The role of IL 1 in the antigen-specific activation of murine class II-restricted T lymphocyte clones

The role of IL 1 in the antigen-specific activation of class II-restricted T lymphocytes was examined by using a model system consisting of cloned WEHI 5 B lymphoma accessory cells and class II-restricted, soluble antigen- or alloantigen-reactive T cell clones. The addition of exogenous recombinant IL 1 to the T cell cultures resulted in a significant enhancement of the antigen-specific T cell proliferation response, but at best, only small increases in IL 2 release. Goat IgG anti-IL 1 antibodies were added to the T cell cultures to assess their effect on T cell activation. The IL 1 enhancement of the T cell proliferation response was inhibited by the anti-IL 1 antibodies in a dose-dependent manner. In contrast, only modest levels (10 to 25%) of proliferation inhibition were observed in T cell cultures containing either WEHI 5 or splenocyte accessory cells but no exogenous IL 1. When the anti-IL 1 antibodies were added to primary mixed lymphocyte cultures stimulated by WEHI 5 cells in the absence of exogenous IL 1, no significant inhibition of proliferation was observed. A small but statistically significant proliferation inhibition was observed when anti-IL 1 antibodies were added to mixed lymphocyte reaction cultures stimulated by splenocytes. Two-color cytofluorometric analysis of the effects of IL 1 on antigen-activated T cell clones demonstrated that under suboptimal stimulation conditions, IL 1 stimulated a small but significant increase in the number of T cells bearing IL 2 receptors. In the presence of optimal numbers of WEHI 5 accessory cells, IL 1 enhanced T cell proliferation in the absence of a detectable increase in the number of T cells bearing IL 2 receptors, the number of IL 2 receptors per T cell, or the levels of IL 2 released. Finally, exogenous IL 1 can be added as late as 18 to 24 hr after culture initiation without significantly reducing its ability to enhance the T cell proliferation response. These data indicate that IL 1 has pleiotropic effects on murine T lymphocytes and can function to enhance T cell activation at multiple points during the activation sequence.     

Development and characterization of a human B cell line that responds to B cell growth factor but not interleukin 2

The human lymphoblastoid cell line we present here proliferated in response to a 14,000 m.w. B cell growth factor (BCGF), and not to interleukin 2 (IL 2). This cell line, designated B-A3, was established by Epstein Barr virus (EBV) transformation of Staphylococcus aureus Cowan I (SAC)-activated spleen B cells, and has been maintained in RPMI 1640 medium complemented with 15% fetal calf serum (FCS) without the addition of other exogenous growth factors. A proliferative response, as measured by [3H]thymidine uptake of B-A3 cells was significantly induced by either commercial IL 2-free human BCGF preparations, or phytohemagglutinin-stimulated mixed lymphocyte culture supernatant at all FCS concentrations used in the assay. The most marked proliferation due to BCGF, however, was observed in the absence of FCS. This BCGF-induced proliferation was not influenced by IL 2 or interferon-gamma (IFN-gamma), because both recombinant IL 2 and IFN-gamma failed to induce proliferation. The response of B-A3 cells to a specific BCGF was additionally indicated by the responsiveness of this cell line to BCGF purified by a series of chromatographic steps. The BCGF to which B-A3 cells responded had a m.w. of 14,000 and was similar to low m.w. BCGF reported from other laboratories. Surface characterization of B-A3 cells, analyzed by flow cytometry with a panel of monoclonal antibodies, demonstrated that the majority of B-A3 cells were stained positively with Leu-12, HLA-DR, and surface IgG markers, whereas staining with surface IgM, IgD markers, pan T cell markers (Leu-4 and Leu-9), and IL 2 receptor (Tac) were consistently negative. Taken together, the human lymphoblastoid cell line we present here responded specifically to a low m.w. BCGF. This cell line may be of value in the purification of BCGF to homogeneity, in studies of the interactions of BCGF with human B cells, and in the identification of the BCGF receptor.     

