Regulation of lymphocyte production in the bone marrow. I. Turnover of small lymphocytes in mice depleted of B lymphocytes by treatment with anti-IgM antibodies

To examine the concept that the genesis of lymphocytes in the bone marrow may be regulated by homeostatic feedback signals from peripheral B lymphocytes or their products, lymphocyte production was measured in mice selectively depleted of B lymphocytes by repeated administration of anti-IgM antibodies from birth. The turnover of small lymphocytes was quantitated radioautographically after DNA labeling by continuous infusion of 3H-thymidine. In the femoral marrow of anti-IgM-treated mice, the number of small lymphocytes was reduced and their turnover time was shorter than in control mice, presumably reflecting the premature elimination from the marrow of maturing cells about to express surface IgM. The absolute number of small lymphocytes being produced per femur in unit time, however, was identical in anti-IgM-treated and control mice. Lymphocyte production in the thymus was also unaffected by anti-IgM suppression whereas in the spleen the turnover of small lymphocytes was reduced due to the lack of young immigrant B lymphocytes from the bone marrow. The results demonstrate that the normal large-scale production of lymphocytes in mouse bone marrow is independent of the magnitude of the peripheral pool of B lymphocytes or the level of circulating immunoglobulins, suggesting the process is not subject to feedback control. Some implications for the genesis and diversity of primary B lymphocytes are discussed.     

Regulation of bone marrow lymphocyte production: IV. Cells mediating the stimulation of marrow lymphocyte production by sheep red blood cells: Studies in anti-IgM-suppressed mice,athymic mice,and silica-treated mice

Some cellular requirements have been examined for the stimulation of lymphocyte production in mouse bone marrow by injected sheep red blood cells (SRBC). The increased genesis of marrow lymphocytes after a single dose of SRBC assayed radioautographically after [³H]thymidine labeling was unimpaired in the marrow of mice treated with anti-IgM antibodies from birth to eliminate B lymphocytes, and in congenitally athymic mice lacking T lymphocytes. However, pretreatment of mice with silica to depress macrophage function completely abolished the SRBC effect both on the total lymphocyte production and on the number of B and null small lymphocytes in the marrow. Comparative studies were performed on the thymus and spleen. The results demonstrate that the stimulation of marrow lymphocyte production by SRBC is mediated by a silica-sensitive mechanism, does not require B or T lymphocytes, and is independent of the humoral immune response. Thus, extrinsic agents may amplify the production of primary B cells and other lymphocytes in the bone marrow by an antigen-nonspecific mechanism, putatively mediated by macrophages.     

Heterogeneity of bone marrow lymphocytes: radioautographic detection of pre-B cells bearing cytoplasmic mu chains, and of B and T lymphocytes, and characterization of null lymphoid cells

A radioautographic immunolabeling technique has been developed to detect pre-B cells bearing cytoplasmic mu chains among populations of bone marrow lymphoid cells identified by conventional hematologic stains. 125I-Anti-mu antibody was applied either to fixed marrow smears, labeling total mu chains both in the cytoplasm (c mu) and at the cell surface (s mu), or to cell suspensions, labeling s mu alone. In stained radioautographs the incidence of c mu+ s mu- pre-B cells was derived both indirectly by subtracting values for s mu+ cells from those for total mu+ cells of various sizes in normal mice and directly by the total mu chain labeling in mice depleted of s mu+ cells by anti-IgM treatment in vivo. Binding specificity was demonstrated by the displacement of labeling by nonradioactive anti-mu antibody. The c mu+ s mu- cells showed a bimodal size distribution. They accounted for 40% of the large lymphoid cells and 30% of the small lymphocytes in the marrow. A further 50% of the small lymphocytes were B lymphocytes (s mu+) and 8% were T lymphocytes (Thy 1.2+). Thus, the technique demonstrates the presence of c mu+ s mu- pre-B cells among both proliferating large lymphoid cells and nondividing small lymphocytes, as classically defined in marrow smears. In addition, the results reveal a broad size distribution of mu- lymphoid cells, including a subset of small lymphocytes which lack c mu, s mu, and Thy 1.2 and thus cannot be assigned to either B or T lineage by these criteria. The findings suggest that in addition to B cells the marrow may produce other types of lymphoid cells, yet to be defined.     

