BCGFII activity on activated B cells of a purified murine T cell-replacing factor (TRF) from a T cell hybridoma (B151K12)

Experiments were performed to examine a growth-promoting activity on B cells or B leukemic cells of T cell-replacing factor (TRF) produced by a murine T cell hybridoma (B151K12) which constitutively produces TRF. The cellfree supernatant (CFS) from B151K12 cells (B151-CFS) could induce terminal differentiation of pre-activated B cells or in vivo passaged chronic B leukemia cells, BCL1, into immunoglobulin-secreting cells, while it did not exert a nominal lymphokine activity such as BCGFI (now known as BSFpl), IL 2, or gamma-interferon. However, it promoted [3H]thymidine uptake of dextran sulfate (DXS)-stimulated normal B cells and in vivo passaged BCL1 cells, suggesting that it also has BCGFII activity. We tried extensively to purify and to separate the TRF active molecule from the BCGFII active molecule by using many types of purification procedures. The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, and gel permeation with fast protein liquid chromatography (FPLC). It was revealed that the BCGFII active molecule was hardly separable from the TRF during the entire purification procedure. The TRF as well as BCGFII active materials were glycoprotein with an apparent m.w. of 50 to 60 Kd on gel permeation chromatography and 18 Kd on SDS-PAGE under reducing conditions. The BCGFII active materials were hardly separable from the TRF active one, even after a reverse-phase FPLC, in which both BCGFII and TRF activities were recovered in the fractions eluted at 44 to 48% acetonitrile in 0.1% trifluoroacetic acid (TFA). Furthermore, the absorption of TRF and BCGFII active materials by using BCL1 cells removed not only TRF but also BCGFII activity. Moreover, B cell-specific monoclonal antibody (9T1), which can preferentially block TRF-dependent plaque-forming cell responses, also inhibited the expression of BCGFII activity to BCL1 cells. Taking all of the results together, we conclude that the TRF from B151K12 cells promotes growth of appropriately activated, such as DXS-stimulated normal cells and BCL1 tumor cells. These results suggest that B151-TRF may act on B cells as B cell growth and differentiation factors.     

T cells produce TRF in response to Con A and factors in T cell hybridoma supernatants

In recent experiments, a soluble factor (TRF) that mediates the differentiation of anti-immunoglobulin (Ig)-activated B cells to Ig-secreting cells has been identified. TRF works in concert with a growth factor, probably IL 2, in the induction of activated B cells. In previous studies, TRF was identified in culture supernatants of activated T cells and accessory cells, and thus the cellular source (T cell or accessory cell) of the factor was not determined. In the present studies, we succeeded in inducing the production of TRF by T cell populations from which accessory cells had been vigorously depleted. Lymph node cells were depleted of accessory cells by nylon wool adherence and anti-Ia and complement treatment; these cells were activated with Con A and a T cell hybridoma supernatant that contains IL 2. Supernatants from these activated T cell cultures supported the differentiation of anti-Ig-activated B cells to Ig secreting cells. These results show that T cells produce the differentiation factor, and further that they do so in response to ligand (Con A) plus a T cell-derived factor.     

Stimulation of B cell growth and differentiation by murine recombinant interleukin 1

Purified splenic B cells from C57BL/6 mice were separated into high-density (resting) and low-density (activated) B cells. Separated B cell populations were cultured at low cell densities (1 X 10(4) cells/well) with recombinant interleukin 1 (r-IL 1) alone or in combination with dextran sulfate (DXS) or anti-IgM monoclonal antibodies (alpha IgM mab), respectively, and proliferative responses were determined. R-IL 1 alone, as well as in synergy with alpha IgM mab or DXS, respectively, stimulated the growth of low-density B cells. Moreover, r-IL 1 and alpha IgM mab costimulated replication of high-density B cells. Separated B cell populations (1 X 10(5) cells/well) were cultured with r-IL 1 alone or in combination with DXS or alpha IgM mab, respectively, and the generation of plaque-forming cells was determined. R-IL 1 alone, as well as in synergy with DXS, stimulated the differentiation of low-density B cells into Ig-secreting cells. These findings suggest that r-IL 1 has B cell growth and differentiation factor activity and is operative on high- and low-density B cells. Thus, IL 1 may play an important role in B cell growth and maturation.     

