important role for CD8 in the phenomenon of T cell cross-reactivity.

Antigens↗

PubMed2005

Characterization of functional and phenotypic changes in anti-Gag vaccine-induced T cell responses and their role in protection after HIV-1 infection.

Worldwide HIV-1 vaccine efforts are guided by the principle that HIV-specific T cell responses may provide protection from infection or delay overt disease. However, no clear correlates of T cell-mediated immune protection have been identified. Here, we examine in a HLA-B27(+) HIV seronegative vaccinee persistent HIV-specific vaccine-induced anti-Gag CD4(+) and CD8(+) T cell responses. Although these responses exhibited those characteristics (multifunctionality, appropriate memory phenotype, and targeting of epitopes associated with long-term nonprogression) predicted to correlate with protection from infection, the subject became HIV infected. After HIV infection, the vaccine-induced CD8(+) T cells expanded, but both CD4(+) and CD8(+) T cell responses acquired the functional and phenotypic patterns characteristic of chronic HIV infection. The virus quickly escaped the vaccine-induced T cell response, and the subject progressed more rapidly than expected for someone expressing the HLA-B27 allele. These data suggest that control of HIV by vaccine-elicited HIV-specific T cell responses may be difficult, even when the T cell response has those characteristics predicted to provide optimal protection.

AIDS Vaccines↗

PubMed2005

Thymic output generates a new and diverse TCR repertoire after autologous stem cell transplantation in multiple sclerosis patients.

Clinical trials have indicated that autologous hematopoietic stem cell transplantation (HSCT) can persistently suppress inflammatory disease activity in a subset of patients with severe multiple sclerosis (MS), but the mechanism has remained unclear. To understand whether the beneficial effects on the course of disease are mediated by lympho-depletive effects alone or are sustained by a regeneration of the immune repertoire, we examined the long-term immune reconstitution in patients with MS who received HSCT. After numeric recovery of leukocytes, at 2-yr follow-up there was on average a doubling of the frequency of naive CD4(+) T cells at the expense of memory T cells. Phenotypic and T cell receptor excision circle (TREC) analysis confirmed a recent thymic origin of the expanded naive T cell subset. Analysis of the T cell receptor repertoire showed the reconstitution of an overall broader clonal diversity and an extensive renewal of clonal specificities compared with pretherapy. These data are the first to demonstrate that long-term suppression of inflammatory activity in MS patients who received HSCT does not depend on persisting lymphopenia and is associated with profound qualitative immunological changes that demonstrate a de novo regeneration of the T cell compartment.

Adult↗

PubMed2005

Immunologic pressure within class I-restricted cognate human immunodeficiency virus epitopes during highly active antiretroviral therapy.

Cytotoxic T lymphocytes (CTL) and highly active antiretroviral therapy (HAART) are known to exert strong evolutionary pressures on the virus population during human immunodeficiency virus (HIV) infection. However, it is not known whether CTL responses continue to substantially affect viral evolution during treatment. To study the effect of immunologic pressure on viral sequences during HAART, we identified 10 targeted HIV-specific CD8+-T-cell epitopes in five treatment-naive patients, sequenced each epitope in plasma-derived viruses, and then identified evidence of immunologic pressure at these epitopes by comparing the frequency of viral variants in plasma to the frequency of the CD8+-T-cell response for each variant identified. For one of the five patients, evidence of viral evolution was found during therapy. The sequence of the CTL-targeted epitope changed from an apparent escape variant prior to the initiation of therapy, to the sequence that is best recognized by the CTL response after the initiation of therapy, and then finally to a new escape variant during continued therapy. These data show that CTL-mediated pressure can continue to affect viral evolution after the initiation of HAART, even when treatment drives the viral load below detectable levels, and suggest that antiretroviral therapy may preferentially inhibit those virus variants that escape the CTL response.

Amino Acid Substitution↗

PubMed2004

T cell cross-reactivity and conformational changes during TCR engagement.

