Thymus-derived Lymphocytes Research Articles

Effect of interchain disulfide bond on hapten binding properties of light chain dimer of protein 315.

The hapten binding characteristics of the covalent light chain dimer, derived from the murine IgA secreted by plasmacytoma MOPC-315, to two nitroaromatic compounds, epsilon-N-(2,4-dinitrophenyl)-L-lysine and 4-(alpha-N-alanine)-m-nitrobenz-2-oxa-1,3-diazole, were investigated by differential spectroscopic titrations. The binding curves for both haptens were found to display sigmoidity similar to that reported earlier for the reduced and alkylated dimer held together by noncovalent bonds only. However, the presence of the interchain disulfide bond in the covalent dimer was found to cause marked changes in its binding properties. The data, like those obtained for the noncovalent dimer, fit the allosteric model of Monod, Wyman, and Changeux in which binding of the first hapten to the dimer causes a conversion of both sites of the protein molecule from a lower to a higher affinity conformation. However, the binding parameters show that both the affinity and the positive cooperativity in the interaction between haptens and the covalent dimer are significantly enhanced. The differences in the parameters of the binding and of the allosteric transition caused by the presence of the interchain disulfide bond demonstrate the existence of longitudinal interactions in immunoglobulin derivatives. These properties of the light chain dimer make it a potential model for the receptors present on thymus-derived lymphocytes.

Prostaglandin E Modulation of the Mitogenic Response of Human T Cells

Prostaglandins (PG) of the E series, PGE(1) and PGE(2) (PGEs), can induce elevations of intracellular cyclic AMP (cAMP) among thymus-derived (T) lymphocytes (T cells) and inhibit their reactivity. For example, 0.1 muM of PGEs induces a two- to threefold increase of intracellular cAMP among human peripheral blood T cells and a 20-30% suppression of their blastogenic response to phytohemagglutinin. However, this suppression actually represents the net reactivity of T-cell populations demonstrating quite different responses to PGEs. Fractionation of T-enriched populations on a discontinuous density gradient yields a population of high density cells whose phytohemagglutinin-induced blastogenic response is suppressed 60%; a population of intermediate density cells whose response is suppressed 20%; and a population of low density T cells whose response is not suppressed, but is enhanced 20% by both of the PGEs. The diametrically opposite responses of low and high density T cells to the PGEs is not related to any difference in their intrinsic mitogen reactivity nor is it influenced by interactions with other T cells, bone marrow-derived (B) cells, or monocytes. Moreover, the distinct blastogenic response of low and high density T cells to PGEs does not simply correlate with PGE-mediated activation of adenylate cyclase. PGE(2) induced comparable absolute and identical relative increases of intracellular cAMP among the low and high density T cells. Cholera toxin, a potent activator of adenylate cyclase, and exogenous 8-bromo cAMP mimicked the effects of the PGEs on these two T-cell populations. These data demonstrate that T cells are heterogeneous with regard to their response to the PGEs. Thus, PGEs should be considered as potential regulators rather than as universal suppressors for T-cell reactivity. Moreover, the effect of PGEs on the blastogenic response of a given T-cell population depends upon intracellular events which occur subsequent to elevations of cAMP.

Participation of monocyte-macrophages and lymphocytes in the production of a factor that stimulates collagenase and prostaglandin release by rheumatoid synovial cells.

Cultured mononuclear cells from human peripheral blood produce a soluble factor (MCF) that stimulates collagenase and prostaglandin E2 (PGE2) release by cultured rheumatoid synovial cells up to several hundred fold. These target rheumatoid synovial cells lack conventional macrophage markers. To determine which mononuclear cells are the source of MCF, purified populations of monocyte-macrophages, thymus-derived (T) lymphocytes, and bone marrow-derived (B) lymphocytes were prepared. The monocyte-macrophages alone produced levels of MCF that were proportional to cell density but unaffected by phytohemagglutinin or pokeweed mitogen. No detectable collagenase activity was produced by the cultured monocyte-macrophages or lymphocytes. Purified T lymphocytes produced levels of MCF approximately or equal to 1--3% those of purified monocyte-macrophages in the presence or absence of the above lectins. Purified T lymphocytes modulated the production of MCF by the monocyte-macrophages, however, in a manner dependent upon relative cell densities and the presence of lectins. For example, at optimal ratios of T lymphocytes: monocyte-macrophages, MCF production was markedly stimulated by pokeweed mitogen. Thus, interactions of T lymphocytes and monocyte-macrophages could be important in determining levels of MCF, which regulate collagenase and PGE2 production by target synovial cells in inflammatory arthritis.