Separable helper factors support B cell proliferation and maturation to Ig secretion

The 24-hr culture supernatant of Con A-activated spleen cells (SN) contains helper factors that enable maturation to high-rate polyclonal Ig secretion and enhance proliferation in cultures of mouse B cells activated with the F(ab')2 fragment of class-specific rabbit antimouse IgM antibody (anti-Ig). When interleukin 2 (IL 2), also called T cell growth factor, is removed from SN by absorption with an IL 2-dependent cell line at either 4 degrees C or 37 degrees C, all the helper activity for anti-Ig-activated B cells is also removed. Partial removal of IL 2 results in partial removal of helper activity for B cells. However, the IL 2-depleted SN appears to contain another helper factor, TRF, that enables anti-Ig-activated B cell cultures to mature to high-rate Ig secretion. This TRF activity is revealed by adding purified human IL 2 or an IL 2-containing supernatant of a cloned, lectin-activated T cell hybridoma line (FS6-14.13) to Il 2-depleted SN, which restores the polyclonal antibody response to anti-Ig. The hybridoma supernatant by itself supports proliferation of anti-Ig-activated B cell cultures, as measured by an increase in cell number, but not maturation to Ig secretion. This proliferative response is likewise IL 2 dependent, although purified IL 2 with anti-Ig is not sufficient. These experiments define separable combinations of factors acting on anti-Ig-activated B cell cultures, one of which (SN) results in both proliferation and maturation to high-rate Ig secretion, whereas the other (hybridoma supernatant) results in proliferation only. IL 2 appears to be an essential component of both combinations, although the target cell for IL 2 action in this system remains to be determined.     

Role of T cell-replacing factor (TRF) in the murine B cell differentiation: induction of increased levels of expression of secreted type IgM mRNA

T cell-replacing factor (TRF) is known to play a critical role in the regulation of B cell growth and differentiation. In this study, the role of TRF in the expression of mRNA for both IgM and IgG1 class was investigated. The TRF was purified from cellfree supernatants from a T cell hybridoma, B151K12. RNA was isolated from chronic B cell leukemia (BCL1) cells, DNP-KLH-primed B cells, or normal B cells cultured with or without LPS, and LPS plus TRF or LPS plus BSF-1. The steady state level of isotype-specific mRNA was assessed by Northern blot analysis with a mu-specific or a gamma 1-specific probe. It was demonstrated that BCL1 and purified B cells cocultured with TRF expresses increased levels (twofold and fourfold, respectively) of secreted forms of mu mRNA. Purified B cells from DNP-KLH-primed mice also expressed increased levels (twofold to fourfold) of mu as well as gamma 1 mRNA for secreted form by stimulation with TRF. Total expression of mu mRNA, however, was approximately threefold higher than that of gamma 1 mRNA. The stimulation of normal B cells with LPS plus TRF induced an increase in the levels of mu mRNA and gamma 1 mRNA expression, fourfold and threefold, respectively. However, the levels of gamma 1 mRNA expression was one-third of that induced in B cells stimulated with LPS plus BSF-1. These results indicate that TRF preferentially induces increased levels of secreted type of mu mRNA and induces less gamma 1 mRNA than BSF-1. The differential role of TRF from BSF-1 in the expression of Ig mRNA will be discussed.     

Identification and characterization of a B cell growth inhibitory factor (BIF) on BCGF-dependent B cell proliferation

