An immunogold cell labelling method for rat T lymphocytes

A method using monoclonal antibodies in conjunction with an immunogold procedure was adapted for labelling T lymphocytes in blood samples obtained from male Wistar rats. The monoclonal antibodies W3/25, MRC/OX8 and W3/13 were used. Changes in the percentages of positively labelled cells were observed in rats dosed with the immunosuppressant cyclosporin.     

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.     

Phytohemagglutinin-lnduced acquisition of T-cell surface markers by rat bone marrow precursor cells in the absence of the thymic microenvironment

Two monoclonal antibodies specific for different rat T-cell subpopulations, the anti-helper-T-cell antibody, W3/25, and the OX8 suppressor cell antibody were used to investigate lectin-stimulated T-lymphocyte differentiation of F-344 rat bone marrow cells in culture. Cytofluorometric analysis of freshly isolated lymphocytes from thymus and spleen revealed that these tissues contained both W3/25? and OX8-positive populations but differed with respect to the number of cells and receptor density distribution. By contrast, bone marrow-derived lymphocytes exhibited negligible W3/25? or OX8-associated fluorescence. However, several days after stimulation of bone marrow lymphocytes with phytohemagglutinin (PHA), cells appeared bearing these markers. Two-parameter histogram analysis of light scatter measurements with cell surface immunoflu-orescence indicated that this phenomenon represented the appearance of a new population of cells, presumably mature T cells, bearing an increased density of marker. These findings suggest an induction of differentiation of bone marrow T precursor cells by nonthymic factors (PHA) since lymphocytes lacking mature T-cell marker expression developed this characteristic after several days in culture.     

Surface phenotype of rat bone marrow-derived mononuclear phagocytes

Utilizing a panel of currently available monoclonal antibodies, the surface phenotype of a pure population of resting rat bone marrow-derived mononuclear phagocytes (BMM phi) was analyzed by means of flow cytometry. The present work provides an extensive list of surface markers expressed by BMM phi and also outlines advantages and limitations of flow cytometric analysis of this cell type. The results show that the majority of surface markers considered to be expressed selectively by T lymphocytes, such as Thy-1, CD2 and CD5 antigens, leukosialin (W3/13), or an alloantigen of peripheral T cells, are not expressed by BMM phi. On the other hand, the CD8 antigen and the leukocyte common antigen recognized by MRC OX-33, considered to represent specific markers of cytotoxic T cells and/or peripheral B cells, are expressed on a variable, often considerable proportion of BMM phi. Monoclonal antibodies W3/25, MRC OX-35, and MRC OX-38, directed against epitopes on the CD4 molecule, labeled a variable proportion of BMM phi. Among the 39 monoclonal antibodies examined, none appeared to recognize an epitope which is expressed selectively by mononuclear phagocytes.     

Characterization of the MRC OX40 antigen of activated CD4 positive T lymphocytes--a molecule related to nerve growth factor receptor.

The antigen recognized by the monoclonal antibody (mAb) MRC OX40 is present on activated rat CD4 positive T lymphocytes but not other cells. cDNA clones were isolated from an expression library using the MRC OX40 mAb and the protein sequence for the OX40 antigen deduced. It contains a typical signal sequence and a single putative transmembrane sequence of 25 predominantly hydrophobic amino acids giving an extracellular domain of 191 amino acids and a cytoplasmic domain of 36 amino acids. The sequence of the extracellular domain includes a cysteine-rich region with sequence similarities with the low affinity nerve growth factor receptor (NGFR) of neurons and the CD40 antigen present on human B cells. Within this region three cysteine-rich motifs can be recognized in OX40 compared with four similar motifs in both NGFR and CD40. OX40, CD40 and NGFR constitute a new superfamily of molecules with expression including lymphoid cells (OX40, CD40) and neuronal cells (NGFR). This is reminiscent of the immunoglobulin superfamily whose molecules are variously found at the surface of lymphoid or brain cells or both.     

