PubMed Health⌕ Search

Biomedical subjects

P E Fields

Publications and source records attributed to P E Fields.

16 recordsLinked to original sources
PubMed2001

A transcriptional block in the IL-2 promoter at the -150 AP-1 site in effector CD8+ T cells.

Both CD4+ and CD8+ T cells that produce IL-2 in response to Ag recognition have been isolated. However, most effector CD8+ T cells recovered after exposure to Ag do not produce sufficient IL-2 to sustain growth, and depend on CD4+ T helper cells for this obligate growth factor. IL-2 expression in CD4+ T cells is primarily controlled at the level of transcription, but mechanisms restricting IL-2 production in CD8+ T cells have not been elucidated. To evaluate transcriptional regulation of the IL-2 gene in CD8+ T cells, we stably transfected reporter genes into Ag-specific CD8+ T cell clones. CD28+ CD8(+) T cells unable to transcribe the IL-2 gene in response to antigenic stimulation had a block in transactivation of the -150 CD28 response element (CD28RE)/AP-1 site of the IL-2 promoter, but did transactivate the composite NFAT/AP-1 and OCT/AP-1 sites, and a consensus AP-1 motif. Mutation of the nonconsensus -150 AP-1 site to a consensus AP-1 site, or insertion of a CD28RE/AP-1 consensus site upstream of the native -150 CD28RE/AP-1 site restored transactivation of the altered promoter. These results suggest that the defect at the -150 site may reflect the absence or inactivity of a required factor rather than repression of the IL-2 promoter.

5' Untranslated Regions↗
PubMed2001

Absence of CTLA-4 lowers the activation threshold of primed CD8+ TCR-transgenic T cells: lack of correlation with Src homology domain 2-containing protein tyrosine phosphatase.

To examine the role of CTLA-4 in controlling Ag-specific CD8(+) T cell activation, TCR-transgenic/CTLA-4 wild-type or -deficient mice were generated in a recombination-activating gene 2-deficient background. Naive T cells from these mice responded comparably whether or not CTLA-4 was expressed. In contrast, primed T cells responded more vigorously if they lacked CTLA-4 expression. We took advantage of the difference between naive and primed T cell responses to approach the mechanism of CTLA-4 function. Single-cell analyses demonstrated that a greater fraction of CTLA-4-deficient cells responded to a fixed dose of Ag compared with CTLA-4-expressing cells, whereas the magnitude of response per cell was comparable. A shift in the dose-response curve to APCs was also observed such that fewer APCs were required to activate CTLA-4-deficient T cells to produce intracellular IFN-gamma and to proliferate. These results suggest that CTLA-4 controls the threshold of productive TCR signaling. Biochemical analysis comparing stimulated naive and primed TCR-transgenic cells revealed no obvious differences in expression of total CTLA-4, tyrosine-phosphorylated CTLA-4, and associated Src homology domain 2-containing protein tyrosine phosphatase. Thus, the biochemical mechanism explaining the differential inhibitory effect of CTLA-4 on naive and primed CD8(+) T cells remains unclear.

Abatacept↗
PubMed2001

Regulation of IL-4 gene expression by distal regulatory elements and GATA-3 at the chromatin level.

Using a transgenic approach, we examined distal regulatory elements located in the IL-4 locus and the role of GATA-3 at these elements. The intergenic DNase I hypersensitive sites (HSS) showed strong enhancement, and the intronic enhancer (IE) and HS5/HS5a sites showed weaker enhancement of the IL-4 promoter. Elements in the 3' region of the IL-4 gene contributed to Th2 specificity. All individual enhancers were T cell activation dependent but not Th2 specific, with the exception of IE. However, when these distal elements were combined into a "minilocus," expression was strongly enhanced and Th2 specific. GATA-3 mediated strong enhancement of IL-4 promoter activity in Th1 cells when the promoter was embedded in the minilocus or linked to HSS and IE, demonstrating that GATA-3 acts through these elements to regulate IL-4 gene expression.

Animals↗
PubMed2000

Cutting edge: TGF-beta inhibits Th type 2 development through inhibition of GATA-3 expression.

