Lymphocyte and lymphoma receptors utilized in differentiation, in homing, and in lymphomagenesis.
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Animals↗
Biomedical subjects
E C Butcher
Publications and source records attributed to E C Butcher.
Enrichment of murine and human Langerhans cells with solid phase immunoabsorption using pan-leukocyte monoclonal antibodies.
Using a solid phase immunoabsorption (panning) technique, we have employed pan-leukocyte monoclonal antibodies to enrich and deplete murine and human Langerhans cells from cell suspensions of normal skin. Langerhans cell-enriched fractions contained 80-99% mononuclear cells, almost all of which had the ultrastructural features of Langerhans cells. These results are comparable to those achieved by panning for human Langerhans cells with anti-Leu-6(T6) antibody. Similarly, less than 1% of these cells were detectable in Langerhans cell-depleted fractions and such fractions were incapable of stimulating allogeneic lymphocytes in the skin cell-lymphocyte reaction. We conclude that panning with pan-leukocyte antibodies is an effective means of enriching or depleting Langerhans cells from heterogeneous skin cell suspensions and can yield results similar to those achieved with more Langerhans cell-specific reagents such as anti-Leu-6(T6). These findings are of particular significance to the enrichment and depletion of murine Langerhans cells since they express no known correlate of the human Leu-6(T6) antigen.
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In vitro analysis of the homing properties of human lymphocytes: developmental regulation of functional receptors for high endothelial venules.
Circulating lymphocytes leave the blood by binding to specialized high endothelial cells lining postcapillary venules in lymphoid organs or sites of chronic inflammations, migrating through the vessel wall into the surrounding tissue. The capacity of lymphocytes to recognize and bind to high endothelial venules (HEVs) is thus central to the overall process of lymphocyte traffic and recirculation. We show that viable human lymphocytes bind selectively to HEVs in frozen sections of normal human lymph nodes, thus defining a simple in vitro model for the study of human lymphocyte homing properties. Optimal conditions for the quantitative analysis of lymphocyte-HEV interaction are described. Furthermore, by using this assay, we demonstrate that the ability of human lymphocyte populations to bind to HEVs parallels their presumed migratory status in vivo. Thus, thymocytes and bone marrow cells, which are sessile in vivo, bind poorly to HEVs in comparison with mature circulating lymphocytes in peripheral blood or in peripheral lymphoid tissues. These results indicate that HEV-binding ability is a regulated property of mature lymphocytes and, as demonstrated previously in animal models, probably plays a fundamental role in controlling lymphocyte traffic in humans. The in vitro model of lymphocyte-HEV interaction thus provides a unique means to assay the migratory properties of normal and neoplastic human lymphocyte subsets, to analyze the role of lymphocyte traffic mechanisms in normal and pathologic inflammatory reactions, and to define some of the molecular mechanisms responsible for the control of lymphocyte migration and positioning in humans.
Adolescent↗
Localization of lymphocyte subpopulations in peripheral lymphoid organs: directed lymphocyte migration and segregation into specific microenvironments.
The distribution of lymphocytes in the peripheral lymphoid organs is controlled by recirculatory and microenvironmental factors. Specific interactions between recirculating lymphocytes and high endothelial venules in various lymphoid organs determine the presence and proportions of the various lymphoid sets and subsets in those organs. Separate endothelial determinants on peripheral node and Peyer's patch endothelium along with complementary lymphocyte receptors mediate this organ specificity. B and T cells also exhibit nonrandom organization within lymphoid tissues; after entry via high endothelial venules they segregate into their respective domains, which appear to be determined by distinct types of nonlymphoid stromal cells. Antigenic stimulation results in changes in lymphocyte phenotype as well as in the lymphoid microenvironment. The response to most complex antigens is the formation of germinal centers (GC) composed primarily of proliferating B cells; the phenotype of the few T cells therein is supportive of the GC as a site of B-T interaction. The phenotype of the B cells in GCs suggest a role for GCs in immunoglobulin class switching and the determination of subsequent homing specificity.
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A homing receptor-bearing cortical thymocyte subset: implications for thymus cell migration and the nature of cortisone-resistant thymocytes.