Protein kinase C mobilization in B lymphocytes. Differential isoenzyme translocation upon activation

The subcellular distribution of protein kinase C has been analyzed in murine B lymphocytes exposed to LPS, anti-IgM antibodies and phorbol dibutyrate. An accurate determination of the enzyme mobilized from the soluble to the particulate fractions by these activators, has been made possible by the use of B cells in which the major part of the activity was present in the cytosol. Upon stimulation, we have analyzed the isoenzymatic forms translocated to the B cell membrane, showing a differential pattern of isoenzyme mobilization between LPS and anti-IgM antibodies. These data, together with the different Ca2+ requirements for the activation of the translocated protein kinase C isoenzymes, might help to unravel the mechanism responsible for the clonal expansion and differentiation of B lymphocytes, induced by the two ligands.     

Respiratory burst in human B lymphocytes. Triggering of surface Ig receptors induces modulation of chemiluminescence signal.

B lymphocytes have been shown to proliferate and release oxygen metabolites when surface Ig is cross-linked and when stimulated with phorbol ester. Biochemical evidence has been provided for the presence of a superoxide generating system in B cells, which seems to be identical to the well-characterized NADPH-oxidase of phagocytes. In this report, we show that normal and EBV-transformed B cells produce superoxide anions after stimulation with phorbol ester and when surface Ig was cross-linked, as detected by lucigenin-dependent chemiluminescence. Anti-surface IgG antibodies induced a significant respiratory burst whereas those directed against surface IgM had no effect on B cell oxidative metabolism. Prestimulated B lymphocytes responded to further triggering by the same or another ligand. Pretreatment with Staphlococcus aureus Cowan I strain (SAC) or anti-IgM antibodies resulted in complete unresponsiveness to subsequent SAC or anti-IgG stimulation, but it did not affect PMA- and ionomycin-mediated B cell chemiluminescence. In contrast to preincubation with anti-IgM antibodies, the pretreatment of B cells with SAC induced a transient inhibitory effect on B cell signaling. In fact, SAC-pretreated B lymphocytes could be restimulated with the same ligand when blast cells were isolated. Furthermore, a 24-h incubation of the pretreated B cells in the absence of SAC completely restored the SAC-mediated respiratory burst. These results suggest that two distinct mechanisms may account for SAC- and anti-IgM-induced inhibition: a transient and reversible modulation of surface Ig, induced by SAC, and a long-lasting desensitization of the surface Ig receptors, respectively. These findings may have interesting implications for understanding the transduction of negative signals in B lymphocytes.     

Experimental autoimmune thyroiditis in mice chronically treated from birth with anti-IgM antibodies

The role of T lymphocytes in the pathogenesis of experimental autoimmune thyroiditis in mice is well established while the role of B lymphocytes is unclear. Mice with thyroid lesions have thyroglobulin antibodies whereas these antibodies can occur in mice immunized with Tg that do not develop thyroid lesions. To determine whether thyroglobulin antibodies are necessary for the development of the thyroid infiltrates with mononuclear cells, which are characteristic for experimental autoimmune thyroiditis, AKR mice chronically treated from birth with goat anti-mouse IgM antibodies were immunized with mouse thyroglobulin in Freund's complete adjuvant when they were 7 weeks old. Control mice, similarly immunized, were chronically injected from birth with normal goat gamma-globulin. Three weeks after immunization, all mice were sacrificed, thyroglobulin antibodies in the serum were measured by hemagglutination assay and enzyme-linked immunosorbent assay, and thyroid pathology was assessed. The serum concentration of IgG and IgM, the percentage of B and T lymphocytes in the spleen (flow cytometry), and the in vitro proliferative response of spleen lymphocytes to stimulation by PHA, LPS, and Tg were also measured. All mice treated with anti-IgM antibodies did not have detectable thyroglobulin antibodies but 63% of these mice and 88% of control mice (all of which had thyroglobulin antibodies) had thyroid lesions. Mice treated with anti-IgM antibodies that did not have thyroid lesions had a more pronounced depression of B lymphocytes than similarly treated mice that had thyroid lesions. These experiments suggest that thyroglobulin antibodies are not necessary for the development of thyroid infiltrates with mononuclear cells. B lymphocytes could still participate in the production of experimental autoimmune thyroiditis by presenting thyroglobulin to helper T lymphocytes.     

Stimulation of B lymphocytes through surface Ig receptors induces LFA-1 and ICAM-1-dependent adhesion.