TGF-beta is an important immunomodulatory cytokine that can inhibit differentiation of effector T cells. In this report, we address the molecular mechanisms through which TGF-beta inhibits differentiation of CD4(+) cells into Th type 2 cells. We demonstrate that TGF-beta inhibits GATA-3 expression in developing Th cells. We also show that inhibition of GATA-3 expression by TGF-beta is a major mechanism of inhibition of Th2 differentiation by TGF-beta as ectopic expression of GATA-3 in developing T cells overcomes the ability of TGF-beta to inhibit Th2 differentiation. TGF-beta likely inhibits GATA-3 expression at the transcriptional level and does so without interfering with IL-4 signaling.

Adjuvants, Immunologic↗
PubMed1998

B7.1 is a quantitatively stronger costimulus than B7.2 in the activation of naive CD8+ TCR-transgenic T cells.

Using a TCR transgenic mouse bred onto a recombinase-activating gene-2-deficient background, we have examined the influence of B7.1 and B7.2 on activation of naive, CD8+ T cells in vitro. We found that B7.1 was a more potent costimulus than B7.2 for induction of proliferation and IL-2 production by naive CD8+ T cells. This difference appeared to be quantitative in nature, as determined using transfectants expressing various defined levels of B7.1 or B7.2, or using purified B7.1 or B7.2 fusion proteins. In contrast to the quantitative differences seen in stimulation of naive T cells, B7.1 and B7.2 were comparable in their ability to costimulate responses in T cells previously primed in vitro. In addition, primed, but not naive, T cells were capable of proliferating and producing IL-2 in response to a TCR stimulus alone, apparently in the absence of B7 costimulation. Lastly, we found that B7.1 and B7.2 were equivalently capable of driving differentiation of naive CD8+ T cells into an IL-4-producing phenotype when exogenous IL-4 was added to the primary culture or to an IFN-gamma-producing phenotype in the presence of IL-12. These results indicate that signals generated by B7.1 and B7.2 are qualitatively similar, but that B7.1 is quantitatively stronger than B7.2. Further, our results indicate that the activation state of the responding T cell may influence the efficiency with which the T cell can respond to a costimulatory signal provided by either B7.1 or B7.2.

Animals↗
PubMed1998

B7-1 engagement of cytotoxic T lymphocyte antigen 4 inhibits T cell activation in the absence of CD28.

Ligation of cytotoxic T lymphocyte antigen 4 (CTLA4) appears to inhibit T cell responses. Four mechanisms have been proposed to explain the inhibitory activity of CTLA4: competition for B7-1 and B7-2 binding by CD28; sequestration of signaling molecules away from CD28 via endocytosis; delivery of a signal that antagonizes a CD28 signal; and delivery of a signal that antagonizes a T cell receptor (TCR) signal. As three of these potential mechanisms involve functional antagonism of CD28, an experimental model was designed to determine whether CTLA4 could inhibit T cell function in the absence of CD28. TCR transgenic/recombinase activating gene 2-deficient/CD28-wild-type or CD28-deficient mice were generated and immunized with an antigen-expressing tumor. Primed T cells from both types of mice produced cytokines and proliferated in response to stimulator cells lacking B7 expression. However, whereas the response of CD28+/+ T cells was augmented by costimulation with B7-1, the response of the CD28-/- T cells was strongly inhibited. This inhibition was reversed by monoclonal antibody against B7-1 or CTLA4. Thus, CTLA4 can potently inhibit T cell activation in the absence of CD28, indicating that antagonism of a TCR-mediated signal is sufficient to explain the inhibitory effect of CTLA4.

Abatacept↗
PubMed1997

Antigenic cancer cells grow progressively in immune hosts without evidence for T cell exhaustion or systemic anergy.

One enigma in tumor immunology is why animals bearing malignant grafts can reject normal grafts that express the same nonself-antigen. An explanation for this phenomenon could be that different T cell clones react to the normal graft and the malignant cells, respectively, and only the tumor-reactive clonotypes may be affected by the growing tumor. To test this hypothesis, we used a T cell receptor transgenic mouse in which essentially all CD8(+) T cells are specific for a closely related set of self-peptides presented on the MHC class I molecule Ld. We find that the tumor expressed Ld in the T cell receptor transgenic mice but grew, while the Ld-positive skin was rejected. Thus, despite an abundance of antigen-specific T cells, the malignant tissue grew while normal tissue expressing the same epitopes was rejected. Therefore, systemic T cell exhaustion or anergy was not responsible for the growth of the antigenic cancer cells. Expression of costimulatory molecules on the tumor cells after transfection and preimmunization by full-thickness skin grafts was required for rejection of a subsequent tumor challenge, but there was no detectable effect of active immunization once the tumor was established. Thus, the failure of established tumors to attract and activate tumor-specific T cells at the tumor site may be a major obstacle for preventive or therapeutic vaccination against antigenic cancer.

