Macromolecular insoluble cold globulin (MICG): a marker for pluripotential hemopoietic stem cells

In embryonic mice pluripotential hemopoietic stem cells (PHSC) originate in the yolk sac and migrate to the fetal liver and from there to the bone marrow. Hemopoietic cells from yolk sac and fetal liver also migrate to the thymic primordium, and within the thymic environment these prothymocytes differentiate into mature T cells. We have recently demonstrated that macromolecular insoluble cold globulin (MICG), a T cell marker, is synthesized and inserted into the plasma membrane of embryonic prothymocytes as soon as these cells appear in the early thymus. In addition, we have shown that MICG+ cells are present within the fetal liver before the thymus has fully formed. In the present study we show that pluripotential hemopoietic stem cells in the fetal liver and bone marrow have MICG on their surface and represent a subpopulation of these MICG+ cells. The implications of these findings in relationship to stem cell differentiation and isolation are discussed.     

Macromolecular insoluble cold globulin (MICG): a novel protein from mouse lymphocytes. III. Relationship to the mixed lymphocyte reaction and effect of anti-MICG antiserum in vivo.

In the present communication we have examined the relationship between the synthesis of macromolecular insoluble cold globulin (MICG) and the mixed lymphocyte reaction (MLR). In addition, we have studied in vivo the effect of antiserum to MICG on the antibody response to sheep red blood cells. The experiments indicate that MICG synthesis compared to either IgM or total protein is selectively stimulated in responder T cells exposed to allogeneic stimulator cells in the MLR. Furthermore, cytotoxicity studies utilizing anti-MICG antiserum demonstrated that T cells bearing MICG on their surface are an essential component of the responder cell population in the MLR. In vivo administration of antiserum to MICG significantly suppressed both the primary and secondary antibody response to sheep red blood cells. A possible mechanism for this suppression is discussed.     

Amino acid transport in thymic- and spleen-derived lymphocytes.

We have examined the transport of amino acids by the sodium-dependent "A" and "ASC" system in thymic- and splenic-derived lymphocytes from the Long-Evans rat. Lymphocytes derived from the thymus transport amino acids by both the "A" and "ASC" systems, whereas lymphocytes from the spleen transport amino acids by the "ASC" system only. Thymic lymphocytes are capable of establishing a steady state distribution ratio of 7.9 for 2-aminoisobutyric acid, but splenic lymphocytes can attain only 3.5. The steady state distribution ratio of alanine was the same in both cell types. Sodium-independent transport is also different in splenic and thymic lymphocytes. But both cells move amino acids by a Na+-independent system for mediated exchange-diffusion. The studies show that lymphocytes derived from the spleen and thymus transport amino acids differently, and that the "T" lymphocytes from the spleen have membrane transport systems different from "T" lymphocytes from the thymus.     

Further studies on Th-B, a cell surface antigenic determinant present on mouse B cells, plasma cells and immature thymocytes.