Interleukin 2 receptors are expressed by alveolar macrophages during pulmonary sarcoidosis and are inducible by lymphokine treatment of normal human lung macrophages, blood monocytes, and monocyte cell lines

Expression of receptors for IL 2 was believed initially to be restricted to T cells after their activation by IL 1 and antigen. However, recently IL 2 receptors (IL 2R) were demonstrated on activated B cells by using an anti-IL 2R monoclonal antibody (anti-Tac). In this study, we examined the capacity of cultured human alveolar macrophages, blood monocytes, and myelomonocytic (HL-60) or monoblast (U937) cell lines to bind three different anti-IL 2R monoclonal antibodies before or after stimulation with the monocyte-activating agents IFN-gamma, LPS, phorbol ester, or lymphokine-containing conditioned medium. For each of the four cell populations examined, resting unstimulated cells bound little or no anti-IL 2R antibody, as shown independently by quantitative cell binding assay and by immunoperoxidase labeling. By contrast, incubation with recombinant IFN-gamma, conditioned medium, or to a lesser extent, native or recombinant IL 2 itself, resulted in a significant enhancement of anti-IL 2 receptor monoclonal antibody binding by all four populations, whereas LPS, PMA, or IL 1 had no effect. In addition, membrane binding of anti-Tac antibody, similar to that seen after stimulation of normal lung macrophages with IFN-gamma, was detected by using macrophages obtained by bronchoalveolar lavage of five patients with active pulmonary sarcoidosis. These findings are consistent with the expression of a functional IL 2R on activated cells of the monocyte lineage, since anti-Tac binding to IFN-gamma-treated HL-60 cells was inhibited by addition of excess IL-2; specific binding of anti-IL 2 monoclonal antibodies was detected in the presence of exogenous IL 2; and a 50 to 55 kD molecule was immunoprecipitated from both activated lung macrophages and T lymphoblasts by using anti-Tac antibody. We conclude that human mononuclear phagocytes can be induced by lymphokines to express IL 2R, and that such IL 2R+ macrophages can be detected in vivo during inflammation.     

Monoclonal antibodies to the human C3b/C4b receptor (CR1) enhance specific B cell differentiation

The addition of monoclonal antibodies against the human C3b/C4b receptor (CR1) to cultures of peripheral blood lymphocytes in the presence of suboptimal amounts of TNP bound to polyacrylamide beads enhanced by 150 to 400% the specific anti-TNP response, as measured by a plaque-forming cell assay on day 7. Anti-CR1 antibodies similarly enhanced the anti-fluorescein antibody response. Enhancement only occurred in cultures performed in the presence of the relevant antigen. No enhancing effect on the anti-TNP response was observed on addition to cultures of monoclonal antibodies directed against other surface antigens of B cells or an anti-T cell antibody of the same subclass as that of anti-CR1 antibodies. Anti-CR1 antibodies alone did not induce nonspecific B cell proliferation and did not provide B cells with a first signal for proliferation in the presence of a source of B cell growth factors. Anti-CR1 antibodies did not enhance the nonspecific proliferative response of B cells to growth factors derived from PHA-stimulated T cells, semi-purified BCGF 20 KD, BCGF 50 KD, or recombinant IL 2 in the presence of anti-mu. In this respect, the effect of anti-CR1 antibodies differs from that of anti-CR2 antibodies which interact with early stages of B cell activation. In contrast, anti-CR1 antibodies enhanced specific differentiation of antigen-activated B cells in the absence of T cells when soluble T cell factors were provided. Similar results were obtained by using either of two sources of differentiation factors, the MLA-144 supernatant or a 30 to 15 KD fraction from PHA-stimulated T cells. These results indicate that triggering of CR1 on B cells positively regulates the specific antibody response to low doses of antigen by enhancing B cell differentiation whether T cell help is provided by intact T cells or by T cell-derived differentiation factors.     

cAMP is an essential signal in the induction of antibody production by B cells but inhibits helper function of T cells

Dibutyryl cAMP and IL 1 were found to stimulate antigen-specific and polyclonal antibody production when added together to cultures of highly purified B cells. We propose that IL 1 and an elevation in cytoplasmic cAMP represent minimal signal requirements for B cell activation. In contrast to its effect on B cells, dibutyryl cAMP inhibited helper T cell activity. Cyclic AMP suppressed the production of IL 2 and T cell replacing factor (TRF) by T cells and thus abrogated the ability of helper T cells to enhance SRBC-specific antibody production by B cells. Cyclic AMP did not inhibit the generation by T cells of B cell growth factor (BCGF). BCGF, not normally detected in Con A supernatant, was found in the culture supernatant of spleen cells that were stimulated with Con A in the presence of cAMP. Our findings indicate that cAMP blocks the production of an inhibitor of BCGF activity. cAMP had no effect on the production by macrophages of IL 1.     