The thymus exports a selected subset of virgin T lymphocytes to the peripheral lymphoid organs. The mature phenotype of these thymus emigrants is similar to that of medullary thymocytes and has been cited as supporting a medullary rather than cortical exit site. Using the monoclonal antibody MEL-14, we identify a 1%-3% subpopulation of thymocytes that expresses high levels of a receptor molecule involved in lymphocyte homing to peripheral lymph nodes. We present evidence that these rare MEL-14hi thymocytes are predominantly of mature phenotype and represent the major source of thymus emigrants. Surprisingly, MEL-14hi thymocytes are exclusively cortical in location, although their mature phenotype may allow them to masquerade as medullary cells in conventional studies. We also demonstrate that unlike medullary thymocytes, many cortisone-resistant thymocytes (CRT) are MEL-14hi. Thus, in contrast to current dogma, CRT do not represent a sample of medullary thymocytes as they are found in situ and their level of immunocompetence does not necessarily reflect that of the medullary population. Our findings refute the hypothesis that phenotypically and functionally mature cells are restricted to the medulla, and support our proposition that most thymus emigrants are derived from the MEL-14hi cortical subset.
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Langerhans cells react with pan-leukocyte monoclonal antibody: ultrastructural documentation using a live cell suspension immunoperoxidase technique.
Langerhans cells are generally regarded as members of an Ia+ dendritic cell system capable of potent accessory cell function in immune responses. While it has been shown that murine Langerhans cells are bone marrow-derived, the ontogenic relationships among human Langerhans cells, other dendritic cells, macrophages, and leukocytes in general have yet to be fully clarified. Recently, several pan-leukocyte monoclonal antibodies have been produced which react with the human leukocyte common antigen. This antigen resembles the murine T200 antigen and is expressed by all leukocyte subtypes but not by nonhematopoietic cells. Using an immunoperoxidase technique for staining suspensions of live skin cells, we have documented Langerhans cell reactivity with pan-leukocyte monoclonal antibody L3B12 at the ultrastructural level. Reactivity with this highly sensitive and specific pan-leukocyte marker supports the concept of the human Langerhans cell as a specialized form of bone marrow-derived mononuclear leukocyte and defines an immunologic feature common to dendritic cells, macrophages, and leukocytes that is not shared by other cell types. This finding is discussed in the context of other recent data concerning the immunologic phenotype of Langerhans cells. Since the immunoultrastructural method employed does not require cell fixation of any kind prior to immunologic staining, it should prove particularly useful for studying cell surface antigens that are adversely affected by fixation.
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The role of carbohydrate in heterotypic cell-cell recognition: lymphocyte-high endothelial cell interaction as a model system.
The studies reviewed here demonstrate that the interaction of lymphocytes with HEV is one of the most approachable models available for the study of heterotypic cell-cell recognition mechanisms. Lymphocyte-HEV interaction is mediated by specific lymphocyte surface receptors recognizing, by as yet unknown mechanisms, determinants expressed by specialized high endothelial cells in lymphoid tissues and sites of chronic inflammation. Both the lymphocyte and endothelial cell surface elements of the interaction are precisely regulated, controlling the traffic of lymphocyte subsets through particular lymphoid organs and into sites of inflammation. Considerable progress, reviewed here, has been made in defining and characterizing the lymphocyte surface molecules mediating this cellular interaction. By contrast, the nature of the endothelial cell determinants recognized by migrating lymphocytes remains a mystery. Future experiments must be designed to identify these endothelial cell determinants, and to examine critically a proposed role of carbohydrate in lymphocyte-HEV interaction.
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Germinal center B cells lack homing receptors necessary for normal lymphocyte recirculation.
Germinal center B cells (GCLC) are a discrete population of antigen-activated lymphoblasts that lack surface IgD and express abundant cell surface binding sites for peanut agglutinin (PNA). These phenotypic features render GCLC easily distinguishable from nearly all plasma cells, T cells, and unstimulated B cells, and have enabled us to identify and isolate GCLC from antigen-stimulated murine lymphoid organs. We have examined the migratory properties of these lymphoblasts in (a) short-term in vivo homing studies, and (b) an in vitro assay of lymphocyte binding to post-capillary, high endothelial venules (HEV) in frozen sections of Peyer's patches and peripheral lymph nodes. In the in vivo experiments, intravenously injected GCLC failed to migrate in significant numbers to peripheral lymphoid organs in comparison with T cells or IgD+ B cells. In the in vitro binding assay, GCLC did not adhere to HEV in either Peyer's patch or peripheral node sections. A variety of factors, such as preferential sequestration in the liver, may operate in vivo to influence the localization of these cells. However, their nearly total failure to migrate into lymphoid organs can best be explained by their inability to recognize and adhere to the specialized HEV which normally mediate the emigration of recirculating lymphocytes from the blood into these sites. The concept that GCLC fail to express functional homing receptors for HEV has been further supported by studies using MEL-14, a monoclonal antibody that appears to recognize the lymphocyte surface receptor for peripheral node HEV: In contrast to most peripheral lymphocytes, GCLC fail to bind MEL-14. These migratory and endothelial-recognition properties of GCLC, when viewed in the context of the possible role of these cells as precursors of plasma cells and/or memory B cells, have led us to propose that the inability of GCLC to recognize HEV may be transient and related to a phase of sessile B cell differentiation.
