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D Vremec

Publications and source records attributed to D Vremec.

16 recordsLinked to original sources
PubMed1998

Does the IL-2 receptor alpha chain induced on dendritic cells have a biological function?

The IL-2 receptor (IL-2R) alpha chain (CD25), but not the IL-2R beta chain, is induced on dendritic cells (DC) by brief periods of culture. To test if this IL-2R alpha is important for DC function, DC were isolated from the spleens of mutant mice with the IL-2R alpha gene disrupted and compared with normal DC for ability to stimulate proliferation of allogeneic CD4 and CD8 T cells in culture. The IL-2R alpha null DC and the normal DC produced nearly identical proliferative responses from CD4 and from CD8 T cells. When the CD8 alpha+ and CD8 alpha- subsets of the IL-2R alpha null DC were separated, they also produced proliferative responses similar to that of their normal DC counterparts. Overall there was no evidence that the inducible IL-2R alpha on DC was required for DC development, for stimulation of T cells or for regulation of T cell responses.

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PubMed1997

Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes.

Freshly isolated, mature dendritic cells (DC) from mouse lymphoid organs were analyzed by immunofluorescent labeling and flow cytometry to determine the number of discrete subpopulations and to assess possible lineage markers. The permanence of surface markers was then determined by overnight culture of the DC. Three DC subtypes were discerned, CD8alpha- DEC-205-, CD8alpha+ DEC-205+, and CD8alpha- DEC-205+, with different tissue distributions. The majority of DC expressed high levels of class II MHC, expressed CD11c, and expressed the costimulator molecules CD80, CD86, and CD40; CD80 and CD40 were further up-regulated on culture. DC also expressed low levels of L-selectin that were up-regulated on culture. Thymus contained predominantly CD8alpha+ DEC205+ CD11b- DC, resembling a major subpopulation of DC in other tissues but unique in expressing BP-1. Spleen contained predominantly two DC populations in equal proportions: one CD8alpha+ DEC-205+ CD11b- as in the thymus, and the other CD8alpha- DEC-205- CD11b+. Lymph nodes contained the same two DC populations as in spleen, but in addition a third population of CD8alpha- DEC-205+ CD11b- DC. The CD8alpha expression of splenic DC subpopulations did not change on culture. Although DEC-205 was up-regulated on culture so all DC became positive, the difference in the level between subpopulations was maintained. However, CD11b was up-regulated on culture, so all subpopulations became positive and finally expressed equivalent levels. Some aspects of this complex, but discrete, pattern of surface marker expression can be correlated with differences in lineage origin and functional activity of the DC.

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PubMed1997

Dendritic cells and T lymphocytes: developmental and functional interactions.

Dendritic cells (DCs) are specialized for presentation of antigen to T cells and are essential for primary T cell activation. Although DCs are generally considered to be myeloid derived, we now have evidence that a subgroup are of lymphoid origin. In particular, the DCs of the adult mouse thymus appear to be derived from the same early, lymphoid-restricted precursor cells that generate T lymphocytes. Purified early thymic T precursors have the capacity to produce T cells, B cells, NK cells and DCs, but not myeloid cells, on transfer to irradiated recipients. They also produce thymic DCs on culture with a mix of cytokines; this mix does not include GM-CSF, needed to generate myeloid-derived DCs. A subgroup of DCs in other lymphoid organs, which like thymic DCs express CD8 as an alpha alpha homodimer, may likewise be of lymphoid origin. These CD8+ DCs in mouse spleen differ functionally from the conventional CD8+ DCs. CD8+ DCs efficiently activate CD4+ T cells but then kill them via Fas ligand on the DC surface. CD8+ DCs efficiently recruit CD8+ T cells into the cell cycle, but their proliferation is then restricted by an inadequate production of interleukin 2. This subgroup of CD8+ DCs therefore appears to have a regulatory role.

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PubMed1997

The influence of granulocyte/macrophage colony-stimulating factor on dendritic cell levels in mouse lymphoid organs.

