Interleukin 4 enhances the ability of antigen-specific B cells to form conjugates with T cells

T cell-B cell conjugates are formed when trinitrophenyl-specific B cells are exposed to trinitrophenyl-ovalbumin and ovalbumin-specific T hybridoma cells. The proportion of conjugates was increased two- to threefold when antigen-pulsed trinitrophenyl-specific B cells, but not T cells, were pre-exposed to interleukin 4. Antigen-specific B cells pretreated with antigen and interleukin 4 and cultured in the presence of specific T helper cells also produced a larger proportion of antibody-secreting cells as compared to cells pretreated with antigen alone. The interleukin 4-induced enhancement of T/B conjugate formation occurred over a wide range of antigen concentrations, was dependent on the concentration of interleukin 4, and was inhibited by the monoclonal anti-interleukin 4 antibody, 11B11. The importance of Ia antigens in the enhancement of conjugate formation and generation of antibody-secreting cells is suggested by a) the fact that the interleukin 4-mediated increase in the density of Ia antigens on the antigen-specific B cells correlated with their enhanced ability to form T/B conjugates, b) the kinetics of the interleukin 4-mediated increase in conjugate formation and surface Ia expression were similar, c) 10- to 20-fold higher concentrations of anti-I-A antibody were required to inhibit T/B conjugate formation by 50% with interleukin 4-treated antigen-specific B cells compared with untreated antigen-specific B cells, and d) interferon-gamma, which inhibits the interleukin 4-mediated increase in Ia antigens, inhibited the interleukin 4-induced enhancement of T/B conjugate formation. These results indicate that the interleukin 4-induced increase in the expression of Ia antigens on B cells plays an important role in the enhancement of T/B cell interactions and the subsequent differentiation of antigen-specific B cells into antibody-secreting cells.     

The modulation of membrane Ia on human B lymphocytes

Using flow cytometry techniques, changes in surface Ia (DR and DS) expression on human B lymphocytes were correlated with changes in the cell cycle following stimulation with anti-mu. The effect of interleukin (IL)-1, IL-2, B-cell growth factor (BCGF), and interferons on Ia expression on resting B cells was also examined. A population of resting B lymphocytes was cultured in vitro with 100 micrograms/ml of anti-mu and immunofluorescently stained for DR and DS at various times following stimulation. Detectable increases in DR and DS expression were found within 8 hr, and the major increases (twofold and fourfold) in DR and DS expression occurred over the next 48 hr. Using cell cycle inhibitors and propidium iodide staining, it was demonstrated that the enhanced DR and DS expression following anti-mu stimulation began during G0 to G1 transition and increased as the cells progressed through G1 phase. During S and G2/M phases, there were minimal further increases in surface Ia. Although prolonged exposure of B cells to anti-mu was required for cellular activation, cell size enlargement, and progression into S phase, a brief exposure to anti-mu, insufficient for cellular activation, markedly enhanced Ia expression. Thus anti-mu-stimulated resting human B lymphocytes rapidly increase their surface Ia expression. This increase occurs predominantly prior to entrance into S phase and can occur in the absence of significant cellular activation. Interferons have been reported to modulate surface Ia expression on a human lymphoid cell line and on monocytes and supernatants with BCGF activity to enhance surface Ia expression on murine B cells; however, neither alpha-interferon, gamma-interferon, IL-1, IL-2, nor BCGF modified surface DR expression on normal resting human B cells.     

Interleukin-1 and interleukin-6 synergize in preparing resting murine B cells to respond to anti-mu: correlation with c-myc expression.

We demonstrate that stimulation with interleukin (IL)-1 and IL-6 prepares high-density B cells to enter the S phase more promptly in response to subsequent stimulation with anti-mu F(ab')2. The stimulatory effect of IL-1 and IL-6 is compared to the one described for IL-4. In contrast to IL-4, preculture in IL-1 and IL-6 does not induce an increase in cell volume or in expression of class II major histocompatibility complex antigens on resting B cells. Similarly, the expression of the p55 subunit of the IL-2 receptor and of the transferrin receptor was not detected on resting B cells stimulated with IL-1 and IL-6. However, the stimulatory effect of IL-1 and IL-6 is correlated with an increased expression of c-myc proto-oncogene mRNA in resting murine B cells.     