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Genetic control of T-cell subset representation in inbred mice.
Lyt-2+ T cells constitute a significantly greater proportion of the total peripheral T-cell population in C57BL mice than in BALB/c and other mouse strains. The inheritance of this differential representation of Lyt-2- vs. Lyt-2+ T cells was studied by two-color immunofluorescence analysis of peripheral T cell subsets in BALB/c, C57BL, F1 and F2 generations, and in CXB recombinant inbred strains. It was shown that the C57BL phenotype (low Lyt-2-/Lyt-2+ ratio) is a dominant Mendelian character. Studies of subpopulations of thymocytes and of early thymus emigrants indicate that the representation of mature Lyt-2- and Lyt-2+ T cells is influenced by mechanisms of selection or differential turnover in the peripheral lymphoid organs, but that thymic and prethymic influences may also play a role.
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Differences in in vivo distribution and homing of T cell subsets to mucosal vs nonmucosal lymphoid organs.
The migratory properties of Lyt-2- and Lyt-2+ T cells in the mouse have been investigated. In short-term in vivo homing studies, Lyt-2- T cells localized consistently more efficiently than Lyt-2+ T cells in Peyer's patches (about 1.5 times as well), whereas both populations localized roughly equivalently in peripheral lymph nodes. These homing characteristics of Lyt-2- and Lyt-2+ subsets are largely independent of their organ source. The specificity of migration appears to be determined by selective recognition of organ-specific determinants on the endothelial cells of high endothelial venules (HEV), specialized venules that mediate the exit of migrating lymphocytes from the blood: In an in vitro assay of lymphocyte binding to HEV in lymphoid organ frozen sections, Lyt-2- cells constituted a significantly and consistently greater proportion of T cells binding to Peyer's patch HEV than of those binding to peripheral node HEV. The homing and HEV recognition preferences of the Lyt subsets are reflected in differences in their in situ representation in mucosal vs nonmucosal lymphoid organs, which suggests that the selective migration of these populations may be an important factor in determining the character of local immune responses.
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Germinal centre B cells: antigen specificity and changes in heavy chain class expression.
Germinal centres are histologically defined aggregates of blast cells that occur in B-cell areas of lymphoid tissues after antigenic stimulation. They are believed to be associated with the development of B-cell memory and plasma cell (especially secondary, IgG and IgA) responses. Recent studies of murine lymphoid tissues have defined cell-surface markers that distinguish germinal centre B cells from other mature B cells, permitting their identification and characterization in cell suspensions. Here we have used these markers to define and study germinal centre cells in lympho nodes, and have found that they constitute a unique population of B cells which (1) arises in response to antigenic stimulation, (2) contains nearly all of the demonstrably antigen-specific B cells in the stimulated organ, (3) bears surface IgM after primary stimulation and (4) as a population, demonstrates isotype switching to a predominant population, demonstrates isotype switching to a predominant surface IgG phenotype after secondary stimulation with specific surface IgG phenotype after secondary stimulation with specific antigen. These findings demonstrate that germinal centres are a major site of proliferation and differentiation of antigen-specific B cells in vivo, and suggest that the germinal centre microenvironment may have an important role in heavy chain class switching during B-cell responses.
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Surface phenotype and migratory capability of Peyer's patch germinal center cells.
Peanut agglutinin (PNA) binds selectively to germinal center cells in mouse peripheral lymphoid organs. Using PNA as a marker, we have determined that Peyer's patch germinal center cells are B cells with a unique phenotype---they express a low level of surface immunoglobulin (about 85% Ig+), predominantly of the IgA class (70% alpha+), with only 10% bearing surface IgM, and few if any expressing IgD. This phenotype identifies murine Peyer's patch germinal center cells as fairly late cells in B cell differentiation, and suggests that they may be precursors of IgA-secreting plasma cells in the gut wall. In addition, we have described a means of purifying PNA+ Peyer's patch lymphocytes, and have demonstrated that these cells lack functional receptors for high endothelial venules and fail to migrate to lymphoid organs in vivo. It is speculated that PNA may be a general marker for nonmigratory lymphocyte populations undergoing local differentiation.
