Inhibitory and stimulatory effects of concanavalin A on the response of mouse spleen cell suspensions to antigen. II. Evidence for separate stimulatory and inhibitory cells.

The addition of concanavalin A to mouse spleen cell suspensions can either inhibit or stimulate the immune response to heterologous erythrocytes according to the experimental conditions. Evidence is presented which is incompatible with the hypothesis that these two effects are mediated by a single cell and which favors the hypothesis of separate inhibitor and stimulatory cells.

Inhibitory and stimulatory effects of concanavalin A on the response of mouse spleen cell suspensions to antigen. I. Characterization of the inhibitory cell activity.

The presence of concanavalin A (Con A) inhibits the immune response of mouse spleen cell suspensions to erythrocyte antigens, stimulates the incorporation of tritiated thymidine, and increases cell recovery. Con A also restores the depressed response of cell preparations treated to remove thymus-derived cells. The dose-response curve for all four effects shows peak activity at 2 microg/ml. The depressed in vitro response of spleen cell suspensions from adult thymectomized, irradiated, bone marrow-restored mice is also restored by Con A. Here the dose-response curve is quite different with activity over a much wider range of concentration. The restoration of thymus-derived cell-depleted cultures by Con A is inhibited by the addition of untreated, unirradiated, mouse spleen cell suspensions, but is not inhibited by untreated, irradiated cells. Small numbers of spleen cells that have been preincubated with Con A and washed will inhibit the response of fresh, untreated cells to antigen. If the mouse spleen cell suspensions are incubated for 24 hr before the addition of Con A, the response to antigen is no longer inhibited but is stimulated instead. The data are compatible with the hypothesis that there are at least two cell targets for the action of Con A. One cell, that mediates the inhibitor effect, is a short-lived, radiosensitive, thymus-derived cell. The other cell, that mediates the stimulating effect, cannot be identified from the data presented here but may also be of thymus origin on the basis of studies by other investigators.

Spleen cell proliferation in response to homologous antigens studied in congenic resistant strains of mice.

The proliferative responses obtained when spleen cell suspensions from two different inbred strains of mice were mixed were investigated further using congenic resistant strain pairs. Strong responses were obtained in 9 cases out of 12 where the two strains differed at a single gene locus controlling an H-2 histocompatibility antigen. No responses were obtained where the difference occurred at loci controlling weak histocompatibility antigens. These findings have been taken to provide additional circumstantial evidence that the response represents an in vitro homograft reaction to homologous tissue antigens.

Competition between concanavalin A-induced stimulatory and inhibitory effects in the in vitro immune response to antigen.

The humoral response of nude spleen cells (b cells) to sheep erythrocytes was measured in the presence of varying numbers of concanavalin A (ConA)-acvated stimulatory spleen T cells (helper) and Con A-activated inhibitory spleen T cells (suppressor) from BDF1 mice. It was found that suppressive effects could be reversed by the presence of additional numbers of stimulatory cells. These results seem incompatible with the hypothesis that suppression is mediated by supraoptimal numbers of stimulatory cells and provides additional evidence that separate populations of T cells mediate stimulation and suppression.

Separation of helper and suppressor T lymphocytes on a ficoll velocity sedimentation gradient.

A 5-20% Ficoll velocity sedimentation gradient has been successfully applied to separate concanavalin A (Con A)-induced helper; and suppressor T cells. When titrated into a constant number of fresh normal spleen cells responding to sheep erythrocytes, cells from the top pool show stimulatory effects while those from the bottom pool show inhibitory activity. Both activities are found to be Con A dependent and anti-theta sensitive. We conclude that Con A-induced helper and suppressor T cells are distinct subpopulations and such separation will allow further characterization of these cell types.

Separation of helper and suppressor T lymphocytes. II. Ly phenotypes and lack of DNA synthesis requirement for the generation of concanavalin A helper and suppressor cells.

Using a Ficoll velocity sedimentation gradient, we have been able to fractionate concanavalin A (Con A)-induced helper and suppressor cells into separate pools. Cells activated by Con A to mediate helper activity are Ly1+, do not require DNA synthesis for induction, and remain as small cells after activation. Suppressor cells are Ly23+, are found in the blast cell fraction and their induction is not inhibitable by prior treatment with mitomycin C or irradiation, both of which inhibit DNA synthesis. The implications of such findings are discussed.

Regulation of the immune response. I. Differential effect of passively administered antibody on the thymus-derived and bone marrow-derived lymphocytes.