Resistance of C58 mice to primary systemic herpes simplex virus infection: macrophage dependence and T-cell independence

The relative contribution of thymus-derived lymphocytes (T-cells) and of macrophages to resistance to primary infection with herpes simplex virus type 1 (HSV-1) was studied in C58 mice. Resistance was dependent on macrophage competence, but was relatively independent of T-lymphocyte competence. Although aging mice became progressively more deficient in functional T-cells, as demonstrated by a decreasing resistance to transplanted line Ib leukemia and by declining responses to T-cell nitogens (concanavalin A and phytohemagglutinin), their resistance to HSV-1 increased with increasing age. Moreover, in mice that were made selectively deficient in T-cells by the combination of adult thymectomy and treatment with anti-thymocyte serum, resistance to HSV-1 did not correlate spleen and mesenteric lymph nodes. However, selective reduction of macrophages by intraperitoneal injection of silica resulted in enhanced susceptibility to HSV-1. Furthermore, in vitro suppression of HSV-1 plaque formation in mouse embryo fibroblast cells was obtained by cocultivation of infected fibroblast monolayers with peritoneal macrophages, but not with splenic lymphocytes, from adult mice. Macrophages from weanling mice failed to suppress the development of plaques, indicating that the increase in resistance to HSV-1 with age is a result of increased macrophage competence.

A Monoclonal Antibody to Viral Glycoprotein Blocks Virus-Immune Effector T Cells Operating at H-2Dd but Not at H-2Kd

Summary A particular monoclonal antibody that binds to the influenza virus HA molecule inhibits HA-specific thymus-derived lymphocytes mediating cytotoxicity in the context of H-2Dd but not of H-2Kd. Another monoclonal antibody blocks both sets of HA-specific effector T cells. This observation, together with related findings from other laboratories, is considered to support the idea that T cell recognition is directed against some association of viral and H-2 glycoproteins, as proposed in the original formulation of the “altered self” concept.

Human lymphocyte antigens: production of a monoclonal antibody that defines functional thymus-derived lymphocyte subsets.

A monoclonal mouse antibody (3A1) that specifically bound to 65% of human peripheral blood (PB) thymus-derived (T) cells but did not bind to complement receptor-positive PB bone marrow-derived (B) cells, polymorphonuclear leukocytes, or human erythrocytes has been produced. The 3AI antibody was synthesized by a stable cloned lymphocyte hybrid cell line. This lymphocyte hybrid line (3AI) was derived from fusion of P3 X 63/Ag8 myeloma cells and spleen cells from BALB/c mice immunized with HSB-2 cells, a human T cell line. The 3A1 lymphocyte hybrid line produced mouse ascites fluid containing 3A1 antibody in saturating titers of up to 1:25,600. Purified PB T cells that carried the 3A1 antigen incorporated tritiated thymidine maximally in response to phytohemagglutinin and concanavalin A stimulation, whereas purified PB T cells that lacked the 3A1 antigen responded suboptimally to phytohemagglutinin and minimally to concanavalin A. Thus, the 3A1 antibody can be easily used to study the role of 3A1-positive and negative T cell subsets in the regulation of normal and abnormal human immune responses.

