In vitro characterization of rat bone marrow-derived dendritic cells and their precursors.

Although the rat is commonly used for basic immunology and transplantation research, phenotypic and functional characterization of rat dendritic cells (DCs) lags behind similar studies in the human and mouse. Therefore, these features were examined using DCs propagated from cultures of rat bone marrow maintained in a medium supplemented with granulocyte-monocyte colony-stimulating factor. Analysis of cytospin preparations of cultured cells showd that DCs arise from OX7+ myelomonocytic precursors. Typical mature rat DCs were morphologically similar to their mouse and human counterparts and expressed major histocompatibility complex (MHC) class II (common part determinant of Ia), OX62 (integrin molecule), OX7 (CD90), ICAM-1 (CD54), and CTLA4 counterreceptor, but were negative for OX8 (CD8), OX19 (CD5), W3/25 (CD4), and ED2, a rat macrophage marker. Functional analysis of OX62+ sorted DCs showed that they could effectively present the soluble antigen ovalbumin to naive T cells in vitro. A combination of anti-MHC class II monoclonal antibody and CTLA4-immunoglobulin inhibited allostimulatory ability more effectively than either reagent alone. Implications for studying the role of DCs in immune responses in the rat are discussed.

Allogeneic hematolymphoid microchimerism and prevention of autoimmune disease in the rat. A relationship between allo- and autoimmunity.

Conventional allogeneic bone marrow transplantation after myeloablation can prevent experimental autoimmunity and has been proposed as treatment for humans. However, trace populations of donor hematolymphoid cells persisting in solid organ allograft recipients have been associated in some circumstances with therapeutic effects similar to replacement of the entire bone marrow. We therefore examined whether inducing hematolymphoid microchimerism without myeloablation could confer the ability to resist mercuric chloride (HgCl2)-induced autoimmunity. Brown-Norway (BN) rats were pretreated with a syngeneic or allogeneic bone marrow infusion under transient FK506 immunosuppression before receiving HgCl2. They were compared with BN rats receiving either no pretreatment (naive) or FK506 alone. Administration of HgCl2 to naive BN rats induced marked autoantibody production, systemic vasculitis and lymphocytic infiltration of the kidneys, liver and skin in all of the animals and a 47% mortality. In contrast, BN rats pretreated with HgCl2-resistant allogeneic Lewis bone marrow and transient FK506 showed less clinical disease and were completely protected from mortality. More specifically, IgG anti-laminin autoantibody production was decreased by 40% (P < 0.05), and there was less histopathological tissue injury (P < 0.005), less in vitro autoreactivity (P < 0.05), less of an increase in class II MHC expression on B cells (P < 0.01), and 22% less weight loss (P < 0.01), compared with controls. Protection from the experimental autoimmunity was associated with signs of low grade activation of the BN immune system, which included: increased numbers of circulating B and activated T cells before administration of HgCl2, and less autoreactivity and spontaneous proliferation in vitro after HgCl2.

A new protocol for the propagation of dendritic cells from rat bone marrow using recombinant GM-CSF, and their quantification using the mAb OX-62.

Bone marrow (BM)-derived dendritic cells (DC) are the most potent known antigen (Ag) presenting cell in vivo and in vitro. Detailed analysis of their properties and mechanisms of action requires an ability to produce large numbers of DC. Although DC have been isolated from several rat tissues, including BM, the yield is uniformly low. We describe a simple method for the propagation of large numbers of DC from rat BM and document cell yield with the rat DC marker, OX-62. After depletion of plastic-adherent and Fc+ cells by panning on dishes coated with normal serum, residual BM cells were cultured in gelatin coated flasks using murine rGM-CSF supplemented medium. Prior to analysis, non-adherent cells were re-depleted of contaminating Fc+ cells. Propagation of DC was monitored by double staining for FACS analysis (major histocompatibility complex (MHC) class II+/OX-62+, OX-19-). Functional assay, morphological analysis and evaluation of homing patterns of cultured cells revealed typical DC characteristics. MHC class II and OX-62 antigen expression increased with time in culture and correlated with allostimulatory ability. DC yield increased until day 7, when 3.3 x 10(6) DC were obtained from an initial 3 x 10(8) unfractionated BM cells. Significant numbers of DC can be generated from rat BM using these simple methods. This should permit analysis and manipulation of rat DC functions in vivo and in vitro.