The effect of passively transfered antiserum against sheep erythrocytes (SRBC) on the antigen stimulated increase of SRBC-specific plaque-forming cells (anti-SRBC-PFC) and SRBC-specific thymus-derived lymphocytes (SRBC-specific T-cells) in the mouse spleen was examined. A dose of antiserum which severely suppressed the development of anti-SRBC-PFC did not prevent the increase in SRBC-specific T-cells, as measured by their ability to cooperate in the in vitro response to trinitrophenylated (TNP) SRBC. It was shown that the insensitivity of these T-cells to antiserum could not be explained by their low antigen requirement as compared to that of PFC. In the in vivo response of mice to TNP-SRBC, antibody specific for TNP suppressed the appearance of both anti-TNP- and anti-SRBC-PFC. The presence of free SRBC specifically prevented the suppression of the anti-SRBC-PFC. These observations are consistent with opsonization by phagocytic cells as the primary means of the observed suppression of PFC development by antibody.

Immunization of dissociated spleen cell cultures from normal mice.

A culture system for cell suspensions from mouse spleens has been described. The system provides adequate conditions for in vitro immunization on initial exposure to heterologous erythrocytes. The in vitro response closely parallels that observed in vivo with respect to size, early kinetics, antigen dose, and the inhibitory effect of passive antibody. The response of cultured cells differs in two respects from that seen in vivo. There is an increase in the ability to discriminate between different varieties of homologous erythrocytes and the in vitro response does not appear to be limited by whatever mechanisms regulate the in vivo response.

Cell populations and cell proliferation in the in vitro response of normal mouse spleen to heterologous erythrocytes. Analysis by the hot pulse technique.

The role of proliferation in the development of 19S antibody-forming cells in the primary response has been investigated in an in vitro system. Spleen cell suspensions from normal, unimmunized mice were cultured in vitro in the presence of mammalian erythrocytes and the number of 19S hemolytic plaque-forming cells that arose 4 days later was measured. The hot pulse technique for the selective irradiation of those cells which synthesize DNA during a defined period of time has been described. The effect of such hot pulses administered at various times after the addition of antigen on the subsequent appearance of antibody-forming cells was determined. The results established that: (a) the onset of DNA synthesis does not start for approximately 24-32 hr after the addition of antigen, (b) essentially all the antibody-forming cells arise by cell division, and (c) different cell populations are involved in the response to two non-cross-reacting antigens.

Production of a B cell growth-promoting activity, (DL)BCGF, from a cloned T cell line and its assay on the BCL1 B cell tumor.

Culture supernatants from a long-term alloreactive T cell line, the Dennert line C.C3.11.75 (DL) contain a B cell-growth-promoting activity. This activity can be assayed on normal B cells or on the in vivo BCL1 tumor line. We have called this activity (DL)BCGF. This activity can be distinguished from the T cell-replacing factor activity we had earlier found in DL supernates [(DL)TRF], which is required together with IL2 for the B cell plaque-forming cell response to erythrocyte antigens. The (DL)BCGF can be absorbed on untreated or glutaraldehyde-fixed BCL1. This absorption does not remove (DL)TRF activity. The production of (DL)BCGF is greatly enhanced when DL is cultured with IL2-containing supernatants. Sublines or clones of DL (DL.B10 and DL.A4) have been obtained that make large amounts of (DL)BCGF in the absence of any stimulator cells or IL2. B cells from the Xid-deficient male (DBA/2 X CBA/N)F1 mice do not respond to (DL)BCGF.

Continuously proliferating allospecific T cells. I. Specificity of cooperation with allogeneic B cells in the humoral antibody response to sheep erythrocytes.

Allospecific mouse T cells, positively selected in one-way mixed lymphocyte culture were maintained for 3 yr in tissue culture by sequential restimulation. Such proliferating T cells were tested for their ability to induce a positive allogeneic effect: activating B cells in an in vitro primary humoral response to sheep erythrocytes. It was found that such T lymphocytes could function as helper cells. Helper activity was shown to be specific in that the B cells activated had to share major histocompatibility complex (H-2) antigens with the strain used for selection of the cell line. Intra H-2 mapping showed that antigens coded in the IAk subregion played an important role in the induction of the positive allogeneic effect. Supernatant factors could substitute for the allogeneic T cells in activation of the in vitro humoral response. However, such supernates exhibited no strain specificity. Therefore, the specificity seen in the positive allogeneic effect is presumably a consequence of the alloantigenic recognition receptors intrinsic to the T cells, and not to any biologically restricting properties of the allogeneic effect factor itself.

Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles.

