Activation of suppressor T cells by low-molecular-weight factors secreted by spleen cells from tumor-bearing mice

The spleens of mice bearing large M-1 fibrosarcomas have been shown to contain several populations of cells which nonspecifically suppress antibody synthesis by cocultured normal spleen cells. It has now been shown that the spleens of tumor-bearing mice also contain inducer cells which secrete soluble factors capable of activating suppressor T cells from unprimed precursor cells. The activated suppressor cells are Thy 1+, Lyt 1+2+ and secrete a soluble suppressive factor. They inhibit the in vitro generation of antibody-forming cells by cocultured normal spleen cells stimulated by T-cell-dependent antigens. They do not, however, suppress the antibody response to T-cell-independent antigens and do not inhibit antibody synthesis by cocultured nude mouse spleen cells cultured with T-cell-dependent antigens and exogenous helper factors. In addition, suppression is blocked if conditioned medium containing T-cell growth factors is added to the suppressor cell assays. These data suggest that cells in the spleens of tumor-bearing mice secrete inducing factors which activate suppressor cells. These activated suppressor cells in turn secrete soluble suppressor factors which inhibit antibody synthesis, possibly by interfering with the synthesis or release of T-cell growth factors.     

Production of a factor inhibiting tumor cell migration by spleen cells from tumor-bearing mice

A factor inhibiting tumor cell migration was found in the culture supernatants of spleen cells of BALB/c mice transplanted with sarcoma cells 20 days before or injected with killed sarcoma cells 14 days before, when the spleen cells were stimulated in vitro for 2 or 24 hr with a 3 M KCl extract of the sarcoma. This factor did not inhibit the migration of guinea-pig macrophages or of theophylline-pretreated sarcoma cells. When the supernatants were fractioned on Sephadex G-100 columns, active fractions with molecular weights lower than 14,000 were distributed broadly.     

Antitumor and therapeutic effects of spleen cells from tumor-bearing mice cultured with T cell growth factor and soluble tumor extract

Summary Spleen cells of BALB/c mice that had been inoculated with syngeneic plasmacytoma MOPC 104E were cultured for 11 days in T-cell growth factor (TCGF) and ultrasonicated tumor extract (USE). Cultured lymphocytes (MOPC-CL) possessed three-fold more lytic units than normal spleen cells cultured in TCGF without USE (N-CL). Moreover, the in vivo neutralization assay suggested that MOPC-CL were composed of at least two populations, one possessing tumor-specific and the other nonspecific antitumor activity. When 2×10⁷ of MOPC-CL were administered IP to mice that had been inoculated IP with 10⁵ MOPC 104E cells 5 days previously marginal prolongation of survival was observed. This effect was not augmented by the single injection of a larger number (5×10⁷) of CL, but was augmented by the repeated daily administration for 4 days (from day 5 to day 8 after the inoculation) of the same total number (5×10⁷) of CL. In addition, IP injection of the streptococcal preparation OK432 before the transfer of CL significantly enhanced the therapeutic efficacy, and resulted in a cure rate of 20%. The mechanism of this combined effect appears to involve the effect of OK432 on interleukin 2 (IL-2) regulation systems in vivo. Our culture system with TCGF and USE and our therapy system with OK432 and CL allow the clinical application of adoptive immunotherapy for the many types of solid cancers.     

Factors regulating stem cell recruitment to the fetal thymus

Colonization of the thymic rudiment during development is initiated before vascularization so that hemopoietic precursors must leave the pharyngeal vessels and migrate through the perithymic mesenchyme to reach the thymus, suggesting that they may be responding to a gradient of chemoattractant factors. We report that diffusible chemoattractants are produced by MHC class II+ epithelial cells of the fetal thymus, and that the response of precursors to these factors is mediated via a G protein-coupled receptor, consistent with factors being members of the chemokine family. Indeed, a number of chemokine receptors are expressed by thymic precursors, and several chemokines are also expressed by thymic epithelial cells. However, these chemokines are also expressed in a tissue that is unable to attract precursors, although the thymus expressed chemokine, TECK, is expressed at higher levels in thymic epithelial cells and we show that it has chemotactic activity for isolated thymic precursors. Neutralizing Ab to TECK, however, did not prevent thymus recolonization by T cell precursors, suggesting that other novel chemokines might be involved in this process. In addition, we provide evidence for the involvement of matrix metalloproteinases in chemoattractant-mediated T cell precursor recruitment to the thymus during embryogenesis.     

