Bone marrow-derived chimerism in non-irradiated, cyclosporin-treated rats receiving microvascularized limb transplants: evidence for donor-derived dendritic cells in recipient lymphoid tissues.

Tolerance to donor transplantation antigens develops when recipients are made chimeric with donor bone marrow. To establish chimerism, the haemopoietic system of recipients typically is severely compromised. We report on a system in which chimerism develops without ablative therapies. Immunosuppression with cyclosporin A allowed the lower limb of a rat to be replaced by a microvascularized transplant from a fully allogeneic donor. Many donor-derived cells populated recipient lymph nodes and spleen, and most had the large size, irregular shape and strong major histocompatibility complex (MHC) class II expression that typify dendritic cells. Donor cells were not found in the macrophage-rich regions of lymphoid tissues, but instead occupied splenic white pulp and lymph node cortex. The donor cells were derived from radiosensitive marrow precursors, as chimerism was abolished if the grafted limb was irradiated, or if muscle and skin flaps devoid of bone were grafted. Donor cells were rare or not detectable in blood, thymus and liver. Whereas lymphoid chimerism was prominent following limb transfer, donor cells were not detected 1-2 weeks after an injection of two femur equivalents of a marrow suspension. We suggest that dendritic cells that undergo rapid turnover in lymphoid organs are replaced from allogeneic precursors in bone grafts. The combination of cyclosporin and vascularized bone provides a means for inducing chimerism in lymphoid tissues of non-irradiated recipients.

Tissue distribution of the DEC-205 protein that is detected by the monoclonal antibody NLDC-145. II. Expression in situ in lymphoid and nonlymphoid tissues.

The rat monoclonal antibody NLDC-145, which has been utilized as a marker for mouse dendritic cells in numerous studies, binds an antigen that is more broadly distributed. This antigen is a unique 205-kDa integral membrane glycoprotein called DEC-205, which we have recently purified in quantities sufficient for basic biochemical studies, N-terminal sequencing, and immunization of rabbits. In cytofluorographic experiments, both the new polyclonal antibody and the original monoclonal detected DEC-205 on many classes of nondendritic murine leukocytes, particularly B cells. The quantities of DEC-205 on the surfaces of these cells were 10 to 50 times lower than those on epidermal and bone marrow dendritic cells. Here we utilize these reagents to reassess the tissue distribution of DEC-205 by immunohistochemical staining of frozen sections from a variety of organs, and by multiple-organ immunoblotting. Abundant expression of DEC-205 was confirmed histologically on thymic and intestinal epithelia and on dendritic cells in the T cell areas of peripheral lymphoid organs. In addition, DEC-205 was visualized in several other locations: B lymphocytes within B cell follicles, the stroma of the bone marrow, the epithelia of pulmonary airways, and the capillaries of the brain. Immunoblotting confirmed the presence of substantial levels of DEC-205 protein in lysates prepared from lymphoid tissues and from lung, marrow, and intestine. Thus, while DEC-205 is expressed at high levels by dendritic cells, it is also expressed by a number of other cell types in situ.

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.

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.

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)

Macrophages, but not dendritic cells, accumulate colloidal carbon following administration in situ.

The dendritic cell system operates in situ to capture and present antigens in a form that is immunogenic to T cells. It is likely that dendritic cells require endocytic activity in order to process antigens. On the other hand, macrophages are considered to be the principal cells that internalize substrates in situ. We therefore investigated the phenotype of cells that scavenge the indigestible endocytic tracer, colloidal carbon, by phenotyping the endocytic cells with monoclonal antibodies that help distinguish macrophages from dendritic cells. Of some importance was the monoclonal N418, an antibody to the p150/90 leukocyte beta 2 integrin. FACS analyses on isolates from blood, spleen and peritoneal cavity showed that N418 reacts primarily with dendritic cells. N418 also stained dendritic profiles strongly in tissue sections of liver and spleen, but most of the cells that actively endocytosed carbon in both organs showed little or no N418 staining. Likewise, carbon could not be identified in cells that react with M342, which stains intracellular granules of dendritic cells. In contrast, the carbon-labeled cells in both liver and spleen were labeled with antibodies (SER-4, F4/80, FA11) that bind primarily to isolated macrophages. Therefore the clearance of colloidal carbon in situ reflects the scavenging activity of macrophages and not the endocytic activity that underlies the antigen presenting function of dendritic cells.

Identification of macrophages and dendritic cells in the osteopetrotic (op/op) mouse.

