Implication of the Thy-1 antigen on mouse thymus cells in the recognition of 'self' structures

Thymus cells of mice form rosettes with autologous and allogeneic erythrocytes. The nature of the thymus cell receptors which mediate the binding of erythrocytes is not known. The aim of the present study was to determine the effect of various antisera to T-cell specific antigens on the formation of rosettes by mouse thymus cells. In all strains of mice tested, the exposure of thymus cells to rabbit anti-mouse brain serum (RABR) was found to inhibit autorosette formation. Similarly, monoclonal Thy-1 antibodies inhibited the formation of autorosettes by thymus cells in all strains tested. Monoclonal antibodies against Lyt-1, Lyt-2, and TL determinants had no such effect. Monoclonal Thy-1 antibodies inhibited the formation of rosettes with allogeneic erythrocytes only when the thymus contained the Lyt-2.2 allele (BALB/c, C57BL), but not when it contained the Lyt-2.1 allele (AKR/J, C3H, DBA/2). These results indicate that Thy-1 determinants on thymus cells are involved in the recognition of 'self' structures, shared by erythrocytes of all strains of mice. Lyt-2 determinants may play a role in the recognition of 'non-self', allogeneic determinants, but the thymus cell surface structures encoded by the two Lyt-2 alleles may differ in their affinity to allogeneic erythrocytes.     

The relationship between secreted and cell surface antigen-binding molecules synthesized by T cells. I. Function-related isotypic determinants on T cells defined by antisera to antigen-specific helper and suppressor factors.

Rabbit antisera have been used to define 'constant region' markers which distinguish between mouse T cell-derived helper and suppressor factors, regardless of their antigen-specificity or strain of origin. These antisera have also been shown to bind to functional T-cell lines. After several absorption steps, rabbit anti-helper factor serum bound specifically to mouse helper cell lines, whereas rabbit anti-suppressor factor serum bound specifically to suppressor cell lines. Neither antiserum bound to cytotoxic T cell lines. The 'isotypic' determinants defined by these antisera were demonstrated to be present on distinct subpopulations of non-transformed T cell populations, such as splenic T cells, cortisone-resistant thymocytes and Con A blasts, but were not found on Thy-1- spleen cells, bone marrow, brain, heart, liver, kidney or heart tissue. The antisera did not stain significant numbers of normal thymocytes, and so expression was restricted to mature T cells. T cells reactive with rabbit anti-helper factor serum were found in the Lyt 2- population of cortisone-resistant thymocytes, and constituted a major subpopulation of in vitro induced helper cells, while rabbit anti-suppressor factor serum stained cells found in the Lyt 2+ population of cortisone-resistant thymocytes, as well as the majority of in vitro induced suppressor cells. Thus, these antisera are potentially of great value in the definition and isolation of functionally-distinct T cell subpopulations.     

Antigen density on target cells determines the immunosuppressive potential of rat IgG2b monoclonal antibodies

The current studies were designed to determine the relevance of T cell antigen density, besides antibody isotype, with regard to the success of antibody serotherapy. We compared the immunosuppressive effects of two rat IgG2b monoclonal anti-Thy-1 antibodies, RmT1 and 30-H12, with distinct binding sites in a graft-vs.-host disease (GVHD) model of fully H-2 and I-A region-mismatched bone marrow transplantation, making use of the difference in Thy-1.2 antigen density between homozygous (BALB/c) and heterozygous (BALB/c X AKR/J)F1 GVHD-promoting donor cells. Antibodies RmT1 (directed against a monomorphic determinant on mouse Thy-1) and 30-H12 (reactive with the Thy-1.2 allele-specific determinant) did not differ in their anti-GVHD activity with regard to Thy-1.2 homozygous grafts. However, in the region of a critical number of binding sites a small difference in the amounts of the two antibodies bound (about 8 X 10(3) IgG molecules/cell) obviously accounts for a great difference in anti-GVHD activity. This is shown in a two haplotype host-graft disparity between C57BL/6 recipients treated with either RmT1 or 30-H12 before challenging them with (BALB/c X AKR/J)F1 grafts, where the Thy-1.2 antigen concentration is approximately 50% compared to the density on BALB/c lymphocytes. Here, mAb 30-H12 loses its remarkable in vivo immunosuppressive quality, whereas RmT1 treatment protects mice against lethal GVHD. Binding sites were quantitated using a computerized approach for the analysis of data from ligand binding experiments of the respective mAb, RmT1 and 30-H12, coated to LN cells of BALB/c and F1 hybrid origin. Furthermore, the in vivo immunosuppressive activity of rat IgG2b antibodies directed against Thy-1 was found to correlate with their ability to generate stable antibody-C1q complexes on the cell surface of immunocompetent T cells.     