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Selective migration of murine lymphocytes and lymphoblast populations and the role of endothelial cell recognition.
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Animals↗
Abnormal migration of T lymphocyte clones.
Several in vitro T cell clones were markedly deficient in their ability to home to peripheral lymphoid tissue. This was found for an alloreactive noncytolytic clone, a soluble antigen- (KLH)specific line, and cytotoxic clones specific for allogeneic cells and for Abelson virus-induced lymphoma cells. This abnormal circulation pattern was probably caused by the lack of the receptors of the lymphocytes for high endothelial venules (HEV), as implied by the lack of binding of these T cells to HEV in frozen sections of mouse lymph node and Peyer's patches. The loss of surface receptors that are necessary for normal lymphocyte migration may thereby alter the in vivo function of adoptively transferred T cells.
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Differences in the migration of B and T lymphocytes: organ-selective localization in vivo and the role of lymphocyte-endothelial cell recognition.
The migration of B and T lymphocytes in the mouse has been studied by using 1) short-term in vivo homing studies, and 2) an in vitro assay of lymphocyte binding to specialized lymphoid organ venules (post-capillary, high endothelial venules (HEV)) in frozen sections of lymph nodes and Peyer's patches. The homing characteristics of B and T cell populations are largely independent of their organ of origin. B cells from any source distribute preferentially to Peyer's patches, whereas T cells home preferentially to peripheral lymph nodes. This organ specificity of migration appears to be determined at the site of lymphocyte exit from the blood by selective recognition of organ-specific determinants on the endothelial cells of HEV. In addition, the in vivo tendency of B cells to migrate preferentially to the spleen, and of T cells to localize better in lymph nodes is confirmed. The results indicate that, in a hypothetical situation in which an equal number of B and T lymphocytes localized in peripheral lymph nodes (or bound in vitro to peripheral node HEV), there would be about 2.5 B cells for every T cell in the mesenteric node, four to six B cells per T cell in Peyer's patches, and seven to nine B cells per T cell in the spleen. Comparison of these homing preferences with the distribution of B and T lymphocyte populations in situ suggests that selective lymphocyte migration may help determine the proportions of functionally distinct lymphocyte classes in particular lymphoid organs or sites of chronic inflammation, and thus may serve to influence the character of local immune responses.
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Surface phenotype of Peyer's patch germinal center cells: implications for the role of germinal centers in B cell differentiation.
The surface phenotype of Peyer's patch germinal center lymphoid cells in the mouse is described. It is confirmed that most germinal center lymphocytes bind high levels of peanut agglutination (PNA), a lectin with specificity for terminal galactosyl residues. It is shown that germinal center lymphocytes can be identified in cell suspensions as a discrete PNAhi population distinct from other B cells, plasma cells, and most T cells, which bind only low levels of PNA. Using fluorescence-labeled PNA as a marker in dual fluorescence studies, we found that the majority of Peyer's patch germinal center cells are B lymphocytes: PNAhi Peyer's patch cells express B220, the B lineage-specific form of the T200 family of molecules, as well as low levels of surface Ig. They do not express the T cell-lineage antigens Thy-1, Lyt-1, or Lyt-2 (only 1 to 3% positive). They bear lower levels of H2-K than PNAlo B cells, but two to three times the level of surface I-A-encoded determinants. A discrete but variable subpopulation of PNAhi Peyer's patch cells bear ThB in AKR/c mice, but BALB/c PNAhi lymphocytes are ThB-. About 10 to 30% bear surface IgM or IgG, but in contrast to essentially all PNAlo B lymphocytes in this site, they express no detectable surface IgD. The majority of Peyer's patch germinal center cells bear surface IgA, and this IgA is allelically excluded in F1 mice, indicating it is synthesized by the germinal center cells themselves. In fact, germinal centers contain most of the IgA-bearing cells in Peyer's patches (70 to 85%). These findings lend considerable support to the concept that germinal centers in Peyer's patches are the site of generation of precursors of the IgA-secreting plasma cells that characterize mucosal immune responses, and also suggest that germinal centers may play an important role in the process of heavy chain class switching.
Alleles↗
Normal and neoplastic lymphocyte maturation.
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Cellular, genetic, and evolutionary aspects of lymphocyte interactions with high-endothelia venules.
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Animals↗