Reactivity of anti-human brain serum with human lymphocytes.

Specific anti-human T-cell serum was prepared in rabbits by multiple subcutaneous injections of human brain homogenates in incomplete Freund's adjuvant. The serum was exhaustively absorbed with human RBCs, lyophilized human liver, lyophilized normal human serum, and peripheral blood lymphocytes from patients with chronic lymphocytic leukemia (CLL). Specificity of the antiserum for human T lymphocytes was tested by indirect immunofluorescence. It stained 70 to 80% of lymphocytes in circulation, 95% of thymus, 27 to 35% of spleen, 5 to 10% of tonsil lymphocytes, and over 90% of phytohemagglutinin-stimulated lymphocytes in vitro. Only T-dependent areas of cryostat-sectioned human lymph nodes stained with the antiserum. It did not stain circulating lymphocytes which formed HEAC rosettes, plasma cells in marrows of multiple myeloma patients or macrophages. After removal of HEAC rosettes by centrifugation in Ficoll-Hypaque, 75% of interface cells formed E rosettes and 65 to 75% stained with the antiserum. The antiserum was used in studies of lymphocytes in chronic and acute lymphocytic leukemias, lymphomas, and other lymphoproliferative diseases. Numbers and distribution in the circulation, spleen and nodes of lymphocytes bearing the T marker were significantly altered in patients with these disorders.     

Cellular response in Salmonella typhimurium-infected mice: evaluation of Salmonella receptors of B lymphocytes.

The cellular response in the course of experimental infection with Salmonella typhimurium was studied in mice. T cells were detected by the presence of theta-antigen, B cells by the binding of fluorescent immunoglobulins, and cells with receptors by labeled Salmonella binding. Lymphocytes were from spleen and lymph nodes. Results have been divided into three groups: group A, including mice with slight symptomatology; group B, including those with serious infection symptomatology; and group C, including mice that died in the course of the experiment. In spleen and lymph nodes of group A mice, an increase in the percentage of T and B lymphocytes was observed. This increase reached a peak 10 days after experimental infection. In lymph nodes, the B-cell percentage was equal to the percentage of T cells, whereas in spleen lymphocytes the B-cell percentage was higher. In spleens of group B mice we observed the same response as in mice of group A, whereas in lymph nodes there was a low response of T and B lymphocytes. In group C mice, there was no significant response of T and B lymphocytes in either spleen or lymph nodes. In B lymphocytes prepared from spleens of surviving mice, a small number of Salmonella receptors was detected: 200 bacterial cells per 10(9) lymphocytes.     

Immunohistochemical study of lymphocytes in rat pineal gland: Selective accumulation of T lymphocytes

Morphologic and immunohistochemical studies were performed to examine the existence of lymphocytes in the brain of rats. Special attention was paid to the time course of the appearance of lymphocytes in and around the pineal gland. Rabbit anti-rat T cell and anti-rat immunoglobulin sera were used for identification of T and B cells in tissue sections. Immunoperoxidase and immunofluorescence techniques were employed to identify cells reacting with anti-T and anti-immunoglobulin sera. No lymphocytes were found in the brain of rats until 20 days after birth. Small clusters of lymphocytes appeared in the pineal region by 30 days of age, after which they gradually increased in number, forming massive clusters in the pineal region by 120 days. Along with an increase in the number of lymphocytic cells, there was a gradual increase of cells reacting with anti-T cell serum. These T cells were only a minority of pineal lymphocytes in younger animals, but 90% or more cells were stained by anti-T cell serum at 120 days after birth. The remaining cells did not react with anti-immunoglobulin sera either. These findings suggest that the gradual increase of T lymphocytes in the rat pineal region is a simple reflection of the normal course of maturation of T cells, and the pineal gland in the rat may have some role in immune responses within the brain.     