Engagement of the surface Ig receptor with anti-IgM antibodies stimulates murine B lymphocytes to markedly increase their expression of the cell adhesion molecules ICAM-1 and LFA-1. Stimulated B cells display increased homotypic adhesiveness and form spontaneous heterotypic conjugates with T lymphocytes. This latter T-B cell interaction is further enhanced if T cells have been previously activated with phorbol esters. In all cases, the formation of cell-cell conjugates is dependent on LFA-1-ICAM-1-mediated interactions as assessed in mAb blocking experiments. B lymphocytes stimulated with anti-IgM display a marked increase in binding to ICAM-1-transfected L cells. This cell-cell interaction is inhibited by anti-LFA-1 mAb binding to the B lymphocyte. Together, these results demonstrate that there is an induction of both ICAM-1 and LFA-1 on stimulated B cells and a corresponding increase in the adhesiveness of these cells. These findings suggest that Ag binding to the surface Ig receptor could prepare a B lymphocyte for subsequent interaction with a T lymphocyte. This provides insight into how efficient T-B collaboration may occur between very infrequent Ag-specific lymphocytes.     

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.     

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.     

A Repopulation Assay For B and T Lymphocyte Stem Cells Employing Radiation Chimaeras

The repopulation of the peripheral lymphoid compartment of lethally-irradiated rats reconstituted with lymphopoietic stem cells was studied. Cell lineages were traced by using genetic markers of cell surface molecules: immunoglobulin allotype for B lymphocytes and peripheral T cell alloantigen for T lymphocytes. Provided the markers had been bred on to a genetic background congenic with the hosts, they conferred neither an advantage nor disadvantage in competition with unmarked cells. the degree of chimaerism measured the lymphopoietic activity of the restorative inoculum. the most potent activity was found in foetal liver and spleen; next was infant spleen and bone marrow; then young adult bone marrow. Peripheral lymphoid tissues showed very little activity and thymus cells were inert. This tissue distribution, the stability of the chimaerism and the substantial expansion of numbers from the injected cells all point to the assay measuring an early stem cell. The overlap of subpopulations of lymphocytes in the rat thoracic duct was studied. A method for the conjugation of fluorescein to antibodies while they are attached to immuno-adsorbent affinity columns is also described.     

B lymphocytes are required for the generation of T cells that mediate healing of cutaneous leishmaniasis

The role that B lymphocytes and/or antibodies play in the healing of Leishmania major infections in genetically resistant C3H/HeN mice was investigated by monitoring the course of infection in animals that had been B cell depleted by treatment from birth with anti-IgM sera (mu-suppressed). L. major infection of mu-suppressed C3H/HeN mice produced lesions that were significantly larger than those induced in control animals, and failed to heal. Moreover, vaccinated mu-suppressed mice also developed chronic nonhealing infections, although their lesions were initially smaller than those developed by nonvaccinated mu-suppressed controls. The enhanced susceptibility of mu-suppressed mice could be completely overcome by adoptive transfer of T lymphocytes from mice that had spontaneously healed their lesions, and to a lesser extent by T lymphocytes from normal animals. Anti-leishmanial antibody responses were completely absent in mu-suppressed mice, regardless of whether they were lymphocyte reconstituted, whereas delayed type hypersensitivity (DTH) to leishmanial antigens was present in normal and mu-suppressed animals. The ability of immune T cells to protect mu-suppressed mice without restoring humoral responsiveness clearly indicates that antibodies are not necessary for healing leishmanial infections. Instead, the observed effect of mu-suppression argues that B lymphocytes are required for the generation of an effector T cell population, apparently unrelated to DTH, which mediates the healing of cutaneous lesions. These results thus provide the first evidence for the B cell and/or Ig dependency of a T cell population that is critical for the development of immunity against a microbial agent.     

The reactivity of frozen B lymphocytes to B cell mitogens and human B cell growth factor: A study of step 1 and step 2 activators

Human B lymphocytes, purified from the peripheral blood of several different donors can be pooled, frozen, and stored in liquid nitrogen to provide an easy and reproducible source of cells for mitogenic assays. These B cell preparations did not show any reactivity to T cell mitogens, but responded to Staphylococcus aureus Cowan strain 1 (SAC) and anti-IgM antibodies to the same extent as freshly purified B cells. When stimulated with either anti-IgM antibodies or SAC, these B cells became responsive to B cell growth factor (BCGF), allowing a quantitative measurement of this important lymphokine activity. In addition, we have studied the reactivity of frozen B lymphocytes to various combinations of activators. We have confirmed that phorbol myristate acetate (PMA) was a very potent mitogenic agent for preactivated human B cells and shown that bacterial lipopolysaccharide (LPS), although not mitogenic by itself, could synergize with anti-IgM antibodies to yield increased levels of stimulation. Furthermore experiments using the lysosomotropic agent leucine methyl ester showed that the action of LPS on anti-IgM-stimulated B cells did not require the presence of functional monocytes. Neither PMA nor LPS could induce BCGF responsiveness and thus these two compounds can be considered exclusive step 2 activators for human peripheral blood B cells.     

