Biomedical subjects

M Witmer-Pack

Publications and source records attributed to M Witmer-Pack.

11 recordsLinked to original sources

PubMed1995

Expression of B7 costimulator molecules on mouse dendritic cells.

Dendritic cells express most known accessory molecules [ICAM's, LFA's, B7's, and CD40] for binding and stimulating T cells. B7 is the most abundant of these, and B7-2 very much predominates relative to B7-1. B7 expression is regulated, not by LPS, but by some signal [s] that parallels maturation. B7 contributes to the T cell stimulatory function of dendritic cells, as do the other accessory molecules. B7-2 is expressed on dendritic cells and macrophages at several sites in situ, especially dendritic cells in the T cell areas.

Animals↗

PubMed1994

TCR selection and allelic exclusion in RAG transgenic mice that exhibit abnormal T cell localization in lymph nodes and lymphatics.

RAG-1 and RAG-2 are developmentally regulated genes that are essential for V(D)J recombination and lymphocyte development. Expression of RAG-1 and RAG-2 by thymocytes is normally limited to cells that have not completed selection. We have previously documented that persistent expression of the recombinase activating genes (RAG) in transgenic mice results in aberrant thymic development, altered lymphatic microanatomy, and a profound immunodeficiency. Here we further document the pathologic changes found in TG.RAG-1,2 mice and examine the role of TCR recombination and positive and negative thymic selection, as well as allelic exclusion, in the etiology of the phenotype. We find that neither selection nor TCR allelic exclusion can be overcome by transgenic expression RAG-1 and RAG-2 under the control of an lck promoter.

Alleles↗

PubMed1994

The tissue distribution of the B7-2 costimulator in mice: abundant expression on dendritic cells in situ and during maturation in vitro.

B7-2 is a recently discovered, second ligand for the CTLA-4/CD28, T cell signaling system. Using the GL-1 rat monoclonal antibody (mAb), we monitored expression of B7-2 on mouse leukocytes with an emphasis on dendritic cells. By cytofluorography, little or no B7-2 was detected on most cell types isolated from spleen, thymus, peritoneal cavity, skin, marrow, and blood. However, expression of B7-2 could be upregulated in culture. In the case of epidermal and spleen dendritic cells, which become highly immunostimulatory for T cells during a short period of culture, the upregulation of B7-2 was dramatic and did not require added stimuli. Lipopolysaccharide did not upregulate B7-2 levels on dendritic cells, in contrast to macrophages and B cells. By indirect immunolabeling, the level of staining with GL-1 mAb exceeded that seen with rat mAbs to several other surface molecules including intercellular adhesion molecule 1, B7-1, CD44, and CD45, as well as new hamster mAbs to CD40, CD48, and B7-1/CD80. Of these accessory molecules, B7-2 was a major species that increased in culture, implying a key role for B7-2 in the functional maturation of dendritic cells. B7-2 was the main (> 90%) CTLA-4 ligand on mouse dendritic cells. When we applied GL-1 to tissue sections of a dozen different organs, clear-cut staining with B7-2 antigen was found in many. B7-2 staining was noted on liver Kupffer cells, interstitial cells of heart and lung, and profiles in the submucosa of the esophagus. B7-2 staining was minimal in the kidney and in the nonlymphoid regions of the gut, and was not observed at all in the brain. In the tongue, only rare dendritic cells in the oral epithelium were B7-2+, but reactive cells were scattered about the interstitial spaces of the muscle. In all lymphoid tissues, Gl-1 strongly stained certain distinct regions that are occupied by dendritic cells and by macrophages. For dendritic cells, these include the thymic medulla, splenic periarterial sheaths, and lymph node deep cortex; for macrophages, the B7-2-rich regions included the splenic marginal zone and lymph node subcapsular cortex. Splenic B7-2+ cells were accessible to labeling with GL-1 mAb given intravenously. Dendritic cell stimulation of T cells (DNA synthesis) during the mixed leukocyte reaction was significantly (35-65%) blocked by GL-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Abatacept↗

PubMed1993

Dendritic cells: antigen presentation, accessory function and clinical relevance.

Because of difficulties in isolation, it has taken some time to arrive at a reasonable outline of the dendritic cell system. With the international effort that is assembled here, the main features of this system are apparent. There are now several criteria that allow for dendritic cell identification, there is understanding of tissue distribution and the interconnections between different compartments, there is new data on the production and maturation components of this system, and there are many observations that help explain antigen presentation, T cell stimulatory function, and behaviour in situ. The contributions of our Dutch hosts should be stressed. Many have energized the study of lymphoid, mononuclear phagocyte, and dendritic cell systems. The beginnings were made by Koenig, Langevoort, Thorbecke and van Furth, continued with Veldman, Nieuwenhuis, Hoefsmit, Drexhage, van Ewijk, Dijkstra, Kraal, Kamperdijk, and now there are many investigators in the biology of antigen presentation, one understands why it is appropriate to be in Holland. Holland even geographically is a "dendritic cell" [Fig 4].