Induction of IL 2 receptor expression and cytotoxicity of thymocytes by stimulation with TCF1

We investigated the role of T cell cytotoxicity inducing factor 1 (TCF1) in the induction of a cytotoxic T cell response. We found that help-deficient thymocyte cultures supplied with saturating amounts of purified IL 2 did not develop CTL in a 5-day culture. The expression of cytotoxicity was dependent on the addition of TCF1 derived from the T cell hybridoma K15. TCF1 also induced proliferation of thymocytes in the presence of IL 2. Only the PNA- thymocyte subpopulation responded to TCF1 with proliferation and cytotoxicity in the presence of IL 2. The monokine IL 1 also induced proliferation in this subpopulation but failed to induce cytotoxicity. IL 1 was further distinguished from TCF1 by inhibition of IL 1-induced but not TCF1-induced proliferation by anti-IL 1 antibodies. In addition, using anti-IL 2 receptor antibodies (AMT 13), we showed that TCF1 in the presence of IL 2 substantially increased IL 2 receptor expression in thymocytes. IL 1 had the same effect on induction of IL 2 receptor expression as TCF1. Because some effects of IL 1 and TCF1 are distinct and some overlap, we discuss whether IL 1 and TCF1 induce different subsets of PNA- thymocytes.     

Production of B cell growth factor by a Leu-7+, OKM1+ non-T cell with the features of large granular lymphocytes (LGL)

The present study reports the characterization of a non-T cell from human peripheral blood which is capable of releasing BCGF. This BCGF-producing non-T cell had a T3-, T8-, Leu-7+, OKM1+, HLA-DR-, Leu-11- surface phenotype and was likely to belong to the so-called large granular lymphocyte (LGL) subset because: after fractionation of non-T cells according to the expression of Leu-7 or HLA-DR markers, it was found in the Leu-7+, HLA-DR- fractions that were particularly enriched in LGL; it co-purified with LGL on Percoll density gradients; and it expressed Leu-7 and OKM1 markers that are shared by a large fraction of LGL. Although co-purified with cells with potent NK capacities, the BCGF-producing cell was not cytotoxic, because treatment of Leu-7+ cells with Leu-11 monoclonal antibody and complement abolished the NK activity but left the BCGF activity unaltered. The factor released by this LGL subset was not IL 1 or IL 2 mistakenly interpreted as BCGF, because: a) cell supernatants particularly rich in BCGF activity contained very little or no IL 1 or IL 2; b) BCGF-induced B cell proliferation was not inhibitable by anti-Tac antibodies (this in spite of the expression of IL 2 receptor by a proportion of activated B cells); and c) BCGF activity was absorbed by B but not T blasts.     

Characterization of a T4+/Leu-8+ T cell clone that directly helps B cell Ig production by secreting B cell differentiation factor

A human T4+/Leu-8+ T cell clone (YA2) was established by phytohemagglutinin activation and interleukin 2 (IL 2) propagation. Functional characterization of this clone demonstrated that it provided potent help towards Ig production by pokeweed mitogen-stimulated B cells in the presence of small numbers of autologous T cells or by Staphylococcus aureus Cowan I (SAC)-activated B cells in the presence of B cell growth factor (BCGF). YA2 provided no help to resting B cells and minimal help to either unactivated B cells cultured with BCGF or SAC-activated B cells. Supernatant generated from clone YA2 by IL 2 stimulation had significant B cell differentiation activity but no BCGF or IL 2 activity. Thus, YA2 is a T4+/Leu-8+ potent direct helper only to B cells that are activated and proliferating due to its selective secretion of a differentiation factor, and not an activation and growth factor. The availability of phenotypically defined cloned populations of T cells with restricted functional helper activity related to the secretion of selected B cell tropic factors should prove useful in the dissection of the role of individual T cell subsets in the regulation of the human B cell cycle.     