Distribution of different cell types in the lymphoid organs of the mouse, as determined with sera against thymus and Peyer's patches

Anti-thymus serum (ATS) absorbed with Peyer's patch cells and anti-Peyer's patch serum (APS) absorbed with thymus cells were prepared. ATS treatment of mice resulted in a suppression of the immune response, measured with Vibrio cholerae as an antigen. APS did not have this effect. ATS and APS were used in the indirect fluorescence test to detect T and B_p cells. The cells staining with APS were called B_p cells to indicate that APS might only detect a special fraction of B cells (mature B cells). In Peyer's patches 66 per cent of the cells reacted with APS (B_p cells), whereas only 19 per cent cells reacted with ATS (T cells). The percentage of T cells in the lymph nodes was high (73 per cent), whereas only 14 per cent B_p cells were found in this organ. In the spleen almost equal numbers of B_p and T lymphocytes (32 per cent and 33 per cent) were detected. However, 35 per cent of the lymphocytes were non-reactive with either anti-serum. Bone marrow contained only small numbers of reactive cells (1 per cent T and 2 per cent B_p cells).

Treatment of mice with dimethylbenzanthracene (DMBA) caused an increase in the number of non-reactive cells in Peyer's patches and a dramatic decrease in the number of B_p cells. The relative increase of the number of T cells in spleen and lymph nodes was not found in Peyer's patches. Cortisone acetate seems also to act primarily on B cells, especially on those of Peyer's patches. In this case also an increase in the proportion of T cells in the spleen was detected.

     

Surface immunoglobulins on the lymphocytes of the skate Raja naevus

Living lymphocytes of the Elasmobranch fish, Raja naevus, have been examined for surface immunoglobulin (Ig) by treatment with a fluorescent anti-Ig system. Large numbers of Ig-positive cells (60-80%) were found in peripheral blood, spleen and thymus. Following modulation of the surface Ig with anti-Ig, resynthesis occurred, showing that the surface Ig is a product of the individual lymphocytes rather than material passively absorbed from the serum. Formation of caps was independent of temperature, occurring as readily at 4 degrees C as at 20 degrees C, a finding which presumably reflects the environmental conditions normally experienced by the skate. The presence of Ig-bearing lymphocytes in the adult skate thymus suggests a similarity to the amphibian larval thymus, which may be a primary lymphoid organ for the production of both T and B lymphocyte analogues.     

Surface markers of lymphocytes in the snake, Spalerosophis diadema. I. Investigation of lymphocyte surface markers.

A specific antiserum was raised in rabbits against thymocytes from snakes, Spalerosophis diadema, and was absorbed repeatedly with snake erythrocytes and kidney cells. In complement-dependent cytotoxicity assays, the absorbed anti-thymocyte serum (ATS) was, at any given dilution, cytotoxic to Sp. diadema thymocytes > peripheral blood lymphocytes (PBL) > spleen cells and could be titrated to a plateau defining a population of about 98% of thymocytes, 80% of PBL and 72% of spleen cells. Antiserum directed against snake immunoglobulins was obtained by injecting rabbits with gamma-globulins separated from snake serum by DEAE-cellulose filtration. the anti-gamma globulin serum was absorbed with snake erythrocytes, and in indirect membrane immunofluorescence stained no thymocytes while reacted with about 15% of PBL and 29% of spleen lymphocytes up to a 1:8 dilution. Fluorescence of positive cells was distributed in spots, patches or caps; cap formation could be inhibited by maintaining the immunofluorescence test at +4 degrees. In each of six separate experiments performed during spring, the percentage of lymphocytes which reacted with anti-snake gamma-globulin serum complemented the percentage of cells recognized by ATS. It was shown, furthermore, that about 3%, 8% and 21% of lymphocytes from thymus, peripheral blood and spleen, respectively, possess a receptor for 2-mercaptoethanol-insensitive antibody-sheep erythrocyte complexes. The results indicate that lymphocyte structural heterogeneity exists in reptiles.     

Antigens on mouse and rat lymphocytes recognized by rabbit antiserum against rat brain: the quantitative analysis of a xenogeneic antiserum.

