Studies on in vitro models of cellular immunity: the role of T and B cells in the secretion of lymphotoxin

When normal murine spleen cells were treated with anti-theta serum and complement, they failed to produce LT or synthesize cellular DNA when stimulated in vitro with PHA. Theta-positive cells were responsible for LT production in spleens removed from X-irradiated and bone marrow- or thymus-reconstituted animals. Finally, spleens from congenitally athymic Nu/Nu mice failed to produce LT when stimulated with pokeweed mitogen or phytohemagglutinin.     

Production of thymocyte-stimulating factor by murine spleen lymphocytes: cellular and biochemical mechanisms.

Cultures of murine spleen lymphocytes treated with Thy 1.2 antiserum plus complement do not produce thymocyte-stimulating factor (TSF). The population of thymocytes composed of immunocompetent, low-density cells produces only small amounts of TSF. Experiments with cyclophosphamide-injected mice and with spleen cells treated in vitro with antiserum to the murine B lymphocyte antigen plus complement and experiments using spleen cells stimulated in vitro with Sepharose-bound phytohemagglutinin indicate that B lymphocytes neither cooperate with T lymphocytes for the production of TSF nor produce TSF. Some lectins (pokeweed mitogen, Lens culinaris hemagglutinins A and B) have been found to induce the production of TSF by spleen cells. Other lectins (wheat germ agglutinin, Agaricus bisporus agglutinin) and sodium periodate do not. Spleen cells of mice immunized in vivo with keyhole limpet hemocyanin bound to bentonite particles or with BCG produce TSF when challenged in vitro with the specific antigen. Experiments using inhibitors of the macromolecular metabolism showed that DNA synthesis is not required for the production of TSF by spleen lymphocytes, whereas RNA and protein synthesis are required. Resolution of spleen lymphocytes on a discontinuous albumin gradient into six subpopulations showed that the TSF activity was rather uniformly distributed among the various subpopulations of cells.     

Leishmania tropica: Association of a B-cell mitogen with hypergammaglobulinemia in mice

Infection of BALB/c mice with Leishmania tropica NIH S strain resulted in splenic enlargement, hypergammaglobulinemia, and polyclonal activation of B lymphocytes as measured by the splenic plaque-forming cell response (PFC) to trinitrophenyl (TNP) and sheep erythrocytes (SRBC). The peak anti-SRBC PFC response occurred 5 weeks after infection; both direct and indirect (facilitated) plaques were significantly increased. The in vitro primary immune response to trinitrophenyl haptenated lipopolysaccharide (TNP-LPS), as enumerated by the anti-TNP PFC response, was also increased on a per-spleen basis beginning 3 weeks after infection. The properties of a lysate of L. tropica promastigotes (LTL) was studied to determine whether polyclonal B-cell activation was related to a parasite-derived mitogen. A B-cell mitogen was identified in LTL which stimulated the proliferation of spleen cells in vitro from uninfected control and congenitally athymic (T-cell-deficient) but not from μ-suppressed (B-cell-deficient) animals. Preliminary characterization of the mitogen material indicated that it was a nonpyrogenic, heat-labile peptide or protein and was probably not bacterial lipopolysaccharide (LPS).     

Subpopulations of human T lymphocytes. VII. Cellular basis of concanavalin A-induced T cell-mediated suppression of immunoglobulin production by B lymphocytes from normal humans.

Purified peripheral blood T lymphocytes from normal subjects were pretreated with varying concentrations of concanavalin A (Con A) for a period of 18 or 48 hr. Following treatment, these T lymphocytes were examined for the proportions of Tμ and Tγ cells and their regulatory effect on immunoglobulin production by normal allogeneic B cells in presence of pokeweed mitogen. A significant decrease in the proportions of Tμ cells and increase in Tγ cells were observed when concentration of Con A, 40 μg/ml, was used to treat purified T cells for either 18 or 48 hr. Significant suppression of in vitro immunoglobulin synthesis was observed at a similar concentration in mixing experiments. The mechanism of Con A-induced T cell-mediated suppression of immunoglobulin secretion by allogeneic B cells is discussed.     

Lymphocyte transformation in casein-induced murine amyloidosis.

Spleen lymphocytes from casein-induced amyloidotic mice demonstrate diminished transformation in vitro to PHA-P, concanavalin A, and pokeweed mitogen but a normal response to lipopolysaccharide. Thymic and peripheral blood lymphocytes respond normally to these mitogens. The amyloidogen casein acted as a mitogen in both normal and casein injected mice. The diminished PHA responsiveness of spleen lymphocytes in vitro could have resulted from an inhibitory effect of amyloid fibrils on lymphocyte proliferation and did not indicate a generalized diminished cellular immunologic responsiveness of amyloidotic mice.     

