Theta-Bearing Cells in the Bone Marrow of Thymus-Deprived Mice

Theta-bearing cells in lymphomyeloid tissues of thymus-deprived and normal mice have been studied by the use of anti-theta. antisenun and cytotoxicity tests in addition to functional tests, In contrast to the findings in peripheral lymphoid tissues, increased percentages and numbers of theta-bearing cells were found in the bone marrow of neonatally thymectomized and nude mice as compared with normal and sham-thymectomized mice. In adult thymectomized mice, percentages comparable to those in sham-perated littermates were found. The findings were not due to irrelevant antibodies in the anti-theta antiserum, and neonatally thymectomized mice grafted with a thymic lobe showed percentages of theta-positive cells in the bone marrow comparable to those of sham-operated animals. Adrenalectomy did not lead to diminished percentages of theta-positive cells in the bone marrow of neonatally thymectomized mice, and the serum levels of hydrocortisone and corticosterone were within normal ranges in thymus-deprived mice. The mitogen responses and graft-versus-host activity of bone marrow cells from neonatally thymectomized mice suggest that most theta-positive cells in the bone marrow of these mice are functionally immature cells.     

Concanavalin-A-induced Suppressor Lymphocytes in Normal Individuals

Adherence of human lymphocytes to allogeneic tumour cell monolayers was found to depend on the presence of monocytes. Adherent lymphocytes could be separated from tumour cells by treatment with lidocaine followed by nylon wool passage. Tumour-adherent cells (70% E-RFC, 45% Fc gamma-R, 23% Fc mu-R, 5% monocytes) exhibited enriched natural killer (NK) activity not only against the tumour cell line used for isolation but also against seven other targets. When T cells were isolated subsequently as E-rosettes by density gradient centrifugation through Percoll, the enrichment in cytotoxicity was even more pronounced. Tumour-adherent T cells were severalfold enriched in both NK and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, this enrichment was not paralleled by a concomitant increase in the number of T gamma cells (tumour-adherent T cells: 17% T gamma, 40% T mu ; tumour-nonadherent T cells: 12% T gamma, 60% T mu). Marked differences could be observed by staining with a monoclonal antibody that was raised against human leukaemic T gamma cells of high NK and ADCC activity. This antibody (T 8-11) stained 60% of tumour-adherent T cells, 20% of nonadherent T cells and 29% of T-cell controls. These results indicate that the spontaneous cytotoxic activity of human T cells resides within a small population, most of which are characterized by a specific surface antigen but not by conventional Fc gamma receptors.     

Idiotypic Lymphocytes in the Rabbit: Occurrence and Nature of Idiotypic Lymphocytes in Normal and Hyperimmunized Rabbits

Rabbits were hyperimmunized with Micrococcus Pysodeicticus, leading to homogeneous antibody responses. Peripheral blood lymphocytes were taken from the rabbits before and monthly (during 3 months) after the start of the immunization. The cells were stored frozen. Lymphocytes were tested with anti-idiotypic conjugates for the presence of surface idiotypic structures. The nature of the idiotype-positive cells was determined with respect to the presence of IgM or T-cell antigenic determinants on their surface. A sharp rise and fall in the percentage of idiotypic lymphocytes was found, ranging between 1/40,000 and 1/1,000. Initially almost all idiotypic lymphocytes were IgM-positive. In the blood taken 2 months after the start of the immunization 20% of the idiotypic cells belonged to the T-cell population and 10% were negative for both IgM and T-cell antigenic determinants.     