A goat antiserum (Goat anti-M104E) has been produced which contains antibodies selectively cytotoxic for mouse B cells and a subpopulation of thymus cells. It reacts with the Th-B antigenic determinant which has been shown by us (1–3) to be present on B cells and on plasma cells and on some cells in the thymus. It also is very cytotoxic for mouse B cells while a previously developed rabbit antiserum was not. The antiserum was obtained by immunization with cells of the BALB/c mouse myeloma MOPC-104E. When the antiserum was purified by in vivo absorption in mice, antibodies remained which were cytotoxic for cells of all of several myelomas at a titer between 1:128 and 1:1024 as determined by an in vitro complement dependent cytotoxicity test. The in vivo purified antibodies were also cytotoxic for about 70% of thymus cells, for about 70% of spleen cells, for about 50% of lymph node cells and for about 20% of bone marrow cells. They were very cytotoxic for splenic or lymph node B cells separated from T cells by a nylon wool column and only slightly cytotoxic for splenic or lymph node T cells. The antibodies were only weakly cytotoxic for one out of five T cell tumors tested and not cytotoxic for the remaining four. Irrespective of target cells used, the cytotoxicity of purified Goat anti-M104E was easily removed by absorption with cell suspensions from tissues which contain B cells, plasma cells or thymus cells. In order to confirm that the same anti-Th-B antibodies recognize the determinant present on spleen cells and on some thymocytes, the purified Goat anti-M104E serum was absorbed with either spleen cells or thymus cells. The absorbed sera were tested for ability to label thymocytes or spleen cells using the fluorescence activated cell sorter (FACS). Either absorption removed essentially all the antibody capable of binding to either cell population. In addition it was shown, using the FACS, that only B cells and not T cells of the spleen contain the Th-B determinant. The anti-Th-B antibodies have now been used for the rapid elimination of B cells from a mixed population of lymphocytes without affecting the function of mature T cells. Thus in vitro treatment of spleen cells from SRBC-immunized donors with purified Goat anti-M104E plus complement results in the killing of a high proportion of the B memory cells as shown by the reduction of PFC produced when the treated cells are transferred to irradiated recipients. The T cell helper function of the transferred cells is not affected by Goat anti-M104E treatment as shown by appropriate cell transfer experiments in which effective B cells are provided by an AKR anti-Thy-1.2-treated spleen cell population and effective T cells are provided by the Goat anti-M104E-treated spleen cell population. Antibodies detecting Th-B may serve as an approach to understanding the ontogeny of lymphocytes. Our results suggest that Th-B is a cell surface marker appearing early in the development of lymphoid cells, on the common precursor of B and T cells and that it is lost from T cells as they mature in the thymus.     

The Croonian Lecture, 1992. The key role of the thymus in the body's defence strategies.

For centuries the thymus has remained a mysterious organ with largely unknown functions. The first demonstration of its crucial role in the development of the immune system was reported in 1961, when it was found that mice thymectomized at birth had poorly developed lymphoid tissues, impaired immune reactivities, and an inordinate susceptibility to develop infections. Although thymus lymphocytes were for a long time deemed immunoincompetent, it was shown in 1967 that they could respond to antigen by proliferating to give rise to a progeny of cells which did not secrete antibody (T cells), but which had a remarkable ability to induce bone marrow cells (B cells) to become antibody formers. This was the first unequivocal demonstration of a major division of labour among mammalian lymphocytes. Tremendous progress in our understanding of the function of the thymus and of the T cells derived from it followed. Distinct T cell subsets were characterized and shown to have an essential role in initiating and regulating a variety of immune responses. The ontogenetic events which occurred during their differentiation were mapped, and this allowed studies of the selection of the T cell repertoire. The major histocompatibility complex and associated peptides were shown to govern T cell selection and antigen activation, and the antigen-specific T cell receptor and the genes which code for it were characterized. Future studies should allow some insight into how to activate T cells more effectively for vaccination purposes, and how to switch them off to prevent autoimmune reactions and to induce tolerance to transplanted tissues.     

An antigen expressed by avian neuronal cells is also expressed by activated T lymphocytes.

A monoclonal antibody, anti-BEN, initially characterized by its reactivity with an epitope present on the surface of avian bursa epithelial cells and neurons, also reacts with membrane molecules on some hemopoietic cells. In this study we examine BEN expression on lymphoid cells in thymus, spleen, and blood. We demonstrate that BEN is an activation antigen on mature T lymphocytes. It is not expressed on peripheral blood or splenic lymphocytes, but following mitogenic or allogeneic stimulation of blood lymphocytes it appears rapidly on a T cell subpopulation in parallel with the appearance of IL-2 receptors. BEN is also expressed on III-C5 cells, an avian IL-2-dependent permanent T cell line, and on immature CD4+CD8+ thymocytes. BEN is not expressed by resting or actively proliferating B cells. Biochemical analyses of the BEN protein on T lymphoblasts shows that the molecule is similar in size to the BEN molecules on bursa epithelial cells and on neurons. The physicochemical properties of the BEN protein and its tissue distribution differs from other known avian and mammalian T cell activation markers, differentiation antigens, and integrins. Thus BEN is a novel marker of activated T cells in birds.     