The in vitro effect of a thymic epithelial culture supernatant on mixed lymphocyte reactivity and intracellular cAMP levels of thymocytes and on antibody production to SRBC by Nu/Nu spleen cells.

Addition of supernatants from rat thymic epithelial cultures (TES) to rat thymocytes stimulated with T-cell-mitogens or allogeneic cells leads to an increase in ¹⁴C-TdR incorporation. Furthermore, in the presence of TES, spleen cells from athymic nu/nu mice exhibit an enhanced in vitro antibody production to SRBC, whereas TES has no such effect on spleen cells from T-cell-deprived mice. If TES is added together with thymocytes to T-cell-deprived spleen cell cultures, the number of plaque-forming cells to SRBC is enhanced, suggesting that TES induces a helper cell function in thymocytes which, if added alone, have no effect. TES also increases intracellular levels of cAMP in thymocytes in vitro and appears to act on a membrane site distinct from the β-adrenergic receptor. TES fails to affect mitogen responses, MLR and cAMP levels of lymphocytes from other lymphoid organs. The biological activity of TES as compared to that of thymic extracts is discussed.     

Scanning electron microscopy of homing and recirculating lymphocyte populations

The surface structure of T and B lymphocytes in vivo was investigated using scanning electron microscopy. For these studies the spleen and mesenteric lymph node of mice enriched for B lymphocytes (adult thymectomized, lethally irradiated, bone marrow reconstituted mice, B mice) and of mice enriched for T lymphocytes (adult, lethally irradiated, thymocyte transferred mice, T mice) were examined. Both types of lymphocytes demonstrated a smooth cell surface when they were situated in their respective microenvironment, whereas recirculating T and B cells exhibited numerous microvilli on the cell surface. In postcapillary venules, known to be the major sites of entry of lymphocytes in lymph nodes, lymphocytes were in contact with the endothelial wall by means of these microvilli. While passing the endothelial lining, lymphocytes withdrew their microvilli and appeared smooth upon arrival in the lymphatic stroma. It is suggested that microvilli on the surface of lymphocytes play a role in cellular recognition mechanisms.     

Subpopulations of splenic T and B lymphocytes producing and regulating leukocyte adherence inhibition factor

Picryl chloride induces contact hypersensitivity in mice, accompanied by spleen cell sensitization that is demonstrable in vitro by specific antigen-induced formation of leukocyte adherence inhibition factor (LAIF). This cellular activity was detected only up to 7 days after sensitization; thereafter the spleen cells appeared to be unreactive with the antigen. The cells were still normally reactive with the mitogen concanavalin A. Antigen reactivity of such “late” cells was restored by passage through a glass-bead column (provided resulting nonadherent cells were reconstituted with normal macrophages), and the restored reactivity was again suppressed by the eluted glass-bead-adherent cells. Suppression was antigen specific. Separation of T and B lymphocytes by affinity chromatography, after glass-bead treatment of sensitized spleen cells, showed that two subpopulations of B cells—those responsible for producing LAIF as well as those suppressing LAIF production by T cells—were glassbead adherent. This was extended by showing directly with anti-Thy-1.2 serum that B cells producing LAIF and suppressor T cells were glass adherent. Thus two suppressive cell populations, and the B cell producing LAIF, were glass adherent while the T-cell LAIF producer was not. Tests for adoptive transfer of cutaneous hypersensitivity in vivo demonstrated the relevance of many of the above observations to conditions in the whole animal. “Late” spleen cells from sensitized mice could not transfer hypersensitivity but this property was restored by glass-bead passage. The eluted adherent cells suppressed transfer. Both adoptive transfer and its suppression were antigen specific.     

Immunoadjuvant activity of pyran copolymer: I. Evidence for direct stimulation of T-lymphocytes and macrophages