Evidence that the Thy-1 molecule is the target for T cell mitogenic antibody against brain-associated antigens

Rabbit anti-mouse brain (RaMBr) antiserum can induce Lyt-1⁺, Lyt-2^?, T cells to proliferate and stimulates the same T cell subset to induce B cell proliferation. The aim of this report is to demonstrate that the mitogenic determinant recognized on the T cell surface by RaMBr antiserum is located on the Thy-1 molecule expressing the products of the Thy-1^a and Thy-1^b alleles. Evidence is drawn from serological and genetic experiments. The brain T cell cross-reactive, mitogenic determinant is not expressed on Thy-1^? mutants of the BW5147 T cell lymphoma that fail to express the Thy-1 molecule but do express other T cell surface proteins such as T-200 and gp 69, 71. Monoclonal anti-Thy-1.1 and anti-Thy-1.2 antibodies block the binding to the appropriate T cells of the majority of the serum antibody from RaMBr antiserum. The absorption of mitogenic antibody was blocked in a similar fashion, thus demonstrating the close association of the determinant and the Thy-1 antigen defined by monoclonal alloantibodies. The mitogenic and Thy-1.1 determinants are probably located on the same molecule because of the data obtained with the BW5147 Thy-1^? mutants and the observation that Thy-1^a T cells, which express a lower level of surface Thy-1 than Thy-1^b T cells, also express lower levels of the determinant recognized by RaMBr antiserum. Furthermore, in (AKR × DBA/2)F₁ mice (Thy-1^a/b) which express less Thy-1.1 antigen than Thy-1.2 at the surface, the mitogenic determinant was found to be prefentially associated with Thy-1.2. The coordinated genetic control of the surface levels of the Thy-1 determinant and the mitogenic determinant suggests that both determinants are situated on the same molecule in the T cell membrane.     

Receptors for the Fc portion of immunoglobulins (FcR) on murine oral mucosal T cells

The incidence and distribution of immunoglobulin receptors on oral mucosal T cells were investigated. Enrichment of either Thy-1.2, L3T4 or Lyt-2.2-positive cells was achieved by the use of a panning procedure following cell extraction by an optimized digestion method that preserved all three T-cell markers. The percentage of cells possessing Fc receptors was determined by using immunoglobulin-coated (IgM, IgG or IgA) fluorescent microspheres in a multipoint rosetting assay. We report that approximately 60% of Thy-1.2, L3T4 and Lyt-2.2 positive cells bear Fc gamma R whereas less than 5% of T cells of any subset bear Fc alpha R or Fc mu R. In frozen tissue sections, Fc gamma R+ cells were mainly scattered throughout the basal and suprabasal epithelium and were observed at a lower frequency in the minor salivary gland network. From their distribution, it is anticipated that Fc gamma R+ cells may be involved in the surveillance of the epithelium while the minor Fc alpha R+ L3T4+ T lymphocyte population may promote the expression of sIgA by resident sIgM-bearing B cells, and their differentiation into IgA plasma cells.     

A new model for investigations of T-cell functions in mice: differential immunosuppressive effects of two monoclonal anti-thy-1.2 antibodies

Two monoclonal anti-Thy-1.2 antibodies were investigated for their activity in eliminating T cells in vitro and in vivo. Both antibodies exert a complement-dependent cell cytotoxicity in vitro. Antibody B that belongs to the IgM class shows a 100-fold higher complement-dependent cytotoxic activity than antibody C, which is of IgG_2a class. However, administration of antibody C into Balb/c mice results in the elimination of T cells as determined by the failure of different T-cell functions. Within 24 hours after administration of antibody C, the reactivity of spleen or lymph-node cells to T-cell mitogens, the antibody response to the Tcell- dependent antigen SRBC and the SRBC-induced delayed-type hypersensitivity are completely abolished. These effects are dose-dependent in a dose range of 0.1–1.0 mg Ig protein per animal and affects only T cells in the peripheral lymphoid organs. The Thy-1.2-bearing cells residing in the thymus are not impaired by the treatment of the animals with this monoclonal antibody and are able to repopulate the peripheral lymphoid organs within 30 to 60 days.Investigations into the mode of action of the removal of peripheral T cells revealed that antibody-C-coated Thy-1.2-bearing cells are rapidly phagocytosed by macrophages, while antibody-B-coated Thy-1.2-bearing cells are not. This might be the reason for the differential in-vivo activities of the two monoclonal antibodies.A model with new qualities for the study of functions and the regeneration of T cells in vivo has been established.     