Quantitative assays for measuring the binding of xenogeneic antiserum to dispersed cell suspensions are described. Cells were incubated with unlabeled xenogeneic antiserum and the antibody bound measured indirectly by a second binding step with 125I-labeled anti-immunoglobulin antibody. This indirect radioactive binding assay was calibrated by measuring, with a radioimmunoassay, the true amount of antibody bound in the first step. With these methods one can measure the strength of antisera, quantitate the number of antigenic sites, and partially differentiate determinants being recognized on cell surfaces. The binding of rabbit anti-rat brain antiserum to rat and mouse lymphocytes was analyzed in detail. After absorption of the antiserum with rat liver, the antibody remaining recognized lymphocyte antigens that were distributed among various rat and mouse tissues in quantities identical to Thy-1.1 antigen. Thus, at saturation, 670 000 Ig molecules from liver-absorbed rabbit antiserum were bound per rat thymocyte, and the antiserum bound to 90%, 21%, 4% and 2% of rat thymocytes, spleen, lymph node and thoracic duct lymphocytes, respectively. With mouse tissues, 90% 24% and 50% of thymocytes, spleen and lymph node cells, respectively, were labeled. In rat brain the concentration of xenoantigen increased with age, while in thymocytes the full adult amount was present at birth. Three antigenic determinants could be defined with the liver absorbed rabbit antiserum: the Thy-1.1 antigen, a rat specific antigen and an antigen cross-reacting between rat and mouse tissues. All 3 may be on the Thy-1 molecule. The anti-brain antiserum contained about 0.05 mg/ml of antibody specific to these xenoantigens.     

Nonspecific suppression of T lymphocyte responses in mice carrying progressively growing tumors

Unfractionated spleen cells from mice with four different types of progressively growing tumors were unable to show enhanced DNA or protein synthesis upon stimulation with phytohemagglutinin (PHA) in vitro. After separation by velocity sedimentation, or passage over an anti-immunoglobulin column, cells were obtained which could respond to this mitogen. An auxiliary population of cells obtained by velocity sedimentation, sedimenting with the characteristics of activated B lymphocytes and sensitive to the cytotoxic effects of anti-immunoglobulin serum and complement, could depress the response of normal spleen cells challenged with PHA. A role for this cell type in the tumor progression seen in the host animals is discussed.     

Maturation of bone marrow lymphocytes. I. Quantitative rosetting methods of detecting Fc and complement receptors and surface immunoglobulin.

Cytocentrifuge rosetting procedures were developed for use with bone marrow cells to quantitate Fc and complement receptors (FcR, CR) and surface IgM on marrow small lymphocytes. Cell suspensions from 9–11-week-old C3H mice were mixed (1 : 30) with washed sheep red blood cells (SRBC) coated with either mouse anti-SRBC serum (for FcR), rabbit anti-SRBC stroma serum plus mouse serum (for CR) or goat anti-mouse IgM serum (for IgM). Centrifuged cell pellets were incubated for 30 min at either 37°C (FcR, CR) or 0°C (IgM) and examined in stained cytocentrifuge preparations. Small lymphocytes were defined as non-DNA-synthesizing lymphocytes smaller than 10 μm nuclear diameter. Many of these cells in the bone marrow formed specific rosettes for FcR (25%), CR (18%) and surface IgM (39%). The incidence of FcR- and CR-bearing small lymphocytes relative to IgM-bearing small lymphocytes was lower in the marrow than in the blood, spleen and lymph nodes. Rosette size ranged from 4 to > 20 SRBC per lymphocyte but tended to be smaller in the marrow and blood than in the spleen and lymph nodes. The methods and results are discussed as a basis for studies of B lymphocyte maturation and lymphocyte heterogeneity in the bone marrow.     

The Polyclonal B-Cell-Activating Property of Protein A Is Not Due to Its Interaction with the Fc Part of Immunoglobulin Receptors

Protein A from Staphylococcus aureus bacteria was found to be a B-cell mitogen and a potent polyclonal B-cell activator (PBA) of antibody synthesis for murine lymphocytes in the absence of macrophages or T lymphocytes. It did not activate T lymphocytes. We investigated whether the interaction between protein A and the Fc part of Ig molecules was responsible for the PBA activity. Protein A failed to induce IgG synthesis in spleen cells from normal mice, even though it binds effectively to IgG molecules. Lymphocytes treated with anti-immunoglobulin antisera followed by protein A were not activated to a larger extent than non-pretreated cells, although only the former cells bound protein A. Finally, direct attempts to suppress the PBA property of protein A by blocking the Fc binding ability with serum or human gamma globulin failed. We concluded that protein A possesses two separate biological properties, namely to interact with the Fc receptor on Ig molecules and to act as a PBA, and these properties are carried out by different parts of the molecule. These findings confirm previous failures to find an active role of the Ig receptors on B lymphocytes in the triggering process.     

