Functional heterogeneity among the T-derived lymphocytes of the mouse. IV. Nature of spontaneously induced suppressor cells.

When spleen cells are cultured for 4 days in the presence of fetal bovine serum, T-cells are generated which nonspecifically suppress the humoral responses of non-precultured cells to a variety of antigens. These T cells can be generated from both short-lived, sessile T1 cells and long-lived, recirculating T2 cells, and thus appear similar to suppressor T cells activated by concanavalin A.     

Functional heterogeneity among the T-derived lymphocytes of the mouse: III. Helper and suppressor T-cells activated by concanavalin A

We have studied the properties of T-cells which when activated by concanavalin A (Con A) either suppress or help the in vitro humoral response of mouse spleen cells. Previously established criteria for the T-cell populations, T₁ and T₂ were applied. T₁ cells were defined by their short half-life (2–3 wk) after adult thymectomy (ATx) and their resistance to small doses of antithymocyte serum (ATS). T₂ cells were defined by their long half-life (~15 wk) and their high sensitivity to ATS. T-cells which could be activated by Con A to help the response to the thymus-dependent antigen, sheep red blood cells, were found mainly in the T₂ subpopulation. T-cells which could be activated by Con A to suppress the response to the thymus-independent antigen, trinitrophenyl-lipopolysaccharide (TNP-LPS), were found within both the T₁ and T₂ subpopulations.These results, our previous results, and those of others suggest that the T-cell responses to phytomitogens distinguish precursors committed to different functions, while the T₁ and T₂ classifications distinguish T-cells at different stages of maturation.     

Functional heterogeneity among the T-derived lymphocytes of the mouse. V. Response kinetics of peripheral T cell subpopulations.

The kinetics of the proliferative response and the appearance of effectors of helper activity after stimulation by antigen were examined in T cell subpopulations. As defined in previous papers of this series, one population, T1, is short-lived after adult thymectomy (ATx), and relatively resistant to elimination by anti-thymocyte serum (ATS). Another population, T2, is long-lived after ATx, but highly sensitive to elimination by small doses of ATS. From precursors within the T2 population, effectors of specific helper activity, after priming with antigen, appeared within 1 to 2 days and reached a maximum on day 4. The responding cells reached their peak proliferative response within 24 hr after stimulation by antigen. In contrast, helper activity arising from T1 precursors first appeared on day 3 and peaked on day 5. These cells did not reach their maximal proliferative response until 60 hr after priming. These findings indicate additional useful markers for distinguishing the T1 and T2 subpopulations and are consistent with models for T cell development in which T1 cells are virgin cells and T2 cells are memory cells.     

Functional heterogeneity among the T-derived lymphocytes of the mouse. VII. Conversion of T1 cells to T2 cells by antigen.

The T1 subpopulation of peripheral T cells was defined in mice by its short half life, insensitivity to anti-thymocyte sera (ATS) in vivo, and slow kinetics of response to antigen. The T2 subpopulation was defined by its long life time, elimination by ATS in vivo, and rapid response to antigen. Mice containing only T1-type T cells were constructed by adult thymectomy (ATx) followed immediately by the elimination of T2 cells by ATS treatment. Immunization of these mice with SRBC led to the production of memory helper cells in the T2 subpopulation. This process depended on the presence of T1 cells and for the most part required SRBC immunization, although a few SRBC-specific T2 cells reappeared in the mice in the absence of antigen. We conclude that T1 cells can give rise to T2 cells in an antigen-driven step and that the two populations correspond to virgin and memory T cells, respectively.     

Functional heterongeneity among the T-derived lymphocytes of the mouse. VI. Memory T cells stored in the T2 subpopulation.

Memory T cells were demonstrated in mice which were the precursors for cells causing sheep red blood cell-(SRBC) specific helper activity in the in vitro response of spleen cells to trinitrophenyl (TNP)-SRBC. These memory cells were distinguished from the effectors of helper activity generated during the primary response and were shown to belong to the T2 subpopulation of peripheral T cells on the basis of 1) their rapid response to a challenge with SRBC, 2) their long lifetime, and 3) their sensitivity to small in vivo doses of anti-thymocyte serum.     

Response of spleen lymphocytes shortly after thymectomy in adult mouse.

