Polyclonal activation of rat B cells. II. Dextran sulfate as a cofactor in mitogen-induced and antigen-induced differentiation of rat B lymphocytes

Salmonella typhimurium mitogen (STM) is a polyclonal activator of rat B lymphocytes, triggering them to proliferate, but not differentiate, to antibody-secreting cells. When lymphokines in the form of a supernatant from Con A-stimulated splenocytes (CAS) are added to B cell cultures activated by STM, only a small number of cells are driven to differentiate. Only with the addition of a third signal provided by the polyanionic polysaccharide dextran sulfate (DXS) is significant rat B cell differentiation observed. In this study, we have shown that this requirement for DXS is not unique to the STM mitogen. LPS, Staphylococcus aureus Cowan I-fixed cells, and anti-Ig antibody all induced rat B cell proliferation with little differentiation, even in the presence of CAS. DXS was necessary to induce differentiation in all cultures costimulated with mitogen and CAS. The requirement for DXS for optimal B cell differentiation is also observed with other lymphokine preparations such as the supernatants from PMA-stimulated EL-4 cells and PHA-stimulated human T cells. Furthermore, this augmentative effect of DXS in rat B cell differentiation was not confined to polyclonal activation systems. Ag-specific IgG secretion was also increased when DXS was added to Ag and CAS costimulated cultures of B cells harvested from the draining lymph nodes of rats immunized with DNP-keyhole limpet hemocyanin. Within the polyclonal activation system, a method of staged additions of STM, DXS, and CAS to B cell cultures was used to investigate the role of DXS during B cell differentiation. Optimal differentiation occurred only when DXS was present in the B cell cultures in conjunction with CAS. The augmentation in differentiation seen with DXS did not appear to be due to the recruitment of an additional CAS-responsive B cell subset, because cycling, low density B cell blasts showed large increases in IgM secretion with subsequent exposure to DXS and CAS. These studies suggest tha DXS acts as a cofactor to various differentiation factors, augmenting polyclonal and Ag-specific rat B cell differentiation. The relevance of DXS to in vivo immune responses is discussed.     

Cell cycle-related expression of the receptor for a B cell differentiation factor

Cloned, neoplastic B cells (BCL1) have been used to evaluate the expression of the receptor for the B cell differentiation factor, BCDF mu. These cells do not secrete IgM before stimulation with BCDF mu-containing T cell supernatants (SN). By inducing cell cycle synchrony in this homogeneous population, the expression of the BCDF mu receptor could be evaluated as a function of the cell cycle. Responsiveness to BCDF mu-containing SN is maximal when the cells are in S and G2 phases of the cell cycle, and a 2-hr exposure of cells to BCDF mu-containing SN during S/G2 results in optimal IgM secretion 5 days later. Cells in S/G2 are also maximally effective in absorbing BCDF mu activity from SN. These data support the hypothesis that B cells do not respond to differentiative signals until after they are committed to at least one round of cell division.     

Synergy between B cell differentiation factors and interleukin 2, using a monoclonal system

By using monoclonal B cell targets, cells derived from patients with chronic lymphocytic leukemia, and B cell differentiation factors (BCDF) derived from monoclonal human T cell hybridomas, we have demonstrated marked synergy for differentiation between interleukin 2 (IL 2) and BCDF. IL 2 alone had no effect on the proliferation of differentiation to immunoglobulin secretion in these cell populations; however, in conjunction with a variety of BCDF, differentiation to plaque-forming cells (PFC) was augmented 10- to 100-fold. There was no increase in proliferation as measured by [3H]thymidine incorporation. These effects could be demonstrated with concentrations of IL 2 as low as 5 U/culture, well within the physiologic range, by using either commercially available or recombinant IL 2. The addition of IL 2 to the B cell and BCDF cultures resulted in almost 100% expression of the IL 2 receptor, Tac, on the surface of these cells, and the augmented PFC response could be inhibited 70 to 80% by the addition of anti-Tac to the culture. Kinetic studies revealed that the addition of IL 2 to the B cell cultures could be delayed for up to 72 hr without a change in the PFC response, suggesting that IL 2 was acting as a secondary or synergistic signal for differentiation. Thus, it appears that IL 2 does have a role in B cell maturation mediated, in part, by IL 2 binding to the IL 2 receptor present on certain B cells.     