Human B cell responsiveness to B cell growth factor after activation by phorbol ester and monoclonal anti-mu antibody

The effect of phorbol ester on human B cell activation was examined. Picomolar to nanomolar concentrations of phorbol ester induced a high level of proliferation in small IgM-positive B cells isolated from peripheral blood by fluorescence-activated cell sorting. The addition of optimal doses of anti-mu antibody resulted in enhanced proliferation of phorbol ester-activated B cells. The addition of B cell growth factor (BCGF) to phorbol ester-activated B cells also resulted in a dose-dependent synergistic effect and maximal enhancement on day 3. BCGF activity could be absorbed with either phorbol ester- or anti-mu-activated B cells, but not with resting B cells, thus confirming the induction of functional BCGF receptor expression. Cell proliferation was not necessary for the induction of functional BCGF receptors. Phorbol ester was a more efficient inducer of BCGF receptor expression than was anti-mu antibody; gamma-interferon treatment had no effect. BCGF enhanced transferrin receptor expression by phorbol ester-activated B cells. The results suggest that phorbol ester-activated small B cells can be used to monitor BCGF activity, and this synergistic combination may be useful in establishing BCGF-dependent B cell clones in culture.     

Characterization of the cell surface receptors for human B cell growth factor of 12,000 molecular weight

Quiescent normal human B cells have been shown to require an activation step before proliferating in response to B cell growth factor (BCGF) of 12,000 m.w. (12 kd). One effect of cell activation has been the putative acquisition of specific cell surface growth factor receptors. In this report, the existence of such receptors has been confirmed by using purified radioiodinated BCGF-12 kd. BCGF-12 kd receptors on activated B cells have been shown to be distinct form those interacting with IL 2. Scatchard analysis revealed both high affinity receptor sites with an apparent Kd of 28.6 pM and low affinity receptor sites with Kd of 1.2 nM on freshly prepared, anti-IgM activated peripheral blood B cells. Human B cells grown in culture for extended periods of time in the presence of BCGF-12 kd also displayed high affinity receptor sites (Kd, 41.4 pM) and low affinity receptor sites (Kd, 0.9 nM). The action of BCGF-12 kd therefore appears to be mediated by binding to its lineage-specific receptors on the cell surface.     

Interleukin 2 produced by activated B lymphocytes acts as an autocrine proliferation-inducing lymphokine

Normal peripheral blood B cells produce a soluble factor after activation that is functionally indistinguishable from interleukin 2 (IL 2) and can support B cell proliferation in vitro. Purified rabbit peripheral blood B cells, when stimulated with a combination of ionomycin (0.5 microgram/mL) and phorbol myristate acetate (PMA) (1 ng/mL), secreted a soluble factor in the culture medium that supported the IL 2-dependent cell line CTLL-2. The ability of these supernatants to support CTLL-2 growth was almost completely blocked by rabbit antibodies against human recombinant IL 2 and by the anti-IL 2 receptor monoclonal antibody 7D4. These data strongly suggest that the growth factor secreted by rabbit B cells is IL 2. To examine the possibility that the IL 2 activity detected in the B-cell cultures may be derived from residual T cells, B cells were further purified by successive panning with a pan-T-cell monoclonal antibody, L11-135, and goat anti-rabbit IgG. These highly purified B cells produced levels of IL 2 activity comparable to those produced by the initial B cell populations. Comparison of IL 2 production by decreasing numbers of purified T cells and purified B cells also indicated that the B cells were the source of IL 2 activity. Supernatants of activated B cells could support proliferation of B-cell blasts, and this activity could be completely absorbed by CTLL-2 cells, indicating that IL 2 is a major growth factor for B cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crosslinking of the CD5 antigen on human T cells induces functional IL2 receptors.