Characterization of T-cell-soluble factors modulating the expression of Ia and H-2 antigens on BALB/c B lymphoma cell lines

The effect of supernatants of concanavalin A-activated spleen cells (CAS) on the expression of various antigens, especially Ia antigens, on BALB/c B lymphoid cells, was examined. This study demonstrates the following: (i) CAS enhanced the expression of Ia antigens on four out of five BALB/c lymphoid cell lines. (ii) CAS selectively modulates the expression of Ia and H-2D, but not sIgM or viral gp70 expression, on X16C 8.5 tumor cells. The enhanced levels of Ia expression on B lymphoid tumor cells were also detected by using anti-Ia monoclonal antibodies. (iii) The molecular weight of soluble factor(s) affecting Ia and H-2 was approximately 40,000 estimated by gel filtration on a Sephadex G-200 column. (iv) Type 1 interferon but not interleukin 1, interleukin 2, or T-cell-replacing factor enhanced the expressions of Ia and H-2D antigens. (v) The activity of CAS-modulating Ia and H-2 antigens was eliminated by acidic treatment. It was concluded from this study that at least one of the factor(s) in CAS, modulating the antigenic expression of B-lymphoid cells, was interferon-like in nature. From our findings, a possible immunoregulatory mechanism by interferon was suggested: T cells, after stimulation of mitogens or antigens, secrete interferons which modulate the expression of Ia and H-2 on B cells. Then B cells, whose Ia and H-2 were modulated selectively by T-soluble factors(s), might interact with T cells much more efficiently.     

Polyclonal B cell activation by B cell differentiation factor B151-TRF2. I. Involvement of self-Ia recognition process mediated by B cells

The present study examined the functional role of Ia antigens on B cells in polyclonal B cell activation induced by a B cell differentiation factor, B151-TRF2. The polyclonal IgM PFC responses by B151-TRF2 were inhibited by monoclonal antibodies specific for class II MHC antigens (Ia antigens) but not class I MHC antigens. Such inhibition by anti-Ia antibodies was haplotype-specific and was observed in the absence of both T cells and accessory cells. Moreover, the anti-Ia antibody-induced inhibition of the B151-TRF2 responses was not due to the blocking of binding of B151-TRF2 to the corresponding B cell receptor. A series of kinetic studies revealed that some Ia-mediated cellular activation process occurs before the resting B cells become responsive to B151-TRF2. Thus, the B151-TRF2-mediated B cell responses consist of at least two distinct phases. The early phase is an Ia-dependent but B151-TRF2-independent process, whereas the late phase is an Ia-independent but B151-TRF2-dependent process. To further characterize the functional role of Ia antigens on B cells, an additional experiment was carried out by using F1 B cells which co-dominantly express both parental Ia antigens on the surface. Interestingly, it was observed that the degree of inhibition of the B151-TRF2-mediated responses of F1 B cells by anti-parental Ia antibody was, at best, one-half that of the parental B cells, suggesting that F1 B cells may be separated into two subpopulations with the restriction specificity for the respective parental Ia antigens. To examine this possibility, (B10 X B10.BR)F1 B cells were separated into adherent and nonadherent cell populations by their ability to bind to either one of the parental B cell monolayers, and the specificity of inhibition of their responses to B151-TRF2 by anti-Ia antibodies was assessed. It was found that the responses of (B10 X B10.BR)F1 B cells adherent to the B10 B cell monolayer or the B10.BR B cell monolayer were almost completely inhibited by anti-I-Ab and anti-I-Ak antibodies, whereas those of nonadherent cells were now selectively inhibited by anti-I-Ak and anti-I-Ab antibodies, respectively. These findings are interpreted as indicating that the B151-TRF2-responsive F1 B cells consist of at least two subpopulations with the restriction specificity for either one of the parental Ia antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-term-cultured mouse B-lymphocyte line. II. Modulation of Ia-antigen expression on B-lymphocyte line