Role of BCGFII in the differentiation to antibody secretion normal and tumor B cells

This report describes the effects of B cell growth factor (BCGFII) and other lymphokines in the differentiation of normal and tumor B cells. We compared BCL1 tumor B cells, normal B cells giving rise to a polyclonal response without the intentional addition of antigen, and an antigen-driven, SRBC-specific response. BCL1 tumor B cells gave maximum PFC responses when partially purified BCGFII was added or when suboptimal doses of BCGFII were mixed with one of several putative terminal differentiation factors we call B cell differentiation factors BCDF. IFN-gamma was not active as any of these factors. Maximum polyclonal responses of B cells were seen when either IL 2 or BCGFII were mixed with BCDF. In contrast, SRBC-specific responses showed a strict requirement for IL 2, and BCGFII and BCDF synergized with IL 2 to give a maximum response. The involvement of BCGFII in all of these responses suggests that BCGFII acts as a growth factor for a population of B cells that has differentiated much of the way towards Ig secretion, and that many B cells become responsive to this growth factor. In addition, the fact that different lymphokine requirements were seen in the different experimental systems raises the possibility that there are multiple pathways to Ig secretion, and suggests that different subpopulations of B cells defined either by different lineages or by different stages of development within a single lineage have requirements for distinct lymphokines that regulate their growth and differentiation.     

Human helper T cell factor(s). IV. Demonstration of a human late-acting B cell differentiation factor acting on Staphylococcus aureus Cowan I-stimulated B cells

At least two distinct B cell stimulatory factors (BSF) were found to be involved in the differentiation of Staphylococcus aureus Cowan I (SAC)-stimulated human B cells to IgG-producing cells. A factor tentatively called B cell differentiation factor I (BCDF I) was found in one fraction, and a second factor, BCDF II was found in another fraction. The BCDF I fraction alone induces IgG-production in SAC-stimulated B cells, but the BCDF II fraction does not. The BCDF II fraction enhances IgG production in SAC-stimulated B cells in the presence of the BCDF I fraction. Studies concerning the time-course of the action of the BCDF II fraction revealed that it contains a late-acting differentiation factor that acts on B cells most effectively when it is added to the SAC-stimulated B cell culture after the addition of BCDF I fraction; it induces IgG plaque-forming cells within 1 day. The pI value of a late-acting BCDF was in the range of 5 to 6; this pI range is different from that of BCDF I but similar to that of BCDF II, which was shown in our previous studies to be able to induce IgG production in Epstein Barr Virus-transformed B lymphoblastoid cell lines. In addition, the m.w. of a late-acting BCDF were about 35,000 and 20,000, which are the same as those of BCDF II, and thus its identity with BCDF II was suggested.     

Demonstration that human B cells respond differently to interleukin 2 and B cell differentiation factor based on their stages of maturation

