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J M Austyn

Publications and source records attributed to J M Austyn.

52 records · Page 3Linked to original sources
PubMed1988

Migration patterns of dendritic cells in the mouse. Homing to T cell-dependent areas of spleen, and binding within marginal zone.

Using quantitative techniques we have shown elsewhere that dendritic cells (DC) migrate from blood into the spleen, under the control of T cells. Here we traced the localization of DC within the spleen and sought to explain the means by which they entered. DC were labeled with a fluorochrome, Hoescht 33342, and injected intravenously. Spleens were removed 3 or 24 h later and DC were visualized within particular areas that were defined by mAbs and FITC anti-Igs. At 3 h most DC were in the red pulp, whereas by 24 h the majority had homed to T-dependent areas of the white pulp and may have become interdigitating cells. Lymphoid DC, isolated from spleen and perhaps normally present in blood, may thus be a migratory stage distinct from the relatively fixed interdigitating cells. We also developed a frozen section assay to investigate the interaction of DC with various lymphoid elements. When DC were incubated on sections of spleen, at 37 degrees C but not at 4 degrees C they attached specifically within the marginal zone and did not bind to T areas; in contrast, macrophages attached only to red pulp and T cells did not bind specifically. However, DC did not bind to sections of mesenteric lymph node, whereas T cells localized in particular regions at 4 degrees C but not at 37 degrees C, probably the high endothelial venules. DC may thus express "homing receptors," similar to those of T cells, for certain endothelia. We propose that T cells can modify the vascular endothelium in certain areas to allow egress of DC from the bloodstream.

Animals↗
PubMed1988

T-cell activation by dendritic cells: CD18-dependent clustering is not sufficient for mitogenesis.

The physical association of dendritic cells and T lymphocytes in clusters is required for primary immune responses. We have used oxidative mitogenesis as a model to examine the requirements for accessory cell-T-cell clustering and T-cell activation; in this polyclonal response, periodate-treated T cells cluster with dendritic cells (DC) and proliferate. Here we observed firstly that macrophages (M phi) but not B cells or B blasts could also cluster with periodate-treated T cells, but they did not stimulate proliferation. Thus, clustering with a cell that can express MHC class II (Ia) molecules and synthesize interleukin-1 (IL-1) is not sufficient for mitogenesis, and Ia expression is not sufficient for clustering. Secondly, that proliferation in oxidative mitogenesis and the allogeneic mixed leucocyte reaction (MLR) was inhibited by CD18, CD4 and CD8 antibodies but not, with one exception, by others of the panel tested. Thirdly, using rapid cluster assays we showed that DC and M phi formed temperature-dependent clusters with syngeneic and allogeneic periodate-treated T cells. Clustering was inhibited by CD18 antibodies, probably at the level of the T cell but not the accessory cell, and this may be a general feature of such inhibition. However, CD4 and/or CD8 antibodies did not affect clustering, showing that these molecules are involved in subsequent stages of T-cell activation. Since clustering of DC and M phi with periodate-treated T cells requires CD18, but only the former leads to proliferation, we conclude that CD18-dependent clustering is not sufficient for mitogenesis.

Animals↗
PubMed1988

Clustering with dendritic cells precedes and is essential for T-cell proliferation in a mitogenesis model.

Several antigen-specific immune responses are known to occur in discrete aggregates of dendritic cells (DC) and lymphocytes. We have used a polyclonal model, the mitogenesis of T cells that have been modified with sodium periodate, to evaluate the significance of cell-cell clustering. Firstly, we found that clustering precedes the onset of DNA synthesis by a day. Within 2 hr, virtually all of the added dendritic cells and most of the T cells that will respond have formed clusters. The T cells then progressively release and become responsive to interleukin-2 over 18 hr and DNA synthesis begins at 24 hr. Secondly, clustering with dendritic cells appears to be essential for mitogenesis. If dendritic cells are eliminated, the clusters disassemble and subsequent proliferation is reduced. Clustering and proliferation can be restored with dendritic cells that are syngeneic or allogeneic with the initial inoculum. DC are inactive if they are treated with ultraviolet light, formaldehyde or heat. Thirdly, the non-clustered cells do not synthesize DNA even when mixed with the clusters. However, non-clusters will respond when supplemented with additional DC. We conclude that clustering with DC precedes and seems essential for T-cell mitogenesis in the periodate model.