Evidence that cyclosporine treatment causes the appearance in rat lymph node of T cells having the antigenic phenotypes of cortical thymocytes.

The antigenic phenotypes and relative size distributions of thymocytes and LN cells from young adult M520 strain rats, injected s.c. with cyclosporine (15 mgm/kg) for 10 consecutive days, were determined by FACS analysis and fluorescence microscopy. In addition, the prothymocyte activity in BM from these CsA-treated rats was assayed by i.v. adoptive transfer into sublethally irradiated, but non-CsA-treated, BUF strain recipients. The results showed that short-term CsA treatment causes a selective and almost complete depletion of both the CD4+8- and CD4-8+ subsets of medullary thymocytes, but that this effect is unrelated to any apparent influence of CsA on the proliferative or developmental potential of prothymocytes in the BM of the treated animals. The results also demonstrated that, early in the course of CsA treatment, there is a marked accumulation of LN T cells that display the antigenic phenotypes and size distribution of cortical thymocytes. Approximately 67% of these T cells had a CD4+8+ TdT+ Thy 1+ phenotype; 17% had a CD4-8+ TdT+ Thy 1+ phenotype. These "immature" lymph node T cells expressed the RT7 pan-T cell alloantigen, but lacked the RT6 postthymic T cell subset alloantigen. All were sIg-. In aggregate they were approximately 30% of the total CsA-LN T cells, as compared with fewer than 3% in controls.

Isolation and characterization of protective T cells induced by Listeria monocytogenes.

Rats convalescing from a recent infection with Listeria monocytogenes generate T cells which can protect recipient rats against a challenge infection with that organism. Using monoclonal antibodies that react with some but not all rat peripheral T cells, the T-cell mediators of acquired resistance to infection (TCRI) were isolated by panning and characterized by using a fluorescence-activated cell sorter. Many L. monocytogenes-immune TCRI were relatively large cells as judged by their light-scattering properties. That finding accords with previously reported cytokinetic data and velocity sedimentation analyses which revealed that the majority of L. monocytogenes-immune TCRI are lymphoblasts. In the current investigation, the surface antigenic profile of these mediator T cells was revealed as W3/25+ OX8+ OX4+ RT6.1-. That phenotype is identical to that of L. monocytogenes-induced prekiller cells which are formed as part of the animal's cell-mediated response to infection. Like prekiller cells and their differentiated counterparts, L. monocytogenes-immune TCRI adhere preferentially to monolayers of syngeneic fibroblasts. The results indicate that L. monocytogenes-immune TCRI belong to a minor subset of peripheral T cells which also contains T cells that have the cytotoxic potential by which L. monocytogenes-induced prekiller cells have been defined.

Monoclonal antibody analysis of Listeria monocytogenes-induced cytotoxic lymphocytes.

An immunizing infection with Listeria monocytogenes provides a potent stimulus for the formation of prekiller lymphocytes. Their cytolytic potential is revealed when the cells are restimulated in vitro by Listeria antigens. Listeria monocytogenes-induced cytotoxic lymphocytes and the prekiller cells from which they are derived were characterized in respect to their surface antigenic markers. Using monoclonal antibodies, B-cell depleted lymphocytes from the thoracic duct of Listeria immune rats were fractionated into subsets by a combination of panning and sorting techniques. Listeria monocytogenes-induced cytotoxic lymphocytes and their prekiller cell precursors were demonstrated to have the phenotype W3/25-, OX8+, OX4+, W3/13+ (high density), OX19+ (low density), RT6.1-. The OX8+, RT6.1- subset, which contained prekiller cells, constituted approximately 6% of lymph-borne T cells. The data indicate that these microbial antigen-induced cytotoxic lymphocytes belong to a minor subset of peripheral T cells whose surface antigenic properties distinguish them from natural killer cells.