Mouse B-cell line, established by culturing anti-Thy-1 and complement-treated splenic B cells with concanavalin A-stimulated conditioned medium, expressed immunoglobulins and Ia antigens on its surface. The long-term-cultured B-cell line was split in two and maintained with or without 3300 R X-irradiated T-cell-depleted syngeneic splenic adherent cells (SAC). Interestingly, the B-cell line cultured without SAC lost its Ia antigen but not its Ig expression, whereas the cell line with SAC maintained both Ia and Ig expression. The ability to express Ia antigens was restored by culturing them only in the presence of Ia-positive feeder cells. Neither recombinant interferon-gamma or lectin-stimulated conditioned medium nor cell-free culture supernatant SAC had the ability to restore Ia antigen expression on the B-cell line. Incubation of Ia-negative B-cell line with phorbol esters restored the Ia expression. It is suggested that the expression of Ia antigen on B lymphocytes was controlled differently from that on macrophage lineage. The B-cell line expressing Ia antigens acts as stimulator cells for alloantigen-activated T lymphocytes and as antigen-presenting cells on the KLH-specific Ia-restricted proliferative T-cell clone in the presence of a specific antigen.     

Role of self carriers in the immune response and tolerance. XII. Effect of epitope density and antigen-presenting cell phenotype on the presentation of hapten-modified self for the induction of immunity or tolerance in vitro

The data presented in the accompanying paper (J. P. Cogswell, R. P. Phipps, and D. W. Scott, Cell. Immunol. 114, 55-70, 1988) indicate that certain macrophage-like and lymphoid dendritic-like (P388AD.2) tumor lines which express major histocompatibility encoded class II (Ia) antigens and produce interleukin 1 (IL-1) are uniquely able to present hapten-modified self (HMS) in an immunogenic fashion in vivo. In the current study, the relationship between phenotype and function has been confirmed utilizing a completely in vitro system. This investigation revealed that B-cell priming required T cells restricted to P388AD.2's I-A antigens. In addition, exogenous IL-1 reconstituted the response of an IL-1-deficient tumor (P388AD.2-ILd), although it had no effect on the other nonimmunogenic Ia+ tumor lines. Unlike the in vivo system, effective B-cell tolerance was induced when P388AD.2 was modified with high concentrations (10 mM) of hapten or when highly haptenated tumor was added to 0.1 mM TNBS-modified P388AD.2. These results suggest that positive regulation of in vitro immune responses to HMS is dependent upon the phenotype of the accessory cell carrier (with lymphoid dendritic-like cells being unusually potent), while negative regulation is associated with high epitope density. This system now allows the dissection of the properties of different accessory cells and the signals required for B-cell priming or tolerance induction.     

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.     

Production of multiple lymphokines by the A20.1 B cell lymphoma after cross-linking of membrane Ig by immobilized anti-Ig

Cross-linking of membrane IgG2a or IgD on the B cell lymphoma A20.1 resulted in the elaboration of lymphokines which were able to support the growth of HT-2 cells and to induce increased Ia expression on resting B cells. Unstimulated A20.1 cell did not produce detectable levels of lymphokine activity. Lymphokine secretion did not occur in response to cross-linking of MHC class II (Ia) or class I (H2K) molecules. The kinetics for secretion were rapid, with detectable levels of lymphokine arising within 3 to 4 h of stimulation. Maximal lymphokine production was reached by 8 to 10 h. Soluble intact anti-Ig antibodies failed to stimulate lymphokine production due to Fc-mediated effects. This was concluded based on the fact that soluble F(ab')2 fragments of anti-IgG, but not soluble intact antibody, stimulated the production of lymphokine by A20.1 cells. Based on serologic criteria, membrane Ig cross-linking by ligand induced secretion of IL-2 but not IL-4 by A20.1 cells. Induction of Ia expression by resting B cells in response to A20.1 supernatant was not mimicked by stimulation with IL-1, -3, -5, or -6 either singly or in combination. Furthermore, preliminary physicochemical characterization revealed that the Ia-inducing factor in A20.1 supernatant has a molecular weight greater than 50,000. These data suggest that the Ia-inducing activity is a novel lymphokine. Thus, this report describes the first evidence for the existence of a B cell tropic lymphokine produced by B cells in response to Ag receptor-mediated signal transduction.     