Studies on the Induction and Expression of T Cell-Mediated Immunity

Abstract Secondary cytotoxic thymus-derived lymphocytes (CTL) with specificity for the priming alloantigen are shown to be generated by in vitro stimulation of allosensitized memory splenocytes with NAGO-oxidized syngeneic, allogeneic, or xenogeneic cells. The cell surface aldehyde resulting from NAGO modification of stimulator cells is required for triggering both lymphocyte proliferation and secondary (2°) CTL activation, since treatment with the reducing agent, KBH4, completely inhibited these responses. Compared to alloantigen stimulation, NAGO-oxidized stimulator cells induced an earlier peak of proliferation (2 to 3 vs 4 to 5 days), whereas the peak of the cytotoxic activity was identical (days 3 to 4). Syngeneic splenocytes, peripheral T cells, plastic and nylon nonadherent cells, and the P388D1 macrophagelike cell line were all efficient stimulators after NAGO modification. Stimulation by syngeneic cells was shown to be mitomycin C resistant, U.V. irradiation sensitive, and ineffective when heat killed cells are used. Therefore, NAGO stimulation is distinct from antigen specific activation whereby U.V. treated cells, heated cells and nonviable membrane fragments have been shown to differentiate memory cells into 2° CTL. In addition, neither NAGO-treated sheep red blood cells nor syngeneic thymocytes were found to be effective stimulators. The addition of specific anti-Ia serum to the culture fluid inhibited both the proliferative and cytotoxic response, suggesting a role for an Ia signal during NAGO stimulation. These results show that the signal(s) required for the activation of 2° CTL from memory populations can be provided by certain subpopulations of NAGO-modified cells and is also dependent on the presence of Ia or an Ia-like molecule.

Surface markers and size of lymphocytes in human umbilical cord blood stimulated into deoxyribonucleic acid synthesis by Epstein-Barr Virus

We characterized subpopulations of lymphocytes in human umbilical cord blood which are stimulated into deoxyribonucleic acid synthesis by Epstein-Barr virus. Lymphocytes were examined simultaneously for deoxyribonucleic acid synthesis by autoradiography and for surface markers by rosette formation with sheep erythrocytes or erythrocytes coated with antibody and mouse complement (EAC). The subpopulation which incorporated [3H]thymidine after exposure to virus consisted mainly of cells which formed rosettes with EAC. Lymphocytes were enriched or depleted of thymus-derived lymphocytes (T cells), null cells, or cells forming rosettes with EAC. The extent of sensitivity of the cells to stimulation by Epstein-Barr virus correlated with the proportion of the population which formed rosettes with EAC. When mononuclear cell populations were depleted of T lymphocytes and then fractionated by size, small lymphocytes showed higher rates of deoxyribonucleic acid synthesis after virus exposure and higher transformation frequency than did larger cells or unfractionated cells. Thus, the cells which are stimulated into deoxyribonucleic acid synthesis by Epstein-Barr virus appear to be the same as cells which are ultimately transformed.

Thymic reconstitution of nude F1 mice with one or both parental thymus grafts

Thymus-derived lymphocytes (T cells) have two outstanding characteristics that distinguish them from other lymphocytes: (a) they express two specificities, one for self-antigens, the major transplantation antigens (H) coded by the major histocompatibility gene complex (MHC), and a second specificity for foreign antigenic determinants. (b) T cells must undergo differentiation or maturation in the thymus (1, 2). Apparently, an important step in T-cell differentiation in the thymus is the selection of T-cells' restriction specificity for self-H. This interpretation stems from experiments with chimeras formed by lethally irradiating parental type mice and reconstituting them with F(1) stem cells: the maturing F(1) T cells expressed predominantly the restriction specificities for the recipient parental MHC type (3-8). Alternatively, adult F(1) mice that were thymectomized, lethally irradiated, reconstituted with bone marrow, and then engrafted with a parental thymus had T cells that were restricted predominantly to the thymus donors' H-2 (4-8). The present study first extends these observations to nude mice that are born without a thymus and therefore do not develop functional T cells and second, attempts to study the possibility that suppression may be responsible for the apparent influence of the radioresistant portion of the thymus on T- cell restriction specificities. We tested the immunocompetence and restriction specificities expressed by lymphocytes from F(1) nude mice reconstituted with both parental thymus grafts; our expectation was that suppression of the expression of T-cell restriction specificity should result either in complete immunoincompetence or emergence of only one of the two possible sets of restriction specificities. Nude F(1)mice that simultaneously received thymus gratis from both parents developed spleen cells restricted to both parental H-2 types. These results are compatible with the idea that the thymus' influence on T- cell restriction is via positive selection rather than by suppression.