Expression of the rat CD26 antigen (dipeptidyl peptidase IV) on subpopulations of rat lymphocytes.

The T cell activation antigen CD26 has been recently identified as the cell surface ectopeptidase dipeptidyl peptidase IV (DPP-IV). DPP-IV is found on many cell types, including lymphocytes, epithelial cells, and certain endothelial cells. The MRC OX61 monoclonal antibody (MAb) which specifically recognises rat DPP-IV was used to examine the expression of CD26/DPP-IV on rat lymphocytes. The molecular nature of the antigen was examined by immunoprecipitation from thymocytes, splenocytes, and hepatocytes. Analysis by one- and two-dimensional gel electrophoresis indicated that the native form of CD26 includes a 220-kDa homodimer. On tissue sections MRC OX61 MAb stained nearly all thymocytes and in the spleen and lymph nodes predominantly stained the T cell areas. However, in immunofluorescence experiments OX61 stained 80 to 87% of lymph node cells and 78 to 85% of spleen cells. Furthermore, two-colour immunofluorescence analysis of the CD4+, CD8+, and Ig+ lymphocyte subsets indicated that only 2 to 5% of each of these subsets lacked OX61 staining. Spleen cells and thymocytes of both CD4+ and CD8+ subsets stained much more intensely with OX61 after these cells were stimulated with phytohemagglutinin. These findings indicate that rat CD26 antigen expression is not confined to the T cell population as has been suggested, but also occurs on B cells, and is increased on T cells following their activation.     

Experimental autoallergic sialadenitis in the LEW rat. III. Role of CD4+ T cells in EAS induction

Experimental autoallergic sialadenitis (EAS) in the LEW rat is an induced autoimmune disease of the salivary tissues. EAS is characterized by a lymphocytic infiltration that consists of both CD4+ (helper/inducer T-cell subset) and CD8+ (cytotoxic/suppressor T-cell subset) T cells and results in the immune-mediated destruction of the exocrine salivary glands. To investigate the role that each of the T-cell subsets may have in the pathogenesis of EAS, LEW rats sensitized with WF SMG homogenate were injected with monoclonal antibodies to deplete or inactivate, in vivo, the CD4, CD5 (OX19; pan T lymphocyte), CD8, or RT6 (70% of peripheral T cells) T-cell populations. Treatment with the OX8 (CD8), OX19 (CD5), or W3/25 (CD4) only partially reduced in vivo the respective splenic or lymph node T-cell subsets when analyzed on Day 14, while treatment with DS4.23 (anti-RT6) resulted in greater than 95% depletion of RT6+ spleen and lymph node T cells. EAS incidence and severity was significantly reduced in the W3/25 (CD4) treatment group (11% incidence rate; histologic score 1.0) as compared to medium-injected controls (88% incidence rate; histologic score 2.9). Although the incidence and severity of EAS in the OX19 (71%; histologic score 1.7), OX8 (55%; histologic score 1.7), and RT6 (67%; histologic score 1.6) treatment groups appeared decreased, the reduction was not statistically significant. These results provide evidence that CD4+ T cells have an important role in EAS induction and demonstrate that in vivo treatment with anti-CD4 can ameliorate and/or prevent EAS in the LEW rat.     

Colony-stimulating factor (CSF-1): its enhancement of plasminogen activator production and inhibition of cell growth in a mouse macrophage cell line

The cell-mediated immune response by the gut-associated lymphoid tissues to antigens within the intestinal tract is poorly understood. Our objective was to investigate the antigen-specific T cell proliferative response and cytotoxic T lymphocyte (CTL) response of cells from the GALT after enteric immunization with vaccinia virus. Lymphocytes able to proliferate in the presence of vaccinia virus in vitro were found in large numbers in mesenteric lymph nodes (MLN) 6 days after the injection of vaccinia virus into the lumen of the small bowel. The MLN at this time also contained vaccinia-specific CTL, but unlike the proliferating cells, which were found for several weeks after immunization, the CTL were demonstrable in the MLN for only a few days. Peyer's patches were found to contain neither antigen-stimulated proliferating cells nor CTL. The viral-specific proliferating lymphocytes from the MLN 10 days after immunization were sIg-, monoclonal antibody W3/25+, MRC OX-8- large lymphoblasts. The vaccinia-specific CTL were also large lymphoblasts, but they belonged to the W3/25-, OX-8+ subset. Thus, a strong T helper and cytotoxic T lymphoblast response is generated within the MLN after viral challenge of the gut.     