Effect of B cell stimulatory factor-1 (interleukin 4) on Fc epsilon and Fc gamma receptor expression on murine B lymphocytes and B cell lines

Culture of murine splenic B cells with interleukin 4 (IL-4) caused the up-regulation of the lymphocyte Fc receptor for immunoglobulin E (IgE) (Fc epsilon R) over a similar dose range as required for Ia up-regulation. However, the expression level of the Fc receptor for immunoglobulin G (Fc gamma R) did not increase, rather IL-4 caused a slight but consistent decrease in the Fc gamma R level on the B cells. Fc epsilon R+ B hybridoma cells also responded to IL-4 by exhibiting increased Fc epsilon R expression; with the hybridoma cells Fc gamma R levels were unaffected. IL-4 caused an increase in the number of Fc epsilon R per cell and the highest levels of expression were obtained by having both IgE and IL-4 present in the culture. The specificity of the increase was demonstrated by blocking IL-4-mediated actions with monoclonal anti-IL-4 (11B11). Experiments following the incorporation of [35S]methionine into the Fc epsilon R demonstrated that IL-4 increased the rate of Fc epsilon R biosynthesis; this provides an explanation for the IL-4-induced increase in Fc epsilon R expression. IL-4, unlike IgE, had no effect on the rate of degradation of the Fc epsilon R. Interferon-gamma (IFN-gamma) totally abrogated IL-4-mediated Fc epsilon R up-regulation; at the same concentration of IFN-gamma Ia up-regulation is also suppressed, although not as effectively. IFN-gamma was shown to directly suppress Fc epsilon R synthesis, thereby explaining the inhibitory action on Fc epsilon R levels. Finally, it was shown that 11B11 inhibited the increased expression of Fc epsilon R on B cells obtained from mice during the early, but not the late, stages of Nippostrongylus brasiliensis infection. This latter finding suggests that the high Fc epsilon R levels seen early in parasite infections are dependent upon IL-4. The results overall provide further insight into the biologic activities of IL-4.     

Activation of human B cells. Comparison of the signal transduced by IL-4 to four different competence signals

The effects of the cytokine IL-4 on resting and activated human B cells were compared with the effects of known "competence" signals able to drive resting B cells into the cell cycle, including anti-Ig, PMA, anti-CD20, and a recently described competence signal, anti-Bgp95. In proliferation assays, IL-4 was costimulatory with anti-Ig and anti-Bgp95 but not with anti-CD20 or PMA. IL-4 alone triggered increases in expression of class II DR/DQ and CD40, but it did not trigger increases in intracellular free calcium [Ca2+]i in resting B cells or induce resting B cells to leave G0 and enter the G1 phase of the cell cycle. Although IL-4 has some characteristics of competence signals, it was most effective if added to B cells up to 12 h after anti-Ig or anti-Bgp95 rather than before, and thus, in this respect, works more like a progression signal. Like IL-4, all four competence signals for B cells triggered increases in class II and CD40, but only IL-4 consistently induced increases in CD23 surface levels. IL-4 was costimulatory only with anti-Ig and anti-Bgp95, each of which can trigger increases in [Ca2+]i and new protein synthesis of the proto-oncogene c-myc, and can increase attachment of protein kinase C to the plasma membrane. IL-4 was not costimulatory with signals that 1) did not affect [Ca2+]i yet induced c-myc protein synthesis (anti-CD20), 2) only stimulated the translocation of protein kinase C (PMA), or 3) only stimulated increases in [Ca2+]i (calcium ionophore). These results suggest that resting human B cells require at least two intracytoplasmic signals before IL-4 can effectively promote B cell proliferation.     