We used a panel of monoclonal antibodies and immunocytochemistry to identify macrophages and dendritic cells in mice that are deficient in macrophage colony stimulating factor (M-CSF or CSF-1) because of the recessive osteopetrotic (op/op) mutation. Prior work had shown that osteopetrosis is associated with a lack of osteoclasts, phagocytic cells required for remodelling in bone. Additional macrophage populations proved to be very M-CSF dependent. op/op mice had few and sometimes no peritoneal cavity phagocytes, splenic marginal zone metallophils, and lymph node subcapsular sinus macrophages. Other populations, however, reached substantial levels in the absence of M-CSF, including phagocytes in the thymic cortex, splenic red pulp, lymph node medulla, intestinal lamina propria, liver (Kupffer cells), lung (alveolar macrophages) and brain (microglia). Dendritic cells, which are specialized accessory cells for T-dependent immune responses and tolerance, were readily identified in skin and in the T-dependent regions of spleen, lymph node and Peyer's patch. The identification of dendritic cells utilized antibodies to MHC class II products and four different antigens that are primarily expressed by these accessory cells. Our findings indicate that only a few macrophage populations are critically dependent upon M-CSF in vivo. With respect to dendritic cells, the data are consistent with prior in vitro work where it was noted that GM-CSF but not M-CSF supported dendritic cell viability, function and growth.

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].

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.

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.

Antigen processing by epidermal Langerhans cells correlates with the level of biosynthesis of major histocompatibility complex class II molecules and expression of invariant chain.

Two prior studies with a small number of T cell lines have shown that the presentation of native protein antigens by epidermal Langerhans cells (LC) is regulated. When freshly isolated, LC are efficient antigen-presenting cells (APC), but after a period of culture LC are inefficient or even inactive. The deficit in culture seems to be a selective loss in antigen processing, since cultured LC are otherwise rich in major histocompatibility complex (MHC) class II products and are active APC for alloantigens and mitogens, which do not require processing. We have extended the analysis by studying presentation to bulk populations of primed lymph node and a T-T hybrid. Only freshly isolated LC can be pulsed with the protein antigens myoglobin and conalbumin, but once pulsed, antigen is retained in an immunogenic form for at least 2 d. The acquisition of antigen, presumably as MHC-peptide complexes, is inhibited if the fresh LC are exposed to foreign protein in the presence of chloroquine or cycloheximide. The latter, in contrast, improves the efficacy of antigen pulsing in anti-Ig-stimulated B blasts. In additional studies of mechanism, we noted that both fresh and cultured LC endocytose similar amounts of an antigen, rhodamineovalbumin, into perinuclear granules. However, freshly isolated LC synthesize high levels class II MHC molecules and express higher amounts of the class II-associated invariant chain. Fresh LC are at least 5-10 times more active than many other cells types in the level of biosynthesis of MHC class II products. These findings provide a physiologic model in which newly synthesized MHC class II molecules appear to be the principal vehicle for effective antigen processing by APC of the dendritic cell lineage. Another APC, the B lymphoblast, does not appear to require newly synthesized MHC class II molecules for presentation.

Use of the fluorescence activated cell sorter to enrich dendritic cells from mouse spleen.

Dendritic cells are a specialized but trace population of antigen presenting cells that always have been enriched by multi-step procedures over a period of 1 or more days in tissue culture. Here we describe the isolation of dendritic cells from fresh mouse spleen suspensions using the FACS and a monoclonal antibody, N418, to the p150/90 member of the leukocyte integrin family (Metlay et al., 1990). By two color fluorescence activated cell sorter (FACS) analyses, the trace N418+ subset expressed most of the surface markers, including the 33D1 antigen, that are characteristic of dendritic cells isolated by other methods. An exception was that small amounts of Fc receptors, CD4 and F4/80 antigen were detected initially, but these diminished upon culture. In functional assays, sorted N418+ cells from fresh spleen were at least 30 times more active than N418- cells in presenting antigen to T cells. The assays were stimulation of the primary mixed leukocyte reaction and presentation of exogenous protein antigens to sensitized populations of lymph node T cells. The viability and MLR stimulating function of the sorted populations both were increased upon exposure to the cytokine, granulocyte-macrophage colony stimulating factor (GM-CSF). These results indicate that dendritic cells can be enriched from fresh isolates of mouse spleen using the FACS, and that when this is done, many of the distinctive features of dendritic cells - phenotype, APC function, and sensitivity to appropriate cytokines - are apparent.

The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies.