Microtubule organization of lymphocytes and its modulation by patch and cap formation.

Microtubule organization of mouse splenic lymphocytes was investigated by immunofluorescence microscopy using specific tubulin antibody. Each resting lymphocyte was shown to contain well organized microtubules which are associated with a microtubule organization center at one end. Microtubule organization of lymphocytes was readily modulated during patch and cap formation of surface immunoglobulins. These modulations induced by antibodies directed against immunoglobulins were prevented when the cells were incubated with 100 μg ml^?1 of concanavalin A before the addition of antibodies. A modulation of microtubule organization was also detected when incubated in hypertonic medium where cap formation of various receptors was nonspecifically induced in the absence of ligand. The degree to which microtubule organization was modulated, however, was larger in cap formation induced with antibodies than in ligand-independent cap formation, probably because the latter does not require specific patch formation. On the cells forming cap in hypertonic medium, strong staining with tubulin antibody was observed beneath the cap, but the region of cap itself was not stained with tubulin antibody. A mitogenic dose of ConA did not readily induce any significant alteration of staining patterns with tubulin antibody but caused extreme changes in microtubule organization of lymphocytes after incubation for 48 hr. Growing lymphoma cells were shown to have poorly ordered microtubules and related structures. These results seem to suggest that the modulations of microtubule organization in lymphocytes are associated with cell surface events and cell growth.     

Studies of the mouse Ly-6 alloantigen system

Three monoclonal antibodies were produced by fusing mouse myeloma cell line NS-1 with spleen cells from C3H/An mice hyperimmunized with B6-H-2^k spleen cells. These antibodies recognized an alloantigen displaying a similar strain distribution pattern to the Ly-6.2 and Ala-1.2 alloantigens. Analysis of C×B and B×H recombinant inbred mice revealed close linkage of genes controlling Ly-m6 and Ly-6. The monoclonal antibodies lysed 70 percent of cells in lymph nodes and 60 percent in spleen in direct cytotoxicity assays, but did not lyse significant numbers of cells of thymus and bone marrow. Separated T and B cells were reactive with the antibodies, but T cells were more sensitive to the antibody and complement than B cells. Virtually all cells in cultures of cells activated in the mixed lymphocyte reaction or by Concanavalin A were reactive with the monoclonal antibodies. Direct plaque-forming cells were completely eliminated by the monoclonal antibody and complement. By absorption tests, cells from all organs tested so far (thymus, lymph node, spleen, bone marrow, brain, kidney and liver) were shown to express the Ly-m6 determinant. Tumor cell lines with T, B or stem cell characteristics were reactive with the monoclonal antibody by direct cytotoxicity and absorption assays.     

Peanut agglutinin. I. A new tool for studying T lymphocyte subpopulations.

Fluorescein-coupled peanut agglutinin (PNA) has been used at the single-cell level to study mouse lymphocyte subpopulations. PNA not only binds to most thymocytes, as has already been shown by other authors, but also binds to a small fraction of peripheral lymphocytes that are all T cells (theta+Ig-) or null cells (theta-Ig-). Most PNA-positive thymocytes are sensitive to in vivo corticosteroids and irradiation (450 rads) treatments. Conversely, the positive spleen cells (5% of total spleen lymphocytes) are essentially resistant to corticosteroids and irradiation. Study of PNA binding during ontogenesis shows the occurrence of PNA-positive cells in the fetal liver before thymus constitution and in the very beginning of embryonic thymus and spleen development. These data indicate that PNA is a marker of early T cell subpopulations but that there are probably several distinct subsets of PNA-positive T cells.     