Further evidence for a human B cell activating factor distinct from IL-4

Supernatants from activated human T cell clones were previously shown to contain B cell-activating factor (BCAF), an activity which results in polyclonal resting B cell stimulation. In the present study, we investigate the relationship between this activity and human interleukin-4 which was also shown to act on resting B cells. The supernatant of the T cell clone TT9 contains IL-4 but anti-IL-4 antiserum does not affect the response of B cells as measured by thymidine uptake or cell volume increase. Furthermore, IL-4 induces Fc epsilon-receptor (CD23) expression on 30% of unstimulated human B cells, whereas BCAF-containing supernatants from clone P2, that do not contain detectable amounts of IL-4, promote B cell proliferation without inducing CD23 expression. Our results therefore establish that IL-4 and BCAF are distinct activities and suggest that they trigger different activation pathways in human B cells. In addition, culture of B cells with T cell supernatants for 72 hr induces a three- to fourfold increase in the expression of HLA-DR, -DP, and -DQ antigens in 50% of B cells. The addition of inhibiting concentrations of anti-IFN-gamma, LT, or IL-4 antisera to the cultures does not change these results. Finally, 30% of B cells cultured with T cell supernatants leave the G1 phase of the cell cycle and 20% reach mitosis. Taken together, our findings further support the existence of a B cell-activating factor responsible for the activation of resting human B cells.     

Single-cell analysis of costimulation by B cells, endothelial cells, and fibroblasts demonstrates heterogeneity in responses of CD4(+) memory T cells.

Human endothelial cells (EC) express MHC class II molecules in vivo and are likely to be involved in presentation of antigens to CD4(+) T cells. We examined, at the single-cell level, EC presentation of superantigens to resting CD4(+) memory T cells. Within 2 h of adherence to class II+ EC early T cell activation is evidenced by translocation of nuclear factor of activated T cells (NFAT), surface expression of CD69, and synthesis of IFN-gamma and IL-2. Naive T cells are not activated. T cell activation is dependent on the prior induction of MHC class II molecules on EC and is blocked by antibodies to LFA-3 (CD58). Our data place EC along a spectrum of antigen-presenting ability. Activated B cells and macrophages trigger more cells to express cytokines than do EC and at lower antigen concentrations; EC are in turn, superior to fibroblasts or smooth muscle cells. Furthermore, the concept of activation thresholds for cytokine synthesis within T cells also extends to earlier activation events: NFAT translocation is relatively easy to trigger, as is CD69 expression; fewer cells can be triggered to express IFN-gamma and fewer still to express IL-2. EC may, therefore, contribute to a graded immune response by inducing qualitatively and quantitatively different responses than professional APC.     

IL-4 down-regulates the expression of J11d on murine B cells.

T cell-derived IL-4 has many effects on murine B cells, including the up-regulation of class II antigens and the induction of isotype switching. The development of memory B cells and the decreased expression of J11d antigens on these cells are influenced by T cells. In this report, we determined whether the decreased expression of J11d can also be mediated by T cell-derived lymphokines and, in particular, IL-4. We found that IL-4 can down-regulate the expression of J11d on both large and small B cells, that this effect becomes significant after 48 hr of culture and occurs at doses of IL-4 that are similar to those required to up-regulate murine class II MHC antigens encoded by I-region alpha genes (IA). Anti-IL-4 antibody completely blocks this effect but IFN-gamma does not. Other lymphokines (IL-1, IL-2, IL-3, IL-5, IL-6) neither induce a decrease in J11d nor alter the ability of IL-4 to down-regulate J11d expression. The decrease in J11d expression on B cells is not due to a preferential survival of cells expressing lower levels of J11d, although IL-4 has a more pronounced effect on these B cells. Finally, the down-regulation of J11d and up-regulation of IA by IL-4 occurs on all inducible B cells.     