We have generated primary effector populations from naive CD8 T cells in response to antigen and determined their patterns of cytokine secretion upon restimulation. The effect of exogenous factors on the effector generation was examined and compared with responses of antigen-specific CD4 effectors generated under comparable conditions. CD8 cells from bm1 mice were stimulated with C57BL/6 (B6) antigen presenting cells (APCs) bearing allogeneic class I and CD8 cells from female severe combined immunodeficiency (SCID) B6 mice, transgenic for a T cell receptor alpha/beta (TCR-alpha/beta) that recognizes H-Y on Db, were stimulated with APCs from male mice. In parallel, CD4 cells from bm12 mice were stimulated with alloantigen and CD4 cells from V beta 3/V alpha 11 TCR transgenics were stimulated with a peptide of pigeon cytochrome c on IEk. T cells from both transgenic mice were of naive phenotype whereas normal mice contained 10-20% memory cells. Effector CD8 populations generated were L-selectin low, CD45RB high, and CD44 high. Naive CD8 cells from SCID anti-H-Y mice made little or no cytokine immediately upon stimulation in contrast to naive CD4 which produced large amounts of interleukin 2 (IL-2). Both populations, however, generated primary effectors over 4-5 d that made substantial quantities of many cytokines upon restimulation. Both CD8 and CD4 effectors produced similar patterns of cytokines with alloantigen or specific antigen. Cytokines present during naive CD8 stimulation influenced the cytokine secretion profile of the effectors, as previously shown for CD4 cells, although secretion by CD8 effectors was generally lower than that of CD4 effectors. CD8 cells cultured with IL-2 alone made predominantly interferon gamma (IFN-gamma) and no IL-4 or IL-5, similar to CD4 cells. Priming with IFN-gamma increased IFN-gamma secretion from CD4 effectors, but had little if any effect on CD8 cells. In contrast, priming with IL-12 generated CD8 effectors, as well as CD4 effectors, producing elevated quantities of IFN-gamma, with similar levels from both the CD4 and CD8 populations. The presence of IL-4 during effector cell generation promoted synthesis of IL-4 and IL-5 from both CD8 and CD4 cells while downregulating IFN-gamma secretion. CD8 cells made only small amounts of IL-4, more than 100-fold less than CD4 cells, whereas significant levels of IL-5 were induced, only 3-10-fold lower than from CD4.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoglobulins on the surface of thymus-derived cells engaged in the initiation of a humoral immune response.

Preculture treatment of normal spleen cells with antiserum against mouse kappa light chains and complement was found to inhibit in vitro responses of these cells to TNP and erythrocyte (carrier) antigens, primarily by elimination of a thymus-derived helper component required for the response. Spleen populations inactivated in this way could be reconstituted with irradiated, carrier-immune spleen cells or with carrier-educated thymus-derived spleen cells. The ability of helper populations (i.e. irradiated, carrier-immune spleen cells or carrier-educated thymus-derived spleen cells) to enhance the response of normal spleen cells to hapten was eliminated by pretreatment of the helper cells with anti-kappa serum and complement. No significant effect of anti-kappa and complement treatment on precursor cell populations in normal spleen or bone-marrow-derived spleen could be demonstrated. The data are interpreted as evidence for the presence of immunoglobulin components. The function of these molecules is not established but it would be reasonable to assume that they are involved in antigen recognition, on the surface of thymus-derived cells.

Cellular events in the immune response : analysis and in vitro response of mouse spleen cell populations separated by differential flotation in albumin gradients.

Cell suspensions from the spleens of normal mice or mice injected with sheep erythrocytes were separated on a discontinous bovine serum albumin density gradient. Four bands or subpopulations were obtained and were assayed for antibody-forming cells, and for antigen-sensitive precursor cells. The antibody-forming cells were assayed by the hemolytic plaque assay and the antigen-sensitive precursors were assayed by the number of plaque-forming cells which developed after 3 or 5 day's culture with antigen. It was found that both antibody-forming cells and their precursors were present in the denser region of the gradient when spleen cell suspensions were taken from unimmunized mice. In contrast, both antibody-forming cells and precursors floated to the top in cell suspensions from mice sacrificed 1, 2, or 3 days after antigen injection. The change in density was detectable as early as 12 hr and was complete by 18 hr. The cell which changed in density was specific for the antigen that brought about that change. The significance of these findings is discussed.

A monoclonal T cell-replacing activity can act directly on B cells to enhance clonal expansion.

We have used a B cell cloning system in which the response of a single isolated B cell to lipopolysaccharide and dextran sulfide can be followed. We have shown that culture supernatants from the Dennert long-term alloreactive T cell line C.C3.11.75 increase the frequency of B cells stimulated to clonal expansion by mitogens. These culture supernatants are devoid of interleukin 1 and 2 but contain the T cell-replacing factor activity (DL)TRF. These experiments provide unequivocal proof that a T cell-derived factor or factors can act directly on a B lymphocyte in the absence of any other cell.

Xenogeneic human anti-mouse T cell responses are due to the activity of the same functional T cell subsets responsible for allospecific and major histocompatibility complex-restricted responses.