Changes in the electrophoretic mobility of thymus, spleen and lymph node cells in tumor-bearing mice during dynamic tumor growth

Using the analytic microscope "Parmoquant-2" (GDR), histograms were obtained demonstrating electrophoretic mobility (EPM) of lymphoid cells of C57BL/6 mice in the course of growth of the Lewis carcinoma (3LL) and melanoma B16 administered under the skin of the femur. Changes in the average values of EPM of thymic, splenic and lymph nodal cells in the process of tumor growth appeared similar. It is shown that the medium thymocyte EMP is growing towards the terminal stage of tumor growth, at the expense of the decrease in the share of PNA+ cells. Splenic cell bimodal distribution according to EPM became, in the course of tumor growth in intact mice, unimodal with some insignificant decrease in the median EPM values. The median EPM of regional and distant lymph nodes in the process of tumor growth is of phase character. It is supposed that investigation of lymph node EPM could be used for studying tumor growth kinetics.     

Radiosensitivity of clonogenic granulocytic macrophage cell precursors in the bone marrow and spleen of tumor-bearing mice

Clonogenic granulocytic macrophagal cells-precursors (CFU-DC) of bone marrow and spleen of intact C57Bl/6 mice and those inoculated subcutaneously with LLC tumor cells do not substantially differ in their radiosensitivity; the concentration of CFU-DC in the spleen markedly varies as tumor grows. The values of Do and extrapolation number n for CFU-DC of the bone marrow are 0.9-1.4 and 1.5-3.0 Gy, and of the spleen, 0.8-1.6 and 1.0-2.6 Gy, respectively.     

Cellular origin of blocking factors from cultured spleen cells of tumor-bearing mice

Spleen cells from mice bearing methylcholanthrene-induced tumors were cultured for 2 days without further stimulation. Blocking factors were consistently detected in culture supernatants by their ability to suppress leukocyte adherence inhibition reactions between soluble tumor antigens and peritoneal cells of tumor-bearing mice. The blocking factors were specific for individual tumors. The cellular origin of these factors was investigated by depleting the spleen cell population of various cell types before culturing. The cells involved were removed by treatment with antibodies to certain membrane markers (Thy-1, Ly-2, Ia, I-J) but not by anti-Ly-1 antibodies. Removal of adherent cells also prevented production of blocking factors, which was restored by reconstitution with syngeneic but not allogeneic cells from normal mice. The normal reconstituting cells were shown to bear Ia, but not I-J or IgM. This indicates that blocking factors (previously shown to have I-J determinants in their molecules) originate from suppressor T lymphocytes (Thy-1+, Ly-1-2+, I-J+), with macrophages (I-J-, Ia+) in the role of accessory cells.     

Antibody in the sera of tumor-bearing mice that mediates spleen cell cytotoxicity toward the autologous tumor.

Pretreatment of MTV-induced BALB/cfC3H mammary tumor cells with autologous serum results in increased spleen cell cytotoxic activity and the recruitment of previously inactive spleen cells to cytotoxic activity against the target cells. These recruiting antibodies are tumor-specific for individual tumors; pretreatment with such serum of target cells of an MTV-induced mammary tumor obtained from a different BALB/cfC3H female results in blocking of spleen cell activity. The autologous recruiting factors are active at dilutions of 1000 or more of whole serum are found in the 19S fraction after gel filtration.     

Activation of T cells in tumor-bearing mice

Subcutaneous transplantation of the syngeneic P815 mastocytoma in DBA/2J mice induced an activation of splenic T cells which resulted in a hyperresponsiveness of the tumor-bearing animal to the unrelated antigens pneumococcal polysaccharide (Pn) and sheep red blood cells (SRBC). These tumor-activated T cells appeared to increase the plaque-forming cell (PFC) potential of suboptimal numbers of spleen cells, caused normal spleen cells to express increased numbers of PFC, and produced lymphokine(s) which also increased PFC responses of normal splenocytes. The tumor-activated T cells responsible for stimulating normal splenocytes in an in vitro antibody response were shown to be Ly+2- cells. The activity of the tumor-activated T-cell supernatants was not genetically restricted and required additional Ly1 T cells in order to induce rigorously clean B cells to produce antibody. The T cells capable of stimulating non-specific antibody responses were also capable of slowing tumor growth when injected with tumor cells in normal recipient mice. These results suggest that T cells activated by tumor antigens release immunostimulatory lymphokines and, at the same time, are capable of leading to inhibition of tumor growth.     