The culture supernatants of Con A-activated human peripheral blood mononuclear cells (PBM) contained at least two regulatory factors upon B cell proliferation. One was B cell growth factor (BCGF), which activated antigen-stimulated B cells to proliferation and clonal expansion, and the other was its inhibitory factor, arbitrarily named B cell growth inhibitory factor (BIF). This BIF inhibited the effect of BCGF on anti-mu-stimulated B cells or the monoclonal mature B cell line (CLL-T.H.) obtained from the peripheral blood lymphocytes of B cell-type chronic lymphocytic leukemia patients, which were activated only with BCGF and without adding other proliferating stimuli (e.g., anti-mu). BIF activity was detected in the 24 hr culture supernatants of Con A-activated human PBM in FCS containing medium and also in serum-free RPMI 1640 medium. This substance with BIF activity could not be derived from FCS. Con A-induced BIF (m.w. of 80,000 and an isoelectric point of pH 5.4) was analyzed by Sephadex G-200 gel filtration and chromatofocusing. BIF was stable at pH 2.0 and at 56 degrees C for 30 min. Partially purified BIF had no effect on cell viability and almost no interferon activity (less than 1 IU/ml). BIF with high titer had a slight but significant inhibition on TCGF-dependent T cell growth and on PHA or Con A responses, but the extent of these inhibitions was far less than that of BCGF-dependent B cell growth. Absorption of BIF with Con A blasts made its inhibition on T cell growth even less. On the other hand, BIF activity could not be absorbed with Con A blasts but was almost absorbed with large numbers of CLL-T.H. cells. BIF had almost no inhibitory effect on the proliferation of a mouse fibroblast cell line (NIH 3T3), a mouse myeloma cell line (NS-1), human lymphoid cell lines (MOLT-4, HSB-2, and Daudi), or a human myeloid cell line (K-562). BIF-producing cells were estimated to be T cells and were identified as T8+ T cells. On the other hand, Con A-induced BCGF was demonstrated to be produced predominantly by T4+ T cells. These results show that human B cell proliferation is regulated by interaction between T4+ and T8+ cells via soluble factors, namely BCGF and BIF, respectively.     

Synergy between T cell-replacing factor and bacterial lipopolysaccharides (LPS) in the primary antibody response in vitro: a model for lipopolysaccharide adjuvant action.

Unfractionated spleen cells, B cells from normal mice, and nu/nu spleen cells respond to the addition of bacterial lipopolysaccharide (LPS) and T-cell-replacing factor (TRF) by production of plaque-forming cells (PFC) in excess of the number expected from the addition of LPS and TRF separately. This synergistic activity is dependent on the presence of the antigen, SRBC. Supernatants of both allogeneic spleen cell mixtures and spleen cells cultured with Con A are effective and synergize best at concentrations suboptimal for their ability to act as TRF alone. Culture supernatants of unstimulated normal or fractionated cell populations are ineffective. Synergy is not dependent on the presence of macrophages in the cultures. Purified LPS free from active contaminants, as well as commercially available LPS, show synergy with TRF. Synergy was seen when TRF was added at initiation of culture or 24 hr later. It is suggested that synergy is the equivalent of LPS adjuvant activity, that the role of T cells in LPS adjuvanticity is that of a conventional cooperating cell, and the LPS acts as an adjuvant by inducing B cells to become more sensitive to T cell helper factors.     

Selective elimination of a B cell subset having acceptor site(s) for T cell-replacing factor (TRF) with biotinylated antibody to the acceptor site(s) and avidin-ricin A-chain conjugate

A covalent conjugate of avidin with ricin subunit A-chain (avidin-RA) was prepared by using N-succinimidyl 3-(2-pyridyldithio)propionate as a coupling agent. Selective cytotoxic activity after the combined treatment of spleen cells with biotinylated antibody and avidin-RA was demonstrated by the fact that the responsiveness to LPS was selectively abrogated by pretreatment of the cells with biotinylated rabbit anti-mouse immunoglobulin (MIg) antibody, but not with biotinylated anti-Thy-1.2 antibody. Neither the biotinylated antibody alone nor avidin-RA alone was effective in decreasing the responses to mitogens. Moreover, a high anti-DNP PFC response elicited by DNP-KLH-primed BALB/c mouse spleen cells stimulated in vitro with DNP-KLH was mostly abrogated by the pretreatment of the cells with biotinylated anti-MIg antibody and avidin-RA. Again, neither the biotinylated antibody alone nor avidin-RA alone was effective in decreasing the anti-DNP PFC response. This cell-killing method with the use of biotinylated antibody and avidin-RA was applied and evaluated in experimental systems in which the helper action of T cells on B cells was mediated by T cell-replacing factor (TRF) or was performed by the direct interaction of T cells with B cells (cognate interaction). When DNP-KLH-primed splenic B cells, pretreated with biotinylated F(ab')2 fragment of DCF1 male anti-BALB/c-B IgG antibody against acceptor site(s) for TRF followed by treatment with avidin-RA, were stimulated with DNP-OVA in the presence of monoclonal TRF, the anti-DNP PFC response was significantly decreased, whereas the same treated B cells responded well to stimulation with DNP-PPD in the presence of Tbc-primed T cells (cognate interaction). These results indicate that B cells responsible for the cognate interaction and those having TRF acceptor site(s) belong to a distinct subpopulation of B cells, and that the cytocidal action of the noncovalent conjugate of the antibody and RA formed from the biotinylated antibody and avidin-RA via an avidin-biotin complex has immunologic selectivity, eliminating only the latter subset of B cells recognized by the antibody.     