T-cell co-operation in the mediation of acquired resistance to Listeria monocytogenes.

Monoclonal antibodies were used to select T-cell subsets that mediate delayed-type hypersensitivity (DTH) and acquired cellular resistance (CRI) in rats infected with Listeria monocytogenes. The mediators of DTH were identified as W3/25+ OX8- T cells. The latter comprised a subset distinct from that which could protect recipient rats against a Listeria challenge. The protective T cells had a W3/25- OX8+ phenotype. The T-cell mediators of cellular resistance to infection (TCRI) failed to augment the expression of DTH; however, the mediators of DTH (TDTH) significantly enhanced the protective capacity of TCRI. This property of TDTH correlated with the ability of the cells to promote the focal deployment of TCRI and macrophages at sites of soluble Listeria antigen injection in skin, and in peritoneal exudates induced by killed L. monocytogenes. These findings illustrate the co-operative interaction of activated T cells in acquired resistance to L. monocytogenes, and imply that DTH has a purposeful role in the host defence against infection.

The mediators of acquired resistance to Listeria monocytogenes are contained within a population of cytotoxic T cells.

T cells from peritoneal exudates induced in rats convalescing from a recent infection of Listeria monocytogenes were fractionated into two subsets based on their ability to bind monoclonal antibodies to cell-surface determinants that are expressed on some but not all peripheral T cells. Two phenotypically distinct subsets, one recognized by the antibody MRC OX8 and the other by W3/25, were assayed for their protective capacity in Listeria-challenged recipients, and for their ability to kill unmodified syngeneic fibroblasts in vitro. The two activities were mediated by the OX8+ subset which comprised approximately half the T cells in the exudates.

The cellular response to Listeria monocytogenes is mediated by a heterogeneous population of immunospecific T cells.

Immunospecific T cells cooperate with macrophages in the expression of delayed type hypersensitivity and cellular resistance to Listeria monocytogenes. The mediator cells involved are generated in response to an immunizing Listeria infection. Immunospecific T cells are formed in lymphoid tissue and appear briefly in the blood. Many localize at sites of microbial implantation, but others leave the blood spontaneously and infiltrate tissues throughout the body. Both circulating T cells and those in tissues participate in the expression of delayed type hypersensitivity and cellular resistance. Several lines of evidence suggest that these two phenomena are mediated by heterogeneous but possibly cooperating T cell populations.

Activation of Listeria monocytogenes-induced prekiller T cells by interleukin-2.

Thymus-dependent lymphocytes, or T cells, of rats infected with Listeria monocytogenes acquire a potent cytolytic capability when the cells are restimulated in vitro by Listeria antigens. Activation of such Listeria monocytogenes-dependent cytotoxic T lymphocytes requires both antigen and histocompatible accessory cells, but only when the responder T cells have been specifically depleted of lymphocytes that are responsive to accessory cell alloantigens. When unselected T cells are used, significant activation occurs in specific-antigen-free cultures containing allogeneic accessory cells. This seeming paradox is explained by the fact that a lymphokine generated in mixed leukocyte cultures can promote the terminal differentiation of cytotoxic T lymphocytes precursors. This lymphokine has tentatively been identified as interleukin-2. The results suggest that a three stage process is involved in the activation of Listeria monocytogenes-dependent cytotoxic T lymphocytes. The first stage occurs in response to an immunizing Listeria monocytogenes infection and itself involves two significant events, the polyclonal priming of prekiller cells and the generation of Listeria antigens-specific helper T cells. During the second stage, helper T cells are stimulated by Listeria antigens to release interleukin-2. This process is efficiently executed only in the presence of histocompatible accessory cells. The third stage in the activation process requires neither accessory cells nor antigen and involves the interleukin-2-driven differentiation of prekiller cells.