The blastogenesis response to the phytomitogens, PHA-P, Con A and PWM was used to assess the effect of adult thymectomy on the spleen lymphocytes of C57B1 mice. The mitogenic response to the phytomitogens was determined by 3H-thymidine uptake. The changes produced in theta-antigen bearing spleen lymphocytes were also evaluated making use of theta antibodies from AKR/S mice previously injected with splenic and thymic lymphocytes from CBA/J mice. The present results show that the response to mitogens PHA-P and Con A is reduced early after thymectomy while the response to PWM was only slightly reduced. There was not any correlation between the disminished response to mitogens and the changes observed in theta bearing spleen lymphocytes.     

Physical heterogeneity of mouse thymus lymphocytes.

Mouse thymocytes have been separated by velocity sedimentation in a density gradient. The resulting fractions have been analyzed using electrophoretic light scattering. The electrophoretic distributions of the individual sedimentation fractions reveal the presence of physically distinct subpopulations. Comparison of the mean mobilities of each fraction indicates that the faster-sedimenting cells tend to have a higher electrophoretic mobility.     

Adult thymectomy promotes the manifestation of autoreactive lymphocytes.

Spleen cells from adult thymectomized mice (ATX) were assayed in a syngeneic graft vs host (GVH) model based upon enlargement of the draining popliteal lymph node following syngeneic cell inoculation into the hind footpad. Spleen cells from ATX mice have been found to induce a significantly higher increase in the weight of the regional lymph node than that induced by the injection of normal spleen cells. Irradiated spleen cells from ATX donors did not cause a similar increase, suggesting either that proliferation of the transferred cells was required at some stage of the reaction or that autoreactive cells are radiosensitive. Autoreactive cells were found in the spleen of mice 2 to 3 months after the thymectomy but were never found in the lymph nodes of such animals or in the thymus of intact mice. They are not phagocytic adherent cells and are not retained on nylon wool columns, which suggests that they belong to the T-cell lineage. Autoreactivity is lost when spleen cells from ATX donors are depleted of autologous rosette-forming cells (A-RFC) by centrifugation on a Ficoll-Hypaque gradient after rosette formation. Autoreactive spleen lymphocytes might belong to the population of A-RFC previously characterized as a population of immature T cells.     

In vitro responses of rat lymphocytes following adult thymectomy. II. Increased inhibition by splenic adherent cells of responses to phytohemagglutinin

Adult thymectomy in rats results in a marked fall of spleen cell responsiveness to PHA over a period of days to weeks. Examination of dose-response curves showed that, with high PHA dose or ≥ 10⁶ cells/ml, there is a profound inhibition of the response with spleen cells from thymectomized animals compared with cells of matched sham-operated controls. However, when adherent cells are removed from the cell suspensions, the remaining nonadherent cells give an almost linear dose-response relationship with increasing PHA similar to that exhibited by the nonadherent cells of the controls or sometimes a slightly decreased response. Similarly, when increasing numbers of spleen cells from these animals are cultured (with admixed thymocytes to make a constant total of 2 × 10⁶ cells/ml) with PHA, the linear portion of the doseresponse curve can be extrapolated to give a similar value for the maximal potential response, which again is the same as or somewhat less than the corresponding value for sham-operated controls. A difference in inhibitory capacity is also shown in mixtures of the two spleen cell populations with LNC or with purified spleen cells. It is concluded that adult thymectomy results in increased “suppressor” activity in the spleen within a few days and may reduce slightly the number of T lymphocytes in the spleen reactive with PHA.     

Functional characterization of mouse T lymphocytes with IgM-Fc receptors. I. Studies on ADCC and helper cell function.

The function of mouse T lymphocytes with receptors for IgM-Fc was analyzed by using immunofluorescence and a column separation method. The cellular uptake of FITC-IgM was inhibited with unlabeled Fc5 mu. IgM-FcR+ cells were active in ADCC against IgM-sensitized target cells, but not against IgG-coated target cells. The cells mediating ADCC with IgM antibody were shown to be distinct and physically separable from those mediating ADCC with IgG antibody. Helper T lymphocytes for humoral antibody formation in a hapten-carrier system were shown to be IgM-FcR+ and Fc-IgG-.     

The antigen-binding capacity of mouse lymphocytes. I. Quantitative estimates

A method is described for molar estimates of the antigen-binding capacity of lymphocytes. The procedure is based on the interaction of cells with a relatively large amount of an intensely radioiodinated ligand to favor saturation of all available receptor binding sites. Some of the parameters governing the reaction are discussed and the specificity of the cell-ligand interaction is evaluated.     