Qa alloantigen expression on functional T lymphocytes from spleen and thymus

The cell-surface expression of the class I alloantigen Qa-2 was analyzed on resting and activated spleen and thymus cells using cytotoxic elimination and immunofluorescence and flow cytometry. Spleen cells activated by mitogens or alloantigen were homogeneously positive for cell surface Qa-2, but activated splenic T cells expressed only about one-third as much Qa-2 per cell as did nonstimulated T cells. These data correlated with the ability to perform cytotoxic elimination with Qa-2-specific monoclonal antibodies (mAbs) in that cytotoxic T lymphocyte (CTL) activity was completely abrogated by pretreatment of spleen cells prior to in vitro culture but was only partially eliminated by treatment of CTL effectors. Qa-2-positive cells constituted only a small subpopulation of fresh normal thymocytes, but were enriched (>40% positive) among cortisone-resistant thymocytes (CRT). These Qa-2-positive CRT contained mature thymocytes as defined by Ly phenotype Ly-2^–, Ly-1^hi. When normal thymocytes were treated with Qa-2-specific mAb and complement prior to in vitro sensitization for generation of allogeneic CTL, CTL activity was completely abrogated despite the fact that the fraction of cells eliminated were undetectable as assessed by cell recovery. CTL effectors from alloantigen-stimulated thymocytes were also susceptible to cytotoxic elimination with Qa-2-specific mAb. These data suggest that the Qa-2 molecule may serve not only as a marker on resting and activated peripheral T cells, but also as a unique marker for functionally mature T cells in the thymus.     

T cell differentiation in the thymus

T lymphocytes arise in the thymus and seed to peripheral lymphoid organs as fully functional cells at the time of exit. In humans, the thymus begins to function very early in ontogeny and releases large numbers of T cells before the time of birth. However, the vast majority of developing thymocytes (>95%) die within the thymus as a result of stringent selection processes. Positive selection imposes self-MHC-restriction on thymocytes and dictates the MHC-restricted repertoire of post-thymic T cells. Negative selection results in deletion of autoreactive cells. Both types of selection depend on cell to cell contracts and on the presence of appropriate growth factors which are still largely undetermined. Cell to cell contacts occur between developing thymocytes and cells of the thymic microenvironment (accessory cells), and are mediated by several receptor/ligand interactions which subserve the function of establishing and stabilizing these contacts. Besides MHC-TCR interactions, adhesion molecules are important for thymocyte maturation, selection and activation, and for the export and peripheral homing of mature T cells produced in the thymus. Here we describe a novel integrin involved in thymocyte-thymic epithelial cell interactions.     

T cell development in the thymus: from periodic seeding to constant output

T cell development occurs in the thymus throughout life. Recent experimental findings show that the seeding of the thymus by multi-potent stem cells from the bone marrow is periodic rather than continuous, as previously assumed. However it is well known that the output rate of cells from the thymus is relatively constant. A quantitative model is used to verify the current hypotheses regarding T cell development in the steady state mouse thymus. The results show that the thymus could be at a periodic steady state with out-of-phase thymocyte populations. Experiments to examine possible periodic fluctuations in the thymus are proposed and methods for further analysis are outlined.     

Lymphokine-induced cytotoxic cell generation in thymus and spleen cell cultures

The effect of a supernatant (SN-A) obtained from PMA-stimulated IL-2 producing EL-4 cells on cytotoxic cell induction in thymocyte and splenocyte cultures was evaluated. SN-A, but not recombinant IL-2 alone, induced cytotoxic cell differentiation in thymus cell cultures, thus indicating that factors distinct from IL-2 are required for effector cell generation. INF-gamma takes part in the process, and Ia+ accessory cell presence is also strictly required in thymus cultures for lymphokine-induced cytotoxic cell generation. Cytotoxic activity in thymocyte cultures was due only to Thy 1+ Lyt 2+ cells having a broad spectrum of target cell specificity, while in spleen cell cultures an effector population with NK activity was also generated.     