Serum Inhibitory Factor in Lepromatous Leprosy: Its Effect on the Pre-S-Phase Cell-Cycle Kinetics of Mitogen-stimulated Normal Human Lymphocytes

The sera of ten Egyptian men with long-standing lepromatous leprosy (LL) (mean duration 17.4 years) that had failed to respond in dapsone treatment were shown to inhibit mitogen stimulation responses of normal human lymphocytes. When first tested, the sera partly inhibited the response to phytohaemagglutinin (PHA) and pokeweed mitogen and virtually abolished that to concanavalin A (Con A): after repeated freezing and thawing, the Con A inhibition had disappeared, whereas the PHA response was still partly Inhibited. The inhibitory serum factor(s) had similar actions on lymphocytes from each of six normal donors. Although the sera varied in potency, they showed similar dose response curves when tested against lymphocytes from a single donor. The principal action of the sera was to reduce the number of cells responding to mitogen, without modifying the kinetics of recruitment or rate of volume growth during G₁-phase in those cells that were unaffected by the inhibitory substances(s). Study of PHA dose-response curves and of the effect of delayed addition of LL serum suggested that the serum factor(s) act by diminishing the responsiveness of the cells, rather than by reducing the concentrations of free mitogen or by blocking cell membrane mitogen receptors The serum from one apparently healthy attendant, who had nursed leprosy patients for 30 years but who did not have leprosy or other chronic infective disease, inhibited completely stimulation by all three mitogens in a manner different from that of LL sera. Serum from the other 13 control patients did not modify the response of normal lymphocytes to stimulation by any of the three mitogens studied. It was concluded that the inhibitory factor(s) in the scrum of patients, with LL were a consequence of the disease and not of the environment in which the patients lived. Microscopy confirmed that the techniques used for recovery of the cultured cells did not introduce bias into the volume spectroscopy measurements.     

T Lymphocytes in Collagen II-Induced Arthritis in Mice

Collagen type II-specific long-term cultured T helper cells, derived from the DBA/1 mouse, have been established and characterized. Clones from these T-cell lines could be shown to recognize either species-specific or species-nonspecific determinants on the collagen type II molecule, including determinants on autologous mouse collagen. Induction of arthritis via transfer to both irradiated and normal syngeneic recipient mice was obtained with both collagen type II-specific T-cell lines and an autoreactive and collagen type II-specific T-cell clone. Fewer cells were needed to evoke arthritis in normal than in irradiated recipients. Cells from lines and the clone used for transfer were by immunocytochemistry shown to have T helper phenotype.     

In Vivo Elimination of Allogeneic Lymphocytes in Normal and T-Cell-Deficient Rats

The distribution of allogeneic and syngeneic thoracic duct lymphocytes was studied over 24 h in normal and T-cell-deficient animals (thymectomized, irradiated rats, 'B rats', or congenitally athymic nude rats). Initial migration from blood was no less for allogenic than for syngeneic cells. After 24 h, however, a marked deficit of radioactively labelled allogeneic cells as compared with syngeneic cells was found in the lymphoid tissue, whereas the allogeneic isotope was recovered in a relatively greater amount in liver, kidneys, and cell-free plasma and lymph. Most of the allogeneic cells are evidently destroyed within the first 24 h and their isotope released into body fluids. Our studies also revealed this process to be even more evident in T-cell-depleted environments. Autoradiographic studies of recipient nude rat spleens showed that allogeneic cells were not found in the great number seen in syngeneic transfers, but a high grain density in the periarteriolar lymphocyte sheath area could be observed. Granula seemed to be predominantly located over large nonlymphoid cells. The elimination of allogeneic lymphocytes is therefore governed by mechanisms independent of an intact thymus and may be due to a cell population or factor more active in nude animals than in their non-nude littermates.     

Lymphocyte Subsets and Mitogen Stimulation of Blood Lymphocytes in Normal Pregnancy