Animals↗
PubMed1987

T cell activation by anti-CD3 antibodies: function of Fc receptors on B cell blasts, but not resting B cells, and CD18 on the responding T cells.

Mouse anti-human CD3 (T3) antibodies can induce T cell proliferation in the presence of Fc receptor (FcR)-bearing accessory cells. Depending on whether the particular antibody can interact with the FcR, it can be mitogenic or otherwise. Previously, some of us (Smith, K. G. C. et al., Eur. J. Immunol. 1986. 16:478) examined human T cell responses to the murine anti-CD3 antibody switch variants UCHT1 (IgG1) and UCHT1B (IgG2b). Using a novel xenogeneic system with mouse macrophages (M phi) and an anti-FcR antibody, 2.4G2, we obtained direct evidence for accessory function of FcR in these responses. However, mouse B cells which also possess FcR were not accessory cells. Here we show that resting B cells do not inhibit anti-CD3 responses in the presence of other accessory cells, and they do not synergize with them. They appear to be inert in these responses but this is not simply because of their radiosensitivity. In contrast, B cell blasts proved to be potent stimulators of responses with UCHT1, UCHT1B and OKT3 (IgG2a). All three responses were inhibited by 2.4G2, whereas we have shown previously that the OKT3 response with M phi was not, in keeping with the known specificities of B cell and M phi FcR. These findings are discussed in relation to the molecular cloning of FcR, and we consider the possibility that distinct FcR could be expressed on resting and activated B cells. A report that anti-CD18 (LFA-1) antibodies blocked the UCHT1 response with human monocytes raised the possibility that this molecule might also be involved in accessory function. However, we show that this inhibition is in fact at the level of the T cell, since anti-human, but not anti-mouse CD18 antibodies, inhibited proliferative responses and clustering with both human and mouse accessory cells. Our results demonstrate that the principal contribution of accessory cells to anti-CD3 responses may be the provision of an FcR, and that CD18 is most probably required at the level of the T cell.

Animals↗
PubMed1987

Inability of dendritic cells to prevent the blood transfusion effect in a mouse cardiac allograft model.

The beneficial effect of blood transfusions before clinical renal transplantation is well established, but this can result in sensitization of some potential first graft recipients. Dendritic cells (DC), present in human blood, are potent stimulators of immune responses in vitro and in vivo, and in different systems they can overcome immune unresponsiveness. We therefore investigated whether DC could prevent the transfusion effect in a murine cardiac allograft model. C57BL/10 (C57) hearts were normally rejected by untreated DBA/2 recipients with a median survival time (MST) of 17 days. Long-term survival (MST greater than 100 days) was induced when the recipients were transfused with C57 blood 14 days before transplantation (d-14). Similar survival times were obtained when up to 1.5 x 10(5) splenic DC were added to the transfused blood. This number of DC or as few as 10(3), in "unsorted" preparations that were 70-85% pure, similarly enhanced when transfused alone at d-14. This enhancement was probably due to the contaminating cells rather than the DC since 10(3) lymphocytes prolonged survival, but an equivalent dose of "sorted" DC (ca. 93% pure, most contaminating cells being removed by sorting on an Ortho Cytofluorograf) did not. Transfusion of unsorted preparations containing 1.5 x 10(5) DC at d-3 led to accelerated graft rejection (MST 4 days). This sensitization was most likely due to the DC because equivalent numbers of lymphocytes were ineffective. Nevertheless, if a blood transfusion was given at d-14 followed by a normally sensitizing dose of DC at d-3, graft survival was still prolonged. Thus in no case were DC able to prevent the blood transfusion effect in this strain combination. Furthermore, DC of donor origin given to DBA/2 recipients with long-surviving C57 hearts, produced by prior blood transfusion, did not trigger rejection of the hearts.