Circulating T and B lymphocytes of the mouse. I. Migratory properties

Studies on the identity of thoracic duct lymphocytes (TDL⁴) from normal and T cell-depleted mice indicated that as many circulating B lymphocytes were produced by healthy T cell-depleted mice as normal mice. Proportions of T and B cells from the thoracic duct of CBA mice changed markedly during the first 4 days of drainage from 82% T cells and 16% B cells at 12 hr to approximately equal proportions of both classes after 3 days. In absolute terms, T cells were mobilized rapidly by thoracic duct drainage and B cells very slowly. Histologically, this was reflected in a rapid depletion of the T cell-dependent areas of the lymphoid organs. The B cell-dependent areas, in contrast, became depleted of lymphocytes only after drainage for a week or more.The homing properties of circulating lymphocytes were investigated using TDL from normal and T cell-depleted mice as relatively pure sources of T and B cells, respectively. Four hours after injection of ⁵¹Cr-labeled T and B cells, a large proportion of both cell classes were found in the spleen. By 24 hr, many T cells had left the spleen and appeared in the lymph nodes. Such redistribution by B cells, however, was minimal.Intravenously injected T and B cells, labeled with tritiated uridine (³HU), localized specifically in the T and B cell-dependent areas, respectively, of the lymphoid tissues.³HU-labeled T cells were found to recirculate rapidly from blood to lymph. Labeled B cells, in contrast, recirculated only very slowly.     

Regulation of contact sensitivity to DNFB in the mouse: effects of adult thymectomy and thymic factor.

The contact sensitivity response to DNFB is decreased after adult thymectomy (ATX). This response decreases to 50% of the control response of normal age-matched mice as soon as 3 weeks after ATX and is not further depressed 9 to 16 weeks after ATX. These results suggest that two T cell subsets of different lifespan are involved in the anti-DNFB response. A circulating thymic factor (FTS) is able to restore the contact sensitivity response to DNFB when injected 3 to 9 weeks after ATX but not 16 weeks later. By contrast, FTS has a depressive effect on the contact sensitivity response to DNFB of normal mice through a cyclophosphamide-sensitive T cell subset. These results suggest that FTS regulates DNFB contact sensitivity by acting on a cyclophosphamide-sensitive T cell subset, still present 9 weeks after ATX but absent after 16 weeks. Thus although the T cell defect, causing a depression of the contact sensitivity reaction to DNFB is quantitatively similar 3 and 16 weeks after ATX, its nature is probably different.     

Functional heterogeneity among peritoneal macrophages. II. Enzyme content of macrophage subpopulations.

Rabbit peritoneal exudate (PE) macrophages were separated into subpopulations on discontinuous density gradients of bovine serum albumin. Four such macrophage subpopulations, referred to as bands A, B, C, and D (from lightest to heaviest buoyant density), were examined for differences in enzyme content. With regard to three acid hydrolases—acid phosphatases, β-glucuronidase, and cathepsin D—cells in bands A and B had greater enzyme activity than cells in bands C and D. A similar distribution of activities was observed for acid p-nitrophenylphosphatase. Peroxidase activity was present only in band D. Lysozyme activity was greatest in band D cells and least in band A cells. Only small differences in cytochrome c oxidase activity were observed among the subpopulations. Arginase activity was found to be greater in cells from band A than cells in bands B, C, and D. Macrophage subpopulations derived from PE macrophages placed in tissue culture for 7 days and macrophage subpopulation cells cultured for 2 days showed differences in acid phosphatase content similar to those seen with freshly obtained subpopulations. These results extend previous work demonstrating heterogeneity among PE macrophages.     

Functional heterogeneity among peritoneal macrophages: I. Effector cell activity of macrophages against syngeneic and xenogeneic tumor cells

Peritoneal exudate cells from immunized and nonimmunized animals were separated into subpopulations by centrifugation on discontinuous bovine serum albumin (BSA) density gradients. Cells in the several subpopulations were then tested for their cytostatic or cytotoxic activity against syngeneic and xenogeneic tumor cells. Nonimmune macrophages isolated at the 8 to 11% BSA interface were highly inhibitory to the growth of syngeneic and xenogeneic tumor cells during coculture for 24 to 48 hr. A second macrophage subpopulation of heavier density was not as effective in preventing tumor growth and frequently augmented it. Cytotoxic activity against (C58NT) D tumor cells could not be detected with macrophages or subpopulations of macrophages from immune as well as nonimmune animals, as determined by a 4-hr chromium release assay. The cytotoxic activity of the immune peritoneal exudate cells observed by this assay could be accounted for by the small percentage of lymphocytes present.     