PROBLEM: In normal pregnancy the maternal immune system should be directed towards tolerance or suppression in order not to reject the partly foreign feto-placental unit. The aim of this investigation was to find hallmarks of systemic immunosuppression during normal pregnancy. METHODS: Five healthy primigravidae were examined during pregnancy and postpartum with flow cytometric analysis to define T and B lymphocyte subsets in peripheral blood. In addition, we studied the proliferative response of lymphocytes to mitogens or interleu-kin-2 (IL-2) alone or in combination with immunomodulating drugs or interleukin-4 (IL-4). The results were compared to healthy, non-pregnant women. RESULTS: During pregnancy and early puerperium we noted an immune balance in favour of suppression, as measured by increased numbers of T “helper/suppressor” (CD4+ CD45RA+) and “suppressor”/effector T cells (CD8+S6F1-), and decreased numbers of T “helper/inducer” (CD4+CD29+), T “helper/memory” (CD4+CD45RO+), killer/effector T cells (CD8+S6F1+), and Natural Killer cells (CD56+), as well as decreased numbers of activated lymphocytes expressing IL-2 receptor (CD25+) and T cells expressing HLA-DR (HLA-DR+CD3+). During pregnancy, lymphocyte proliferation was impaired in autologous serum with concanavalin A (ConA), phytohemagglutinin (PHA), or IL-2. A difference in proliferative response to PHA or IL-2 between cultures with AB serum and autologous serum is suggestive of an immunosuppressor factor in serum during pregnancy. Indomethacin significantly increased lymphocyte proliferation in autologous serum with ConA, indicating PGE₂ mediated suppressor activity during pregnancy. Chlorambucil and cimetidine modulated the proliferative response to ConA, indicating an alkylating agent sensitive and a histamine dependent suppressor activity during pregnancy. CONCLUSIONS: During normal pregnancy, a state of systemic suppression of the maternal immune system seems to be present.     

The Effect of Prostaglandin E1 or E2 OH the in Vitro Blastogenic Response of Lymphocytes from Normal and Tumor-Bearing Mice

The in vitro effect of exogenously added prostaglandin (PG) El or E₂ over concentrations ranges of from 1 × 10^--⁴ to 1 × 10-9 M were studied in order to determine their effect on the in vitro lymphocyte proliferation of thymic and splenic T and B cells from normal and tumor-bearing CD₂ F ₁ mice. It was found that PGE₁ generally caused greater inhibition of blastogenesis than did PGE₂ when reacted with splenic lymphocytes from normal mice. Indeed, PGE₂ was found to be stimulatory for both Con A^- and LPS-sensitive normal splenic lymphocytes. Both PGE_l and PGE₂ caused potent inhibition of Con A^- and PHA-sensitive splenic lymphocytes from the tumor-bearing mice. Additionally, PGE₂ was found to stimulate the LPS-reactive lymphocytes from the tumored mice. PGE₁ and PGE₂ both inhibited the Con A- and PHA-reactive thymic lymphocytes from normal mice at the lower concentrations studied, i.e., 10^--⁴ to 10^--⁶ M. Thereafter, at concentration ranoes of from 10^--⁷ to 10^--⁹ M both PGE₁ and PGE₂ were both found to be stimulatory. Finally, both PGE₁ and PGE₂ at all concentrations studied, strongly inhibited the thymic lymphocytes from tumored mice.     

Surface-Bound Immunoglobulin on Lymphocytes from Normal and Immunodeficient Humans

By means of immunofluorescence technique, lymphocytes with surface-bound immunoglobulin were detected in the blood of normal humans F(ab')2, IgM, IgG, IgA, the four IgG subclasses, and the Gm markers Gm(f) and Gm(Z) were demonstrated on cells. IgM was the dominating class, and IgG2 the dominating IgG subclass. The data suggest that the Ig on any one cell belongs to a single class and IgG subclass. Very few lymphocytes stained for antigens on the C-terminal half of ihe IgG molecule. Ig-positive lymphocytes were also demonstrated in cord blood and in spleen cell suspensions. An almost total lack of Ig-positive lymphocytes was demonstrated in the blood of 8 patients with severe hypogammaglobulinemia. The Ig-positive lymphocytes are thought to be analogous to B-lymphocytes in other species.     

Contact Sensitivity in Mice Measured with Thymidine Labeled Lymphocytes

An ear assay was used to detect delayed responses in mice. Animals were sensitized and subsequently injected with ³H thyminedeoxyryribose to label antigen sensitive cells. Each animal was given a control and a test ear challenge and the respective responses were assessed by scintillation counting of ³H in cells which had accumulated at the reaction site. Delayed responses were consistently observed in animals sensitized to dinitrochlorobenzene (DNCB) but not in animals sensitized to sheep red blood cells. Reactivity to DNCB was minimal in tolerant animals and animals sensitized to the control agent. Both peritoneal exudate and spleen cells of antigen-sensitized animals were found to contain more label than cells from non-sensitized animals.     