Lymphokine-mediated activation of a T cell-dependent IgA antipolysaccharide response

Immune responses to bacterial polysaccharides are important to host immunity at mucosal surfaces. We previously showed that BALB/c mice produce substantial T cell-dependent IgA responses to alpha (1,3) glucan determinants on the bacterial capsular polysaccharide dextran B1355. The data in this study demonstrate that the requirement for T cells for the activation of the IgA anti-alpha (1,3) dextran B1355 response can be replaced by T cell-derived nonantigen specific helper factors that appear to act during the late stages of B cell differentiation. Supernatants from the activated T cell lines cr-15 and (DL)C.C3.11.75, which contain interferon and late-acting T cell replacing factor activity, supported terminal differentiation of dextran-stimulated B cells to IgA anti-alpha (1,3) glucan antibody-forming cells and substantially increased IgM anti-alpha (1,3) glucan responses in culture. Although supernatants with interleukin 2 activity did not support optimal antigen-driven plaque-forming cell responses, they synergized with supernatants having interferon and T cell replacing factor activity in the production of IgA and IgM anti-alpha (1,3) glucan responses and IgM anti-SRBC responses. Supernatants from the T cell lines B6.11 and (DL)A.4 contained B cell growth factor activity but did not support activation of IgA anti-alpha (1,3) glucan PFC. These studies suggest that interferon and/or T cell replacing factor play an important role in the antigen-driven differentiation of B cells of the IgA and IgM isotypes to antibody-forming cells.     

Mitogens for murine embryo cell lines

The growth-promoting activities of fetal bovine serum, cortisol, phorbol myristate acetate, prostaglandin F2α, insulin, epidermal growth factor, and fibroblast growth factor were evaluated on four murine embryo cell lines (Swiss 3T3, Balb 3T3, M2, and C3H10T1/2). Each cell had an unique response spectrum to this collection of reported mitogens. Phorbol myristate acetate and prostaglandin F2α were active only on selected cell lines; cortisol was inactive on all four lines. Serum, epidermal growth factor, and fibroblast growth factor were able to stimulate cell division in all four lines, albeit to varying degrees for the different target cells.     

Analysis of the activation of lymphoid cells by mitogens in vitro with limiting dilution methods: adherent peritoneal cells suppress B-cell activation and synergize with WEHI-3 in the activation of T cells by Con A

Adherent peritoneal cells (APC) have often been used as a pure and effective macrophage population. Using partition analysis and small numbers of lymphoid cells activated by mitogens (concanavalin A for T cells (in the presence of TCGF) and LPS + DxS for B cells) we found that APC were accessory cells for T cell activation and growth but were not effective for B cells. Although APC were effective in assisting T-cell mitogenesis, they were not especially efficient. However, when APC were mixed with irradiated WEHI-3 cells (a tissue culture line previously shown to exhibit accessory cell activity in vitro for mitogenic activation T and B cells), the APC and WEHI-3 showed apparent synergy. One reason for failure of APC to assist B-cell mitogenesis was traced to the presence of a suppressor cell population which overcame the accessory cell help given by irradiated WEHI-3 cells to LPS-DxS stimulated murine B cells. It is thus possible to find "helper" effects (synergy of APC and WEHI-3 assisting the mitogenesis of T cells), as well as suppressor effects within the range of cells found in adherent accessory cells.     