The mechanisms whereby interleukin 2 (IL 2), interferon-gamma (IFN-gamma), and B cell differentiation factor (BCDF) alone or in combination modulate human B cell differentiation are currently under intensive study. To dissect out the effects of individual lymphokines contained in mixed lymphocyte reaction-culture supernatants (MLR-CS) on B cell differentiation, we employed pure factors that possessed the same activity as factors contained in MLR-CS (IL 2: 50 U/ml, IFN-gamma: 7 U/ml, BCDF-Nal: 5 pM/ml, BCDF-YA2: 12.5% v/v) singly and in combination to human B cells. By activating purified human B cells with Staphylococcus aureus Cowan I (SAC) for 3 days, separating B blast cells by the Percoll centrifugation method, and then either using these B blast cells as B cells in the earlier stage after SAC-activation, or further culturing these B blast cells for 4 more days without any stimuli and using these B cells as B cells in the later stage after SAC-activation, we could define two different populations of cells. Disparity in the populations could be demonstrated by the observation that B cells in the earlier stage were 81.2% Tac-antigen+, 23.2% B2+, 68.9% transferrin receptor+, and 90.5% HLR-DR+, whereas B cells in the later stage were observed to be less positive for each surface antigen: 36.1% Tac-Ag+, 8.3% B2+, 45.3% transferrin receptor+, and 58.7% HLR-DR+. By adding each factor to both B cell fractions, we also demonstrated functional differences in the two populations. B cells in the earlier stage of activation only differentiated in response to IL 2 or IL 2 + IFN-gamma but not to BCDF, which was in contrast to B cells in the later stage that did not differentiate in response to IL 2 but did differentiate to BCDF. However, B cells in both stages proliferated in response to IL 2 but not to BCDF. Finally, we separated B cells in the later stage into two populations by the Percoll discontinuous gradient centrifugation. Lower density (larger) B cells were observed to proliferate but not to differentiate in response to IL 2, whereas higher density (smaller) B cells were observed to differentiate in response to BCDF. Therefore, we conclude that activated B cells initially become large and gain Tac-Ag and differentiate in response to IL 2 alone as well as the combination of IL 2 and IFN-gamma, whereas later in the more mature stage they become smaller again and differentiate into Ig-secreting cells only in response to BCDF.     

The role of interleukin 2 in inducing Ig production in a pokeweed mitogen-stimulated mononuclear cell system

Cyclosporin A (CsA) has been found previously to block mitogen-stimulated T cell proliferation and production of discrete T cell-derived lymphokines such as interleukin 2 (IL 2) and interferon (IFN)-gamma. In addition, CsA blocks pokeweed mitogen (PWM)-driven T cell-dependent differentiation of B cells into immunoglobulin (Ig)-secreting cells. Recently, we reported that CsA (1 microgram/ml) inhibited PWM-induced T cell production of IL 2 and IFN-gamma, but supernatants retained B cell differentiation factor (BCDF)-like activity. The present study demonstrates the ability of CsA to suppress T cell functions in PWM-driven Ig production in mononuclear cells (MNC), and the capacity of exogenous T cell lymphokines to reverse CsA-induced suppression. CsA profoundly suppressed PWM-driven PFC formation (greater than 95%). However, Ig production was substantially reconstituted by the addition of IL 2 at concentrations of 10 to 50 U/ml. In contrast, no effects were observed by the addition of IFN-gamma or BCGF. The kinetics of CsA inhibition of Ig production and IL 2 secretion were found to be closely related. In addition, to obtain effective reconstitution in the CsA-treated PWM-MNC system it was necessary to add IL 2 at the initiation of culture. T cells themselves were also required for B cell differentiation in this system. However, surface Ig+ cells obtained by cell sorting after 3 days of culture could differentiate in the absence of T cells but only in response to IL 2, not in response to IFN-gamma or BCDF. Thus, in PWM-driven B cell differentiation T cells are necessary early in culture, whereas IL 2 is essential from the initial stage of B cell activation through the final stage of B cell differentiation.     

Characterization of lymphokines mediating B cell growth and differentiation from monoclonal anti-CD3 antibody-stimulated T cells