Cell-Cycle Kinetics of Proliferating Mouse B Lymphocytes In Vitro

Resting mouse B lymphocytes were stimulated in vitro with Upopoiysaccharide, Sepharose-coupled anti-γ antibodies or a combination of the two. B lymphocytes stimulated with anti-γ entered the cell-cycle with more rapid kinetics and at a higher frequency than did the corresponding cell population stimulated with lipopolysaccharide. Using cell cycle analysis after DNA staining combined with an M phase block, the cell-cycle kinetics of in vitro cultured B-lymphocytes was studied. The labelling index of lipopoly saccharide stimulated B lymphocytes was 60% while that for anti-γ Sepharose stimulated cells was 85%. The generation time of the actively cycling population from both types of cultures was constant and was of the order of 18 h. Thus, the fraction of B lymphocytes induced to proliferate in vitro varies depending on the stimulus, while the growth kinetics of the actively proliferating populations are remarkably constant.     

PHA-Induced Activation of Suppressor Cells in Normal Human Peripheral Blood Lymphocytes

Normal human peripheral blood and tonsil lymphocytes can be stimulated to proliferate by phytohemagglutinin (PHA). When cells cultured with this mitogen for 3 days were transferred fo fresh autologous lymphocytes in fresh medium with PHA, the mitogen response of the fresh lymphocytes was suppressed. The suppression required the presence of viable cells, in that culture supernatants alone were not inhibitory and cell extracts showed only marginal inhibition. Approximately equivalent numbers of previously stimulated cells were required to produce optimal suppression of the PHA response of fresh cells. Cells irradiated after PHA stimulation were as effective as nonirradiated cells in causing suppression. PHA-stimulated cells also inhibited concanavalin A-induced proliferation and a mixed lymphocyte reaction. However, PHA-stimulated cells only partially inhibited the response to pokeweed mitogen. The suppressive effects were fully retained by a nylon-wool-enriched T-cell fraction but not by a B-cell-enriched fraction.     

Effects of Induced Anemia in Normal and Autoimmune Mice

Normal and autoimmune mice were studied with regard to signals eliciting differentiation and division of bone marrow stem cells. The erythropoiesis induced by anemia following serial bleedings was analyzed in young autoimmune New Zealand Black (NZB) mice and non-autoimmune strains. No difference in the response to the stimulus created by anemia was noted between the strains. After serial bleedings as a stimulus to stem cell proliferation, a five-fold increase in numbers of proliferating spleen cells occurred in both NZB and DBA/2 strains; the increased proliferating spleen cells in both strains were non-lymphoid. The bled animals had decreased percentages of B cells. The production of autoantibodies was not significantly altered by the experimentally induced anemia. In contrast, anti-immunoglobulin activation of resting B cells was increased in response to anemia. Young mice which had experimentally induced anemia had several characteristics in common with old autoimmune NZB mice. Both old NZB mice and young anemic animals had splenomegaly, increased numbers of proliferating spleen cells, decrease in splenic Ly 5⁺ cells and an increase in splenic colony forming units (CFUs). The anemic normal strains of animals lacked other characteristics of old NZB mice such as hyperimmunoglobulinemia or autoantibody production or elevated CD5+B cell numbers. This work supports the concept that the increase in spleen cell number, proliferating spleen cells, CFUs and the increased percentages of non-Ly-5 cells (which include erythroid precursors) found in the spleens of old NZB mice may in part result from their autoimmune hemolytic anemia.     