A single injection of pyran copolymer has been shown to greatly increase the number of hemolytic plaque forming cells to sheep erythrocytes (sRBC). Pyran given from 1 day before to 2 days after sRBC inoculation increased both specific activity and plaques/spleen, suggesting that macrophage activation was probably not responsible for the enhancement seen. In addition, pyran given 1 day prior to the primary injection of sRBC was found to increase the secondary response to SRBC given alone. As similar experiments using thymectomized irradiated bone marrow reconstituted mice showed no increase in specific activity following pyran administration, it was unlikely that pyran was acting directly on B cells. Furthermore, experiments measuring the antibody response to Escherichia coli lipopolysaccharide, a thymic independent antigen, pyran did not increase the response to this antigen. In contrast to the above, pyran delayed and depressed cell mediated cytotoxicity to the allogeneic DBA/2 P815 mastocytoma. However, no difference in the titers of cytotoxic antibody against mastocytoma cells was seen between pyran-treated and normal animals. Pyran was mitogenic for spleen cells in vitro. However, following the administration of pyran in vivo, mitogen induced blastogenesis in vitro to PHA and LPS was inhibited and this inhibition was determined to be macrophage-dependent. These results are consistent with a model in which the immunoregulatory effects of pyran act through macrophages and T-lymphocytes.     

Thymectomized, irradiated, and bone marrow-reconstituted chimeras have normal cytolytic T lymphocyte precursors but a defect in lymphokine production

A model system has been developed to study extrathymic T cell differentiation; mice have been thymectomized, lethally irradiated, and reconstituted with bone marrow cells depleted of Thy-1+ cells. After 8 wk, the spleen cells of these athymic, bone marrow-reconstituted chimeras contain Thy-1+ precytolytic T lymphocytes (CTL) that are able to respond to antigen only if supernatant from Con A-activated T cells is added to culture. The phenotype of these pre-CTL is similar to that of thymocytes, suggesting that they may be immature T cells. Initial evaluation of the CTL repertoire of these athymic mice demonstrated that the CTL generated to trinitrophenyl-modified syngeneic cells are H-2-restricted, and that the CTL generated to alloantigens have many of the cross-reactivities observed in normal mice but not in nude mice. In this report, we demonstrate a helper T cell defect in these thymectomized chimeras. These chimeras lack an Ly-1+ helper cell required for thymocytes to differentiate to CTL. Further studies revealed that when spleen cells from these thymectomized chimeras were stimulated with Con A, they produced normal levels of interleukin 2. However, these splenocytes were defective in the production of another factor needed for CTL differentiation.     

A reappraisal of the effector cells mediating mitogen induced cellular cytotoxicity.

The ability of mitogens to induce cytotoxic effector reactions in vitro has been studied to investigate basic mechanisms of cell mediated cytotoxicity. The type of mitogen, the source of effector cells, and the nature of the target cell are all critical variables in determining the characteristics of the cytotoxic event in this system. Spleen cells and bone marrow cells from congenitally athymic nude mice as well as from their heterozygous control littermates were capable of mediating lysis of RBC targets in the presence of either PHA or Con A. Removal of macrophages from these effector populations by adherence columns, density gradient centrifugation, and carrageenan treatment failed to abrogate this cytotoxic capacity. However, purified macrophages themselves also were capable of mediating mitogen induced killing of RBC targets, although the kinetics of this cytotoxicity were substantially different from that induced by lymphocytes. In contrast to these observations, the capacity of mitogen stimulated cells to kill metabolically active complex targets like the P815 mastocytoma or cultured L cells appears to be exclusively a T lymphocyte dependent function. In addition, blastogenic transformation of the effector cells with the T cell mitogens PHA and Con A, but not with the B cell mitogen LPS, leads to enhanced killing of these complex targets. These data suggest that mitogen or lectin induced cellular cytotoxicity can detect at least three different active effector cell types (B cells, T cells, and macrophages) acting via at least four different mechanisms.     

20alpha-hydroxysteroid dehydrogenase: a T lymphocyte-associated enzyme.

20alpha-Hydroxysteroid dehydrogenase (20alpha-SDH), an enzyme which reduces progesterone to 20alpha-dihydroprogesterone, was found to be associated with T lymphocytes. 20alphaSDH activity was present in spleen cells bearing theta antigen, spleen cells nonadherent to nylon wool (T lymphocyte-enriched population), and in thymocytes. Almost no enzymatic activity was found in bone marrow cells from normal mice and in spleen cells from neonatally thymectomized or athymic nude mice. T cell mitogens (PHA and Con A), but not the B cell mitogen LPS, induced high levels of enzymatic activity 48 hr after addition to spleen cell cultures. The level of 20alphaSDH activity in lymphocytes was age dependent. At the age of 4 weeks 20alphaSDH activity in thymocytes, spleen cells, and lymph node lymphocytes was 3 to 5 times higher than at 8 and 16 weeks. Progesterone (5.0 X 10(-7) M) was found to inhibit thymocyte proliferation after exposure to mitogens, but not 20alpha-dihydroprogesterone (10(-6) M). 20alpha SDH may protect the embryonic thymocytes against high concentrations of progesterone.     