Addition of anti-CD3 mAb 147 (IgG1), 446 (IgG1), or 454 (IgG2a) to cultures of T plus non-T cells can result in both B cell growth and differentiation. To determine whether lymphokines mediating these activities were similar to those described from conventional mitogen-induced T cell activation, normal peripheral blood T cells were stimulated with anti-CD3 mAb for 48 h. The supernatants were assayed for factors inducing B cell growth or differentiation (BCDF). A marked increase in Ig secretion was observed when either EBV-transformed B cell lines or normal B cells, pre-activated with Staphylococcus aureus Cowan I strain, were cultured in the presence of mAb 446 (anti-CD3) stimulated T cell supernatant whereas no significant increase in Ig secretion was noted with either mAb 454- or 147-induced T cell supernatant despite equivalent T cell proliferative responses to these antibodies. In contrast, IL-2 secretion was detectable in T cell supernatants from T cells stimulated with either mAb 454 or 147 but not 446. Factors promoting B cell proliferation were detected in all antibody-stimulated T cell supernatants but, contrary to BCDF, appear to act only on non-activated B cells. To determine whether these effector activities were due to distinct lymphokines, supernatants were pooled and concentrated by ammonium sulfate precipitation. Superose 12 permeation chromatography revealed BCDF activity with an apparent Mr of approximately 30,000 Da. The growth factor activity eluted over a wider and higher molecular weight range which overlapped the differentiation factor activity. Fractions containing BCDF activity were pooled, dialyzed, applied to a Mono Q anion-exchange column, and eluted with a linear NaCl gradient. The growth factor activity came off in a single-peak while BCDF was found divided into two major areas. The growth factor eluted at an ionic strength between the two BCDF activities. BCDF has an apparent isoelectric point (pI) of 6, in contrast to the reported pI 5 for IL-6 and more acidic than the documented basic pI of IFN-gamma. Lastly, peaks with BCDF activity were not active in assays for either IL-2 or IL-4. In addition, a rabbit anti-IL-6 heteroantiserum failed to inhibit the pI 6 BCDF, suggesting non-identity between IL-6 and anti-CD3 induced BCDF. Thus, anti-CD3 activated T cells generate both growth factor activity and BCDF as separate molecular entities distinct from IFN-gamma, IL-2, IL-4, and conventional IL-6.     

The necessity for T cell help for human tonsil B cell responses to pokeweed mitogens: induction of DNA synthesis, immunoglobulin, and specific antibody production with a T cell helper factor produced with pokeweed mitogen.

Human B lymphocytes obtained from tonsils do not proliferate when stimulated with pokeweed mitogen. A soluble factor produced from T cells cultured with pokeweed mitogen stimulates B cells to synthesize DNA and differentiate into immunoglobulin producing cells. This PWM produced supernatant induced a PFC response to SRBC. The T cell supernatant activity is produced within 12 hr of stimulation in the presence of serum and without a requirement for T cell division. Optimal stimulation of B cells occurred at 7 to 9 days of culture. This helper factor activity eluted postalbumin from a column of Sephadex G-200. Insolubilized pokeweed mitogen was not mitogenic for B cells. The continuous presence of the lectin in culture was not required for B cell proliferation or for immunoglobulin synthesis.     

MLC-derived human helper factor(s) that promote B cell differentiation: induction, functional characterization, and role of Ia antigens

Utilizing a PFC assay to quantitate the polyclonal activation of human peripheral blood B lymphocytes, we have investigated the induction and functional activity of MLC-derived human helper factor(s). Our data demonstrate that highly purified responder T cells, but not B or null cells, are required for the elaboration of MLC helper factor(s) that trigger the in vitro differentiation of B lymphocytes into PFC. Helper factor can trigger B cell maturation in the absence of helper T cells, since complement- (C) mediated lysis of the small (less than 5%) fraction of T cells present in anti-F(ab)2 immunoabsorbent column purified B cell population eliminates the PWM induced, but not the helper factor-induced PFC response. Responder T cells required for helper factor production do not bear surface membrane Ia, since alpha p23,30 + C treatment of this population does not affect helper factor generation. In contrast, alpha p23,30 + C treatment of the allogeneic stimulator cell population eliminates helper factor production. Taken together, these results demonstrate that interaction between Ia-bearing stimulator cells and Ia- responder T cells is required for the production of MLC-derived helper factor. In additional experiments, we determined that alpha p23,30, in the absence of C, totally abrogates the PFC response triggered by MLC helper factors. This result suggests an important role for Ia antigens in the functional activity of preformed helper factor molecules.     

