Potentiation of the T-lymphocyte response to mitogens. II. The cellular source of potentiating mediator(s).

Effective supernatants (SUP), which potentiate mouse T-cell responses to phytohemagglutin (PHA), are obtained from cells of several species (human, rabbit, rat, mouse) and indeed from syngeneic spleen, thymus, or bone marrow cells. Unstimulated cells release some SUP activity but more is produced after stimulation. Lipopolysaccharide (LPS) produced very active SUP in all cultures tested. PHA was similarly active on human leukocytes only, whereas concanavalin A (Con A) gave highly efficient SUP only with mouse spleen cells. SUP production is not correlated with a mitotic response of the donor cells and is observed in cultures unable to respond mitotically to the stimulant. Adherent mouse spleen cell populations, consisting largely or entirely of macrophages, produce active SUP, while nonadherent cells do not. Similarly, purification of human peripheral leukocytes on nylon columns, with removal of macrophages and other adherent cells, destroys their ability to produce SUP. The importance of indirect effects in stimulating mitotic responses of T cells is emphasized by the fact that the mitotic response of mouse thymocytes to LPS and its ability to potentiate the response of these cells to PHA disappears with removal of adherent cells from the thymocyte population. Conversely the production of SUP from spleen cells stimulated by Con A requires the presence of T cells.

Origin and fate of IgE-bearing lymphocytes. I. Peyer's patches as differentiation site of cells. Simultaneously bearing IgA and IgE.

Peyer's patches (PP) from adult conventionally raised (C) and germ-free (GF) rats of the same age differed strikingly in the distributions of cells bearing membrane-bound immunoglobulin of the different isotypes. High concentrations of cells with membrane-bound IgE (approximately 20% of total cells determined by indirect immunofluorescence) were found in PP of rats, whereas IgE+ cells were absent from PP of C rats. The numbers of IgA+ cells were almost threefold higher in PP of GF rats when compared with C rats. In contrast, PP of GF rats had greatly reduced numbers of IgM-bearing cells (4%) when compared with C rats (23%); in some experiments virtually no IgM-bearing cells were detected. The levels of IgA- and IgE-bearing cells in spleen of GF rats were increased (to 11% and 7%, respectively). Of the IgE-bearing cells in PP and spleen of GF rats, approximately one-half were simultaneously positive for IgA. When these PP cells were treated with pronase to remove membrane bound immunoglobulins and maintained in culture, both IgE and IgA reappeared within 12 h. The proportion of doubly labeled cells was similar to that of the untreated population. No IgE+ cells were detected in bone marrow of C of GF rats at any time, in agreement with the findings of Ishizaka et al., although up to 20% of bone marrow cells bore other immunoglobulin isotypes, suggesting that IgE-bearing cells arise in the PP either de novo or by switching from precursors carrying IgM or IgA.

Role of the thymus in tolerance. 3. Tolerance to bovine gamma globulin after direct injection of antigen into the shielded thymus of irradiated rats.

Rats subjected to high doses of whole-body irradiation, with simultaneous shielding of the thymus or spleen, recovered at 3 wk the ability to develop delayed sensitization and to form hemagglutinating and precipitating antibody following foot-pad injection of BgammaG in complete adjuvant. Injection of BgammaG into the shielded thymus immediately after irradiation, in amounts between 20 gammag and 40 mg, inhibited these response to later challenge partially or completely. A comparable effect on immune responses to BgammaG was not seen after injection of heterologous antigen (Ea) intrathymically, BgammaG intraperitoneally, or BgammaG into the shielded spleen. However high doses (20 or 40 mg) of antigen given by the latter routes resulted in some diminution of later response. Arthus reactivity recovered partially in the spleen-shielded group and was readily suppressed by intrasplenic administration of antigen.

Bone marrow as a source of cells in reactions of cellular hypersensitivity. I. Passive transfer of tuberculin sensitivity in syngeneic systems.

Splenectomy or thymectomy of adult Lewis rats following sensitization with tubercle bacilli did not affect their ability to develop delayed skin lesions upon skin testing with PPD. The presence of the thymus also had no significant effect on reactivity of the recipients in passive transfer experiments. The passive transfer of tuberculin hypersensitivity with sensitized lymph node cells to thymectomized, irradiated recipients depended on the simultaneous or prior injection of normal bone marrow cells. When lymph node cell transfer was performed shortly after irradiation and injection of marrow, high doses of marrow cells (3.5-4.0 x 10(8) were required to permit eliciting reactions of reasonable intensity. If, however, periods of 7-10 days elapsed between the injection of bone marrow and sensitized lymph node cells, lower doses of marrow were sufficient for comparable reactions. Normal thymus, spleen, lymph node, or peritoneal exudate cells, even at high doses could not be substituted for the bone marrow in producing good tuberculin reactions.

Bone marrow as source of cells in reactions of cellular hypersensitivity. II. Identification of allogeneic or hybrid cells by immunofluorescence in passively transferred tuberculin reactions.