Receptors for the FC Portion of Immunoglobulins (FCR) on Murine Oral Mucosal T Cells

The incidence and distribution of immunoglobulin receptors on oral mucosal T cells were investigated. Enrichment of either Thy-1.2, L3T4 or Lyt-2.2-positive cells was achieved by the use of a panning procedure following cell extraction by an optimized digestion method that preserved all three T-cell markers. The percentage of cells possessing Fc receptors was determined by using immunoglobulin-coated (IgM, IgG or IgA) fluorescent microspheres in a multipoint rosetting assay. We report that approximately 60% of Thy-1.2, L3T4 and Lyt-2.2 positive cells bear FcyR whereas less than 5% of T cells of any subset bear FcaR or FcjiR. In frozen tissue sections, FcyR+ cells were mainly scattered throughout the basal and suprabasal epithelium and were observed at a lower frequency in the minor salivary gland network. From their distribution, it is anticipated that FcyR+ cells may be involved in the surveillance of the epithelium while the minor FcocR+ L3T4+ T lymphocyte population may promote the expression of slgA by resident slgM-bearing B cells, and their differentiation into IgA plasma cells.     

Stimulation and expansion of a human T-cell subpopulation by a monoclonal antibody to T-cell receptor molecule

A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing T-cell line HPB-ALL. This antibody, C37 (an IgG1,K) also reacted with a small (2-5%) population of normal peripheral blood T (PBL-T) cells. These C37-positive (C37+) cells were found in both the T4/Leu3+ and T8/Leu2+ subsets. Like OKT3 antibody, C37 induced T-cell mitogenesis with a peak proliferative response at day 3. In long-term cultures containing irradiated autologous feeder cells and IL-2, C37 antibody caused the selective expansion of C37+ T cells. On HPB-ALL cells C37 induced comodulation of the T3 molecule. C37 precipitated a disulfide-linked dimer characteristic of the T-cell antigen receptor consisting of an alpha-subunit (45-48 kD) and a beta-subunit (38-42 kD) from both C37+ T-cell blasts of a normal individual and HPB-ALL cells that were surface radioiodinated. However, the precipitated molecule isolated from C37 antibody-activated T-cell blasts exhibited a different pI from that isolated from HPB-ALL cells. Our studies indicate that C37 recognizes an epitope on the T-cell receptor molecule that is shared by a subpopulation of human T cells, which raises the possibility that multiple variable-region associated and/or framework-like determinants of the T-cell antigen receptor can be defined serologically and used in functional and molecular studies of T-cell subsets.     

Receptors for the FC Portion of Immunoglobulins (FCR) on Murine Oral Mucosal T Cells

The incidence and distribution of immunoglobulin receptors on oral mucosal T cells were investigated. Enrichment of either Thy-1.2, L3T4 or Lyt-2.2-positive cells was achieved by the use of a panning procedure following cell extraction by an optimized digestion method that preserved all three T-cell markers. The percentage of cells possessing Fc receptors was determined by using immunoglobulin-coated (IgM, IgG or IgA) fluorescent microspheres in a multipoint rosetting assay. We report that approximately 60% of Thy-1.2, L3T4 and Lyt-2.2 positive cells bear FcyR whereas less than 5% of T cells of any subset bear FcaR or FcjiR. In frozen tissue sections, FcyR+ cells were mainly scattered throughout the basal and suprabasal epithelium and were observed at a lower frequency in the minor salivary gland network. From their distribution, it is anticipated that FcyR+ cells may be involved in the surveillance of the epithelium while the minor FcocR+ L3T4+ T lymphocyte population may promote the expression of slgA by resident slgM-bearing B cells, and their differentiation into IgA plasma cells.     