CD5 is a 67-kDa antigen that is expressed on the membrane of the majority of human T cells, and on a subset of B cells. Previous studies have demonstrated that anti-CD5 monoclonal antibodies (mAb) can provide a helper signal for T cell activation through the TCR/CD3 complex. We now demonstrate that when CD5 is crosslinked by immobilized anti-CD5 mAb in the absence of other activating stimuli, the T cells proliferate in response to recombinant interleukin 2 (rIL2) (but not to rIL4). Four different anti-CD5 mAb (anti-Leu1, 10.2, anti-T1, and OKT1) had a similar effect. IL2 responsiveness could be induced with immobilized anti-CD5 mAb in cultures of purified T cells, but was enhanced by the addition of monocytes, by monocyte culture supernatant, or by the combination of IL1 and IL6. Staining with an anti-IL2 receptor (p55) mAb demonstrated expression of IL2 receptors on about 10% of the anti-CD5-stimulated T cells. Both virgin (CD45RA+) and memory (CD45RO+) T cells were responsive. Our data provide further evidence for the involvement of CD5 in T cell activation.     

A mitogenic factor, released by stimulated human mononuclear cells and distinct from interleukin 2 (IL 2), B cell growth factor (BCGF), and interleukin 1 (IL 1)

Two lymphocyte mitogenic factors, interleukin 2 (IL 2) and blastogenic factor (BF), are generated concomitantly in human mixed lymphocyte cultures (MLC). The latter mitogenic factor is directly mitogenic for unstimulated lymphocytes, whereas the former mitogenic factor acts only on previously activated lymphocytes. Both factors had a m.w. range, as determined by gel filtration, of 18,000 to 30,000. Thus, these two factors were inseparable on the basis of m.w. size. However, BF and IL 2 were separable during ion exchange chromatography on the DEAE cellulose and phenyl-Sepharose chromatography. In addition, BF activity in the supernatants of MLC reached a maximum after day 5, whereas IL 2 activity peaked at day 3, thus distinguishing BF from IL 2 kinetically. These results clearly indicate that BF activity is mediated by molecules distinct from IL 2. The biochemical relationship between B cell growth factor (BCGF) and BF was also examined. Because BF was readily separable from BCGF by Con A-Sepharose chromatography, BF is distinguishable from BCGF. No augmentation of PHA-stimulated C3H mouse thymocyte proliferation was associated with the preparation of partially purified BF, demonstrating that BF and IL 1 are distinct molecules. Taken together, these results indicate that BF is clearly distinct from IL 2, BCGF, and IL 1. BF-containing MLC supernatants have direct mitogenic activity on both T and B cells. Both T and B cell blastogenic activities copurified during ammonium sulfate precipitation, gel filtration, DEAE cellulose ion exchange chromatography, and hydrophobic chromatography. Thus, these two activities appear to be biochemically inseparable. Monoclonal anti-Tac, that has been suggested to recognize the receptor for human IL 2, was highly inhibitory to the T cell response to the phenyl-Sepharose preparations of BF (IL 2-free). In contrast, this antibody had minimal or no effect on BF-induced B cell proliferation. However, when MLC supernatants were absorbed with a cloned IL 2-dependent T cell line, only IL 2 activity, but not BF activity, was removed, demonstrating that BF and IL 2 have different binding specificities. The precise mechanism(s) by which anti-Tac inhibits BF-induced proliferation of T cells is unknown at present. Additionally, during the course of these experiments, we observed that Con A-Sepharose chromatography could be used as a simple one-step method of separating BCGF from IL 2.     

B cell-derived BCGF functions as autocrine growth factor(s) in normal and transformed B lymphocytes

This paper demonstrates that B cell lines, as well as normal activated B cells generate and respond to B cell-specific growth factor(s) (BCGF). BCGF derived from B cells (B-BCGF) appears to be distinct from interleukin 1, interleukin 2 (IL 2), B cell stimulatory factor, BCGF-II, interferon-gamma, or transforming growth factor. It acts on activated B cells, but not on resting G0 phase B cells to induce proliferation. B cell lines, immortalized by Epstein-Barr virus, constitutively secrete 10-fold higher level of B-BCGF compared with normal activated B cells, suggesting that an activated autocrine loop might be operating in immortalized B cells. On the basis of our observations, we postulate that B cell clonal expansion may occur, at least in part, through a B-BCGF-dependent autocrine pathway similar to IL 2 effect on T cells.     