Qualitative and Quantitative Analysis of T Lymphocytes During Normal Human Pregnancy

PROBLEM : Human reproduction involves contact between cells which are allogeneic to one another, however the fetus not only survives but thrives. METHODS : Aspects of T-cell-mediated immunity during normal human pregnancy were studied. PBMNCs of pregnant and nonpregnant women were stimulated with PHA and cytomegalovirus antigens (CMV). The capacity of stimulated cells to proliferate, to produce IL-2 and IFN-γ, to express IL-2 receptor (IL2R1) and the effect of rIL2 on the proliferation rate of lymphocytes were examined. FACS was utilized for T-cell subset comparisons. RESULTS : The proliferation rate, IL-2, and IFN-γ synthesis were all significantly impaired at suboptimal concentration of PHA throughout pregnancy. Exogenous rIL-2 corrected this depression of cell-mediated immunity (CMI). At optimal concentration of PHA, proliferation rate and production of IFN-γ and IL-2 were all decreased. Exogenous rIL-2 corrected these deficits only in the third trimester. Third trimester pregnant women demonstrated a significant depression of proliferation as well as IL-2 and IFN-γ production after CMV stimulation, which was partially corrected by exogenous rIL-2. FACS analysis suggested that after stimulation by CMV and optimal concentration of PHA, T cells were activated and both CD4+ and CD8+ lymphoblasts expressed normal density of IL-2R1. With suboptimal PHA, the number of activated CD4+ and CD4+IL2R1+ cells were diminished and CD4+ and CD8+ T lymphoblasts expressed lower number of IL2R1. CONCLUSIONS : CD4 T helper (Thl) cell function is down regulated progressively during the three trimesters of pregnancy without changes in the quantity of T cell subsets.     

Growth of Pasteurella multocida in Vaccinated and Normal Mice

Specific pathogen-free CD-1 mice are highly susceptible to infection by Pasteurella multocida strain 5A whether introduced intravenously, intraperitoneally, subcutaneously, or aerogenically. The growth of the challenge organism in the blood, liver, spleen, lung, and peritoneal cavity was quantitated hourly for up to 12 h. Unvaccinated mice died 9 to 12 h after intravenous challenge due to the uncontrolled growth of the organism in all tissues tested. The rate of removal of the bacteria from the blood and of phagocytosis by peritoneal macrophages was extremely slow. In the absence of specific opsonins, more than 90% of the unopsonized challenge inoculum remained in the extracellular growth phase throughout the challenge period. Vaccination of mice with two doses of 10(8) heat-killed (60 C for 60 min) P. multocida given 7 days apart protected the mice against 100 to 1,000 lethal challenge doses. Survival data and growth curves obtained for both actively and passively immunized mice indicated that a humorally mediated immune mechanism was involved. Peak resistance to challenge occurred 21 to 28 days after the mice received the second dose of antigen, and this correlated with an 8- to 16-fold increase in specific agglutinin titers over the same time. Resistance to aerogenic challenge by vaccinated mice was less effective than when other routes of infection were used. The significance of these findings is discussed.     

Concanavalin-A-binding Proteins on the Surface of Human Malignant and Normal Lymphocytes

The concanavalin-A-binding cell surface glycoproteins from normal and certain leukaemic human lymphocytes were radiolabelled and then solubilized with detergent, isolated by affinity chromatography on Con A insolubilized on agarose beads, and subsequently analysed by SDS-polyacrylamide gel electrophoresis. Leukaemic T cells from patients with Sezary syndrome were found to express major concanavalin-A-binding glycoproteins on their outer surface similar to those of normal T lymphocytes. Leukaemic B cells from patients with chronic lymphocytic leukaemia expressed Con-A-binding proteins similar to those of B-cell lines. HLA antigens were predominant among the major Con-A-binding proteins on the surface of the normal and the malignant T cells studied. Human Ia-like antigens, HLA antigens, and the cell surface immunoglobulins IgD and IgM represented the major Con-A-binding proteins on the B cells studied. beta 2-microglobulin was found associated with HLA antigens on both leukaemic and non-leukaemic T and B cells. The presence of additional Con-A-binding proteins expressed on the surface of the different cell types studied is discussed along with some physical characteristics of the human Ia-like antigens isolated.