Selective T cell killing of human lymphocytes by ultraviolet radiation

The effects of ultraviolet radiation (uv) on human B and T lymphocytes were studied. In vitro studies showed that T lymphocytes were more sensitive to uv than B lymphocytes as assessed by eosin-dye exclusion. Following uv exposure, the viable lymphocytes responded to mitogens (PHA, PWM), and functional B lymphocytes were present at a time when no viable T cells were detected. Varying doses of uv were required to abrogate different in vitro responses (proliferative response to antigen or allogeneic cells, MIF production, and cell-mediated lympholysis). In vivo, uv was able to diminish an established cutaneous delayed hypersensitivity response. In vitro uv treatment of parental mouse spleen cells eliminated a graft-versus-host reaction in F₁ recipients as determined by the spleen index. The basis for the differential effect of uv on B and T lymphocyte viability and functional responses is unknown.     

Role of self carriers in the immune response and tolerance. III. B cell tolerance induced by hapten-modified-self involves both active T cell-mediated suppression and direct blockade.

The induction of B cell unresponsiveness with hapten-modified syngeneic murine lymphoid cells (hapten-modified self, HMS) can be achieved in vivo and in vitro. Tolerance in vivo in mice required a latent period of 3 to 4 days. Moreover, B cell unresponsiveness could not be induced by HMS in athymic nude mice, although their nu/+ littermates were rendered hyporesponsive by HMS. Pretreatment of normal mice with cyclophosphamide (cyclo) prevented their susceptibility to tolerance induction by haptenated lymphoid cells. Nude mice became sensitive to HMS-induced suppression if they were first reconstituted with spleen cells from normal (but not cyclo-treated) donors.Interestingly, labeling of H-2 antigens was not necessary for tolerance induction by HMS since haptenated teratoma cells (lacking H-2) were tolerogenic in normal recipients.In contrast, suppression of the in vitro response to haptenated flagellin occurred equally well with nude, nu/+ and anti-Ly 2 + C-treated spleen cells. These data suggest that cyclo-sensitive modified self-reactive (T) cells may regulate the immune response and mediate tolerance to HMS in vivo. However, the in vitro “blockade” of B cell reactivity may be directly mediated on hapten-specific PFC precursors.     

Lymphocyte membrane receptors in cultures treated with mitogens

Lymphocyte membrane receptors for sheep erythrocytes (E) and human erythrocytes sensitized with antibody and complement (HEAC) were used as markers for human T and B cells, respectively. Combining the method of rosette formation with E and HEAC with radioautography, we have studied the effect of in vitro stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), staphylococcal filtrate (SF) and mercuric chloride (HgCl₂) on the proportion of small lymphocytes and blasts presenting these receptors. After mitogenic stimulation, small lymphocytes as well as blasts were found forming rosettes with E or HEAC, in variable proportions. PHA, Con A, SF and HgCl₂ showed a similar effect in vitro since most of the blasts obtained after stimulation had receptors for E and a smaller proportion for HEAC.The stimulation with PWM led to a blast population made up of a higher percentage of HEAC than E rosette-forming cells.     

Induction of interferon production in mouse spleen cell cultures by corynebacterium parvum.

Spleen cells of CS7BL/6 mice produced considerable amounts of interferon (IF) in vitro when tested 5 to 20 days after injection of killed Corynebacterium parvum. Interferon was also produced when C. parvum was added in vitro to spleen cell cultures of previously untreated mice. High levels were detected after 1 day of culture with some increment during subsequent days. In a number of experiments IF was also produced in untreated control cultures but only after prolonged cultivation and not after 1 day. The highest levels of IF were usually obtained when spleen cells of C. parvum-treated mice were challenged with additional C. parvum in vitro. The IF induced by C. parvum shared certain physicochemical properties with a tested immune IF and was not neutralized by an antiserum raised against a type I IF. Spleen cells of nu/nu mice and spleen cells treated by anti-θ serum plus complement did not differ from their respective controls, indicating that production of IF did not require mature T lymphocytes. Removal of B lymphocytes by nylon wool columns abolished the capacity of spleen cells to produce IF. When spleen cells were freed of adherent cells by the use of plastic surfaces, they no longer produced IF. Peritoneal exudate macrophages (PEC), which by themselves did not produce IF, in small numbers reconstituted nonadherent spleen cells. Nylon column-treated spleen cells, however, could not be restored by PEC. It is concluded that IF upon challenge with C. parvum is produced by B lymphocytes and requires the help of macrophages.     