Acquired Immunodeficiency Syndrome↗

PubMed1992

Two populations of splenic dendritic cells detected with M342, a new monoclonal to an intracellular antigen of interdigitating dendritic cells and some B lymphocytes.

A monoclonal has been isolated that labels an intracellular antigen in dendritic cells and some B cells. The M342 hamster immunoglobulin was selected because it stained cells in the periarterial sheaths of spleen, the deep cortex of lymph node, and the thymic medulla--the same regions in which one finds interdigitating cells, the presumptive in situ counterparts of isolated lymphoid dendritic cells. M342 labeled an antigen within granules of isolated dendritic cells, but only in cells that had been cultured for a day and not in fresh isolates. This extends recent findings that most freshly isolated spleen dendritic cells are located in the periphery of the white pulp nodule and may serve as precursors for the periarterial pool of interdigitating cells, the site for M342 staining in situ. By electron microscopic immunolabeling, the M342 antigen was found exclusively in a type of multivesicular body. M342 staining was not found in mononuclear phagocytes from blood and peritoneal cavity. Peritoneal B cells expressed M342+ granules, and upon appropriate stimulation splenic B cells developed reactive granules as well. We conclude that M342 is a strong marker for interdigitating cells. Its existence reveals intracellular specializations in the vacuolar system of antigen-presenting cells including subsets of dendritic cells.

Animals↗

PubMed1990

Migration and maturation of Langerhans cells in skin transplants and explants.

The behavior of Langerhans cells (LC) has been examined after skin transplantation and in an organ culture system. Within 24 h (and even within 4 h of culture), LC in epidermal sheets from allografts, isografts, and explants dramatically increased in size and expression of major histocompatibility complex class II molecules, and their numbers were markedly decreased. Using a new procedure, dermal sheets were then examined. By 24 h, cells resembling LC were found close to the epidermal-dermal junction, and by 3 d, they formed cords in dermal lymphatics before leaving the skin. In organ culture, the cells continued to migrate spontaneously into the medium. These observations establish a direct route for migration of LC from the epidermis into the dermis and then out of the skin. These processes are apparently induced by a local inflammatory response, and are independent of host-derived mediators. The phenotype of migratory cells was then examined by two-color immunocytochemistry and FACS analysis. The majority of migratory leukocytes were Ia+ LC, the remainder comprised Thy-1+, CD3+, CD4-, CD8- presumptive T cell receptor gamma/delta+ dendritic epidermal cells, which clustered with the LC, and a small population of adherent Ia-, FcRII+, CD11a/18+ macrophages. In contrast to the cells remaining within the epidermis of grafted skin at 1 d, the migratory cells were heterogeneous in phenotype, particularly with respect to F4/80, FcRII, and interleukin 2 receptor alpha expression, which are useful markers to follow phenotypic maturation of LC. Moreover, cells isolated from the epidermis of grafts at 1 d were more immunostimulatory in the allogeneic mixed leukocyte reaction and oxidative mitogenesis than LC isolated from normal skin, though less potent than spleen cells. The day 1 migratory cells were considerably more immunostimulatory than spleen cells, and day 3-5 migratory cells even more so, suggesting that functional maturation continues in culture. Thus, maturation of LC commences in the epidermis and continues during migration, but the cells do not need to be fully mature in phenotype or function before they leave the skin. In vivo, the migration of epidermal LC via the dermis into lymphatics and then to the draining nodes, where they have been shown previously to home to T areas, would provide a powerful stimulus for graft rejection.

Animals↗

PubMed1990

Dendritic cells as antigen presenting cells in vivo.

The biology of antigen presenting cells (APC) traditionally is studied in tissue culture systems using T cells that have been expanded beforehand by stimulation with antigen. Here we consider the distinctive roles of dendritic cells for sensitizing or priming T cells both in vitro and in vivo. Several functions of dendritic cells have been identified in tissue culture that are pertinent to T cell sensitization. These include the ability to a) capture and retain foreign antigens in an immunogenic form, b) bind antigen-specific resting lymphocytes, and c) activate T cells to produce lymphokines and undergo long term clonal growth. Dendritic cells have several properties in vivo that also would contribute to APC function. These are a) their widespread tissue distribution permitting access to antigens in most organs, b) the capacity to home via the blood stream and afferent lymph to the T-dependent areas of spleen and lymph node, and c) the ability to capture antigen in antigen-pulsed animals. Dendritic cells bearing antigen have been administered in situ to initiate responses like contact sensitivity, graft rejection, and antibody formation. A most striking recent example is that, when dendritic cells are pulsed with protein antigens in vitro and administered to immunologically naive mice, there is direct priming of antigen-specific T cells that are restricted to the MHC of the injected APC.

Animals↗

PubMed1989

A small number of anti-CD3 molecules on dendritic cells stimulate DNA synthesis in mouse T lymphocytes.