Anti-immunoglobulin M induces both B-lymphocyte proliferation and differentiation in Xenopus laevis

Anti-IgM induced the proliferation of spleen lymphocytes of the amphibian Xenopus laevis as determined by 3H-thymidine uptake. The responding cells were B lymphocytes, since lymphocyte populations enriched in surface-Ig-positive cells exhibited an increased proliferative response, and spleen cells from larvally thymectomized animals still responded to anti-IgM. Immunofluorescence analysis and gel electrophoresis of biosynthetically labeled Ig polypeptides revealed that lymphoblasts induced by anti-IgM differentiated into plasmablasts that synthesized and secreted mainly IgM and small amounts of IgY. The in vitro differentiation of B lymphocytes also occurred in spleen cells obtained from thymectomized animals. These findings are in contrast with those obtained in mammals and suggest that the differentiation of B lymphocytes in X. laevis is subject to different regulatory mechanisms.     

Pre-B cells: bone marrow persistence in anti-mu-suppressed mice, conversion to B lymphocytes, and recovery after destruction by cyclophosphamide.

Chronic treatment of mice from birth with anti-mu antibodies aborts development of B lymphocytes and plasma cells. In these studies we show that bone marrow from anti-mu-treated mice contains a population of cells with cytoplasmic IgM, but which lack detectable cell-surface IgM. These cells are analogous to pre-B cells, defined in ontogenetic studies as the immediate precursors of B lymphocytes. Pre-B cells from bone marrow of anti-mu treated mice retain their functional integrity, as evidenced by their ability to give rise to sIgM+, LPS-responsive lymphocytes in culture. We also show that cyclophosphamide treatment destroys pre-B cells and that recovery of pre-B cells in bone marrow precedes the regeneration of sIgM+ B lymphocytes. Generation of B lymphocytes in adult mice apparently occurs exclusively in the bone marrow because induction of extramedullary hemopoiesis in spleen was not accompanied by the appearance of pre-B cells in that organ.     

Regulation of B-lymphocyte activation, proliferation, and immunoglobulin secretion

Lymphocyte growth and differentiation are controlled by signals resulting from the interaction of antigen and cellular products, such as lymphokines, with specific cell membrane receptors. Resting B lymphocytes can be activated by low concentrations (1-5 micrograms/ml) of antibodies to membrane IgM, which is the B-lymphocyte receptor for antigen. The binding of anti-IgM to B cells causes a rapid increase in intracellular free calcium concentration ([Ca2+]i), in inositol phosphate concentration, and in protein kinase activity. Moreover, the effects of anti-IgM on B cells are mimicked by the combined use of calcium ionophores and phorbol esters. Since phorbol esters activate protein kinase c, this suggests that the increase in [Ca2+]i and in phosphatidylinositol metabolism stimulated by anti-IgM are critical events in B-cell activation. The entry into S phase of B cells stimulated with anti-IgM depends on the action of a T-cell-derived factor designated B-cell stimulatory factor (BSF)-1. This is a 20,000-Da protein which is a powerful inducer of class II major histocompatibility complex molecules. Although an important cofactor for B-cell proliferative responses to anti-IgM, its major locus of action is on resting B cells. B cells stimulated with anti-IgM and BSF-1 do not synthesize secretory IgM. However, if two additional T-cell-derived factors, B151-TRF and interleukin-2, are added to cultures, a substantial proportion of stimulated B cells produce secretory IgM. BSF-1 has also been shown to participate in the "switch" in Ig class expression. Resting B cells cultured with lipopolysaccharide will switch to IgG1 secretion in the presence of purified BSF-1.     

Changes in the populations of null, NK1.1+, and Thy1lo lymphocytes in the bone marrow of tumor-bearing mice: effect of indomethacin treatment.