Inverse relationship between proliferation and differentiation in a human TNP-specific B cell line. Cell cycle dependence of antibody secretion

Studies of an EBV-transformed and TNP-specific human B cell line revealed that, unlike myeloma or hybridoma cell lines that consist mainly of fully differentiated cells, most of the cloned EBV-transformed cells were not fully differentiated, as judged by inability to bind TNP-SRBC and to secrete anti-TNP antibody. The minority of more differentiated cells were selected by TNP-SRBC rosetting. They were found to proliferate to a lesser extent than nonrosetting cells and to contain increased numbers of antibody-secreting cells. This inverse relationship between proliferation and differentiation was also shown to be cell cycle related in that the TNP-SRBC rosetting cells resided, to a greater extent than the nonrosetting cells, in the G1 phase of the cell cycle. The finding that the G1 phase of the cell cycle was associated with differentiation into anti-TNP secreting cells was confirmed by demonstrating that treatment with hydroxyurea, which arrests the cells in G1, resulted in decreased proliferation and an increased proportion of antibody-secreting cells. Similarly, addition of phorbol ester resulted in increased antibody secretion and decreased proliferation, suggesting a role for protein kinase C in this differentiation pathway. The strategy of increasing the number of antibody-producing cells in this human EBV line, by promoting differentiation of the cells in G1, may be relevant to the large scale production of specific human mAb for the treatment and diagnosis of human diseases.     

The direct effects of interleukin 1, interleukin 2, interferon-alpha, interferon-gamma, B-cell growth factor, and a B-cell differentiation factor on resting and activated human B cells

A wide variety of cytokines have been demonstrated to affect B-cell function. However, it is unclear which of these mediators actually exert direct effects on the B cells themselves. In the present study, the direct role of interleukin (IL) 1, IL-2, Interferon-gamma, or Interferon-alpha in human B-cell activation, proliferation, or differentiation was examined and compared with the effects of a B-cell growth factor (BCGF) or a B-cell differentiation factor (BCDF). Highly purified human B lymphocytes were separated according to size into two nonoverlapping populations. The fraction of small B cells was incubated with IL-1, IL-2, Interferon-gamma, Interferon-alpha, BCGF, or BCDF, and cell size changes, RNA synthesis, DNA synthesis, or supernatant immunoglobulin (Ig) production were measured. Neither IL-1, IL-2, Interferon-alpha, Interferon-gamma, nor the BCGF induced substantial cell size changes, RNA synthesis, DNA synthesis, or Ig production by the small fraction of B lymphocytes; however, the BCDF could directly activate a proportion of resting B lymphocytes to secrete Ig. The fraction of large B cells was also incubated with these cytokines. While neither IL-1, Interferon-alpha, nor Interferon-gamma enhanced DNA synthesis or Ig production by the fraction of large B lymphocytes, DNA synthesis was augmented 23-fold by BCGF and IgG production was increased 7-fold by BCDF. Additionally, IL-2 slightly enhanced both proliferation and differentiation of large B cells but substantially less so than BCGF and BCDF; DNA synthesis was increased 4-fold, while Ig production in the presence of IL-2 was increased by approximately 50%. Thus, the most important lymphokines modulating the function of these two fractions of tonsillar lymphocytes were a BCGF and a BCDF.     