Effect of Cigarette Smoke Exposure on Maturation of the Antibody Response in Spleens of Newborn Mice

Balb/c mice were exposed to smoke from Kentucky Reference IRI cigarettes within 24 hr after birth on a Prototype Mark II Walton Horizontal Smoke Exposure Machine for various periods of time up to 10 wk of age. There was no difference in the magnitude of the splenic plaque-forming cells (PFC) response in smoke-exposed and untreated control animals immunized with sheep red blood cells (SRBC) on sequential days up to day 9 postpartum. However, beginning at day 10 and for each subsequent time period assayed thereafter, there was a statistically significant reduction in the number of splenic PFC observed in the smoke-exposed animals. On day 10, the PFC response of smoke-exposed mice was reduced by 33%, on day 14, there was a 60% reduction, whereas animals exposed to smoke from 4-10 wk showed a 90% reduction of the splenic PFC response.

Bovine dialyzable lymph node extracts have antigen-dependent and antigen-independent effects on human cell-mediated immunity in vitro

We have studied the effects of dialyzable lymph node extracts (DLNE), from bovines immune or nonresponsive to tuberculin, or human cell-mediated immunity to purified protein derivative of tuberculin (PPD) in vitro. The effects of bovine DLNE (B-DLNE) on the immune responsiveness of human cells were evaluated using both the direct and indirect agarose leukocyte migration inhibition (LMI) assays to measure the migration of human leukocytes, and by determining the effects of B-DLNE on the rate of regeneration of receptors for sheep red blood cells (SRBC) on human thymus-derived (T) lymphocytes after trypsinization. The effects of human dialyzable leukocyte extracts (H-DLE) were also evaluated in both systems, as controls. The results indicated that both B-DLNE and H-DLE contain a “transfer factor-like” activity which can be detected in vitro with the agarose LMI assay. Both B-DLNE and H-DLE contained components which promoted measurable specific antigen responsiveness in previously nonimmune lymphocytes; specificity was shown by analyses of the migration of human leukocytes in the presence of PPD, coccidioidin, or candida antigen in the presence and absence of H-DLE or B-DLNE from donors specifically responsive or nonresponsive to the various antigens. The components promoting specific antigen responsiveness were dialyzable and were derived only from lymphoid tissues. Dialysates from brain, liver, or skin failed to promote similar effects. When the amount of B-DLNE tested was less than that found to be optimal for promotion of antigen-specific responsiveness, pronounced enhancement of migration of human polymorphonuclear neutrophils (PMN) was seen, similar to the results obtained previously with H-DLE. Both B-DLNE and H-DLE significantly increased the rate of SRBC receptor generation on human T cells (up to a threefold increase at optimum concentrations). This activity, termed “mononuclear leukocyte-derived maturation factor” (LDMF), was found preferentially in lysates of lymphoid or reticuloendothelial organs and cells.

Loss of reactivity of a myeloma tumor with H-2 compatible thymus-derived lymphocytes

It was previously shown that MOPC-315-EL, a subline of the BALB/c myeloma tumor MOPC-315, reversibly alters its reactivity with T cells that recognize H-2 d, 2,4,6-trinitrophenyl, and minor histocompatibility antigens. This report demonstrates (a) that CTLs directed against vesicular stomatitis virus and Sendai virus are unable to recognize viral antigens associated with the unreactive tumor cell, and (b) that incorporation of Sendai virus antigens into the same membrane as the H-2 gene products is required for effective recognition by cytotoxic T lymphocytes.

Role of protein and RNA synthesis in the development of insulin binding sites on activated thymus-derived lymphocytes.