Phenotypic analysis of splenic lymphocytes and immunohistochemical study of hepatic granulomas after a murine infection with Salmonella abortusovis.

Infection in mice with an attenuated strain of Salmonella abortusovis (SAO), a specific pathogen for sheep, was used as a convenient model to understand further the induced immunity against SAO. The hypovirulent Rv6 strain, subcutaneously inoculated in salmonella-susceptible BALB/cby (Itys) mice, colonized the spleen and the liver in less than 6 days post-infection (PI) to be cleared after Day 28 PI. Simultaneously, an increase in spleen cell numbers, splenomegaly and hepatic granulomatous lesions developed to a maximum level on Day 9 PI. In spleen of uninfected mice, the number of Thy-1.2+ cells represents twice the number of surface immunoglobulin-positive cells (sIg+). Cytofluorometric analysis of the spleen lymphoid cell subsets showed a significant increase (10 times, P less than 0.05) in the number of sIg+ cells from Day 6 to Day 28 PI compared to control values. The number of Thy-1.2+ cells also significantly increased, to a lesser degree than the sIg+ cells, on Day 2 and on Day 16 PI (twice control values, P less than 0.05), but decreased on Day 6 PI compared to Day 2 PI. The highest L3T4+:Lyt-2+ ratio was observed on Day 2 PI and the lowest on Day 9 PI. On Day 28 PI, the number of sIg+ cells was still greater than the number of Thy-1.2+ cells. The granulomatous lesions were observed in the liver as early as Day 2 PI and their frequency was maximal on Day 9 PI. Immunohistochemical analysis of the granulomatous lesions showed that macrophages (F4/80+, Mac1+) were the basic cells and that L3T4+ cells were the predominant T cells. In well-developed granulomas observed on Day 9 PI, macrophages were in the centre whereas L3T4+ T cells were preferentially located at the periphery. T cells expressing Lyt-2 antigen were rarely detected. Variations in the proportion of lymphoid cells in the spleen and in hepatic granulomatous lesions suggest different and complementary effector mechanisms in induced immunity against SAO.     

Analysis of the Thy-1 pathway of T cell hybridoma activation using 17 rat monoclonal antibodies reactive with distinct Thy-1 epitopes

Seventeen monoclonal anti-Thy-1 antibodies (mAb) derived from LOU/M rats immunized with mouse T cell clones were used to study the role of Thy-1 in antigen-independent T cell activation. These mAb identified Thy-1.2 or monomorphic determinants and immunoprecipitated a molecule of 25-28 kDa from detergent-solubilized, 125I-labeled T cell surface proteins. Competitive cross-inhibition binding assays demonstrated that these reagents defined 3 epitope groups including either Thy-1.2 (group A) or Thy-1 monomorphic (groups B and C) determinants. Experiments using high titered culture supernatants revealed that all 6 IgG mAb defining the epitope group C, and one IgG2c mAb directed at a determinant in group A were capable of stimulating the terpolymer-L-glutamic acid60-L-alanine33-Ltyrosine10 (GAT) plus I-Ad-reactive BALB/c T cell hybridoma T14-117.9 to produce interleukin 2 (IL2) in the absence of accessory cells. Cross-linking of cell-bound rat mAb by a BALB/c anti-rat kappa chain mAb, or the presence of B cell lymphomas in the culture resulted in an increase of the Thy-1-mediated IL2 responses of this hybridoma. Some mAb from group B required antibody doses exceeding 80 micrograms/ml in order to activate T cells, while others remained nonstimulatory at any dose tested. Striking synergy in mAb-mediated T cell activation was observed when nonmitogenic doses of mAb group groups A and C were mixed in the same culture. Analysis of a panel of GAT plus I-Ad-specific T cell hybridomas revealed that these cells markedly differed in the magnitude of their IL2 responses induced by a given amount of stimulating anti-Thy-1 mAb. Such reagents also stimulated normal thymocytes to express IL2 receptor on their surface. These studies show that the epitopic specificity and the amount of anti-Thy-1 mAb, and the susceptibility of the T cell examined represent important parameters for the triggering of the Thy-1 pathway of T cell activation.     