Evidence for effects of interleukin 4 (B cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages

We have studied the effects of recombinant mouse interleukin 4 (IL 4) (previously known as B cell stimulatory factor 1) on the antigen-presenting ability of murine splenic B cells and bone marrow macrophages. Our assay is based on the induction of antigen-presenting ability in these cells after incubation with IL 4 for 24 hr. The presenting cells were then used to stimulate IL 2 production by antigen-specific, I-Ad-restricted T cell hybridomas, a response mainly dependent on the induction of Ia antigens. Consistent with our previously published data using partially purified natural IL 4, we show here that recombinant IL 4 (but not interferon-gamma (IFN-gamma) or IL 1) induces antigen-presenting ability in B cells. Recombinant IL 4 was also found to induce antigen-presenting ability in a cloned, bone marrow derived-macrophage cell line (14M1.4), and in normal bone marrow-derived macrophages. These macrophage populations also respond to IFN-gamma showing enhanced antigen-presenting ability (mediated by increased Ia antigen expression). A small but significant increase in Ia antigen expression was also detected in 14M1.4 macrophages induced with IL 4. However, additional analysis suggested that the effect of IL 4 on 14M1.4 is different from that of IFN-gamma, because IL 4 (but not IFN-gamma) is able to maintain the viability and increase the size of and metabolic activity of bone marrow macrophages. However, IL 4 may not affect all macrophages because the macrophage cell line P388D1, which responds to IFN-gamma, failed to show enhanced antigen-presenting function after stimulation with IL 4. These observations indicate that IL 4, a lymphokine previously considered to be B cell lineage specific, has effects on macrophages and may be involved in their activation.     

Differential effect of antigen-nonspecific T-cell factors and lipopolysaccharide on the Ia antigens and surface immunoglobulins of BALB/c lymphoma cell lines

In order to study the mechanism of B-cell differentiation using B lymphoid tumor cells as models, we investigated the effects of antigen-nonspecific T-cell factors in combination with lipopolysaccharide (LPS) on the expression of surface markers on B lymphoid cell lines. This study demonstrated that culture supernatant from concanavalin A-activated spleen cells (CAS) gave 2- to 3.5-fold enhancement of the expression of Ia antigens. The effect of CAS was dose dependent and as little as 2% CAS gave maximum enhancement of Ia antigen expression. The CAS effect was due to concanavalin A-activated cell products and was not due to the concanavalin A. The effects of allogeneic effect factor (AEF) on Ia antigens were similar to those of CAS. In contrast to CAS and AEF, LPS did not affect the expression of Ia antigens on ×16C 8.5. LPS enhanced 1.5- to 3-fold the expression of sIgM on this cell line. The expression of sIgM was minimally affected by T-cell factors; CAS induced 20 to 70% enhancement of sIgM expression while AEF induced no significant effects. This study showed that antigen-nonspecific factors (CAS and AEF) influenced mainly Ia expression on the B-cell lymphoma, ×16C 8.5, while LPS selectively affected sIgM expression. Therefore, it was concluded that the mechanisms by which B cells are activated by T-cell factors and mitogens are different.     

Characterization and antigen-presenting function of a murine thyroid-derived epithelial cell line

We have developed and evaluated the antigen-presenting function of a murine thyroid-derived epithelial cell line M.5 in order to further investigate the possible role of the thyroid follicular epithelium in the inductive phase of autoimmune thyroiditis. M.5 cells did not express class II major histocompatibility complex encoded (Ia) antigens constitutively, but these could be readily induced with interferon-gamma. We found that Ia expressing M.5 cells were ineffective in stimulating T cell proliferation when tested in a 4-day primary mixed leukocyte reaction (MLR). However, significant T cell stimulation was obtained when phorbol myristate acetate (PMA) was added either to the M.5-T cell co-cultures, or for a brief period to the M.5 cells prior to adding the responder T cells. Cytofluorographic analysis of M.5 cells disclosed that PMA did not significantly alter the expression of Ia antigens. Additional experiments indicated that interleukin 1 (IL-1) was unlikely to represent the co-stimulatory factor generated by PMA. This was based on the observations that M.5 cells failed to secrete significant IL-1 either spontaneously, or in the presence of various stimuli, and that murine recombinant IL-1 failed to substitute for PMA in the activation of T cells. The nature of the co-stimulatory signal is as yet unknown. We conclude from these experiments that a pure population of thyroid-derived epithelial cells may be able to function, under the described circumstances, as antigen presenting cells.     