Quiescent T (thymus-derived) lymphocytes have no insulin receptors. Lymphocyte activation by alloantigen or mitogens stimulates receptor appearance. There are two theories that may explain the mechanism by which cellular activation leads to newly measurable receptors: 1) activation uncovers nascent receptors by alteration of membrane configuration or 2) activation initiates synthesis of the receptor or substances crucial to the integrity of the receptor. This study was designed to test the latter theory directly. T cells were activated with the mitogen phytohemagglutinin (PM) and treated with either mitomycin C (100 pg/ml), cycloheximide (0.5 mu), or actinomycin D (1 PM). Controls consisted of cells cultured with mitogen alone or with medium alone. Inhibitors reduced DNA synthesis 83% (r3H]thymidine), RNA synthesis 90% (r3H]uridine), and protein synthesis 88% (13H]leucine) in separate series of studies. Insulin binding after PHA interaction was 4.0 f 0.6 pg/106 in control and 3.93 + 0.1 in mitomycintreated cultures. In contrast, binding after PHA activation (75 gg/ml) fell from 6.3 + 1.3 to 0.4 + 0.2 pg/106 cells after RNA synthesis inhibition. Inhibition of DNAdependent, RNA synthesis by 96%, as measured by assay of RNA polymerase II, by cx-amanitin (1

Allogeneic cytolysis of reconstituted membrane vesicles.

The successful use of lipid bilayer model membranes as targets for cytotoxic lymphocytes is described. Lipid vesicles were made from a mixture of dipalmitoyl lecithin, dimyristoyl lecithin, and cholesterol. Membrane proteins of LSTRA or EL4 tumor cells (as source of H-2 antigens), human eye muscle membrane proteins (as supporting proteins), and 51Cr marker were inserted into the lipid vesicles. Incubation of the reconstituted vesicles with lymphocytes sensitized in mixed lymphocyte cultures against allogeneic cells resulted in the specific release of intravesicular 51Cr. Vesicle damage was mediated by thymus-derived lymphocytes. H-2 antigens could be incorporated into vesicles without eye muscle proteins. However, immune damage of the vesicles could not be demonstrated when vesicles inserted with H-2 antigens in the absence of eye muscle proteins were used as targets.

43 ANTIBODY TREATMENT OF MARROW GRAFT IN VITRO: A PRINZIPLE FOR PREVENTION OF GvH DISEASE

Graft-versus-Host disease (GvHD) is still a frequent complication in clinical marrow transplantation. Advances in immunology have delineated the caual role of thymus-derived (T) lymphocytes in GvH reactions. Attempts have been made to reduce T-cells by treating the bone marrow itself in vitro after preparation. Experimental studies of our group showed hat in mice an in vitro treatment of incompatible sonar cells with T-cell specific antibodies before transplantation could supress an otherwise lethal GvH-reaction completely. The GvH-reactive T-lymphocytes were removed by a specific xenogenic antiserum against T-cells which had been purified from antibodies cross reacting with hemopoietic stem cells by an extensive absorption procedure. The present report will summarize the application of this principle to clinical bone marrow transplantation: A case of a 11 year old girl with a second elapse of common acute lymphoblastic leukemia was successfully transplanted after marrow incubation with anti-T-cell globulin.

T-Cell Subpopulations in Chronic Lymphocytic Leukemia: Abnormalities in Distribution and in In Vitro Receptor Maturation

Purified human thymus-derived (T) lymphocytes were analyzed by detection of Fc receptors for either IgG or IgM in healthy controls and in patients with chronic lymphocytic leukemia (CLL). There was a significant and persistent increase in the numbers of T cells bearing receptors for IgG (Fcγ) in CLL patients in comparison to the controls. After an in vitro culture period, there was a significantly decreased appearance of cells with IgM receptors (Fcμ) in CLL patients in comparison to the control group. These results indicate an imbalance in circulating T-cell subpopulations for CLL patients. In addition, an in vitro defect in CLL T-cell membrane receptor appearance is present.