A human helper T cell clone secreting both killer helper factor(s) and T cell-replacing factor(s)

A human helper T cell clone (d4), which showed its helper effect on the differentiation of both T and B cells, was established by MLC reaction of normal T cells against a B lymphoblastoid cell line (CESS) followed by cloning in the presence of IL2 and x-irradiated CESS and autologous non-T cells. d4 cells helped the induction of cytotoxic T cells against UV-treated CESS cells. Antigen-stimulated d4 cells secreted helper factor(s) involved in the induction of cytotoxic T cells (killer helper factor(s), KHF), and KHF activity could be separated into two fractions, one with the m.w. of 15,000 to 20,000 and the other with the m.w. of 45,000 to 50,000. The factor with 15,000 to 20,000 m.w. showed IL 2 activity; the other factor showed gamma-interferon activity without IL 2 activity, suggesting that both IL 2 and gamma-interferon exerted KHF activity. d4 cells or their culture supernatant showed helper activity in the induction of IgG in a B cell line (CESS). The helper activity of the supernatant (TRF) was absorbed with CESS cells but not with IL 2-dependent CTLL, whereas KHF activity was absorbed with IL 2-dependent CTLL but not with CESS cells. The results showed that TRF and KHF were distinct molecules and a single helper T cell clone could secrete helper factors for both B and T cells.     

Evidence for existence of two distinct TNP-Ficoll-responsive B cell subpopulations preferentially reactive either to a T cell-replacing factor (B151-TRF) or to a signal from accessory cells

The ability of B cells to respond to TNP-Ficoll has been shown to correlate with their ability to respond to T cell-replacing factor (TRF). The present study analyzed the relationship of TNP-Ficoll-responsive B cells to a TRF-responsive B cell subpopulation. The B cells from normal, unprimed mice responded to TNP-Ficoll in the presence of accessory cells. Such responses were notably augmented by the addition of TRF derived from a monoclonal T cell hybridoma, B151K12(B151-TRF). Interestingly, B cells of mutant X-linked immunodeficient DBA/2Ha which failed to respond to B151-TRF gave anti-TNP PFC responses to TNP-Ficoll comparable to those of normal mice, depending on the presence of accessory cells. However, under this condition, the addition of B151-TRF did not augment the TNP-Ficoll responses. One explanation of the augmentation of TNP-Ficoll response by TRF for the B cells from nondefective mice was that two distinct B cell subpopulations exist which differ in their respective activation requirement for TRF and accessory cells. To examine this possibility, syngeneic accessory cells were pulsed with TNP-Ficoll and were assayed for their ability to activate normal B cells in the presence or absence of B151-TRF. The results revealed that TNP-Ficoll-pulsed accessory cells were able to induce primary anti-TNP PFC responses in normal B cells to the same magnitude as soluble TNP-Ficoll. However, these B cell responses induced by the TNP-Ficoll-pulsed accessory cells were not augmented by the addition of B151-TRF to the culture. These results support the notion that two distinct TNP-Ficoll-responsive B cell subpopulations exist; one requires accessory cell-B cell interaction to be activated by TNP-Ficoll but fails to respond to TRF, and the other can be activated by TRF in a totally accessory cell-independent manner.     

Establishment of an assay system for the detection of translated materials of T-cell-replacing factor mRNA in Xenopus oocytes

We established an assay system for detecting T cell-replacing factor (TRF) activity of translated materials in Xenopus oocytes of poly (A)-positive mRNA extracted from a T cell hybrid cell line, B151K12 (B151) which constitutively produces TRF. Since it was difficult to detect TRF activity of the translated products of B151-mRNA, partly because of low TRF activities, we developed the following two systems. First, RNA was prepared from B151 cells stimulated with phorbol myristate acetate and calcium ionophore A23187 because such stimulations augmented TRF production by approximately three to five-fold. Second, interleukin 2 (IL-2, 125 U/ml) was added to the culture of BCL1 cells to detect a small amount of TRF-active materials since IL-2 synergizes with a suboptimal dose of TRF to induce IgM secretion in TRF-responding BCL1 cells (chronic B cell leukemic cells). Here we describe TRF activity of translation products of B151-mRNA in Xenopus oocytes. B151-TRF mRNA was detected in the fractions sedimented between 15 and 18S by analysis using sucrose density gradient centrifugation.     