Effects of the removal of adherent and phagocytic cells on the spleen cell lymphoproliferative response of tumor-bearing mice

Summary Cell-mediated immunity was investigated in two BALB/c mouse tumor systems using the lymphoblastogenesis test with phytohemagglutinin as the mitogen. This lymphoproliferative response was quantitated using the Stimulation Index (SI). There was little evidence for suppressor cell activity in cell mixing experiments in which spleen cells from #51 cell-injected mice were mixed with spleen cells from normal mice. Following macrophage removal by Sephadex G-10 columns and carbonyl iron ingestion, there were no significant changes in the SI values for spleen cells from the #51 cell-injected mice. In contrast, spleen cells from mice injected with H238 cells, a herpes virus-transformed cell line, had a significantly lower SI value than that of normal mice. Suppressor cell activity was demonstrated in cell mixing experiments in which spleen cells from H238 cell-injected mice were mixed with normal spleen cells. Removal of adherent cells from spleen cells from H238 cell-injected mice by Sephadex G-10 columns restored the SI value to that of normal mice. An increased SI value was also seen after removal of phagocytic cells by carbonyl iron. These results suggested that cells with the functional properties of macrophages played an important part in the immunosuppression observed in the H238 tumor system. Comparison of the two macrophage depletion methods suggested that another cell population was also involved in the suppressive effect. Results of immunofluorescent techniques with anti-Lyt-1 and anti-Lyt-2 monoclonal antibodies show these cells to be Ly 1^–, Ly 2,3⁺ phenotypes of T-lymphocytes.     

T and B lymphocyte populations of the spleen and thymus in normal and immunosuppressed BALB/c mice.

The antituinor agent 1,3 bis (2-chloroethyl)-1-nitrosourea (BCNU) has been studied in order to determine its effect on thymic and splenic T and B lymphocytes in normal and immunosuppressed BALB/c mice. Utilizing indirect immunofluorescence and lymphocyte proliferation studies we detected an initial reduction of splenic T and B cells as a result of the administration of an optimal dose, 30 mg/kg, of BCNU. The population dynamics of the thymic lymphocytes are totally different in their mitogenic reactivity than that of the splenic lymphocytes. An initial decline in the PHA and LPS-sensitive splenic lymphocytes of BCNU-treated mice was temporary. However, there was no return to normal levels detected for the Con A-sensitive splenic lymphocytes. On the other hand, the PHA-sensitive thymic lymphocytes of BCNU-treated mice not only failed to repopulate but were totally depleted by the tenth day.     

Reconstitution of the thymus dependent area in the spleen of lethally irradiated mice

Cell and Tissue Research - To study the submicroscopical morphology of the microenvironment for T-lymphocytes in the spleen, mice were lethally X-irradiated and injected intravenously with...     

Immunosuppression of T lymphocyte function by fractionated serum from tumor-bearing mice.

Sera from mice with transplanted 3-methylcholantrene-induced tumors have been shown previously to inhibit the function of normal lymphoid cells. When chromatographed on Sephadex G-150, the fraction eluting with immunoglobulin has been shown to inhibit the proliferative response of normal spleen cells to concanavalin A and to inhibit the in vitro antibody response to a T-dependent antigen, but has a lesser effect on the antibody response to a T-independent antigen. This paper deals with studies on the mode of action of the serum factor. The immunoglobulin containing fraction of serum from tumor-bearing mice inhibited the in vitro generation of both allogeneic and syngeneic cytotoxic lymphocytes. Time course studies demonstrate that the serum fraction inhibits the generation of antibody-producing and cytotoxic lymphocytes if added during the first 2 days of a 5-day culture. Serum fractions added after day 2 had no effect on the in vitro response. The serum factor appears to inhibit the generation of specific T cell function during the proliferative stage of development but has no effect on the differentiation stage which leads to either antibody-producing cells or cytotoxic lymphocytes.