Sensitization of Human Lymphocytes Against Autologous or Allogeneic Lymphoblastoid Cell Lines: Characteristics of the Reactive Cells

Normal human peripheral blood lymphocytes were sensitized to autologous or allogeneic lymphoblastoid cells in vitro. Purified T lymphocytes were found to be able to respond both by performing DNA synthesis and by functioning as killer cells. Surface marker analysis of blast-transformed lymphocytes in the in vitro cultures showed a high proportion (around 50%) of blasts lacking any surface marker attributable to b or T blasts; such 'null' blasts have previously not been found in conventional mixed leukocyte culture or after phytohemagglutinin or concanavalin A activation Since the 'null' blasts could be shown to be produced in a high percentage from originally almost pure sheep erythrocyte(SRBC) binding lymphocytes and displayed a similar killing capacity per unit cell number is SRBC-binding lymphoblasts, we consider the 'null' blast to be of T origin.     

Functional and binding activity of monoclonal anti-Thy-1 antibodies: evidence for different expression of the two alleles

Monoclonal anti-Thy-1.1 and anti-Thy-1.2 antisera selected for complement-dependent cytotoxicity have high cytotoxic and binding titers on thymocytes and peripheral T cells of mouse strains bearing the appropriate Thy-1 allele. The effect of both anti-Thy-1.1 and anti-Thy-1.2 monoclonal antisera plus complement on cytotoxic T cell effectors is to abrogate their activity. On the functional activity of precursor cytotoxic T cells, monoclonal antisera against the two alleles have different effects: anti-Thy-1.2 plus complement removes precursor activity of Thy-1.2-bearing strains, including (Thy-1.1 × Thy-1.2)F₁ heterozygotes. In contrast, six different anti-Thy-1.1 monoclonals, including four of the IgM class and two of the IgG class, failed to remove cytotoxic precursor activity from the splenic T cells of AKR, A. Thy-1.1 or (CBA × AKR)F₁ mice. Analysis by fluorescence-activated cell sorting of in vitro cultured AKR spleen cells shows that Thy-1.1 antigen appears on the cell surface during the five-day culture period.     

Protective immunity in murine histoplasmosis: functional comparison of adoptively transferred T-cell clones and splenic T cells.

In this study, I examined whether a murine T-cell line and three clones that recognize Histoplasma capsulatum antigens in vitro could confer protection in vivo against a challenge of Histoplasma yeasts. C57BL/6 mice were each inoculated with 5 X 10(4) yeasts intravenously; 1 h later, 5 X 10(6) or 2 X 10(7) resting T cells were inoculated intravenously. At week 1 of infection, the T-cell line and all clones failed to reduce the number of H. capsulatum CFU in the spleens of mice compared with numbers in infected controls. Administration of recombinant interleukin 2 or cyclophosphamide to infected mice did not potentiate the functional activity in vivo of either the T-cell line or the clones. In contrast, inoculation with 2 X 10(7) CD4+ but not CD8+ cells isolated from the spleens of mice immunized with 10(6) viable yeast cells sharply diminished the number of CFU in the spleens of infected animals. Moreover, splenic CD4+ cells from immune mice transferred a delayed-type hypersensitivity response, whereas the T-cell line and clones did not. Injection Injection of an equal number of cloned T cells and CD8+ splenocytes from immune mice did not transfer resistance to infected mice. Additional studies were undertaken to determine if the ineffectiveness of cloned T cells was associated with a failure to migrate to and survive within spleens of infected mice. B6.PL Thy-1a/Cy mice, which are genetically identical to C57BL/6 mice except that T cells of the former bear Thy-1.1 rather than Thy-1.2, were inoculated with Histoplasma yeasts and then injected with immune CD4+ splenocytes or a T-cell clone. At days 1 and 7 of infection, virtually no Thy-1.2+ cells were detected in the spleens of infected mice given cloned T cells. However, the spleens of animals inoculated with immune CD4+ cells contained a small but significant (P less than 0.01) proportion of Thy-1.2+ cells at both day 1 and day 7 postinoculation of H. capsulatum. Thus, the failure of T-cell clones to transfer protection against H. capsulatum may be explained by defective trafficking or poor survival in vivo or both.     