Functional and biochemical characterization of B-cell differentiation factor (BCDF) produced by an HTLV-I-transformed human T-cell clone and demonstration of specific binding of the factor to a BCDF responsive cell line

We have previously described YA2, a human T-cell clone that secretes B-cell differentiation factor (BCDF) but not B-cell growth factor (BCGF). The BCDFs secreted by YA2 and HTLV-I-transformed YA2 (TYA2) were functionally similar in their ability to stimulate Ig secretion by Staphylococcus aureus Cowan strain I-activated B cells and IgM secretion by SKW6.4 cells. In addition, they were biochemically similar with a MW of 30 kDa by high-performance liquid chromatography (HPLC) sieving, and a pI of 6.0-6.8 by isoelectric focusing. The BCDF activity was not blocked by antibodies to interleukin 2 and BCGF. BCDF was purified from TYA2 supernatant by sequential media protein immunoadsorption, flat bed isoelectric focusing, HPLC TSK 2000 sieving, and repeated immunoadsorption and was then iodinated. The iodinated material had functional BCDF activity and migrated to a single band at MW 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at pI of 6.8 by polyacrylamide gel isoelectric focusing. 125I-BCDF purified in this manner bound specifically to a BCDF-responsive cell line and not to phytohemagglutinin-activated T cells. 125I binding to the BCDF-responsive cell line was competitively inhibitable by the addition of cold BCDF. Thus we have purified and characterized a factor with BCDF activity and demonstrated that this factor binds specifically to a BCDF-responsive cell line.     

Immortalization of BGDF (BCGF II)- and BCDF-producing T cells by human T cell leukemia virus (HTLV) and characterization of human BGDF (BCGF II)

Human peripheral T cells were transformed by human T cell leukemia virus (HTLV), and T cell lines producing BGDF (BCGF II) and BCDF were established. Among these cell lines, a cell line, TCL-Na1, secreted the highest level of both BGDF and BCDF, and the amount of BCDF secreted by TCL-Na1 cells was 900-fold more than that produced by PHA-stimulated T cells. Within the limits of our examination, none of the HTLV-transformed T cell lines produced IL 2 or BSF-p1 (BCGF I). BCDF produced by TCL-Na1 cells had a m.w. of 35,000 and a pI value of 5.5, being separated from BGDF, which was eluted in the fractions corresponding to m.w. of more than 60,000 and pI values of 5 to 6. BGDF induced both proliferation and IgM secretion in a mouse leukemic B cell line, BCL1, and these activities were not separated by either isoelectric focusing or gel filtration in the presence or absence of 0.1% Triton X-100, suggesting that the molecule designated BGDF exerted both growth and differentiation activities. BGDF acted on normal mouse B cells to induce proliferation as well as IgM secretion. The target cells of BGDF were in vivo activated B blast cells. BGDF acted on DXS-activated murine B cells to induce both proliferation and IgM secretion but not anti-Ig-activated B cells, indicating that BGDF and BSF-p1 were different molecules.     

Induction of IgG secretion in a human B cell clone with recombinant IL 2

A human B cell clone (SGB3) responsive to IL 2 was established. Both recombinant IL 2 and B cell differentiation factor (BCDF) induced IgG secretion in SGB3 cells in a dose-dependent manner, but IL 2 did not affect the proliferation of SGB3 cells. FACS analysis showed that SGB3 cells expressed Tac antigen and anti-Tac antibody inhibited IL 2-induced IgG secretion without any inhibitory effect on BCDF-induced IgG secretion. These results showed that IL 2 could directly act on B cells and provide a differentiation signal through IL 2 receptors distinct from BCDF receptors. Physiologic relevance of IL 2 in the antibody response was discussed.     

Letter from the Guest Editor

Understanding the mechanisms of stem cell proliferation, self-renewal and differentiation is fundamental for stem cell biology. Stem cells proliferate by either symmetric division or asymmetric division. Through asymmetric division, stem cells self-renew and differentiate to mature cells. Stem cells could also divide symmetrically to give rise to differentiated cells. Besides intrinsic cues, proliferation and self-renewal of most stem cell types also rely on extrinsic signals from niche or surrounding cells. Failure in any of these factors may result in disturbed stem cell proliferation, self-renewal or differentiation and/or generate cancer stem cells that drive cancer development.     