To ascertain whether the development of dendritic cells (DC) in mouse lymphoid organs is dependent on granulocyte/macrophage colony-stimulating factor (GM-CSF), we determined the number of DC in the thymus, spleen and lymph nodes of normal mice, of mice with the genes coding for GM-CSF or its receptor inactivated, and of transgenic mice with excessive levels of GM-CSE DC were extracted from the tissues and enriched prior to flow cytometric analysis. The total DC level and the incidence of DC expressing lymphoid-related markers (CD8(hi) CD11b(lo)) and myeloid-related markers (CD8(lo) CD11b(hi)) were monitored. Both in GM-CSF null mice, and GM-CSF receptor null mice, DC of all surface phenotypes were present in all lymphoid organs; only small decreases in DC levels were recorded, except for the lymph nodes of GM-CSF receptor null mice which showed a more pronounced (threefold) decrease in DC numbers. Since the GM-CSF receptor null mice lacked the beta chain common to the GM-CSF, interleukin (IL)-3 and IL-5 receptors, the development of DC in the absence of GM-CSF was not due to common beta chain mediated developmental signals elicited by IL-3 or IL-5. In GM-CSF transgenic mice, there was only a 50 % increase in DC numbers in thymus and spleen, paralleling an increase in overall cellularity, but a more pronounced (threefold) increase in DC numbers in lymph nodes. There was no evidence that GM-CSF had a selective effect on any particular DC subpopulation defined by CD8 or CD11b expression. We conclude that the development of most lymphoid tissue DC can proceed in the absence of GM-CSF, although this cytokine can produce some elevation of DC levels. It is not clear whether the enhancing effect of GM-CSF is direct or an indirect effect mediated by other cytokines.

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PubMed1997

Are CD8+ dendritic cells (DC) veto cells? The role of CD8 on DC in DC development and in the regulation of CD4 and CD8 T cell responses.

The CD8-expressing dendritic cells (DC) present in mouse spleen have been shown to have a regulatory effect on the CD4 and CD8 T cells they activate, restricting subsequent T cell proliferation by either inducing apoptotic T cell death (CD4 T cells) or by limiting endogenous cytokine production (CD8 T cells). To determine the role of the CD8 molecule itself in these regulatory phenomena, the DC from CD8 null mice were studied. The DC marker DEC-205 (NLDC 145) was used as a surrogate marker for CD8, since the expression of these two molecules on splenic DC was closely correlated. DC levels were normal, and the incidence of DEC-205+ and DEC-205- DC was normal in CD8 null mice, indicating that the absence of CD8 did not affect DC development. The proliferative response of T cells to allogeneic DEC-205+ DC from either CD8-/- or CD8+/+ mice was similar and was much less than the response to DEC-205- DC from these mice. This applied to both the CD4 and the CD8 T cell responses. Thus the lack of the CD8 molecule did not affect the stimulatory or regulatory properties of the DC. The regulatory CD8+ DEC-205+ DC therefore differ in that respect from antigen-presenting 'veto' cells, where CD8 itself is involved in transmitting negative signals to the T cells. DEC-205 may prove to be a more pertinent marker of the regulatory DC population.

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PubMed1996

Effects of excess GM-CSF levels on hematopoiesis and leukemia development in GM-CSF/max 41 double transgenic mice.

Double transgenic mice were produced by mating granulocyte-macrophage colony-stimulation factor (GM-CSF) transgenic mice with max 41 transgenic mice that exhibit excess granulopoiesis and a predisposition to thymic lymphoma development. Although only two-thirds of the double transgenic mice had elevated circulating GM-CSF levels, double transgenic mice maintained significantly higher blood granulocytes and monocytes and more extreme granulopoietic hypercellularity in the marrow and spleen than max 41 transgenic mice. In double transgenic mice, early death occurred from the GM-CSF transgenic syndrome. Because of these early deaths, the incidence of thymic and generalized lymphomas was artificially lower than in max 41 mice but those lymphomas that did develop occurred earlier than in max 41 mice. While the excess GM-CSF levels in double transgenic mice stimulated increased granulocyte and monocyte formation and peritoneal dendritic cells were excessive, this failed to prevent the spontaneous development of T lymphomas, suggesting that dendritic cell-initiated suppression of tumor development may not be effective with this type of tumor.

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PubMed1995

Intermediate steps in thymic positive selection. Generation of CD4-8+ T cells in culture from CD4+8+, CD4int8+, and CD4+8int thymocytes with up-regulated levels of TCR-CD3.