Characterization of a monoclonal rat anti-mouse interleukin 2 (IL-2) receptor antibody and its use in the biochemical characterization of the murine IL-2 receptor

Anti-murine interleukin 2 (IL-2) receptor monoclonal antibodies (mAb) were made from rats immunized with murine cytotoxic lymphocytes. One mAb, designated M7/20, strongly inhibited the proliferation of both IL-2 dependent CTLL-2 cells and concanavalin A (Con A)-induced T-cell blasts. Inhibition was linearly dependent on the concentrations of both M7/20 and IL-2. Utilizing FACS analysis, M7/20 was shown to bind selectively to mitogen-activated T lymphocytes and, to a lesser degree, to activated B lymphocytes. 125I-Labeled M7/20 binding assays indicated that 48-hr Con A-induced T-cell blasts possessed 89,000 binding sites/cell with a Kd of 1.2 X 10(-9) M. Competitive binding analyses indicated that M7/20 and IL-2 occupy the same or overlapping cell surface sites. Preliminary biochemical characterization of M7/20 immunoprecipitates of detergent extracts from both surface-iodinated and internally D-[3H]glucosamine-labeled T lymphoblasts indicated that the murine IL-2 receptor is an N-glycosylated 58,000-Da glycoprotein. Together these results suggest that mAb M7/20 binds at or near the IL-2-binding epitope on the murine IL-2 receptor and, thus, upon manipulation may act as an IL-2 agonist.     

The poor accessory cell function of macrophages in the rat may reflect their inability to form clusters with T cells

The accessory cell functions of Ia+ alveolar and peritoneal macrophages were compared to those of splenic cells in the rat. Whereas splenic mononuclear cells and dendritic cells were excellent supporters of both MHC-restricted and nonrestricted T-cell mitogenic responses, Ia+ macrophages were inefficient antigen-presenting cells and poor supporters of lectin mitogenic responses. Binding of antigen-primed T-cell blasts by splenic cells in the presence of Con A or antigen occurred within 30 min and subsequently led to the formation of nonadherent clusters of "dendritic-like cells" and proliferating T-cell blasts. Unstimulated Ia- macrophages failed to bind T cells during 30 min of coculture but formed conjugates with T-cell blasts within 24 hr. Delayed binding did not require the presence of antigen or lectin, or the expression of Ia antigens by the macrophage, and did not lead to T-cell proliferation. Antigen-specific binding and antigen presentation, but not lectin mitogenesis, were enhanced by treating antigen-pulsed Ia+ macrophages with neuraminidase for 30 min at 37 degrees C. Neuraminidase did not augment splenic accessory cell function. Antigen-specific binding of T cells to Ia+ macrophages and accessory cell function may be enhanced by desialation of glycoproteins on the cell surface membrane.     

Chicken T-Cell Growth Factor: Use in the Generation of a Long-Term Cultured T-Cell Line and Biochemical Characterization

Supernatants from concanavalin A (Con A)-stimulated chicken spleen cells were used to generate a long-term cultured cell line from antigen-primed chicken peripheral blood leukocytes. This line has been kept in continuous proliferation in vitro for more than 25 weeks. Morphologically these cells were lymphoblastoid and expressed class I and class II antigens of the major histocompatibility complex as well as T-cell (but not B-cell or macrophage) antigens. In addition they contained no peroxidase or non-specific esterase activity, neither were they phagocytic. Proliferation of the line was totally dependent on exogenous T-cell growth factor (TCGF) activity provided by the Con-A-stimulated spleen cell supernatant, comparable with the proliferation of Con-A-induced T-cell blasts. TCGF activity from the supernatant was absorbed both by the long-term cultured T cells and by Con A blasts, demonstrating the presence of receptors for the same TCGF species on the two populations. We have used the long-term cultured cell line to characterize chicken TCGF further. The molecular weight of the biologically active fractions found by gel filtration on Sephadex G-100 was approximately 13,000 and isoelectric focusing showed chicken TCGF to have a pI of pH 5.9. We propose that the TCGF described here is the chicken analogue to the mammalian interleukin 2.     