Animals↗
PubMed1986

T cell activation by anti-T3 antibodies: comparison of IgG1 and IgG2b switch variants and direct evidence for accessory function of macrophage Fc receptors.

The human T3 antigen is closely associated with the T cell receptor. Some anti-T3 antibodies cause T cell proliferation in the presence of monocytes which have Fc receptors (FcR) that bind particular antibody subclasses. Such an interaction is thought to determine whether or not an anti-T3 antibody is mitogenic. We examined the mitogenicity of an IgG1 antibody, UCHT1, and an IgG2b switch variant of identical specificity, UCHT1B. With autologous monocytes, 76% of individuals responded to UCHT1 and 9% to UCHT1B, falling into three patterns of responsiveness. Both antibodies in the absence of monocytes induced responsiveness to recombinant interleukin 2, even for UCHT1B nonresponder T cells. The proliferation induced by UCHT1B, however, was always less than that induced by UCHT1. These findings demonstrate the critical role played by the Fc region for mitogenesis, and suggest a possible role for the hinge region. We then obtained direct evidence that mitogenicity can be mediated exclusively via FcR. Mouse macrophages have distinct FcR: FcRI binds IgG2a but FcRII binds IgG1 and IgG2b and its function can be inhibited by the specific antibody 2.4G2. Because UCHT1 and UCHT1B were of the correct subclass to interact with FcRII we examined the accessory function of mouse peritoneal macrophages. Without exception, human T cells now responded to both antibodies. Proliferation was drastically inhibited by 2.4G2 but not by an irrelevant anti-macrophage antibody, F4/80, nor by an anti-human neutrophil FcR antibody, 3G8. Furthermore, 2.4G2 did not inhibit the accessory function of mouse macrophages for OKT3, an IgG2a antibody that presumably interacts with FcRI, and did not inhibit the function of human monocytes for UCHT1 and UCHT1B. Mouse B cells, in contrast to macrophages, have an FcR which binds all three subclasses, but which can be inhibited by 2.4G2. B cells, however, were not accessory cells for mitogenesis with UCHT1, UCHT1B or OKT3. These findings are discussed in relation to other requirements for T cell activation by anti-T3 antibodies.

Antibodies, Monoclonal↗
PubMed1983

Dendritic cells initiate a two-stage mechanism for T lymphocyte proliferation.

T cells oxidized with sodium periodate proliferate polyclonally in response to accessory cells. We confirmed previous work showing that DC are potent stimulators of this response. In addition, the accessory function of unfractionated mouse spleen and spleen adherent cells was markedly reduced after elimination of DC with a specific monoclonal antibody and complement. Therefore oxidative mitogenesis was used as a model to study the mechanism by which DC stimulate T cell proliferative responses. A two-stage mechanism was identified. The first stage occurred during the first 20 h of culture, required live DC, and involved the progressive release of interleukin 2 (IL-2) into the medium and acquisition of responsiveness to this growth factor. The second stage occurred between 20 and 40 h, did not require live DC, and involved DNA synthesis in response to IL-2. Similar events occurred during culture of DC with unmodified T cells (syngeneic MLR) but were quantitatively reduced. The experimental approach was to co-culture DC and T cells for up to 20 h and then kill the DC with specific antibody, or anti-Ia antibody, and complement. Subsequent proliferation was inhibited if the T cells were cultured in fresh medium. However, proliferation was restored when the lymphocytes were cultured in the original DC-T cell medium, or with a crude or a purified preparation of IL-2. IL-2 did not induce the proliferation of T cells that had been cultured in the absence of DC, and did not synergize with viable DC. We conclude that DC induce proliferation by tightly coordinating the release of, and responsiveness to, T cell growth factor or IL-2.

Animals↗
PubMed1981

Expression of the macrophage-specific antigen F4/80 during differentiation of mouse bone marrow cells in culture.