Comparison of the expression of IL 2 receptors by human T and B cells: induction by the polyclonal mitogens, phorbol myristate acetate, and anti-mu antibody

It is well established that IL 2 plays an important role in the proliferative response of T cells. Activated B cells were also recently found to express IL 2 receptors. The present studies were designed to compare qualitative, quantitative, and functional aspects of IL 2 receptor expression by activated T and B cells. Phorbol myristate acetate (PMA)-activated human T and small resting B cells and enhanced the expression of HLA-DR, HLA-DC/DS, and transferrin receptors while reducing Leu-4 antigen expression by T cells and IgM and IgD expression on B cells. PMA induced both T and B cells to express functional IL 2 receptors before cellular proliferation. Immune interferon did not participate in this induction. The m.w. of the IL 2 receptors expressed by activated T and B cells was identical: 54,000 to 59,000. Several differences were noted in the expression of IL 2 receptors by activated T and B cells on stimulation with PMA; T cells expressed IL 2 receptors sooner than B cells and in higher density, and the enhanced proliferative response of T cells to IL 2 was more difficult to inhibit with antibody to IL 2 receptors. In addition, IL 2 enhanced the expression of transferrin receptors by activated T cells but did not have a similar effect on activated B cells. Small B cells from the blood could also be induced by a mitogenic monoclonal anti-IgM antibody to express functional IL 2 receptors. Relatively large B cells in fresh blood samples were found to express functional IL 2 receptors and were capable of a modest proliferative response to IL 2. The intensity of the IL 2 receptor expression and the proliferative response by large B cells were enhanced by PMA stimulation. The data suggest that IL 2 receptors may play an auxiliary role in the B cell proliferative response and that IL 2 may exert its effect at a late phase in the B cell activation process.     

Production of interleukin 1 by adult T cell leukemia (ATL) cell lines

The accessory function for T cell activation and the production of interleukin 1 (IL 1) of adult T cell leukemia (ATL) cell lines were studied in vitro. ATL cell lines such as Hut-102, MT-1, and MT-2 functioned as accessory cells for the stimulation of human T cell proliferative response induced with concanavalin A (Con A) and induced allogeneic mixed lymphocyte reaction. Cell lysates of three ATL cell lines and the culture supernatant of MT-2 cells had activities to stimulate murine thymocyte proliferative response. Then we studied physicochemical properties of the factors produced by MT-2 cells. The m.w. of the factors were approximately 15,000 by Sephacryl S-200 column chromatography, and their isoelectric point values were 5.4 and 4.8 by chromatofocussing technique. No fraction contained interleukin 2 (IL 2) activities to stimulate IL 2-dependent murine cytotoxic T cell line. The thymocyte-stimulating activities of the factors were absorbed with rabbit anti-IL 1 alpha antiserum, but not with anti-IL 1 beta antiserum. Furthermore, messenger RNA extracted from MT-2 cells hybridized to complementary DNA of IL 1 alpha, but not of IL 1 beta, by Northern blot hybridization analysis. The factors from MT-2 cells could stimulate the production of IL 2 and the expression of IL 2 receptors of human T cells in the presence of Con A as well as recombinant IL 1 alpha and IL 1 beta did, and these activities were also blocked by rabbit anti-IL 1 alpha antiserum, but not by anti-IL 1 beta antiserum. These results suggest that the factors produced by MT-2 cells correspond to IL 1 alpha. However, the accessory function of MT-2 cells for T cell activation was not blocked by rabbit anti-IL 1 antiserum. These results suggest that ATL cell lines produce IL 1-like factors, but the accessory function of ATL cell lines for T cell activation is mediated by some other mechanisms rather than by secreted IL 1-like factors.     