The capacity of microsomally-activated cyclophosphamide to induce immunosuppression in vitro.

Cyclophosphamide (CY) was activated in vitro with washed rat liver microsomes and cofactors. Pretreatment of mouse spleen cells in vitro with the activated drug abolished their capacity to give a primary antibody response to SRBC and levan on transfer to irradiated syngeneic recipients. However, responsiveness returned if challenge was delayed for 7 or more days after transfer. Part of this was shown to be of donor origin by an allotype marker. The treatment of normal spleen cells with activated CY in vitro also prevented B cells from regenerating their immunoglobulin receptors after capping with anti-immunoglobulin serum. The induction of suppression required contact between lymphocytes and activated CY for at least 30 min at 37 degrees and did not appear following incubation for 1 h at 0 degrees. Since the antibody response of drug-treated spleen cells to SRBC could not be restored with purified normal B or T cells, it is probable that B and T lymphocytes are both susceptible to suppression by activated CY in vitro. Similar pretreatment abrogated the graft-versus-host (GVH) reactivity of spleen cells as measured by survival and in a popliteal lymph node assay. B cell chimerism in F1 recipients of drug-treated parental spleen cells was demonstrated by the presence of congenic allotype markers. This suggests a possible approach for the attenuation of GVH disease which is associated with bone marrow transplantation in man.     

Anti-lymphocytic antibodies and marrow transplantation. III. Effect of heterologous anti-brain antibodies on acute secondary disease in mice

Antisera against murine thymus-derived lymphocytes were raised in rabbits injected with mouse brain. It was possible to remove contaminating antibodies which cross-reacted with B cells and hemopoietic stem cells by absorption with liver and plasmacytoma followed by purification and fractionation over DEAE-cellulose. T cell specificity of the absorbed anti-mouse brain serum (AMBS) and its purified globulin fraction (AMBG) was demonstrated by cytotoxicity, complement fixation and unaltered numbers of plaque-forming cells and colony-forming units. In vivo, the effect of absorbed AMBG on the graft-versus-host reaction was investigated in an H-2-incompatible parent-to-F₁ hybrid donor-recipient combination. (C57BL/6 × CBA)F₁ mice irradiated with 900 rad and transfused with C57BL/6 spleen cells died from acute secondary disease within 20 days. They survived as complete chimaeras beyond day 300 without symptoms of secondary disease when the donor cells had been preincubated with absorbed AMBG. The implications of the suppression of secondary disease in this model are discussed.     

Non-specific suppression of delayed-type hypersensitivity by intrathymic injection of killed bacteria

Previous intrathymic injection of 50 micrograms of killed BCG as well as killed Listeria in mice produced suppression of delayed-type hypersensitivity responsiveness induced with BCG cell walls (CW) or live Listeria. This finding was analysed in vitro by the macrophage migration inhibition test. Non-adherent spleen cells from mice that had been injected intrathymically with killed BCG or killed Listeria were tested for the ability to regulate the migration inhibition response of peritoneal exudate cells from BCG-CW immunized mice. No migration inhibition was observed, indicating that suppressor cells were induced in the spleen by intrathymic injection of killed BCG or killed Listeria. Suppressor cells were shown to be T cells since they were eliminated by treatment with anti-brain associated theta serum and complement. Thus intrathymic injection of bacteria induced antigen nonspecific suppressor T cells in the spleen.     

Virus-specific T cell response prevents lymphoma development in mice infected by intrathymic inoculation of Moloney leukaemia virus (M-MuLV).

Previous work on mice neonatally injected with Moloney-murine leukaemia virus (M-MuLV) had shown that T cell lymphoma development correlates with virus infection of lymphoreticular cells (T and B lymphocytes and macrophages) as well as with a lack of generation of virus-specific cytotoxic T lymphocytes (CTL) due to clonal deletion of CTL precursors. In the present report, viral antigen expression and T cell response in mice injected as adults with M-MuLV intrathymus (i.t.) was investigated. Only thymic and splenic T lymphocytes from these mice express virus-induced antigens since they were lysed by virus-specific CTL, and stained by anti-M-MuLV fluorescent serum. In addition, the percentage of M-MuLV-infected T cells increased with increasing post- inoculation times. However, these mice could mount a strong cellular immune response against M-MuLV-infected cells, as detected by massive mixed leucocyte tumour cell culture and by evaluation of virus- specific CTL precursor frequency. Finally, i.t. injected mice were not viraemic and did not develop lymphomas during an observation period of 12-15 months. These data, in contrast with the recent hypothesis that T cell lymphoma development depends on a chronic stimulation of virus-specific T lymphocytes, indicate that the cellular immune response is sufficient for prevention of neoplastic transformation, despite a persistent viral infection of the thymus and peripheral T lymphocytes.     