Animals↗
PubMed1997

CD28 costimulation promotes the production of Th2 cytokines.

CD28 ligation augments TCR-mediated proliferation, IL-2 production, and T cell survival. However, the role of CD28 costimulation in T cell differentiation remains controversial. To address this issue, CD28+ and CD28-deficient TCR alphabeta transgenic (Tg) mice were used to examine cytokine production by T cells following antigenic stimulation. Increasing CD28 ligation resulted in increased production of IL-4 and IL-5, consistent with differentiation toward a Th2 phenotype, in both CD4+ TCR Tg T cells and CD8+ TCR Tg T cells. The same result was obtained with CD4+ TCR Tg mice bred to RAG2-deficient mice, indicating that the Th2 differentiation observed with increased CD28 ligation was not due to the presence of memory T cells. Although CD28 costimulation is an essential factor regulating IL-2 synthesis, differentiation toward a Th2-like phenotype by CD28 ligation was not an indirect effect of enhanced IL-2 production. In contrast, blockade of IL-4 during the primary cultures of the T cells resulted in a profound inability to produce Th2-type cytokines upon restimulation. The critical role of IL-4 was confirmed by the finding that CD28-deficient TCR alphabeta Tg+ T cells cultured with rIL-4 differentiated into Th2-like T cells. Therefore, CD28 ligation promotes the production of Th2-type cytokines by naive murine T cells via an IL-4-dependent mechanism.

Animals↗
PubMed1996

Blocked Ras activation in anergic CD4+ T cells.

T cell anergy is a state of functional unresponsiveness characterized by the inability to produce interleukin-2 (IL-2) upon T cell receptor stimulation. The mitogen-activated protein kinases ERK-1 and ERK-2 and the guanosine triphosphate-binding protein p21ras were found to remain unactivated upon stimulation of anergic murine T helper cell 1 clones. The inability to activate the Ras pathway did not result from a defect in association among Shc, Grb-2, and murine Son of Sevenless, nor from a defect in their tyrosine phosphorylation. This block in Ras activation may lead to defective transactivation at activator protein 1 sites in anergic cells and may enable T cells to shut down IL-2 production selectively during anergy.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Immunosuppression of thyroiditis.

Immunization of mice with 50 micrograms human thyroglobulin (TG) in complete Freund's adjuvant leads to histological thyroiditis; production of IgG, IgA, and IgM anti-TG antibodies; and in vitro proliferative responses after incubation of lymphocytes with TG. Oral administration of 500 micrograms TG at four intervals before Tg immunization and once afterward causes up to 80% suppression of these responses. The effect is antigen specific and dose dependent. Feeding TG after immunization produces a 40% reduction in responses. We wished to define the mechanism of this antigen-specific oral tolerization. Popliteal lymph nodes (PLN) of orally tolerized animals (T) are reduced in size compared to those in immunized (I) animals not fed TG. PLN and mesenteric lymph nodes (MLN) of I animals produce interleukin-2 (IL-2) and interferon-gamma (IFN gamma) after in vitro incubation with TG, typical of an inflammatory immune response. PLN and MLN of tolerized animals do not proliferate in response to antigen, do not produce IL-2 or IFN gamma, but do not produce the cytokines IL-4 and transforming growth factor-beta (TGF beta). Mixing in vitro of spleen cells from T and I animals causes a reduction in the immune response when incubated with TG, but no reduction in response to purified protein derivative (PPD) (the antigen in complete Freund's adjuvant). When T splenocytes are incubated with TG and PPD together, the response to TG and PPD is suppressed. Partially purified CD8+ cells from tolerized animals produce IL-4 and TGF beta after exposure to human TG and induce suppression, whereas partially purified CD4+ cells produce IL-2 and IFN gamma and do not cause suppression. MLN cells do not proliferate in response to antigen, but do produce inhibitory cytokines. T animals appear to shift the immune response from a Th-1 helper cell subset response to a Th-2 helper cell immunosuppressive response. In this model, oral tolerization produces a dramatic reduction in the immune response. Exposure of MLN to oral TG appears to cause the production of regulatory cells that migrate to spleen and PLN. In vitro studies demonstrate that on exposure to antigen, these regulatory cells produce IL-4 and TGF beta, which suppress all aspects of specific immune responsiveness and nonspecifically suppress other ongoing immune responses (bystander effect). Oral tolerization may include some element of T cell deletion or anergy. This model defines an experimental system with possible relevance to immunosuppression of human autoimmune thyroid disease.