The precise origin of cells infiltrating tuberculin skin reactions was studied with the technique of immunofluorescence. Thymectomized, irradiated Lewis rats were restored with bone marrow from allogeneic or F(1) donors. They were passively sensitized to tuberculin by a subsequent transfer of Lewis lymph node cells and were given intradermal skin tests with tuberculoprotein. In 24 hr reactions the majority of cells were shown to be derived from the infused marrow. These results were the same regardless whether the lymphocyte transfer was performed on the day of irradiation and marrow injection or 7 days later. The cells in the tuberculin reactions, marrow, spleen, and lymph nodes not derived from the bone marrow were found to originate in the transferred lymph node cells. The relative percentages of marrow-derived and lymph node-derived cells in the tuberculin reactions remained the same during the 9-24 hr period following skin test.

Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. II. Correlation of the in vitro response with skin reactivity.

Damage of rat embryo fibroblasts in the presence of sensitized lymph node cells reacting with specific antigen was shown to be closely correlated with delayed hypersensitivity in the animals from which the lymph node cells were taken. The phenomenon was not correlated with Arthus reactivity. In. animals sensitized with picryl conjugates of ovalbumin or human serum albumin, skin reactivity and the in vitro cytotoxic effect could be elicited only with the homologous conjugate or the protein carrier alone and not with picryl conjugates of heterologous proteins. Lewis rats developed more intense delayed sensitivity than BN rats, and Lewis lymph node cells were correspondingly more effective in producing specific damage of both syngeneic and allogeneic fibroblasts.

Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. I. Characterization of the phenomenon.

In the presence of specific antigen, lymph node cells from inbred rats with delayed hypersensitivity to tuberculoprotein, bovine gammaglobulin, and egg albumin produced progressive destruction of monolayers of rat embryo fibroblasts in tissue culture, first apparent at 48 hr and maximal at 72 hr. The effect was specific and did not depend on a genetic difference between the lymph node cells and target cells. It required antigen concentrations equal to or greater than 1.25 microg/ml and lymphocyte: target cell ratios of approximately 10 or 20:1. It could be evaluated both by a plaquing technique and by cell enumeration with an electronic particle counter.

Regulation of lymphocyte responses in vitro. I. Regulatory effect of macrophages and thymus-dependent (T) cells on the response of thymus-independent (B) lymphocytes to endotoxin.

DNA synthesis of normal rat spleen cells in response to endotoxin increases markedly if adherent cells are first removed from the cell suspension. Addition of small numbers of purified macrophages to the cultures restores the response to a low level. T-deprived cells show these effects in very much lesser degree. Large numbers of macrophages completely suppress the response of both normal and T-deprived spleen. We conclude that two mechanisms of suppression are at work: a direct effect of macrophages and a macrophage-dependent "suppressor T cell" effect.

Potentiation of the T-lymphocyte response to mitogens. I. The responding cell.

Human and mouse lymphoid cells, stimulated by phytohemagglutinin (PHA) or lipopolysaccharide W (LPS), release supernatant factor(s) which are mitogenic for mouse thymocytes and which potentiate their responses to PHA or concanavalin A (Con A), The term LAF (lymphocyte-activating factor) is proposed for this activity. LAF not only enhances the mitotic responses of the less dense thymus subpopulations (A, B, and C) separable on discontinuous bovine serum albumin (BSA) gradients but also gives substantial responses in the otherwise inert cells of the denser fractions D and P. LAF does not exert a potentiating stimulatory effect on the responses of unfractionated mouse spleen cells, but does act synergistically with PHA on nonadherent spleen cells and on spleen cells of mice of several strains 5 days after irradiation and injection of thymocytes. Similarly LAF, which has no visible effect on unfractionated human peripheral blood cells, strongly potentiates the PHA response of column-purified lymphocytes, when these are cultured at low concentration. We conclude that LAF stimulates both central and peripheral T lymphocytes and enhances their responses to other stimulants.

Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. 3. Analysis of mechanism.

The cytopathic effect of lymph node cells from tuberculin-sensitized rats on rat embryo fibroblasts in the presence of PPD was not enhanced by admixture of normal (nonsensitized) lymph node cells. Preincubation studies showed that this in vitro response is initiated by the reaction of lymphocytes with specific antigen, beginning within 30 min, rather than uptake of antigen by the fibroblasts. The supernatant fluids from suspensions of sensitized cells incubated with PPD for 17 hr or more possessed cytotoxic activity. The target fibroblasts showed a marked increase in acid phosphatase content within 48 hr after the addition of sensitized lymph node cells and antigen.

Kinetic study of T lymphocytes after sensitization against soluble antigen. III. Potentiation and suppression of the PHA response by antigen-activated lymphocytes of low density.

Lymph node cells of ovalbumin-sensitized rats were separated on the basis of buoyant density into fractions reciprocally enriched in cells responsive to ovalbumin or phytohemagglutinin (PHA). Recombination of high density and low density fractions in varying proportions resulted in potentiation or suppression of the DNA synthetic response to PHA in culture. The response of cultures containing equal numbers of high and low density cells was markedly greater than the sum of the two populations stimulated separately. However, when decreasing numbers of low density cells were cultured with a constant number of high density cells, profound suppression was observed.

The thymic suppressor cell. I. Separation of subpopulations with suppressor activity.