Human T cells respond strongly to mouse major histocompatibility complex (MHC) antigens. The response is directed predominantly to the polymorphic determinants of the MHC antigens and there is little or no response to the nonpolymorphic determinants or to non-MHC antigens. Human cytotoxic T lymphocytes (CTL) are generated specific for the mouse class I MHC antigens and the CTL effectors are blocked by anti-Leu-2a antisera. Human interleukin 2-producing T cells are generated specific for mouse class II antigens and their induction is blocked by anti-Leu-3a antisera. These and other considerations lead us to propose a model for the T cell receptor that provides an explanation for several of the features of T cell recognition. In this model, the recognition of the "class" (I or II) of MHC antigen is separate from the recognition of the polymorphic determinants. We suggest that the initial recognition of the conserved "class" determinants positions another domain of the receptor so that it can only engage with the part of the MHC molecule carrying the polymorphic determinants.

Migration kinetics and final destination of type 1 and type 2 CD8 effector cells predict protection against pulmonary virus infection.

The requirements for CD8 T cells to provide protection against a localized virus infection in models of adoptive immunotherapy are not well defined. Here we investigated the protective value of defined in vitro-generated hemagglutinin (HA) peptide-specific primary CD8 T cell effectors from the clone 4 T cell receptor transgenic mice, secreting type 1 or type 2 cytokines, against pulmonary infection with whole influenza virus. Cytotoxic T lymphocytes producing type 1 and type 2 cytokine (Tc1 and Tc2) populations were equally cytolytic, but Tc1 effectors and not Tc2 effectors reduced the pulmonary virus titer early during infection. Host recovery mediated by Tc1 effectors was found to be independent of interferon gamma production. Tc2 effectors entered the lung with delayed kinetics as compared with Tc1 effectors, and after lung entry Tc2 effector cells did not localize near the infected airway epithelium as did Tc1 effectors but were found within clusters of inflammatory cells distant from the epithelium. We also show that the expression of several chemokine receptors was selectively regulated in the Tc1 and Tc2 subsets. Thus, the protective value of a CD8 cell population against pulmonary influenza virus infection is strongly correlated with its ability to exert its effector potential at the site of virus infection.

Analysis of histocompatibility requirements for proliferative and helper T cell activity. T cell populations depleted of alloreactive cells by negative selection.

T cell populations were prepared from donors immunized with hapten-carrier conjugates and were depleted of alloreactive cells by negative selection. This was accomplished by injection of the cells into H-2-disparate irradiated recipients and recovery from the thoracic duct after 18-40 h. The genetic requirements for the proliferative and helper activity of these populations was determined. The proliferative response to antigen presented on adherent, Thy-1-negative cells was determined, and a requirement for syngeneic antigen-presenting cells (APC) was demonstrated. The same T cells were assayed for their ability to give help to hapten primed B cells. It was shown that there was a requirement for syngeneic APC and for linked recognition of hapten and carrier determinants on the same molecule by the B cell and T cell. There was no requirement for the B cell to be H-2 compatible with the T cell. The requirement for linked recognition was taken as evidence that the responses in allogeneic combinations were not a result of positive allogeneic effects. Precisely comparable restrictions were found with positively selected cells.

B cell stimulatory factor 1 (interleukin 4) is sufficient for the proliferation and differentiation of lectin-stimulated cytolytic T lymphocyte precursors.

In this report, we demonstrate that IL-4 is sufficient to stimulate both the proliferation and differentiation of Lyt-2+, Ia- splenic CTL precursors stimulated with the mitogenic lectin Con A. The response to IL-4 and Con A was not dependent on a putative endogenous production of IL-2 within the cultures, as demonstrated by an absence of an inhibitory effect by an anti-IL-2-R blocking mAb. Our results indicate that IL-2 and IL-4 can support an equivalent proliferative response by lectin-stimulated Lyt-2+ T lymphocytes, while IL-4 is more efficacious in stimulating their differentiation into mature cytolytically active cells.

Evidence for two distinct classes of murine B cell growth factors with activities in different functional assays.

Several previously described B cell growth factor (BCGF) activities from a number of mouse monoclonal T cell sources were compared in different functional assays. The results indicate that there are two distinct classes of BCGF defined by functional activity and source. BCGF I, whose prototype is (EL4)BCGF, synergized with anti-Ig in the proliferation of normal splenic B cells but had no activity when dextran sulfate (DXS), rather than anti-Ig, was used to costimulate the same source of B cells. BCGF I also failed to directly stimulate BCL1 tumor B cells. In contrast, BCGF II, whose prototype is (DL)BCGF, showed a reciprocal pattern of activity. BCGF II failed to synergize with anti-Ig-costimulated normal B cells to give good proliferative responses. Sources of BCGF II also directly stimulated (no anti-Ig or DXS added) B cells of the BCL1 tumor-carrying mice. These results suggest that the two BCGF may have activity on two subsets of B cells that respond differentially to induction with the two polyclonal B cell activators, anti-Ig and DXS. The possibilities that these different patterns of response occur in separate lineages of B cells and/or in B cells in different states of differentiation is discussed.