Minor thymus subpopulations representing possible intermediates in thymic positive selection were isolated by cell sorting from bcl-2 transgenic mice, and cultured 1 to 4 days in simple medium to assess their ability to spontaneously develop the surface phenotype of mature T cells. Recovery of cells was in the 60 to 80% range, and no cell proliferation occurred. Only cells originally expressing high, near mature T cell levels of CD3 developed further in culture by down-regulation of CD4 or CD8. The main mature cell product was CD4-8+, regardless of whether the starting phenotype of the CD3high intermediates was CD4+8+, CD4int8+, or CD4+8int; only an intermediate subpopulation expressing the highest levels of CD4 (CD4high8int) produced a dominance of CD4+8- mature progeny. Partial down-regulation of CD8 was therefore not a good indicator of CD4+ T lineage commitment. These and previous results indicate that maturation to the CD8+ T lineage involves a rapid up-regulation of the TCR-CD3 complex, but a relatively slow down-regulation of CD4; it may also involve a partial, transient reduction in surface CD8. In contrast, maturation to the CD4+ T lineage involves a relatively rapid down-regulation of CD8, with maintenance of high levels of CD4. There appears to be a marked asymmetry in the developmental steps leading from CD4+8+ thymocytes to the CD8+ or to the CD4+ T cell lineage.

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PubMed1995

Mouse thymus dendritic cells: kinetics of development and changes in surface markers during maturation.

The early thymus precursor population of adult mice has the capacity to generate T cells, B cells and dendritic cells (DC). These precursors were injected into the thymus of irradiated recipients in order to follow the kinetics of thymic DC development. The resultant cohort of T-lineage cells developing in the thymus was accompanied by a parallel cohort of DC, present at 10(3)-fold lower frequency. The intrathymic lifespan of these DC was as short as that of T-lineage thymocytes. As the thymic DC matured, some markers characteristic of the original precursor population gradually declined (Ly-5, c-kit, Sca-2) whereas markers characteristic of thymic DC appeared and were maintained (major histocompatibility complex class II, CD11c, NLDC-145 and CD8 alpha). Some thymic DC expressed the early B-cell marker BP-1, and BP-1 mRNA, throughout their maturation. The surface markers on thymic DC could be divided into two groups. Some markers, including class I and class II MHC, CD8 alpha and BP-1, appeared to be integral components of the DC surface. In contrast, other markers, including Thy-1, CD4 and CD8 beta, had probably been picked up from associated thymocytes.

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PubMed1994

CD4 and CD8 expression by human and mouse thymic dendritic cells.

Dendritic cells (DC) from human and mouse thymus were compared. DC from both sources were isolated by digestion with collagenase, disruption of cellular complexes with a chelating agent, selection of light density cells, immunomagnetic bead depletion of other cell types (without depletion with anti-CD4 or anti-CD8) and finally sorting for cells expressing high levels of class II MHC. Yields of DC from human and mouse thymus were comparable (around 1 DC/10(3) thymocytes), they displayed similar DC morphology, and both showed strong expression of CD11c. DC from the human thymus all expressed very high levels of CD4 but low levels of CD8. In contrast, DC from the mouse thymus expressed high levels of CD8 but only low levels of CD4. Human thymic DC were also substantially larger than mouse thymic DC. The biological significance of CD4 and CD8 expression by DC is discussed in view of this major species difference and the possibility that human thymic DC may be targets for HIV infection.

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PubMed1993

Characterization of thymic nurse-cell lymphocytes, using an improved procedure for nurse-cell isolation.

Thymic nurse cells (TNC), multicellular complexes consisting of lymphoid cells enclosed within cortical epithelial cells, were isolated from mouse thymus by a modified procedure allowing immunofluorescent labeling and flow cytometric analysis of their lymphoid contents (TNC-L). Collagenase was the only protease used for tissue digestion, to ensure that surface antigen markers remained intact. Zonal unit-gravity elutriation was used to enrich the TNC on the basis of their high sedimentation rate, followed by immunomagnetic bead depletion to remove residual mononuclear cell contaminants and a density separation to remove debris. The TNC-L were then released from inside TNC by a short period of culture. The measured contamination of TNC-L with exogenous thymocytes was around 0.5%. Three-color immunofluorescent labeling revealed that TNC-L included, as well as a majority of immature CD4+8+3low thymocytes, about 12% of apparently mature CD4+8-3high and CD4-8+3high thymocytes. TNC are located in the cortex, where mature cells are rare; the occurrence of mature phenotype cells within these structures suggests that they represent a microenvironment for the selection and generation of mature T cells.