This study confirms the finding that washed thymocytes from rats given 100 mg of BGG 48 h earlier, when transferred to syngeneic recipients, exert a specific suppressor effect on immunological responses to BGG in the latter. The active cells are found in a subpopulation of low density, making up less than 10% of the total thymocytes, and are partially resistant to hydrocortisone.

Cellular differentiation in the thymus. 3. Surface properties of rat thymus and lymph node cells separated on density gradients.

Young adult rat thymus and lymph node cell subpopulations were obtained by differential flotation on discontinuous BSA density gradients and assayed for properties characteristic of mature thymus-derived lymphocytes. One such subpopulation (C) of thymocytes was enriched in its ability to respond mitotically to a hemiallogeneic MLR stimulus, to localize in the parenchyma of lymph nodes and spleen, and to initiate a GVH reaction in a suitable host. These cells did not respond well to mitotic stimulation by PHA, they were lighter in density than the majority of mature lymph node thymus-derived lymphocytes, and they possessed a thymus-specific antigen (RTA) not present on peripheral lymphoid cells. We conclude that the acquisition of peripheral properties occurs sequentially, during an intrathymic differentiation cycle or shortly after the cells leave the thymus.

Regulatory substances produced by lymphocytes. VI. Cell cycle specificity of inhibitor of DNA synthesis action in L cells.

IDS inhibits DNA synthesis and mitosis of L cells only when present during the late G1 phase of the cell cycle, as shown with L cells synchronized by a variety of methods. This corresponds well with earlier findings that IDS inhibits DNA synthesis in mitogen-stimulated lymphocytes when present between 16 and 24 h after adding mitogen. In both cell types, the inhibition produced by IDS appears to be totally the result of elevation of cAMP level. Thus, inhibitors of cAMP phosphodiesterase work synergistically with IDS, and activators of cAMP phosphodiesterase overcome the inhibition by IDS. This paper shows that IDS raises cAMP levels in L cells only within a narrow interval of the cell cycle, around 6-8 h after mitosis. This cell cycle specificity, which may be related to appearance of receptors for IDS only at discrete times, may be important in limiting IDS action to suppression, as elevated cAMP levels have a variety of other effects during other phases of the cell cycle.

Cytochalasin B inhibits lymphotoxin production by antigen-stimulated lymphocytes.

Lymph node cells of rats sensitized with hen ovalbumin produced lymphotoxin after 6 to 12 hours of exposure to specific antigen. Lymphotoxin was assayed by its cytotoxicity for fibroblasts from syngeneic embryos during a 72-hour incubation. Cytochalasin B inhibited lymphotoxin production, as well as later DNA synthesis, at concentrations (0.1 to 5.0 micrograms per milliliter) comparable to those which affect microfilament function and cell motility in other systems, and this inhibition was reversible. Binding of antigen was not affected.

Delayed hypersensitivity: bone marrow as the source of cells in delayed skin reactions.

Adult Lewis rats were thymectomized, irradiated, and restored with bone marrow from allogeneic (or F(1)) donors. They were passively sensitized to tuberculin by a subsequent transfer of Lewis lymph node cells and were given intradermal skin tests with tuberculoprotein. In 24-hour skin reactions the majority of cells, in successive experiments, were shown to be allogeneic (or F(1)) with the use of isoantibody against the antigens of the transplanted marrow cells and by the indirect fluorescent antibody technique. Our results demonstrate that the non-specific cells making up a large proportion of the infiltrating elements in tuberculin skin reactions probably originate in the bone marrow.

Cytotoxic effect of lymphocyte-antigen interaction in delayed hypersensitivity.

Lymph node cells from inbred rats having delayed sensitivity to soluble proteins inhibit growth of syngeneic or allogeneic fibroblasts in the presence of specific antigen. A relation is suggested between this in vitro phenomenon and other systems believed to be specific manifestations of delayed hypersensitivity.

Cytokine inhibition of DNA synthesis: effect on cyclic adenosine monophosphate in lymphocytes.

A greater than twofold increase in intracellular adenosine 3',5'-monophosphate (cyclic AMP) inhibited DNA synthesis, in stimulated rat lymphocytes. A two- to fourfold rise of intracellular cyclic AMP, starting at 16 hours, was produced by purified inhibitor od DNA synthesis added to such cells either at 0 or 16 hours, in close association with the initiation of DNA synthesis.

The importance of IgA in nonmucosal immune responses.

IgA may be an important component of nonmucosal immune responses, like those occurring in the central nervous system, in certain virus infections, and in multiple sclerosis. Mucosal immune responses, in appropriate, natural, and experimental situations, may be predominantly IgE or IgG, or indeed T-cell mediated.

Autoimmunity in demyelinating diseases.

Demyelinative diseases of the CNS and peripheral nervous system can be distinguished on the basis of primary mediation by antibody or T lymphocytes (or failure of the T-cell-mediated response) and on the basis of chronicity. The principal mechanisms are autoimmunization to myelin antigens after actual immunization with tissue or infection with cross-reactive viruses or, alternatively, persistent infection of the nervous system (viral or spirochetal) with an associated immune response to the pathogen.