We have defined the expression of the macrophages (m phi)-specific antigen (Ag) F4/80 during differentiation in culture. The progenitor cells-the colony-forming unit in culture and cluster-forming cell-lacked Ag F4/80 but gave rise to colonies of F4/80-positive adherent m phi, as shown by fluorescence-activated cell sorting and clonal assays with L cell-conditioned medium as the source of growth factor. Ag F4/80 first appeared on a nonadherent precursor found in mass liquid BM cultures after 3 d. Once adherent, m phi expressed high levels of Ag F4/80 and other markers. The role of L cell-conditioned medium and of adherence on expression of Ag F4/80 was also examined. Clonal analysis of F4/80 and other Ag, Mac-1, and 2.4G2 (FcR) showed that all cells in all independent colonies come to express these markers. These studies establish that F4/80 is a marker for the more mature stages of m phi development and that Ag expression increases progressively during maturation in vitro. Heterogeneity of Ag expression can be ascribed to variation in development and not to independent subsets of the m phi.

Animals↗
PubMed1981

F4/80, a monoclonal antibody directed specifically against the mouse macrophage.

A hybridoma clone which secretes a macrophage (M phi)-specific monoclonal antibody, F4/80, was produced by fusing spleen cells from a rat hyperimmunized with cultured thioglycollate-induced mouse peritoneal M phi with a mouse myeloma, NS1. Binding of antibody to primary cells and cell lines was detected by radioimmune indirect binding assay, autoradiography or fluorescence-activated cell sorter analysis. F4/80 binds to mouse M phi from the peritoneal cavity or other sources, blood monocytes, M phi derived from bone marrow precursors in culture and M phi-like cell lines, but not to other cells, including polymorphonuclear leukocytes, lymphocytes or fibroblasts. F4/80 does not bind to M phi via Fc receptors, is not cytotoxic and is of the rat IgG2b subclass. Since F4/80 binds to all M phi defined by adherence, morphology and immune phagocytosis, it provides a new marker to define the M phi in the mouse. Large differences in expression of antigen F4/80 were found, depending on intraperitoneal stimulation, time in culture and stage of maturation. Immunoprecipitation experiments demonstrated that the antigen F4/80 is part of a component of Mr 160000 which is synthesized by the M phi and, at least in part, exposed on the cell surface.

Animals↗
PubMedDate not supplied

The role of dendritic cells and T cell activation in allograft rejection.

The lineage of dendritic leukocytes (DL) comprises cells that are widely distributed throughout lymphoid and non-lymphoid tissues, and which are phenotypically and functionally heterogeneous. These cells play a central role in the initiation of immune responses. Immature DL, which may be situated primarily in non-lymphoid tissues, are responsible for uptake and processing of native antigens, after which they migrate via blood and/or lymph into secondary lymphoid tissue, and develop into mature dendritic cells (DC). These cells can present foreign peptide-MHC complexes to resting T cells and deliver signals for T cell activation, thereby initiating the response. There is good evidence that DL can migrate from transplanted tissues into the recipient's lymphoid organs and initiate allograft rejection. Interruption of this process, by depleting DL before transplantation or by inhibiting their function, may contribute to the prevention of rejection in clinical transplantation.

Animals↗
PubMedDate not supplied

The dendritic cell system and anti-tumour immunity.

Dendritic cells (DC) are essential for the initiation of T- and T-dependent immune responses by virtue of their capacities to acquire, process and present antigens and to deliver activation signals to mature resting T cells. In contrast, antigen presentation by other types of antigen-presenting cells (APC) may lead to peripheral T cell tolerance. This paper reviews the immunobiology of DC, and considers the possibility that immune unresponsiveness of tumour-bearing hosts may be due to clonal anergy of T cells. An important issue is whether it is possible to use DC to vaccinate tumour-free individuals, to overcome the immunological unresponsiveness of tumour-bearing hosts, and ultimately to cause rejection of primary and metastatic cancers.

Animals↗