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PubMed1992

The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells.

A new procedure for rapid isolation of dendritic cells (DC) was devised, involving collagenase digestion of tissues, dissociation of lymphoid-DC complexes, selection of light-density cells, then depletion of lymphocytes and other non-DC by treatment with a mixture of lineage-specific monoclonal antibodies (mAbs) and removal with anti-immunoglobulin-coupled magnetic beads. This enriched population (approximately 80% DC) was further purified when required by fluorescence-activated cell sorting for cells expressing high levels of class II major histocompatibility complex (MHC). The isolated DC were characterized by immunofluorescent staining using a panel of 30 mAbs. Thymic DC were surface positive for a number of markers characteristic of T cells, but they were distinct from T-lineage cells in expressing high levels of class II MHC, in lacking expression of the T cell receptor (TCR)-CD3 complex, and having TCR beta and gamma genes in germline state. Splenic DC shared many markers with thymic DC, but were negative for most T cell markers, with the exception of CD8. A substantial proportion of DC from both thymus and spleen expressed CD8 at high levels, comparable with that on T cells. This appeared to be authentic CD8, and was produced by the DC themselves, since they contained CD8 alpha mRNA. Thymic DC presented both the CD8 alpha and beta chains on the cell surface (Ly-2+3+), although the alpha chain was in excess; the splenic DC expressed only the CD8 alpha chain (Ly-2+3-). It is suggested that the expression of CD8 could endow certain antigen-presenting DC with a veto function.

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PubMed1991

The kinetics of T cell antigen receptor expression by subgroups of CD4+8+ thymocytes: delineation of CD4+8+3(2+) thymocytes as post-selection intermediates leading to mature T cells.

Cortical thymocytes from adult mice, separated on the basis of coexpression of CD4 and CD8 or of binding of high levels of peanut agglutinin (PNA), were subdivided according to the level of expression of the T cell receptor (TCR)-CD3 complex. The incidence of dividing cells in the resultant subpopulations was determined by DNA staining. Precursor-product relationships and the timing of TCR-CD3 acquisition were studied using continuous in vivo [3H]TdR labeling and radioautography. The extent of intrathymic selection for TCR specificity in the subpopulations was determined from the incidence of cells bearing V beta 6 or V beta 17a in different mouse strains. The majority of dividing CD4+8+ blast cells expressed extremely low levels of TCR-CD3, indicating that TCR expression and specificity selection generally occurred after division ceased. The [3H]TdR-labeling studies indicated that postdivision TCR expression was rapid, and that those nondividing cortical thymocytes which had not expressed significant levels of TCR by day 1, remained extremely low or negative for their entire 3.6-d lifespan. Small cortical thymocytes which expressed moderate levels of TCR-CD3, were predominantly an unselected population with a lifespan of 3.8 d. A small subgroup of CD4+8+ PNA+ cortical thymocytes expressing high levels of TCR-CD3 was identified as a nondividing intermediate between the small cortical thymocytes expressing moderate levels of TCR and mature medullary thymocytes. These intermediates showed a 1-d lag in [3H]TdR labeling, then a 3.4-d transit time. The cell flux through this intermediate subpopulation was approximately 10(6) cells/d, similar to the rate of turnover of mature thymocytes; thus, although only 3-4% of thymocytes progressed to this intermediate state, once reaching it most then progressed to full maturity. In accordance with this, the incidence of the V beta selection markers within the intermediate subpopulation indicated that both positive and negative selection had already occurred. Selection for TCR specificity in the systems studied appeared to take place among CD4+8+ thymocytes expressing intermediate levels of TCR.

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PubMed1991

Does negative selection involve accumulation of self-reactive thymocytes in thymic rosettes?