Tissue distribution and cytofluorometric analysis of oral mucosal T cells in the BALB/c mouse

The incidence and distribution of Thy-1.2+, Lyt-2.2+ and L3T4+ cells in the murine oral mucosa were investigated using qualitative and quantitative approaches. From immunostaining of frozen tissue sections, it appeared that the majority of oral T cells are located either in the epithelium or within the minor salivary gland network. The occurrence of Thy-1.2+, L3T4+ and Lyt-2.2+ cells at these sites points to two strategic lines of defence in the event of mucosal infections or aggression. A quantitative analysis of oral T-cell subsets was made possible by optimizing an enzymatic digestion procedure which preserves all three T-cell surface markers. Flow cytometric analysis of oral mucosal cells demonstrated that the helper phenotype is about twice as numerous as the cytotoxic/suppressor phenotype in the mucosa. Furthermore, in single cell suspensions, virtually all Thy-1+ cells were either L3T4+ or Lyt-2.2+ in the mucosa and in the spleen. From this frequency analysis and our previous studies, we conclude that T cells are a major component of the oral immune system, being 2-3 times as numerous as B cells or macrophages. Present data on the spatial distribution and characteristic ratio of T-cell subsets assess the basal activity of the local T-cell populations in healthy animals and lay the basis for comparative studies of both qualitative and quantitative variations occurring during mucosal infections or autoimmune reactions.     

Bovine gammadelta T-cell responses to the intracellular protozoan parasite Theileria parva

T cells bearing the gammadelta antigen receptor (gammadelta T cells) can constitute up to 50% of T cells in the peripheral blood and lymphoid organs of young cattle. We present data showing that gammadelta T cells are involved in immune responses against Theileria parva. gammadelta T cells isolated from peripheral blood mononuclear cells (PBMC) of T. parva-naive and -immune cattle proliferated in the presence of fixed or unfixed autologous T. parva-infected lymphoblasts (TpL) and heat-stressed concanavalin A (ConA)-induced blasts (ConA blasts) but not untreated ConA blasts. The specificity of response was further evaluated with a panel of gammadelta T-cell lines and clones. T-cell reactivity was blocked by GB21A, a monoclonal antibody (MAb) specific for the gammadelta T-cell receptor, but not by MAbs specific for class I and class II major histocompatibility complex (MHC) molecules. In addition, TpL but not ConA blasts from a variety of MHC-mismatched animals induced proliferation of the gammadelta T-cell lines and clones. These gammadelta T cells were found to respond to TpL infected with several different parasite stocks and failed to recognize TpL after elimination of the parasite by the theilericidal drug BW 720C. Assays for cytotoxic activity of gammadelta T cells sorted from bulk cultures of immune PBMC restimulated several times with autologous TpL demonstrated that effector cells whose specificity is similar to that of proliferating cells are generated. These results suggest that bovine gammadelta T cells are activated by and lyse T. parva-infected cells by recognizing conserved parasite-induced or parasite-derived antigens in an MHC-unrestricted fashion.     

Detection of Thy-1.2 membrane complexes shed from thymocytes and lymphoma cells by an immunoradiometric assay

A direct binding immunoradiometric assay (IRA) for Thy-1 antigen was developed to study the properties of membranous complexes shed from murine thymocytes and lymphoma cell lines. Monoclonal anti-Thy-1.2 antiserum was iodinated and used to study the shedding from AKR (Thy-1.1) and C3H(Thy-1.2) thymocytes, and S49.1(Thy-1.2), S49-Thy-1^? and BW5147(Thy-1.1) continuous lymphoma cell lines. Culture supernatant fluids or purified shed complexes were allowed to bind to microtiter plates followed by measurement of the binding of iodinated anti-Thy-1.2. The assay was found to be completely specific for the Thy-1.2 allotype, and in conjunction with antibody coated wells could detect Thy-1 solubilized from cells with N-P40 detergent. Shed complexes containing Thy-1 from thymocytes and lymphoma cell lines were analysed by isopycnic centrifugation with continuous potassium tartrate gradients (5–40%). Shed complexes had a buoyant density of 1.06–1.10 g/cm³ as compared to 1.15–1.17 g/cm³ expected for murine leukemia virus or 1.20–1.24 g/cm³ expected for mycoplasma. We concluded that the shed membranous complexes had a buoyant density similar to plasma membrane and the complexes were similar from both thymocytes and lymphoma cell lines. Thy-1 was not associated with virus, mycoplasma or other particles found in the gradients and Thy-1 was not found in the unsedimented fraction. The release of Thy-1 from thymocytes and cultured cell lines results in only one defined density of particles which may participate in cellular communication or in the survival of malignant cells.