Characterization of monoclonal B cell growth factor (BCGF) produced by a human T-T hybridoma

We previously demonstrated the development of a cloned human T cell hybridoma that secretes B cell growth factor (BCGF) in the absence of demonstrable interleukin 2 or B cell differentiation factor. Sephadex gel filtration chromatography demonstrated the m.w. of this factor to be 18 to 20K. The present studies were performed to further characterize the biochemical properties of the molecule and to determine its target cell specificity. Temperature stability studies showed the monoclonal BCGF to be stable at 37 degrees C for 12 hr and at 70 degrees C for 15 min; however, most (93%) of the activity was lost after incubation at 70 degrees C for 30 min. Aliquots of hybridoma supernatant were exposed to buffer solutions with variable pH with no diminution in activity over a pH range of 4.0 to 10.0 BCGF activity was not affected by 2-mercaptoethanol, neuraminidase, or nucleic acid denaturing enzymes. In contrast, all activity was destroyed by 10 M urea, trypsin, and chymotrypsin. Chromatofocusing demonstrated the isoelectric point of BCGF to be 6.3 to 6.6. Finally, absorption experiments demonstrated that BCGF activity was absorbed by large, activated B cells. Mitogen-stimulated T cell blasts, small resting B cells, and CESS cells failed to absorb BCGF activity from the hybridoma supernatant. These and future studies with purified monoclonal human BCGF should enhance our understanding of its immunochemical properties and of its role in the immunoregulation of human B cell responses.     

Alkaline phosphatase activity as a membrane marker for activated B cells

Alkaline phosphatase activity was assayed by a microtiter assay (with p-nitrophenylphosphate used as substrate) in the plasma membrane of mouse spleen cells activated in vitro by the B cell mitogen LPS and the T cell-dependent B cell mitogen, PWM. No activity was detected in spleen cells cultured in the presence of the T cell mitogens PHA and Con A. This alkaline phosphatase activity was detected in the blast-enriched 30 to 40% fraction of discontinuous Percoll gradients in LPS-treated cultures, and the enzymatic activity assayed was susceptible to inhibition by the alkaline phosphatase inhibitors EDTA and L-phenylalanine. These data support the idea that the membrane alkaline phosphatase activity could be used as a marker for activated B cells.     

Partial purification and characterization of a BCGFII from EL4 culture supernatants

Two B cell growth factor activities have been previously described. One activity, present in the culture supernatants of PMA-induced EL4 is active in a co-stimulator assay with normal B cells and anti-immunoglobulin. The other activity is present in the culture supernatants of the alloreactive T cell line C.C3.11.75 and can be assayed in a co-stimulator assay with normal B cells and dextran sulfate or with BCL1 in vivo line B cell tumor. We have termed the first activity BCGFI and the second BCGFII. We have now shown that a very similar BCGFII activity can be obtained from EL4 culture supernatants induced by PMA. This (EL4)BCGFII has an apparent m.w. of 55,000, is eluted from DEAE Sephacel at 0.05 M NaCl, and has a pI of 5.5, which is clearly distinct from the properties of (EL4)BCGFI activity. (EL4)BCGFII activity is similar to but not identical to (DL)BCGFII. It differs from (DL)BCGFII in chromatographic behavior and in the kinetics of the response of BCL1 to the two factors. (EL4)BCGFII activity can be detected in 18 to 24 hr by virtue of its ability to cause in vitro proliferation of in vivo BCL1 tumor B cells.     

Suppressor factor secreted by T hybridoma established from peripheral blood lymphocytes of a bone marrow transplantation patient. I. Establishment of human T-cell hybridoma and partial characterization of suppressor factor

A stable human T-cell hybridoma was established by cell fusion between activated human peripheral blood lymphocytes from an allogeneic bone marrow transplantation patient and the JD1-17 cell line, a subclone of the human T leukemia Jurkat cell line. This hybrid clone 1-8, which bore the surface phenotype of suppressor cells (CD8+HNK1+), spontaneously secreted a factor which, at high dilutions, suppressed the responses of T and B cells induced by mitogens and alloantigens. This suppressor factor was found to be heat-resistant (56 degrees C, 30 min), stable at alkaline but not acid pH, unaffected by 2-mercaptoethanol, and sensitive to trypsin. Preparative isoelectric focusing revealed an isoelectric point of 5.35. The suppressor activity was selectively absorbed by blast T cells. By gel filtration on Sephacryl S-200 and HPLC, the suppressor activity was found in two peaks corresponding to 40-45 kDa (monomer) and 90-95 kDa (dimer).