小鼠乳腺表达抗PD-1抗体对其脾脏T细胞的影响

为从细胞水平研究小鼠乳腺表达抗PD-1抗体对转基因鼠脾脏T细胞表面抗原蛋白、细胞因子表达、脾脏CD4⁺T细胞增殖以及增殖相关通路的影响,将8周龄未经历过怀孕的和18周龄经历过哺乳的表达抗人PD-1抗体的转基因小鼠分成两组,每组以转基因阴性鼠为对照,提取脾脏淋巴细胞,检测脾脏淋巴细胞的变化。与转基因阴性小鼠相比,乳腺表达抗PD-1抗体的转基因小鼠的免疫系统中的脾脏T细胞的效应T细胞比例上升,Treg细胞比例下降,CD4⁺T细胞表达的IFN-γ、IL-17以及IL-2有不同程度的增加。IL-4、IL-10以及TGF-β都没有发生变化。与T细胞刺激相关的一些细胞表面的蛋白分子也没有引起变化。转基因阳性鼠和转基因阴性鼠中T细胞增殖没有显著性差异,转基因阳性鼠中PI3K/Akt/mTOR和Ras/MEK/ERK这两条通路上的磷酸化蛋白只有部分表达上调,整个通路没有完全上调。结果表明,转基因小鼠作为表达抗PD-1抗体这类免疫系统相关单克隆抗体的宿主是可行的。     

Ontogenetic analysis of some surface markers on pig lymphocytes using fluorescence-activated cell sorter

Surface markers were demonstrated on pig lymphocytes using anti-T cell-IgG and anti-Helix pomatia (HP) IgG during prenatal and postnatal development. A fluorescence-activated cell sorter analysis of T-cell surface markers was accompanied by an image analysis to prove the association of T antigenic determinants with the plasma membrane only. We found development-dependent changes in both anti-T cell and HP surface markers in both primary and secondary lymphatic organs. The number of T-positive (T+) cells estimated by anti-T cell-IgG was very similar to the results obtained by spontaneous E-rosette forming tests. At all selected age intervals, changes in the number of T+ cells were not significant in the thymus, but a marked increase in T+ cells was found in both spleen and lymph nodes. The image analysis confirmed the expression of T cell markers on the cell surface. The distribution of T cell markers was uneven, i.e. various degree of fluorescence intensity on whole ring-pattern projection of the cell surface image was estimated. In second lymphatic organs especially, fluorescence intensity of cells, i.e. total number of T cell markers estimated by anti-T cell-IgG, increased with age. On fetal day 73, T cell markers were slightly expressed, but very high fluorescence intensity and heterogeneous distribution of T cell markers on lymphocytes were found on fetal day 107 and postnatal day 56. The results indicate the possibility of functional maturation of various T cell markers on T cell subsets, furthermore a different degree of expression of T cell markers on various T cell subsets can be suggested. The number of HP+ cells increased with age in both primary and secondary lymphatic organs. In the prenatal period, the expression of HP receptors was very weak in both primary and secondary organs in contrast to the marked increase in HP+ cells during the postnatal interval. Differences in fluorescence intensity of cells were found, representing the increase by 22% in thymus cells comparing to cells of secondary lymphatic organs. Heterogeneity of HP+ cell populations in thymus was shown by the Scatchard plot, indicating at least two subpopulations of HP+ cells with different avidity to HP. Cells with low HP avidity could include a subset with cytolytic activity.     