Role of DNA synthesis in secretion of immunoglobulin from murine B cells stimulated by T cell derived lymphokines

We have investigated whether cell division is required for induction of Ig secretion from three types of B cells, which represent distinct activation states: normal splenic B cells, anti-Ig-treated B cells, and a monoclonal murine B cell tumor, BCL1. Polyclonal Ig secretion was stimulated in vitro by LPS or by lymphokines produced by EL-4 cells (EL-4 SN), which includes B cell growth factor II (BCGF II). LPS and EL-4 SN were mitogenic for all three cell populations and stimulated substantial IgM secretion from both B cells and anti-Ig blasts. Aphidicolin, a reversible inhibitor of DNA synthesis, abolished IgM secretion from B cells and anti-Ig blasts induced by either mitogen, indicating that Ig-secreting cells in these cultures are part of a cycling population. BCL1 tumor cells respond to BCGF II (but not to interleukin 2 or B cell stimulatory factor 1) with IgM secretion and cell division, allowing a direct assessment of the influence of BCGF II-stimulated cell division on secretion of IgM. Secretion by these cells during the first 24 hr of culture was not substantially affected by aphidicolin, but secretion at 48 or 72 hr was markedly inhibited. Culture of BCL1 cells for 48 hr with aphidicolin alone had no effect on cell viability or on subsequent responsiveness if the drug was removed, eliminating non-specific toxicity as an explanation of the drug's effect. Addition of aphidicolin during the last 24 hr of culture to either normal B cells or BCL1 cells was much less effective at inhibiting IgM secretion. These results indicate that the cells that secrete IgM in response to BCGF II also synthesize DNA when exposed to this factor. Thus, induction of high-rate Ig secretion from murine B cells by some stimuli, including BCGF II, may require at least one round of cell division.     

Production of B cell growth factor by normal human B cells

Although it has been demonstrated that malignant human B cell lines are capable of producing B cell growth factor (BCGF), production of BCGF by normal B cells has not been shown. In this study, we demonstrate BCGF production by normal B cells, achieved by using human peripheral blood B cells prepared by a positive selection technique and stimulated with Staphylococcus aureus Cowan I (SAC) for 12 hr. SAC was removed from the supernatants by anti-SAC-coupled Sepharose. Supernatants absorbed with this antibody were functionally free of SAC, as demonstrated by their inability to activate resting B cells. B cells stimulated with SAC for 12 hr produced BCGF activity that was generally unmeasurable in supernatants by 36 hr. Characterization of BCGF produced by SAC-stimulated B cells revealed a m.w. of 32,000 by high-performance liquid chromatography sieving and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; this BCGF was found to have an isoelectric point of 6.7. Furthermore, this BCGF lacked interleukin 1, interleukin 2, interferon, and B cell differentiation factor activity. This observation that BCGF can be produced by normal human B cells is significant because it demonstrates for the first time that normal B cells have the ability to provide their own growth factors or the growth factors for other B cells.     

Identification and partial purification of a human natural killer cell proliferation-inducing factor

Culture supernatants of Concanavalin A activated human peripheral blood mononuclear cells were found to contain a factor which induced proliferative response in normal peripheral blood mononuclear cells. This proliferation-inducing factor specifically induced and sustained proliferation of purified human NK cells but not of T or B cells. Although interleukin 2 (IL12) also has proliferation-inducing effects on NK cells, the partially purified proliferationinducing factor preparations contained no measurable IL2 contamination. Moreover, neutralizing anti-IL2 antibodies did not block the growth effect of proliferation-inducing factor on purified human NK cells. Other cytokines which were tested, including IL4, IL6, IL7, IL12, TNF and IFN, were all found to be inactive in the proliferation-inducing factor assay. While proliferation-inducing factor by itself had no effect on T-cell proliferation, IL2-induced proliferation of T cells was significantly enhanced in the presence of proliferation-inducing factor, as was IL2-induced NK-cell proliferation. NK cells could be maintained in culture for at least a month in the presence of proliferation-inducing factor alone, but the cells lost their cytolytic activity after 3–4 weeks in culture. Addition of IL2, to NK cells which had been cultured in the presence of proliferation-inducing factor, restored their cytotoxicity. Proliferation-inducing factor activity was partially purified on an anion exchange HPLC column. The molecular weight of proliferation-inducing factor appeared to be about 10 kDa, based on its elution profile on a sizing HPLC column. Our results indicate that proliferation-inducing factor is a novel NK-cell proliferationinducing factor.