Cellular interactions in the generation and expression of histamine-induced suppressor activity

The tissue, anatomic, and developmental distribution of Maclura pomifera (MP) lectin binding to rat lymphoid cells was examined. Analysis was performed by immunofluorescence microscopy and by the fluorescence-activated cell sorter. Comparison with anti-rat Ig to detect B cells and the monoclonal antibodies W3/13, W3/25, and OX 8 to detect T cells revealed that the MP lectin reacted with all T cells and not B cells in spleen and lymph node of young adult rats. The lectin also bound selectively to the thymus-dependent areas in frozen sections of spleen and lymph node. Using the MP lectin in conjunction with anti-Thy1 antibody and the monoclonal antibodies, W3/25 and OX 8, four T-cell subpopulations in spleen and lymphnode were identified on the basis of their cell surface antigenic phenotype. The T-cell developmental distribution of MP binding revealed that 100% of normal and neoplastic thymocytes bound the lectin whereas approximately 25% of TdT⁺ bone marrow cells, putative thymocyte progenitors, were MP⁺. Thus, the MP lectin is a nonimmunoglobulin reagent which binds to prethymic, thymic, and post-thymic cells of the T-lymphocyte lineage. Affinity chromatography experiments indicated that the MP lectin binds, at least in part, to the major thymocyte cell surface glycoprotein which is recognized by the W3/13 monoclonal antibody.     

Membrane antigen phenotype of sensitized T lymphocytes mediating tuberculin-delayed hypersensitivity in rats

The membrane antigen phenotype of immune lymph node cells (LNC) which mediate tuberculin-delayed hypersensitivity (DH) in Lewis rats was examined. The results show that the T-cell population which expresses the RT7.1 alloantigen defined by the BC 84A monoclonal antibody contained cells capable of transferring DH. Separation of the RT7.1-positive T-cell population with the monoclonal antibodies W3/25, MRC OX-8, or DS 4.23 (which defines the RT6.1 alloantigen) revealed that either the W3/25-positive or the RT6.1-negative T-cell subpopulations contained DH effector cells, whereas the corresponding MRC OX-8-positive or RT6.1-positive T-cell subpopulations did not. Moreover, when the W3/25-positive T-cell subpopulation was divided into either RT6.1-positive or RT6.1-negative T-cell subsets, only the W3/25-positive, RT6.1-negative subset transferred DH. These results indicate that the effector cells that mediate tuberculin DH are contained within the immune T-cell subset which bears both the RT7.1 and the W3/25 markers, but lacks both the MRC OX-8 and the RT6.1 markers.     

Reversal of ageing changes in the thymus of rats by chemical or surgical castration

Summary Differences in the thymus of young and old male CSE Wistar rats were examined by use of routine histological stains on paraffin-embedded sections. There was a highly significant loss of thymic weight and disruption of architecture with age. Both surgical castration and chemical castration induced by a luteinizing hormone-releasing hormone analogue (Goserelin) caused a significant increase in thymic weight and the reappearance of a well-defined cortex and medulla in ageing rats. Cell surface antigens were detected on cryosections after incubation with a range of monoclonal antibodies. The Pan T cell marker (detected with antibody W3/13) showed fewer positive cells in ageing rats, and an increase after chemical castration. The smaller glands of old rats had fewer positive T cells with CD4 (MRC OX35) and CD8 (MRC OX8) antigens, and more after chemical castration in both young and ageing rats, but the greatest changes were seen in the intensity of Class II major histocompatibility complex (MRC OX6) immunoreactivity. In both young and ageing chemically-castrated rats, the numbers of cells and the intensity of immunoreactivity were greatly increased in the medulla.     