Cell surface antigens expressed by subsets of pre-B cells and B cells

A large number of monoclonal antibodies, produced by immunizing rats with mouse pre-B cell lines, have been analyzed for their ability to define cell surface antigens expressed by B cells at early stages of differentiation. Whereas many antibodies recognized antigens on pre-B cell lines, only two clones detected cell surface antigens that were distinguished by their restricted distribution among a panel of continuous cell lines and cells from various tissues. Monoclonal antibody clone AA4.1 recognized a cell surface antigen found on all pre-B lymphomas and on one of three B lymphomas tested. This antigen was found on cells at highest frequency in the bone marrow. Adult spleen and fetal liver also have detectable numbers of AA4.1+ cells. Cells that did not express this antigen include plasmacytomas, two of three B lymphomas, T lymphomas, a stem cell line, adult liver, brain, thymus, and lymph node cells. Clone GF1.2 detected an antigen on some pre-B cell lines, one of three B lymphomas tested, and a small fraction of cells from adult bone marrow, spleen, lymph node, and fetal liver. Plasmacytomas, some pre-B lymphomas, two B lymphomas, T lymphomas, adult liver, brain, and thymus cells were negative. In adult bone marrow, AA4.1 bound to all cytoplasmic IgM+ pre-B cells, whereas GF1.2 detected one-half of these cells. Both antibodies recognized approximately 50% of surface IgM+ (sIgM+) bone marrow cells. A small population of bone marrow cells lacking any detectable Ig (surface or cytoplasmic) also reacted with these antibodies. Depletion of AA4.1 or GF1.2 antigen-bearing cells from bone marrow reduced the ability of bone marrow B cells to respond to LPS by 50 to 65%. Experiments with a cloned pre-B lymphoma demonstrate that AA4.1+ pre-B cells become sIgM+ GF1.2+ B cells after activation with LPS. These antibodies recognize cell surface determinants with restricted distribution among the B lymphocyte lineage because they detect antigens displayed by normal and transformed immature B lymphocytes.     

Human macromolecular insoluble cold globulin (MICG). II. Immunologic definition of T cell and null cell MICG and the biologic effect of antiserum to MICG.

Thymus cell-derived macromolecular insoluble cold globulin (T-MICG) is a 225,000-dalton protein, selectively synthesized in human T cells. Null cell-derived macromolecular insoluble cold globulin (N-MICG) is a 185,000-dalton protein, synthesized in null cells, and antigenically distinct from T-MICG. Evidence to support these conclusions was provided by using isolated cell preparations that were radiolabeled, lysed in desoxycholate, and precipitated with monospecific antiserum to each component. These studies demonstrated that antiserum to T-MICG precipitated a 225,000 dalton protein from PBL and T cells, but not from B or null cells. Antiserum to N-MICG reacted with a 185,000 dalton protein present in PBL and null cells, but not with lysates from either T or B cells. The plasma membrane distribution of these proteins was shown by absorption of antiserum to T + N-MICG with either isolated T or null cells. Antibody-induced cytotoxicity and immunofluorescence confirmed the cell surface location of T and N-MICG. Divergent biologic effects of these antisera were also noted. Antiserum to T-MICG inhibited T cell rosette formation and the one-way mixed lymphocyte reaction, although anti-N-MICG antiserum had no such effect. The potential importance of these proteins is discussed.     

Leu-1+ (CD5+) B cells. A major lymphoid subpopulation in human fetal spleen: phenotypic and functional studies

Examination of the cell surface phenotype of fetal splenic lymphocytes demonstrated a major, novel subpopulation of B cells that co-express Leu-1 (CD5) in addition to B cell differentiation antigens (Leu-1+ B cells). These cells are similar to some conventional B cells in that they express HLA-DR, Leu-12, and B1, as well as both immunoglobulin (Ig) M and IgD. They comprise 40 to 60% of total splenic B cells in the fetus but are infrequent in fetal liver and adult spleen. Fetal Leu-1+ B cells do not respond to pokeweed mitogen with either proliferation or Ig secretion, and in contrast to the murine counterpart, Ly-1 B cells, they do not constitutively produce Ig. Leu-1+ B cells were incapable of augmenting Ig production of Leu-1- B cells when suboptimal numbers of T cells were present; however, they did require the presence of T cells to secrete antibody. They do not cap either the CD5 protein or surface Ig. These cells are a unique subpopulation of fetal splenic B cells that do not function as conventional B cells. Their role in the humoral immune response is unknown. They may represent the normal stage of B cell development, which is reflected in the phenotype of B cell CLL cells.     