Genetic heterogeneity among the founders of laboratory populations ofDrosophila. I. Scutlllar chaetae

The incidence of flies with mor than four scutellar chaetae (additional chaetae) has been followed for 45 generations in three strains set up from single inseminated females ofDrosophila melanogaster collected in the wild from the same locality at the same time. Each strain differed in the incidence of additional chaetae over this time, and the differences between strains were found to be controlled largely by additive genes.Fifteen further such strains were followed for 9 generations and each maintained consistent incidences of additional chaetae.Thus there may be genetic differences between populations derived from single inseminated females present in a given wild population, so lending support to a role for genetic drift (as a founder effect) in these situations. These differences may be maintained for many generations.Some possibilities for obtaining rapid responses in directional selection experiments based on the initial selection of favourable strains derived from single individuals are discussed briefly.     

Clonospecific structural heterogeneity in the Thy-1 molecule from mouse T lymphocytes

The Thy-1 molecule immunoprecipitated from detergent-solubilized, ¹²⁵I-labeled cell-surface proteins was shown to be processed in two distinct ways by mouse T lymphocytes: one leading to the expression by thymocytes, concanavalin A-activated spleen blasts, and six of nine T-cell clones of a molecule of 25–28 kd, and another, observed in three other T-cell clones, leading to the expression at their surface of a so far undescribed low M _r (23 kd) form of Thy-1. The results of two-dimensional gel electrophoresis and neuraminidase, endoglycosidase H, and endoglycosidase F treatment revealed that the observed heterogeneity of Thy-1 molecules from peripheral cloned T cells was due to major differences in the maturation and sialylation of their N-linked complex-type oligosaccharide residues. It was also found that a given T-cell clone could express T200, LFA.1, and transferrin receptor molecules with a low or high M _r. Furthermore, and in contrast to previously reported results, this study revealed that the differences in cell-surface glycoprotein profiles could not be correlated with the Lyt-2,3/T4 phenotypes, the specificity for allo-H-2, allo-I-A, allo-I-E, or GAT + I-A^k determinants, nor with the cytolytic or helper/amplifier potential of the various T-cell clones examined. The possible implications of these findings are discussed.     

The human LT system. I. Physical-chemical heterogeneity of LT molecules released by mitogen activated human lymphocytes in vitro.

Molecules with LT activity which can be identified in supernatants from PHA stimulated human lymphocytes by lysis of murine L-929 cell in vitro are heterogeneous. They can be separated by MW on sephadex or ultrogel columns into four separate classes: (a) complex (>200,000 daltons); (b) α (70–90,000 d); (c) β (25–50,000 d); and (d) γ (10–20,000 d). The amount of activity in a supernatant due to each class varies but is approximately: Cx—5 to 20%, α—40 to 60%, β—20 to 40%, and γ—0 to 10%. These classes differ one from another in their stability and kinetics of appearance in culture. Furthermore, they may aggregate together with the complex class under conditions of low ionic strength. Each class, except γ, can be further separated into subclasses by ion exchange chromatography and polyacrylamide gel electrophoresis. Alpha class can be separated on DEAE into the three subclasses, termed, α₁ (rf 0.25), α₂ (rf 0.37), and α₃ (rf 0.50). Alpha₂ subclasses can be further separated on phosphocellulose at pH 6.6 into α_2a (rf 0.37) and α_2b (rf 0.30). However, α₂ contains additional subclasses, which were resolved on PC columns at pH 5.5. Beta class activity can be resolved by DEAE and PAGE into two subclasses, termed β₁ (rf 0.28) and β₂ (rf 0.49). Gamma class activity was not studied, because of its instability. The complex class of LT activity is a macromolecular aggregate greater than 200,000 daltons which appears to contain smaller MW LT class(es). This study demonstrates that materials with LT activity in supernatants from PHA activated human lymphocytes in vitro are very heterogeneous.