Resting T cells enter cell cycle when challenged with anti-CD3 mAb and accessory cells that bear required Fc receptors (FcR). Presentation of anti-CD3 is thought to be a model for antigens presented by accessory cells to the TCR complex. We have obtained evidence that the number of anti-CD3 molecules that are associated with the accessory cell can be very small. We first noticed that thymic dendritic cells and cultured, but not freshly isolated, epidermal Langerhans cells (LC) were active accessory cells for responses to anti-CD3 mAb. DNA synthesis was abrogated by a mAb to the FcR but not by mAb to other molecules used in clonally specific antigen recognition, i.e., class I and II MHC products or CD4 and CD8. The requisite FcR could be identified on the LC but in small numbers. Freshly isolated LC had 20,000 FcR per cell, while the more active cultured LC had only 2,000 sites, using 125I-anti-FcR mAb in quantitative binding studies. Individual LC had similar levels of FcR, as evidenced with a sensitive FACS. FcR could not be detected on T cells or within the dendritic cell cytoplasm, at the start of or during the mitogenesis response. When the response was assessed at 30 h with single cell assays, at least 20 T cells became lymphoblasts per added LC, and at least 8 T cells were synthesizing DNA while in contact with the LC in discrete cell clusters. To the extent that anti-CD3 represents a polyclonal model for antigen presentation to specific T cell clones, these results suggest two conclusions. First, only 200-300 molecules of ligand on dendritic cells may be required to trigger a T cell. Second, the maturation of LC in culture entails "sensitizing" functions other than ligand presentation (anti-CD3 on FcR) to clonotypic T cell receptors.

Animals↗

PubMed1989

Presentation of exogenous protein antigens by dendritic cells to T cell clones. Intact protein is presented best by immature, epidermal Langerhans cells.

The capacity of dendritic cells to present protein antigens has been studied with two MHC class II-restricted, myoglobin-specific, T cell clones. Spleen dendritic cells and cultured epidermal Langerhans cells (LC) presented native myoglobin weakly and often not at all. These same populations were powerful stimulators of allogeneic T cells in the primary MLR. Freshly isolated LC were in contrast very active in presenting proteins to T cell clones but were weak stimulators of the MLR. Both fresh and cultured LC could present specific peptide fragments of myoglobin to the clones. These results suggest that dendritic cells in nonlymphoid tissues like skin can act as sentinels for presenting antigens in situ, their accessory function developing in two phases. First antigens are captured and appropriately presented. Further handling of antigen then is downregulated while the cells acquire strong sensitizing activity for the growth and function of resting T lymphocytes. The potent MLR stimulating activity of cultured epidermal LC and lymphoid dendritic cells probably reflects prior handling of antigens leading to the formation of allogeneic MHC-peptide complexes.

Animals↗

PubMed1989

The cell surface of mouse dendritic cells: FACS analyses of dendritic cells from different tissues including thymus.

The surface of dendritic cells from mouse spleen, thymus, and epidermis has been compared with a panel of monoclonal antibodies and the FACS. A method was first developed to isolate populations of large, adherent, thymic dendritic cells that were greater than 90% pure. These were released by collagenase digestion and separated from adherent macrophages after overnight culture. Enrichment was based on the facts that most macrophages remained plastic adherent and rosetted strongly with antibody-coated erythrocytes. As in spleen, thymic dendritic cells were stellate in shape, had abundant class I and II MHC products, lacked many standard macrophage and lymphocyte markers, and actively stimulated the mixed leukocyte reaction. Most spleen and thymic dendritic cells could be lysed by the 7D4 mAb, to the low-affinity IL-2 receptor, and complement but the levels of 7D4 by FACS were low and sometimes not above background. Differences among dendritic cells from different tissues were noted with other mAb. Adherent dendritic cells from thymus all expressed the J11d "B cell" antigen and the NL145 interdigitating cell marker, but lacked the 33D1 spleen dendritic cell antigen. Eighty to ninety percent of spleen dendritic cells were J11d-, NL145-, 33D1+ but the remainder expressed the J11d+, NL145+, 33D1- thymic phenotype. The latter phenotype also was identical to that of epidermal Langerhans' cells. We postulate that the major 33D1+ cell in spleen represents a migratory stage in which dendritic cells are moving from tissues to lymphoid organs.

Animals↗

PubMed1988

Macrophages phagocytose thymic lymphocytes with productively rearranged T cell receptor alpha and beta genes.

The thymus gland is important for the formation of competent T lymphocytes. However, there is long-standing evidence that greater than 95% of newly formed thymocytes do not emigrate to peripheral lymphoid tissues but instead die locally. We have identified a rapid and selective pathway for thymocyte turnover in vitro. The mechanism entails binding, uptake, and digestion by macrophages. The susceptible cells are a subpopulation of double-positive thymocytes. These thymocytes can be enriched by virtue of their high buoyant density in Percoll and prove to have low levels of surface CD3 and little or no surface TCR. However TCR-alpha and -beta genes have undergone rearrangement, and full length alpha and beta transcripts are abundant. Therefore many double-positive cells rearrange and express TCR genes but do not have normal levels of TCR on the cell surface. We propose that thymocytes that undergo high turnover in situ are unable to form receptors that can be selected by MHC molecules in the thymus, and that these cells are recognized and cleared by the macrophage.

Gene Rearrangement, T-Lymphocyte↗