Mouse bone marrow produces many "null" lymphocytes which lack B and T lineage markers (B220-Thy1-). A subset of these cells expresses the natural killer (NK) cell marker, NK1.1. In addition, some rapidly renewed bone marrow lymphocytes express low intensities of Thy1 (Thy1lo). In view of their possible implication in tumor-host interactions these various cell populations have now been examined in mice injected with either the nonmetastatic Ehrlich ascites (EA) tumor or the Lewis lung carcinoma (LLc), a highly metastatic solid tumor. In each case, the number of null lymphocytes, as defined by a lack of radioautographic labeling of either B220 glycoprotein or Thy1, increased markedly in both the bone marrow and spleen. Treatment with the prostaglandin inhibitor, indomethacin, enhanced the increase in null cells in the bone marrow and spleen of LLc-bearing mice. The number of null small lymphocytes expressing NK1.1, as detected by combined radioautographic and immunoperoxidase techniques, increased almost 30-fold in LLc-bearing mice. The number of Thy1lo small lymphocytes increased in parallel with null cells during EA tumor growth. The findings accord with the hypothesis that the null lymphocyte population produced in mouse bone marrow includes newly formed NK lineage cells which sequentially express NK1.1 and Thy1lo. The present work demonstrates that the populations of null, NK1.1+, and Thy1lo lymphocytes in mouse bone marrow expand rapidly during the early growth of transplanted tumors, the initial increase in null lymphocytes apparently being curtailed by prostaglandin production. The results suggest that the production of null lymphocytes in mouse bone marrow is responsive to tumor development, possibly providing cells to be involved in tumor-host interactions.     

Growth regulation of a human mature B cell line, B104, by anti-IgM and anti-IgD antibodies

An EBNA- human B lymphoma cell line, B104, was established. B104 cells express IgD as well as IgM on their surface, which is thought to be a basic characteristic of mature B cells. The growth of B104 cells was inhibited by treatment with a panel of anti-IgM antibodies. Cell cycle analyses revealed that the transition of B104 cells from the G2/M to the G0/G1 phase of the cell cycle was markedly inhibited by treatment with anti-IgM antibodies. Progression of B104 cells to the M phase of the cell cycle was found to be suppressed in the presence of anti-IgM antibodies. In contrast, both the entrance of G0/G1 phase cells into the S phase and the progression of S phase cells to the G2/M phase of the cell cycle did not seem to be inhibited significantly by treatment with anti-IgM antibodies. These results indicate that the mechanism of the inhibition of growth of B104 cells by anti-IgM antibodies is blockage of the transition from the G2 to the M phase of the cell cycle. In contrast to anti-IgM antibodies, anti-IgD antibodies could not cause growth inhibition of B104 cells at all. B cell growth factors such as IL-4 and IL-6 had no effect on the inhibition of growth of B104 cells by anti-IgM antibody. IFN-alpha and -beta, which have no B cell growth factor activity, did increase the number of cells that survived the treatment with anti-IgM antibodies. B104 is an excellent experimental model for the study of the mechanism of signal transduction through sIg as well as the functional difference between sIgM and sIgD.     

T-cell function in B-lymphocyte-deprived mice

Previous work has identified selective defect(s) in T cells in mice deprived of B lymphocytes by the chronic administration of anti-IgM antibody. Experiments described in the present communication revealed that anti-IgM-treated mice do not possess T cells with surface Ia and FcR, and, unlike T cells from normal animals, they also fail to bind these molecules in vitro. Functional assays disclosed that an anti-suppressor pathway which relies on Ia+ donor and acceptor T cells is interrupted in these mice at both levels. These observations may provide an insight to explain the selective failure of some T cells when B lymphocytes have been deleted.     

Signal requirements for growth and differentiation of activated murine B lymphocytes

Mouse B lymphocytes were stimulated at high cell concentrations with goat anti-IgM antibodies, which leads to the induction of B cell proliferation without the addition of any growth factors. After 48 hr, blast cells were purified and cultured at low cell concentrations. Proliferation and differentiation of purified B lymphocyte blasts is then dependent on the addition of either mitogens (e.g., LPS) or certain lymphokines derived from activated T cells or macrophages. One such lymphokine was isolated from supernatants of various activated T cells and characterized by gel filtration as a material with an apparent m.w. of 40,000 to 50,000, similar to BCGF II. It supports the proliferation of the B cell blasts and induces their differentiation into plaque-forming cells. Lymphokines such as BCGF I, interleukin 2, and BCDF gamma could neither maintain growth nor induce differentiation of B lymphocytes preactivated by goat anti-IgM.