The immune response to Schistosoma mansoni infections in inbred rats. VI. Regulation by T cell subpopulations

These studies assess the roles of subpopulations of T lymphocytes in inducing and modulating resistance to Schistosoma mansoni. CDF rats were depleted of RT 7.1+ (anti-Pan-T), W3/25+ (anti-T helper/inducer), or OX8+ (anti-T suppressor) cells by the in vivo administration of monoclonal antibodies (mAb). The development of parasites and immunity to challenge by S. mansoni were compared with results in undepleted normal and congenitally athymic rats. Discrete subpopulations of T lymphocytes were adoptively transferred to ascertain effects upon parasite development and the protective immune response. In vitro studies, involving utilizing cocultivation of cell subpopulations +/- cyclosporin A, were utilized to dissect mechanisms. Depletion of T lymphocytes by anti-RT7.1 mAb and anti-W3/25 mAb resulted in augmented initial worm development, suboptimal resistance, and decreased antibody and delayed-type hypersensitive reactivity directed against schistosome antigens. Depletion with OX8 mAb produced opposite effects. The adoptive transfer of T cell subpopulations produced concordant results with T cell regulation expressed B cell-dependent effector mechanisms. The coadoptive transfer of cells resulted in the suppression of resistance afforded by the W3/25+ cells by OX8+ cells, which could be augmented in vitro by cyclosporin A. Thus, protective immunity to S. mansoni in rats is regulated by discrete subpopulations of T lymphocytes. The findings suggest the possibility of selective immune regulation of resistance based on the manipulation of specific T cell subpopulation.     

The effects of genetics and age on expression of MHC class II and CD4 antigens on rat cardiac interstitial dendritic cells

Interstitial dendritic cells (IDC) in normal hearts of inbred rat strains and congenic and congenic recombinant lines were identified and quantitated by immunohistologic methods, on the basis of cellular reactivity with MRC-OX6, a monoclonal antibody (MAb) directed against MHC class II determinants, and with W3/25, a MAb directed against a CD4 epitope. In all strains and lines examined, the W3/25+ IDC frequency was uniformly high with little interstrain variation. In contrast, all rat strains and lines examined showed either high or low OX6+ IDC frequency. Double staining by two color immunofluorescence indicated that strains with a low OX6+ IDC frequency were characterized by a high frequency of W3/25+ OX6- IDC and a low frequency of W3/25+ OX6+ IDC. In strains with high OX6+ IDC frequency, the majority of IDC coexpressed both markers. Comparative analysis of (i) MHC identical background disparate strains and lines, (ii) MHC disparate background identical strains and lines, and (iii) F2 segregation analysis of intercrosses and backcrosses derived from an original cross between high and low frequency OX6+ IDC strains, all indicated that OX6+ IDC frequency is dependent upon both MHC- and non-MHC-linked genetic factors. It is suggested that rat cardiac OX6+ IDC frequency is influenced by a minimum of two autosomal genes, one of which is MHC linked. The frequencies of both W3/25+ IDC frequency reached adult levels by 10 days of age; adult levels of OX6+ IDC were not attained until Day 21. It is postulated that in the rat heart, W3/25+ OX6- IDC are potential precursors of W3/25+ OX6+ IDC, and that the cellular frequency of coexpression in the adult is under genetic influence. Whether these genetic factors modify constitutive or physiologic levels of class II-inducing lymphokine activity, or influence cellular susceptibility of IDC to induced class II expression is unclear.     