Cooperation of nonsyngeneic tolerant lymphocytes: genetic restriction.

We have used a previously devised in vivo experimental model to investigate the ability of mouse thymus-dependent (T) and bone marrow-derived (B) lymphocytes to cooperate in immune (humoral) rejection of rat Yoshida ascites sarcoma (YAS). Because of conflicting reports in the literature concerning the effectiveness of T-B cooperation across major histocompatibility complex (MHC) barriers, we explored the interaction of T and B lymphocytes from mutually tolerant animals. Tolerance was achieved by establishing radiation chimeras of B6 → B6D2F₁ and D2 → B6D2F₁ constitutions. Chimeras' erythrocytes and spleen cells were shown by serological analysis to be the donor type. When the chimera was to serve as the tumor host or B-cell source, it was thymectomized prior to irradiation and reconstitution (TIR). Tolerance was evaluated by noting the inability of chimeric spleen cells to effect graft-versus-host damage upon injection into TIR host-type mice and the markedly reduced anti-host-type reactivity in short-term [³H]thymidine-uptake tests. Successful cooperation, manifested by YAS rejection, was seen whenever donor T and host B lymphocytes were syngeneic. Parental (P) T cells enabled F₁ TIR mice to reject YAS, but the reciprocal was not true: F₁ donor T cells did not cooperate with B cells in parental TIR mice. However, when the host B lymphocytes were tolerant P cells, i.e., in a P → F₁ TIR chimera, injected F₁ T lymphocytes did cooperate successfully. The final test of allogeneic T and B cells gave the clear-cut negative answer that, even when tolerant mice are used as sources of lymphocytes, cooperation does not occur. These results therefore confirm that T and B lymphocytes must at least share one MHC haplotype in order to cooperate.     

Spontaneous adherence and rosette formation of lymphocytes to Leydig cells: an in vitro technique

A spontaneous rosette phenomenon occurs in vitro when suspensions of mouse testicular cells are mixed with suspensions of lymphocytes. The core cell of these rosettes are the Leydig cells, and the coronas are formed by lymphocytes. No other testicular cell, either seminal or Sertoli, participates in lymphocyte binding.Autogeneic, syngeneic, allogeneic, and xenogeneic lymphocytes from thymus, spleen, and peripheral blood similarly react with mouse Leydig cells. Lymphocytes from spleens of athymic nude mice and from chicken bursa also give Leydig rosettes. It is suggested that mouse Leydig cells carry membrane receptors spontaneously recognized by lymphocytes.     

The induction in vitro of immunological tolerance in T lymphocytes: an inhibitory role of macrophages.

Hapten-reactive helper T cells were generated in the spleen of C57BL/6 mice primed with sulfanilated syngeneic IgG (S-MGG). Specific immunological tolerance was induced in vitro in these helper T cells, when spleen cell suspension was passed through Sephadex G-10 column to remove adherent cells and cultured in the presence of soluble S-MGG for 21 to 24 hours. On the other hand, tolerance was not inducible in unfractionated, primed spleen cells. When G-10-passed spleen cells were added to the culture dishes containing phagocytic, adherent cells of the spleen, tolerance was no more inducible in these reconstituted cell population. From these experimental results, it was concluded that macrophages played an interfering role in tolerance induction. The experimental data were also discussed in terms of macrophage function in the recognition of antigen by T lymphocytes.     

Regulation of bone marrow lymphocyte production: IV. Cells mediating the stimulation of marrow lymphocyte production by sheep red blood cells: Studies in anti-IgM-suppressed mice,athymic mice,and silica-treated mice

Some cellular requirements have been examined for the stimulation of lymphocyte production in mouse bone marrow by injected sheep red blood cells (SRBC). The increased genesis of marrow lymphocytes after a single dose of SRBC assayed radioautographically after [³H]thymidine labeling was unimpaired in the marrow of mice treated with anti-IgM antibodies from birth to eliminate B lymphocytes, and in congenitally athymic mice lacking T lymphocytes. However, pretreatment of mice with silica to depress macrophage function completely abolished the SRBC effect both on the total lymphocyte production and on the number of B and null small lymphocytes in the marrow. Comparative studies were performed on the thymus and spleen. The results demonstrate that the stimulation of marrow lymphocyte production by SRBC is mediated by a silica-sensitive mechanism, does not require B or T lymphocytes, and is independent of the humoral immune response. Thus, extrinsic agents may amplify the production of primary B cells and other lymphocytes in the bone marrow by an antigen-nonspecific mechanism, putatively mediated by macrophages.