Hybridoma fusions with hamster hosts were undertaken to generate mAbs to mouse spleen dendritic cells. Two mAb were obtained and used to uncover the distinct integrins of these APC. One, 2E6, bound a determinant common to all members of the CD11/CD18 family, most likely the shared 90 kD CD18 beta chain. 2E6 immunoprecipitated the characteristic beta 2 integrin heterodimers from lymphocytes (p180, 90; CD11a) and macrophages (p170,90; CD11b), but from dendritic cells, a p150,90 (presumably CD11c) integrin was the predominant species. 2E6 inhibited the binding function of the CD11a and CD11b integrins on B cells and macrophages in appropriate assays, but 2E6 exerted little or no inhibition on the clustering of dendritic cells to T cells early in primary MLR, suggesting a CD11/CD18-independent mechanism for this binding. The second mAb, N418, precipitated a 150, 90 kD heterodimer that shared the 2E6 CD18 epitope. This N418 epitope may be the murine homologue of the previously characterized human CD11c molecule, but the epitope was only detected on dendritic cells. N418 did not react with peritoneal macrophages, anti-Ig-induced spleen B blasts, or bulk lymph node cells. When used to stain sections of spleen, N418 stained dendritic cells in the T-dependent areas, much like anti-class II mAbs that were also generated in these fusions. In addition, N418 revealed nests of dendritic cells that punctuated the rim of marginal zone macrophages between red and white pulp. This localization positioned most dendritic cells at regions where arterial vessels and T cells enter the white pulp. We conclude that the p150, 90 heterodimer is the major beta 2 integrin of spleen dendritic cells, and we speculate that it may function to localize these APC at sites that permit access to the recirculating pool of resting T cells.

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.

The surface of dendritic cells in the mouse as studied with monoclonal antibodies.

A family of dendritic cells has been identified in situ and in vitro by microscopy and immunolabeling. The members of this family include the dendritic cells isolated from lymphoid organs, Langerhans cells [LC] of the epidermis, veiled cells in afferent lymph, and interdigitating cells [IDC] in the T-cell areas. Some common features to all members of the family are high levels of MHC class II antigens, a lack of most B and T cell markers, and an absence or low levels of macrophage/granulocyte antigens. This review summarizes the markers of mouse dendritic cells as assessed by a panel of monoclonal antibodies, and stresses a few recent findings. 1) In spleen, there are two populations of dendritic cells. More than 75% of isolated cells are 33D1+, NLDC145-, and J11d-, while the remainder have the reciprocal phenotype and thus share the NLDC145 antigen of IDC. Thymic dendritic cells, released by collagenase digestion, and epidermal LC also are 33D1-, NLDC145+, J11d+. 2) When epidermal LC are placed in culture, there are changes in cell function and phenotype. There is a decrease in Fc gamma receptors and the F4/80 macrophage antigen, an increase in class I and II MHC products and p55 IL-2 receptors, and persistence of the NLDC145 IDC antigen. The cultured LC thereby resembles the IDC. 3) A new antibody N418 shows that dendritic cells express the p150/90 member of the leukocyte beta 2 integrin family. Immunolabeling of tissue sections of spleen indicates that N418+ dendritic cells not only are present in the periarterial sheaths, the location of IDC, but also in "nests" at the periphery of the T area where 33D1 has been found. The peripheral collections interrupt the marginal zone of macrophages that separates white and red pulp, and places the dendritic cells in the path of T cells as they move through the white pulp. Therefore the members of the dendritic cell family have important markers in common, as well as differences that are associated with state of immunologic function and location.

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.

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.

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.

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.

Quantitation of surface antigens on cultured murine epidermal Langerhans cells: rapid and selective increase in the level of surface MHC products.

It was recently discovered that murine epidermal Langerhans cells (LC) changed significantly in function and phenotype when maintained in culture. Notably, accessory cell function for primary immune responses increased while cytologic markers like ATPase, nonspecific esterase, and Birbeck granules were lost. To further analyze LC differentiation, we used flow cytometry and a panel of 22 monoclonal antibodies to quantitate changes in surface antigens at the single-cell level. A striking change was a fivefold increase in the amount of Ia antigens (which are expressed on class II MHC products) during the first day of culture. The increase was evident within 3 h and reached a plateau at 15-24 h. Both I-A and I-E products behaved similarly. The increase in Ia was blocked by 1 microgram/ml cycloheximide. Expression of other surface antigens was then monitored on Ia+ LC by two-color flow cytometry. Low levels of class I (H-2D and H-2K) MHC products were detected on freshly isolated LC, and these antigens also increased severalfold during the first day of culture. Fc receptors (identified with the 2.4G2 mAb) and the F4/80 macrophage antigen decreased, as reported previously. Three antigens that were detected in fresh suspensions were expressed at constant levels in culture. These were the C3bi receptor and the pan leukocyte and interdigitating cell antigens. Several leukocyte antigens that were not found initially on LCs did not appear, including B220 anti-B cell, 33D1 anti-dendritic cell, and CD4, CD5, CD8 T-cell specificities. We conclude that the surface of cultured LCs undergoes selective changes in culture. As a result, the cells are rich in Ia and H-2 and have detectable C3bi receptors, but have little or no LFA-1, Ti, CD4, 5, and 8, 33D1, 2.4G2, F4/80, and B220 antigens.