Thymic rosettes, the natural associations between thymocytes and either macrophages or dendritic cells, were isolated from the thymus by collagenase digestion and unit-gravity elutriation. Rosettes from mouse strains where either the V beta 6-bearing thymocytes are deleted because of reactivity with products of the Mlsa allele of the minor lymphocyte stimulating locus, or where V beta 17a-bearing thymocytes are deleted because of reactivity with IE class II MHC molecules, were compared with rosettes from appropriate control strains to test if a selective association with stromal cells preceded deletion. Rosettes from an Mlsa-bearing strain were able to stimulate an Mlsa-reactive T-hybridoma, but much of this stimulatory activity was attributable to the few B cells associated with the rosette preparations; the stromal components of the rosettes appeared to be poor presenters of Mlsa gene products. There was no enrichment of thymocytes bearing high or low levels of V beta 6 TcR in the rosettes from the Mlsa-bearing strain, which might have reflected the poor presentation by the stromal cells. However, nor was there detectable selective association of thymocytes bearing C beta 17a in the rosettes from an IE-positive mouse strain. This argues against binding and immobilisation on stromal cells as part of the deletion process, but not against the stromal cells delivering a rapid signal during a transient association, leading later to deletion.

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PubMed1991

Different subpopulations of developing thymocytes are associated with adherent (macrophage) or nonadherent (dendritic) thymic rosettes.

Thymic rosettes (ROS), structures consisting of thymic lymphoid cells attached to a central stromal cell, were isolated from mouse thymus by collagenase digestion and unit-gravity elutriation. The ROS were then separated into those where the stromal cells were either macrophage-like (M-ROS) or dendritic cell-like (D-ROS), on the basis of the differences in adherence properties or in the level of MAC-1 surface antigen. The ROS were then dissociated and the thymocyte content analyzed by immunofluorescent staining and flow cytometry. M-ROS and D-ROS differed in thymocyte composition, although the major component of both was the CD4+CD8+ cortical thymocyte. D-ROS were enriched in thymocytes expressing high levels of surface T-cell antigen receptor (TcR) and the associated CD3 complex, and these included both CD4+CD8-CD3++ and CD4-CD8+CD3++ mature thymocytes. M-ROS were enriched in CD4-CD8- thymocytes and had a reduced content of thymocytes expressing high TcR-CD3 levels; they nevertheless contained some mature thymocytes, but only of the CD4+CD8-CD3++ category. Several lines of evidence indicated that the mature thymocytes in ROS were cells recently formed in the cortex, and were not from the medullary pool. ROS-associated mature thymocytes expressed lower levels of H-2K than free, mature thymocytes. The CD4+CD8+CD3++ subpopulation, believed to be a developmental intermediate between cortical thymocytes and mature T cells, was present in both ROS populations. Further, late intermediates leading to both mature T-cell categories were evident in D-ROS, but only those leading to CD4+CD8-CD3++ T cells were evident in M-ROS. The results are compatible with a role for ROS in TcR-specificity selection and in the final maturation steps in the thymic cortex.

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PubMed1989

Nature of the thymocytes associated with dendritic cells and macrophages in thymic rosettes.

Thymic rosettes, structures consisting of 3-30 thymic lymphoid cells attached to a central macrophage or dendritic cell, were released from mouse thymus tissue by collagenase digestion. They were shown to be preexistent structures within the thymus, but to be subject to extensive exchange with free thymocytes under certain conditions. An isolation procedure was developed, using a new technique of zonal unit-gravity elutriation, which minimized exchange and produced a completely pure sample of the larger rosettes. The rosette-associated thymocytes were analyzed by two- and three-color immunofluorescent staining and flow cytometry. The dominant cell type was a small, CD4+CD8+, cortical-type thymocyte. However, all of the established thymus subpopulations defined by CD4 and CD8, including CD4-CD8+ and CD4+CD8- mature thymocytes and CD4-CD8- early thymocytes, were also present in rosettes. Very few of the cells present were of an intermediate or transitional phenotype. Rosette-associated thymocytes were somewhat enriched in large dividing thymocytes, in CD4-CD8- thymocytes, and in mature thymocytes expressing the T-cell antigen receptor-CD3 complex. Their most striking characteristic was a marked depletion in small thymocytes lacking surface H-2K expression, a major population among free thymocytes. The physiological role of the rosette structure is discussed, and it is suggested that the heterogeneity of the associated thymocytes in part reflects the existence of different types of rosettes in different areas of the thymus.

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