Cellular basis of allograft rejection in vivo. V. Examination of the mechanisms responsible for the differing efficacy of monoclonal antibody to CD4+ T cell subsets in low- and high-responder rat strains

MRC OX35, an anti-CD4 mAb, was used to treat high responder Wistar Furth (W/F) (RT1u) and low responder DA (RT1a) rats which had been grafted with directly vascularized hearts from PVG (RT1c) rats across a full MHC plus non-MHC incompatibility. Four doses of mAb at 7 mg/kg given in the first 2 wk postgrafting induced indefinite graft survival (greater than 150 days) in DA hosts, but only delayed rejection to 18 to 42 days in W/F as compared to rejection times of 6 to 8 days in untreated rats. The extension of MRC OX35 treatment to 6 wk in W/F rats induced indefinite graft survival in three of six rats. During treatment MRC OX35 therapy only partially depleted CD4+ cells, and all circulating CD4+ cells were coated with MRC OX35. The capacity of naive CD4+ and CD8+ cells from W/F and DA to be activated to PVG alloantigen was compared both in vitro in an MLC assay and in vivo by an adoptive transfer assay of their capacity to restore rejection of PVG heart grafts in irradiated syngeneic hosts. CD4+ cells from both W/F and DA proliferated in MLC and restored graft rejection. W/F CD8+ cells both proliferated in MLC and restored rejection, but DA CD8+ cells neither proliferated nor reconstituted rejection. Examination of lymphocytes from MRC OX35 treated hosts with long-surviving grafts showed that they were neither depleted of CD4+ T cells nor did they lack the capacity to proliferate to PVG Ag in MLC, this response being similar to that to third-party Ag or by naive lymphocytes. Compared to first-set rejection, PVG skin graft rejection was delayed 2 to 3 days in W/F and 10 to 12 days in DA rats with long-surviving grafts after MRC OX35 therapy, whereas they rejected third-party skin grafts in first-set tempo. These studies show that differences in graft survival in anti-CD4 treated low and high responder strains may be due to the inherent capacity of CD8+ cells to be activated to effect rejection independent of CD4+ cells in W/F but not in DA. In those hosts that accept grafts, there is no evidence of clonal deletion, but there appears to be a form of unresponsiveness akin to that induced in adult rats by other immunosuppressive therapies that protects the graft from rejection.     

The membrane phenotype of in vivo induced tumor selective cytolytic lymphocytes of the rat: a distinction from NK cells

The phenotype of tumor selective cytolytic lymphocytes of the rat is defined by staining of peritoneal cells of tumor-immunized donors with the monoclonal antibodies OX19, OX8, and W3/25, sorting in a cytofluorometer and evaluating cytolytic capacity in a 51Cr release assay. It is shown that the tumor selective cytolytic lymphocytes bear the OX19 and OX8 markers but are lacking the W3/25 marker. It is also shown that the OX19+ lymphocytes do not contain any NK-like activity. The OX19 marker can therefore distinguish cells that execute selective cytolysis from cells with NK-like activities.     

T-lymphocyte heterogeneity in the rat: Separation of distinct rat T-lymphocyte populations which respond in syngeneic and allogeneic mixed lymphocyte reactions

These experiments were designed to determine if separate subpopulations of T cells were involved in the syngeneic and allogeneic mixed lymphocyte reaction. Rat lymph node T cells were separated into W3/25⁺ and W3/25^? subpopulations by panning with the monoclonal antibody W3/25 and tested for their ability to proliferate in both syngeneic (SMLR) and allogeneic (MLR) mixed lymphocyte responses, as well as to develop cytotoxicity against allogeneic, syngeneic, and trinitrophenol (TNP)-modified syngeneic targets. The W3/25⁺ T cells reacted strongly in the SMLR and the MLR whereas the W3/25^? fraction proliferated only in response to allogeneic stimulation and with a kinetic pattern distinct from W3/25⁺. Furthermore, addition of W3/25 monoclonal antibody directly to the cultures was shown only to inhibit the proliferation of the W3/25⁺ T-cell fraction. The W3/25^? subpopulation contained cytotoxic T cells (CTLs) against both allogeneic determinants and TNP-modified self. However the requirements for the activation of allospecific CTLs were distinct from those for CTLs for TNP-self in that W3/25^? allospecific CTLs required no detectable help from W3/25⁺ T cells but generation of the CTL response against TNP-self required the presence of W3/25⁺ helper T cells (Th). These data suggest that in the rat, there exist subsets of T cells recognized by their cell surface phenotype that distinguish between self and nonself determinants and the requirements for activation are different for each of these populations.     