All thymically selected T cells are inherently cross-reactive, yet many data indicate a fine specificity in antigen recognition, which enables virus escape from immune control by mutation in infections such as the human immunodeficiency virus (HIV). To address this paradox, we analyzed the fine specificity of T cells recognizing a human histocompatibility leukocyte antigen (HLA)-A2-restricted, strongly immunodominant, HIV gag epitope (SLFNTVATL). The majority of 171 variant peptides tested bound HLA-A2, but only one third were recognized. Surprisingly, one recognized variant (SLYNTVATL) showed marked differences in structure when bound to HLA-A2. T cell receptor (TCR) recognition of variants of these two peptides implied that they adopted the same conformation in the TCR-peptide-major histocompatibility complex (MHC) complex. However, the on-rate kinetics of TCR binding were identical, implying that conformational changes at the TCR-peptide-MHC binding interface occur after an initial permissive antigen contact. These findings have implications for the rational design of vaccines targeting viruses with unstable genomes.

Amino Acid Sequence↗

PubMed2004

Imatinib inhibits T-cell receptor-mediated T-cell proliferation and activation in a dose-dependent manner.

The tyrosine kinase inhibitor imatinib (imatinib, STI571, Glivec, and Gleevec) is increasingly used in patients undergoing allogeneic transplantation for leukemia. However, little is known regarding its potential immunoregulatory effects. Here, we investigate the effect of imatinib on T-cell receptor (TCR)-mediated activation of human T cells. Following stimulation with the anti-CD3 antibody 12F6, proliferation of activated T cells was almost completely inhibited by 10 microM imatinib. Furthermore, antigen-triggered expansion of CD8+ T cells in response to immunodominant cytomegalovirus (CMV) and Epstein-Barr virus (EBV) peptides was significantly reduced. Up-regulation of the activation markers CD25 and CD69 in response to TCR cross-linking was suppressed by imatinib at a mean inhibitory concentration 50% (IC50) of 5.4 microM and 7.3 microM, respectively; interleukin 2 (IL-2) production was also impaired. Analysis of the TCR-induced signaling cascade showed that imatinib substantially reduced tyrosine phosphorylation of ZAP70 and LAT in response to activation through the TCR. Sequence comparisons of all 90 tyrosine kinase genes in the human genome for homology in the adenosine triphosphate (ATP) binding pocket identified LCK, which is required for ZAP70 activation, as a likely target for imatinib. The IC50 for LCK inhibition by imatinib was 0.6 microM to 0.8 microM in an in vitro tyrosine kinase assay. In summary, imatinib can interfere with T-cell activation in vitro, and its impact on the frequency of opportunistic infections and graft-versus-host or graft-versus-leukemia reactions after transplantation should be investigated in clinical trials.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Design of soluble recombinant T cell receptors for antigen targeting and T cell inhibition.

The use of recombinant T cell receptors (TCRs) to target therapeutic interventions has been hindered by the naturally low affinity of TCR interactions with peptide major histocompatibility complex ligands. Here, we use multimeric forms of soluble heterodimeric alphabeta TCRs for specific detection of target cells pulsed with cognate peptide, discrimination of quantitative changes in antigen display at the cell surface, identification of virus-infected cells, inhibition of antigen-specific cytotoxic T lymphocyte activation, and identification of cross-reactive peptides. Notably, the A6 TCR specific for the immunodominant HLA A2-restricted human T cell leukemia virus type 1 Tax(11-19) epitope bound to HLA A2-HuD(87-95) (K(D) 120 microm by surface plasmon resonance), an epitope implicated as a causal antigen in the paraneoplastic neurological degenerative disorder anti-Hu syndrome. A mutant A6 TCR that exhibited dramatically increased affinity for cognate antigen (K(D) 2.5 nm) without enhanced cross-reactivity was generated; this TCR demonstrated potent biological activity even as a monomeric molecule. These data provide insights into TCR repertoire selection and delineate a framework for the selective modification of TCRs in vitro that could enable specific therapeutic intervention in vivo.

Antigens↗