T-cell subpopulations in chronic lymphocytic leukemia: abnormalities in distribtuion and in in vitro receptor maturation

Purified human thymus-derived (T) lymphocytes were analyzed by detection of Fc receptors for either IgG or IgM in healthy controls and in patients with chronic lymphocytic leukemia (CLL). There was a significant and persistent increase in the numbers of T cells bearing receptors for IgG (Fc gamma) in CLL patients in comparison to the controls. After an in vitro culture period, there was a significantly decreased appearance of cells with IgM receptors (Fcmu) in CLL patients in comparison to the control group. These results indicate an imbalance in circulating T-cell subpopulations for CLL patients. In addition, an in vitro defect in CLL T-cell membrane receptor appearance is present.

Thy-1 antigen on astrocytes in long-term cultures of rat central nervous system.

THE THY-1 ANTIGEN has been exploited by immunologists as a marker for thymus-derived lymphocytes in mice1. Since its first description by Reif and Allen, it has been known to be present in equivalent amounts in brain and thymus2. However, in contrast to thymus, where adult levels of Thy-1 are present at birth, Thy-1 in mouse and rat brain increases with physiological maturation of the brain from 1–2% at birth to 100% of the adult level by 3–6 weeks of age2–5. It is widely assumed that neurones are the principal cell type which express Thy-1 and by implication that the increase in brain Thy-1 during neonatal development is related to increases in neuronal membrane as a result of dendritic arborisation5. I describe here the use of monoclonal anti-Thy-1 antiserum in combination with other recently defined neural cell markers6 to study the distribution of Thy-1 on cells grown in long-term cultures of rat central nervous system. I have found that Thy-1 appears on astrocytes as well as neurones and fibroblasts but not on oligodendrocytes, and that the rate of appearance of Thy-1 on cultured astrocytes parallels that of Thy-1 in brain in vivo. This is the first report to identify unambiguously Thy-1 on astrocytes. It settles a controversial point about the Thy-1 distribution on neural cells and clearly demonstrates that previous implications (see discussion) that neurones are the major Thy-1-positive cells in the brain is incorrect.

Studies on the induction and expression of T cell-mediated immunity: VIII. Effector-target junctions and target cell membrane disruption during cytolysis

Target cell destruction following contact of the target cell by specific alloimmune cytotoxic thymus-derived lymphocytes (CTL) has been examined by time-lapse film (TLF), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Effector-target conjugates of murine CTL with leukemia cells were prepared for use in these studies. TLF shows that contact of the two cells results in tumor cell zeosis involving violent membrane blebbing, and subsequent tumor cell death. TEM of the contact region shows that the CTL-tumor cell junction is extremely adherent. Examination of conjugates incubated at 37 °C to permit tumor cell lysis shows tumor cell membrane stretching and rupture, and tumor cell membrane fragments adhering to CTL. Close examination of the contact region has revealed electron-lucent junctions spanning the gap between the two cell membranes, but no packaging or secretory apparatus was prominent. The results are consistent with the mechanism of cell-mediated cytolysis being a membrane phenomenon involving junctions connecting the CTL and target cell and the initial target cell lesion observable as a stretching and rupture. The shear force of vigorous cell movements is most likely responsible for this target membrane tearing, creating a target cell lesion which results in loss of osmotic integrity and cell death.

Thymus-derived lymphocytes control the expression of immunogenic properties of peritoneal macrophages.

Macrophages, given keyhole limpet hemocyanin in vitro, from normal mice initiate in culture antigen-specific T cell-mediated immune reactions. On the other hand, macrophages from nude, from adult-thymectomized, or from neonatal-thymectomized mice are impaired with respect to their capacity to signal such an antigen-specific T cell reaction. Thymocytes from hydrocortisone-treated donors, added in culture to such impaired macrophages, rendered them immunologically potent. The capacity of macrophages from adult thymectomized mice to promote the activation of antigen-specific "helper" T cells which, cooperating with B lymphocytes, would lead to antibody production, was also impaired. Thus, it appears that short-lived T lymphocytes control the maturation of macrophages up to a stage at which they can present antigen-specific T cells with antigen in an immunogenic form. We found that such T lymphocytes also control the phagocytic properties of macrophages, yet the impairment of their immunogenic properties does not seem to be derived from decreased phagocytosis.