Stimulation of pig lymphocytes with anti-immunoglobulin serum and mitogens.

Mitogenic stimulation of pig lymphocytes by anti-immunoglobulin serum and by Con A, PHA, PWM and LPS has been measured by 3H-thymidine incorporation. Lymphocytes from blood, spleen and lymph node were readily stimulated by antiglobulin. Specific anti-micron serum was mitogenic for blood lymphocytes, indicating that those B cells with surface IgM can be stimulated by complexing of this surface component with antibody. An initial 2-hour incubation with anti-Ig was as effective as continuous incubation. Differentiation of stimulated cells into high rate synthesising plasmablasts was not observed. Pig blood lymphocytes were reactive to Con A, PHA, PWM and LPS. LPS was also tested against spleen and lymph node cells, which were responsive to this mitogen.     

Spontaneous interaction in vitro between lymphocytes and syngeneic peritoneal macrophages of mice.

Ficoll-purified lymphocytes (peritoneal, splenic, or thymic) and macrophages (peritoneal) from Toxoplasma-immune and normal female NMRI mice were used. Suspensions of washed cells were made in medium 199 containing 20% heat-inactivated normal calf serum. Sixty minutes after the adherence of 10(5) macrophages to cover slips in Leighton tubes, lymphocytes were added in various concentrations. The mixed cellular population was then incubated at 37 C. Eighteen hours later, most of the lymphocytes were firmly attached to macrophages to form rosettes. This cellular interaction, which was temperature, cell ratio, and time dependent, occurred in the absence of any particular antigenic stimulation. Morever, the reaction was cytotoxic only for adhered lymphocytes as judged by staining with 0.2% trypan blue. Splenic and thymic lymphocytes were bound in significantly greater number than peritoneal lymphocytes. Incubation of macrophages for more than 48 h at 37 C before the addition of fresh lymphocytes markedly reduced rosette formation. Treatment of macrophages and lymphocytes with mouse anti-immunoglobulin did not affect the reaction. The labeling of lymphocytes with fluorescent anti-mouse sera and the use of nude NMRI mice showed that both B and T cells can form spontaneous rosettes with syngeneic peritoneal macrophages.     

Nomarski differential interference contrast studies of murine lymphocytes

Nomarski differential interference contrast microscopy (DIC) of lymphocyte surface morphology was combined with immunofluorescence studies of T and B cell markers on the thymus, lymph nodes, spleen, peripheral blood lymphocytes and thoracic duct lymph of female CBA mice. DIC identified smooth cells and several categories of villous cells; more extreme forms were present in lymph. Most B cells seemed to belong to the smooth group and most peripheral T cells to the villous group. Thymus cells were almost entirely smooth, but treatment with cortisone increased the proportion of villous cells to 50%. The surface morphology of lymphocytes was highly labile preventing direct identification or separation of T and B cells. In vivo removal of T cells by adult thymectomy, lethal irradiation and bone marrow reconstitution caused the villous cells to decrease. During recovery from irradiation, T lymphocytes tended to parallel villous cells, B lymphocytes smooth cells, but there were differences between the spleen and lymph nodes. Mice deprived of T1 cells by adult thymectomy showed a modest decrease of smooth cells in the spleen and blood; mice depleted of T2 cells by anti-lymphocyte serum, or which were naturally deficient in T2 cells, were markedly lacking in villous cells. Thoracic duct lymph, which is rich in T2 cells, had a high proportion of extremely villous lymphocytes. Exposure to lymph induced extreme villous features in lymph node cells, and it was found that the thoracic duct lymph was markedly hypertonic to serum, although varying in osmolarity throughout the day. It is suggested that the villous shape of T2 cells is a circulatory adaptation, necessitated by the peculiar character of the lymphatic system in mice.     