Complement subcomponent C1q stimulates Ig production by human B lymphocytes

The regulation of Ig production by human B lymphocytes is a complex process involving interactions among B cells, APC, T lymphocytes and soluble factors including activation, growth, and differentiation factors. Components of the complement system, including C3a, C3b, C3d, and C5a, have been shown to influence various stages in this process. In this study, we demonstrate that the C1q subcomponent of complement binds to both small resting and large activated B cells and stimulates immunoglobulin production by Staphylococcus aureus Cowan-activated tonsillar B lymphocytes. This effect is present whether C1q is added to the B cells either at the beginning or near the end of a 7-day culture period and is not associated with enhancement of proliferation. The C1q stimulation of Ig production is, however, associated with increased steady state levels of mRNA for the mu Ig H chain. Furthermore, C1q stimulated IgM production by the human B cell line SKW 6.4, which is capable of secreting IgM in response to B cell differentiation factors (BCDF). SLE is a disorder frequently associated with polyclonal activation of B lymphocytes. We studied the effect of C1q on B cells from two patients with this disorder and one with an SLE-like illness, all selected for the predominance of either IgM or IgG in serum. Spontaneous or BCDF-stimulated Ig secretion was of the isotype predominant in vivo, whereas C1q selectively stimulated B cells to produce the other isotype (IgG vs IgM). Thus, C1q interacts with B lymphocytes in a manner distinct from that of BCDF found in mixed lymphocyte supernatants. C1q may be an important factor influencing the production of Ig by B lymphocytes in normal individuals and in patients with abnormalities of B cell activity.     

Development and regulation of chlamydia-responsive murine B lymphocytes

We have examined characteristics of chlamydia-stimulated mouse B cells as well as cells that regulate polyclonal responses in vitro. B lymphocyte proliferation stimulated by chlamydia arises at a similar time as Escherichia coli lipopolysaccharide (LPS)-induced proliferative responses during ontogeny. In contrast, development of immunoglobulin (Ig)-secreting cells after chlamydia stimulation is delayed by several weeks relative to ontogeny of LPS-inducible plaque-forming cells (PFC). The lack of Ig secretion by immature B cells is not due to a deficiency of Lyb5+ B lymphocytes, since X-linked immunodeficient (xid) NBF1 mice that lack this B lymphocyte population respond well to chlamydia stimulation. Adherent cells are important for chlamydia-stimulated B lymphocyte differentiation, but are not as necessary for their proliferation. Neither adult adherent cells nor T cells can correct the inability of immature spleen cells to develop into Ig-secreting cells; spleen cells from 2-wk-old mice (i.e., immature B cells) will not suppress adult B lymphocyte responses to chlamydia. When B lymphocytes are separated according to their buoyant densities, chlamydia stimulates low density (activated) B cells to proliferate and differentiate better than high density (resting) cells. Proliferative responses to chlamydia arise earlier during ontogeny, do not require adherent cells, and can proceed to a relatively greater extent in resting B cell population (compared with activated B cells) than induction of Ig-secreting cells.     

In vitro analysis of allogeneic lymphocyte interaction. III. Generation of a helper allogeneic effect factor (AEF) across an I-J subregion disparity.

Allogeneic effect factors (AEF) were produced across an I-J subregion incompatibility. The helper activity of these AEFs is H-2 restricted since they help B cells only of the stimulator haplotype and of other haplotypes that carry the same I-J subregion gene(s) as the stimulator haplotype. Immunoadsorption studies demonstrate that they consist of I-J determinants derived initially from the GVHR host and MLR stimulator cells and not the GVHR donor and MLR responder cells used to generate AEF. It is postulated that the genetic restriction of AEF helper activity is mediated in part by the ability of the GVHR activated donor T cells to acquire, in vivo, recipient T cell and/or macrophage derived I-J determinants. Cellular adsorption studies indicate that AEF helper activity may be adsorbed by B cells, but neither T cells nor macrophages, of the stimulator haplotype. The results suggest that an I-J-positive AEF interacts with an I-J subregion controlled complementary recognition structure on a target B cell and, after antigenic stimulation, activates that B cell to IgG antibody synthesis.