A membrane antigen of rabbit bursal equivalent cells.

Rabbit cells, bearing a specific antigen for bursal equivalent cells, could be detected with a suitably absorbed heterologous antiserum (goat). The antigen, detected with this antiserum, was designated RABELA. In the presence of complement, the RABELA antiserum lysed 80% of appendix cells, 50% of tonsil cells, 50% of spleen cells, and 25% of blood lymphocytes. It did not lyse a significant number of bone marrow or thymus cells.After complement-mediated kill with RABELA antiserum, cell populations lost the ability to respond to B mitogens. The responsiveness to the T mitogen, Concanavalin A, was reduced, but could be restored by addition of B cells which, alone, did not respond to Concanavalin A.RABELA-bearing subpopulations of spleen cells were characterized by velocity sedimentation analysis and were distinguished from subpopulations which took up thymidine after treatment with antibody directed against light chain allotypic specificity.     

The glycoprotein surface coat on different classes of murine lymphocytes

Both thymus and spleen lymphocytes of mice have been shown to possess a surface coat visible when the cells are stained with ruthenium red. Measurements on high-magnification photographs showed that the coat on thymus lymphocytes is significantly thicker than on spleen lymphocytes from genetically athymic nu/nu mice, which form a pure B cell population. Most of the coat on thymus cells is removed by treatment of the cells with neuraminidase or with trypsin, indicating that the coat is glycoprotein in nature. Functional implications of the difference in the cell coats of thymus and B cells are discussed.     

IgM complex receptors on subpopulations of murine lymphocytes.

Murine lymphocytes from spleen, lymph node, and thymus were examined for IgM complex receptors. Lymphocytes from all three organs were found to bind SRBC sensitized with IgM from various sources including: primary anti-SRBC serum, murine and rabbit anti-Escherichia coli LPS sera, and a murine IgM myeloma (MOPC 104E). Rosette formation by lymphocytes with IgM-sensitized SRBC was inhibited by soluble antigen-IgM complexes but not by IgM or antigen alone. Rosette formation was also inhibited by human IgM (Fc)5mu but not by Fab mu. Antiserum and complement treatment of the cells and subsequent recovery of the viable cells by trypsinization, filtration, and washing revealed the IgM rosette-forming cell (RFC) in the thymus to be a T cell. Spleen on the other hand was found to contain both B and T cells capable of binding IgM sensitized SRBC. Removal of both B and T cells from spleen cell suspensions eliminated all IgM RFC. The IgM complex receptor was found to be trypsin insensitive. Anti-Ig column fractionation enriched IgM RFC in spleen and lymph node suspensions passed through the columns, whereas cells bearing surface Ig, IgG complex receptors, and C3 receptors were retained in the columns.     

The separation of different cell classes from lymphoid organs. X. Preparative electrophoretic separation of lymphocyte sub-populations from mouse spleen and thoracic duct lymph

Conditions have been established for the separation of viable mouse lymphoid cells by continuous free-buffer film preparative electrophoresis. The detailed electrophoretic distribution profiles of T and B lymphocytes from mouse spleen and thoracic duct have been determined. Cell surface θ-antigen was used as a marker for T cells, and high surface-density of immunoglobulin as a marker for B cells. Spleen cells from athymic “nude” mice were also studied. In the unselected normal spleen cell populations B lymphocytes are heterogeneous, about 60% being of low mobility with the remainder distributing broadly, and extending into the highest mobility fractions. T lymphocytes are predominantly of high mobility. Lymphoid cells lacking markers of either the B or T lineage are of intermediate mobility. There is only partial separation of T and B cells because of the extensive overlap between the populations. The high mobility B cells, which separate along with T cells, include a substantial proportion of large cells, and include cells with high surface density of immunoglobulin. The majority of these large B cells can be selectively eliminated by their adherence on passage through a glass-bead column. By pre-selecting the 50% non-adherent lymphocytes from spleen as the starting material, a very sharp and more extensive separation of B and T cells can be achieved, with 100% pure B cells and 90% pure T cells in many fractions. However these samples are not representative of the total T and B cell populations of spleen. In thoracic duct lymph high mobility B-cells are absent, there is little overlap between T and B cell mobility. 100% pure T and B cells can be isolated.     