Enumeration of polyclonal mitogen-responsive cells in different lymphoid tissues of the mouse.

The relative number of cells capable of responding to Con A, PHA and LPS in the spleen, blood, lymph node and Peyer's patches of CBA mice has been quantified by means of a cytological analysis technique. No difference has been found between Con A- and PHA-responsive cells in spleen and lymph node. The lymphoid tissues of T cell-deprived mice have a reduced content of PHA responsive cells, but LPS responsiveness is within normal limits. Pretreatment of peripheral lymphocyte populations with high concentrations of anti-O antiserum and complement abolishes the response of the treated cells to PHA, but not to LPS, whereas similar treatment with a cytotoxic anti-immunoglobulin serum, which has no effect on PHA-responsive cells, only partially reduces the response to LPS. The results for mitogen responsiveness are discussed with reference to other methods of quantifying T and B cells using cell-surface markers.     

Lymphocyte activation: IV. The ultrastructural pattern of the response of mouse T and B cells to mitogenic stimulation in vitro

Thymocytes from cortisone-treated mice (`T' cells), `B' spleen cells (B lymphocytes from thymectomized, irradiated, marrow reconstituted mice) and normal spleen (T + B) cells were examined by electron microscopy after 60 hours stimulation by Concanavalin A (a T cell specific mitogen), endotoxin (B cell specific mitogen), and pokeweed mitogen (which stimulates both T and B cells). Stimulation of T cells by Con A or PWM induced the appearance of lymphoblasts (Type I) and only PWM or endotoxin stimulated B cells developed `plasmablast' features (dilated, vesicular rough endoplasmic reticulum; Type II). A few stimulated B cells also had lymphoblast morphology. Large cells from normal (T + B) spleen stimulated by PWM were heterogeneous consisting of 55–60 per cent plasmablasts and 40–45 per cent lymphoblasts. It was concluded that the ultrastructure of stimulated lymphocytes depended on whether T or B cells were stimulated and not primarily on the mitogen used. In general, the response evoked by mitogens paralleled at the ultrastructural level that induced by antigens. It was also found that multivesicular bodies and glycogen particles occurred predominantly in the cytoplasm of stimulated T cells (lymphoblasts).     

Natural killer cells and B lymphocytes in L-selectin and Mac-1/LFA-1 knockout mice: marker-dependent,but not cell lineage-dependent changes in the spleen and bone marrow

The majority of B lymphocytes, virgin T lymphocytes and a subpopulation of memory T cells express the addressin, L-selectin. Natural killer (NK) cells in rodents and humans also express L-selectin. We have shown that a similar proportion (40%) of NK cells in mouse spleen also express the integrin, CD18Mac-1, and moreover, that NK cells express both the addressin and the integrin constitutively. It was the aim of the present study to quantify, in knock-out mice deficient for either the L-selectin addressin, or the CD18:Mac-1/LFA-1 integrins, NK cells and B cells in both the spleen and their bone marrow birth site. These cells, in both organs, were immunophenotypically stained with FITC-conjugated anti-NK1.1 (to identify NK cells), and FITC-conjugated anti-mouse B220 (to identify B lymphocytes) and subjected to flow-cytometric analysis using a FACScan equipped with a doublet discrimination module. From the known total organ (spleen, femurs) cellularity, obtained by means of an electronic cell counter, at the time of extraction of each organ, the absolute numbers of NK cells and B lymphocytes from each mouse were obtained. The results revealed that there are significantly more NK cells and B lymphocytes in the spleens of CD18:Mac-1/LFA-1 knockout mice than in control (same strain) mice. Moreover, in L-selectin knockout mice spleens, NK cells and B lymphocytes were elevated by 26.2% and 17.8% respectively. NK cells and B lymphocytes in the bone marrow of the integrin knockout showed no difference from control, however, both cell types in the bone marrow of the L-selectin knockout mice fell to only 3/4 their control levels. Collectively, the results demonstrated that there are organ-specific, but not cell lineage-specific differences in the absolute numbers of NK cells and B lymphocytes, in integrin-deficient (CD18:Mac-1/LFA-1 knockout) mice and addressin-deficient (L-selectin knockout) mice.