Role of HLA class I and class II antigens in activation and differentiation of B cells

The monoclonal antibodies (MoAb) CR10-214, CR11-115, and Q1/28 to distinct monomorphic determinants of HLA class I antigens, the MoAb CL413 and PTF29.12 recognizing monomorphic determinants of HLA-DR antigens, the anti-HLA-DQw1 MoAb KS11, the anti-HLA-DPw1 MoAb B7/21, and the anti-HLA-DR,DP MoAb CR11-462 were tested for their ability to modulate human B-lymphocyte proliferation and maturation to IgM-forming cells. Purified tonsillar B cells were stimulated with Staphylococcus aureus bacteria of the Cowan first strain (SAC) or anti-human mu-chain xenoantibodies, as well as in growth factor- or T-cell-dependent activation cultures. The B-cell proliferative responses induced by SAC or by mitogenic concentrations of anti-mu-chain xenoantibodies were inhibited by some of the anti-HLA class I and anti-HLA class II monoclonal antibodies tested. The same antibodies were effective inhibitors of the proliferation of B cells stimulated with interferon-gamma (IFN-gamma) or interleukin-2 (IL-2) and with submitogenic concentrations of anti-mu-chain xenoantibodies. The proliferation induced by IL-2 of SAC-preactivated B cells was inhibited by some of the anti-HLA class II monoclonal antibodies, but not by the anti-HLA class I monoclonal antibodies tested. This inhibition appeared to reflect at least in part a direct effect on later events of the B-cell activation cascade, since some anti-HLA class II monoclonal antibodies still exerted considerable inhibitory activity when added together with IL-2 to SAC-preactivated B cells after the third day of culture. Anti HLA-DR, DQ, and DP monoclonal antibodies consistently inhibited the IgM production induced in B cells by T cells alone, T cells plus pokeweed mitogen (PWM), SAC plus IL-2, or IL-2 alone. In contrast, two of the three anti-HLA class I monoclonal antibodies tested inhibited the IgM production in cultures stimulated with SAC plus IL-2 and one the IgM production induced by IL-2 alone, but none of them had inhibitory effects on T-cell dependent IgM production. The results reported herein indicate that HLA class II molecules directly participate in different phases of the B-cell activation cascade. In addition, our data also suggest that HLA class I molecules can be involved in the events leading to B-cell proliferation and differentiation into immunoglobulin-secreting cells.     

Interleukin 5 and interleukin 4 produced by Peyer's patch T cells selectively enhance immunoglobulin A expression

Considerable evidence suggests that the high frequency of B cells committed to the IgA isotype in Peyer's patches is regulated by T lymphocytes. To understand more accurately the mechanism of this immunoregulation, an autoreactive T cell line from Peyer's patches was generated by culturing L3T4+ Peyer's patches T cells with syngeneic B cell blasts. The resulting T cell line, designated PT-1, and a clone derived from this line, PT-1.14, stimulated immunoglobulin secretion in spleen B cells with a preferential enhancement of IgA and IgG1 isotypes. Supernatant derived from concanavalin A-stimulated PT-1 or PT-1.14 cells could also enhance IgA secretion if spleen B cells were preactivated with lipopolysaccharide. Peyer's patches T cell supernatant did not contain IgA-specific binding factors. PT-1 supernatant scored positive in lymphokine assays for interleukin (IL)-2, IL-4 (B cell stimulatory factor 1), IL-5 (B cell growth factor II), and interferon-gamma, whereas PT-1.14 supernatant was positive for IL-4 and IL-5 and negative for IL-2 and interferon-gamma. Only IL-5 enhanced IgA secretion in lipopolysaccharide-activated B cells and this response was increased two- to three-fold by IL-4. These results suggest that the type 2 T helper subset which produces both IL-5 and IL-4 plays a primary role in regulating IgA expression.