T cell subsets mediating lethal graft versus host disease: demonstration that synergy between CD4+ and CD8+ T cells is the predominant mechanism in low responder rat strains

T cell subsets from rat strains that have been characterized as high and low responders to alloantigen were examined for their capacity to mediate lethal graft versus host disease (GVHD) across strain combinations incompatible for class I, class II, and non-MHC antigens. Inocula of 5 X 10(7) lymph node and spleen cells (LC) from low responder DA (RT1a) and high responder W/F (RT1u) strains caused lethal GVHD in (W/F X DA)F1 hybrids given 6 Gy whole body irradiation. W/F CD4+ (W3/25+) cells (2 X 10(7], equal to the number in 5 X 10(7) LC mediated lethal GVHD but 10(8) DA CD4+ cells were required to cause lethal GVHD. CD8+ (MRC OX8+) cells (5 X 10(7] from W/F rats alone caused lethal GVHD but those from DA rats could not. Mixtures of CD4+ and CD8+ DA T cells, equivalent to the number in 5 X 10(7) LC, did mediate lethal GVHD, demonstrating that synergy between the subsets was the predominant mechanism with DA cells. These results suggest that differences in alloreactivity between the strains tested may be due to alternate requirements for the alloactivation of T cell subsets; the high responder subsets being self-sufficient and the low responder subsets being dependent upon each other.     

Regulation of the immune response to alloantigens: suppressor and helper T cells generated in the primary MLR of the rat

The primary MLR of the rat was used to generate suppressor, cytotoxic, and helper T cells from lymph node cells of the WF (RT1 mu) inbred strain. They were assayed in 51Cr-release cytotoxic assays and by their effect on proliferation of fresh unprimed responder cells. Suppression by MLR cellular products was antigen-specific and generation and functional expression were directed to class II (RT1.B,D) antigens of stimulator cells in the strains tested. In contrast, help was not antigen-specific. The monoclonal antibodies OX8 and W3/25 were used to separate the primed products of the MLR into the constitutive subsets, suppressor/cytotoxic (OX8+) and helper/inducer (W3/25+). Gamma irradiation of OX8+ MLR-primed cells caused modest reductions in suppressive activity, but had no effect on the helper activity of W3/25+ cells. MLR-derived suppressor cells are effective only when added in the early stages of the test primary MLR, whereas helper cells can augment proliferation even when added late. Feedback suppression is not mediated by classical cytotoxic T cells, because of differences in kinetics of development, cell numbers required, susceptibility to freezing, and expression of the RT6 differentiation antigen.     

Selective induction of OX19+ (CD5+) or OX19- (CD5-) alloreactive cytolytic lymphocytes in the rat

The phenotypes of alloselective cytolytic lymphocytes of the rat are defined by staining of peritoneal cells of alloimmunized donors with monoclonal antibodies, sorting in a cytofluorometer and evaluating cytolytic capacity in a 51Cr-release assay. We demonstrate that alloimmunization of BN rats can result in either OX19+ (CD5+) or OX19- (CD5-) cytolytic alloselective lymphocytes and show that the OX19- (CD5-) cytolytic cells are OX34+ W3/25- (CD4-) OX8+ (CD8+) lymphocytes not exposing surface Ig. It is further demonstrated that the appearance of CD5+ and CD5- cytolytic alloselective lymphocytes are mutually exclusive; immunization with (WF X BN) F1 cells leading exclusively to appearance of OX19+ effector cells while immunization with WF cells leads to OX19- effector cells. Alloimmunization of WF rats only results in appearance of OX19+ cytolytic lymphocytes.