Administration, Oral↗
PubMed1993

Major histocompatibility complex class II-restricted presentation of an internally synthesized antigen displays cell-type variability and segregates from the exogenous class II and endogenous class I presentation pathways.

Although reported examples of endogenous antigen (Ag) presentation by major histocompatibility complex (MHC) class II molecules have increased, the mechanisms governing this process remain poorly defined. In this communication, we describe an experimental system designed to examine the mechanisms governing class II presentation of internal Ag. Our target peptide is processed from a transmembrane protein constitutively expressed by a variety of nucleated cells (MHC class I, H-2Ld), is naturally displayed by MHC class II molecules in vivo, and is recognized by a class II-restricted, CD4+ T cell hybridoma. Our results indicate that presentation of the Ld target Ag is independent of its plasma membrane expression, may not involve endosomal proteolysis, and thus may be distinct from the classically defined class II presentation pathway. In addition, the observations that Ld presentation does not require a functional TAP-1 complex, is not blocked by invariant chain, and cannot utilize cytoplasmic forms of H-2Ld, suggest that a classical class I pathway is not involved in this presentation event. Finally, our data suggest that different cofactors participate in MHC class II presentation of exogenous and endogenous Ag, and that disparate Ag presenting cells, such as B, T, and pancreatic islet cells, may differentially express these two class II pathways of Ag presentation.

Animals↗
PubMed1992

Metabolic requirements for macrophage presentation of Listeria monocytogenes to immune CD8 cells.

Though ingested Ag are readily degraded into peptides within endocytic vesicles, APC usually cannot present these fragments to CD8 cells. Despite this generalization, some exceptions have been noted. For example, murine macrophage targets readily process heat-killed Listeria monocytogenes (HKLM) into a form recognizable by immune CD8 CTL. Using an assay of Listeria-specific, CD8-mediated cytotoxicity to quantitate Ag presentation by C57Bl/6 macrophage targets, we have examined some of the cellular requirements for this form of Ag processing. To assess whether the physical form of the Ag is an important determinant of processing, we compared the ability of macrophages to present intact HKLM, fractionated L. monocytogenes (LM) membranes, and octyl-beta-d-thioglucopyranoside-solubilized extracts of LM membranes. Macrophages presented each Ag form in a similar manner indicating that processing is not critically dependent on the presence of intact bacteria or even on the introduction of Ag in a particulate form. To gain insight into the metabolic requirements for Ag processing, we examined the effects of several inhibitors. As might be expected, listerial Ag presentation was blocked by brefeldin, a known inhibitor of the endogenous pathway of Ag processing. LM Ag presentation, however, was also blocked by inhibitors of endosomal acidification (chloroquine, ammonium chloride, and monensin) and by the acid protease inhibitor pepstatin A, suggesting that endocytic processing may play an essential role in CD8 recognition of this Ag. To formally establish that this pattern of exogenous Ag processing requires the presence of a class I MHC product, we demonstrated that beta-2 microglobulin-deficient macrophages, which lack class I MHC product expression, cannot present HKLM to CD8 cells. However, we could not block Ag presentation by incubating macrophages with monoclonal anti-H-2K or H-2D antibodies, suggesting that LM Ag presentation may be mediated by some other class I MHC product. Additional characterization of this pathway of Ag presentation is warranted in view of its possible role in initiating CD8-mediated immunity against microbial Ag.

Animals↗
PubMed1981

Histoplasmosis in the eastern Caribbean: a preliminary survey of the incidence of the infection.

A preliminary survey of the incidence of histoplasmosis in the eastern Caribbean was carried out. The percentages of positive histoplasmin skin test reactors in the populations studied were as follows: Barbados (4%), Trinidad (42%) and Guyana (29%). In Trinidad 69% of individuals under 60 had positive test. However a lower proportion (4%) had serological evidence of past infection with Histoplasma capsulatum. The organism was not isolated from the soil although it had been found previously in Trinidad. On this island five previously diagnosed cases of histoplasmosis were identified. The study confirmed the presence of the infection in the area and further investigations are suggested.

Health Surveys↗