Glycolipid markers of murine lymphocyte subpopulations.

We have shown previously that purified antibodies to ganglioside GM1 react with peripheral T cells and most thymocytes in several strains of mice, independent of Thy-1 phenotype. GM1 and the Thy-1.2 antigen cap independently on C3H thymocytes, which provides additional evidence that GM1 is not the Thy-1.2 antigen. In C3H and nude mice antibodies to GM1 also react with a population of cells, comprising about 25% of lymphocytes from lymph nodes or spleen, that bear surface immunoglobulin. After removal of immunoglobulin from these cells by digestion with proteolytic enzyme, the GM1+ cells regenerate their surface immunoglobulin during 18 hr in culture, which indicates that these double-labeled cells synthesize their surface immunoglobulin. Protease treatment of lymphocytes reveals receptors for antibodies to GM1 on most cells. These data indicate that T and B cells differ in the accessibility of GM1 to antibody, and not necessarily in their content of GM1. Purified antibodies to asialo GM1 react with mature T cells in all strains of mice tested. In contrast to anti-GM1, these antibodies do not react with most thymocytes, with immunoglobulin-bearing lymphocytes of C3H or nude mice, nor with pronase-treated B cells.     

Production of BSF-1 during an in vivo, T-dependent immune response

BSF-1, a cytokine produced by some T lymphocyte tumors, has been shown to act with anti-Ig antibodies to stimulate B lymphocyte proliferation, to independently induce resting B lymphocytes to increase their expression of surface Ia antigen, and to induce some activated B lymphocytes to differentiate into IgG1- or IgE-secreting cells. To determine whether BSF-1 might be secreted by normal lymphoid cells in the course of a physiologic immune response, BALB/c mice were injected with an affinity-purified goat antibody to mouse IgD (GaM delta), which induces the generation of a large, polyclonal T-dependent IgG1 response; 4-hr culture supernatants of spleen cells from these mice were prepared, and these supernatants were assayed for BSF-1 activity by analyzing their ability to induce BALB/c nu/nu spleen cells to increase their expression of cell surface Ia in vitro. Culture supernatants of unfractionated spleen cells removed from mice 4 to 8 days after GaM delta antibody injection induced substantial increases in B lymphocyte surface Ia expression; these increases were blocked by a monoclonal anti-BSF-1 antibody. Culture supernatants of spleen cells from untreated BALB/c mice or from untreated or GaM delta antibody-treated BALB/c nu/nu mice induced small to moderate increases in B cell surface Ia expression, and GaM delta antibody itself induced large increases in B cell surface Ia expression; however, these increases were not significantly blocked by a monoclonal anti-BSF-1 antibody. A culture supernatant of T cell-enriched spleen cells from untreated mice induced small increases in B cell surface Ia expression that were inhibited by anti-BSF-1 antibody, as was the larger increase in B cell Ia expression induced by a culture supernatant of T cell-enriched spleen cells from mice sacrificed 3 days after GaM delta injection. On the other hand, T cell-depleted spleen cells from BALB/c mice injected with GaM delta antibody 7 days before sacrifice failed to generate culture supernatants with BSF-1 activity. Supernatants prepared from spleen cells taken from untreated mice or mice treated with GaM delta antibody 1 to 3 days before sacrifice did not block the ability of purified BSF-1 to induce an increase in B cell surface Ia expression, and thus did not contain inhibitors of BSF-1 activity.(ABSTRACT TRUNCATED AT 400 WORDS)