Evaluation of a novel particle-based assay for detection of autoantibodies in idiopathic inflammatory myopathies.

BACKGROUND: Myositis specific antibodies (MSA) represent not only important diagnostic tools for idiopathic inflammatory myopathies (IIM), but also help to stratify patients into subsets with particular clinical features, treatment responses, and disease outcome. Consequently, standardization of MSA is of high importance. Although many laboratories rely on protein immunoprecipitation (IP) for the detection of MSA, IP standardization is challenging and therefore reliable alternatives are mandatory. Recently, we identified significant variation between IP and line immunoassay (LIA) for the detection of MSA and myositis associated antibodies. In this study we aimed to compare the results from our previous study to the results obtained with a novel fully automated particle-based technology for the detection of MSA and MAA. METHODS: A total of 54 sera from patients with idiopathic inflammatory myopathy (IIM) were tested using three methods: IP, LIA (Euroimmun, Germany) and a novel particle-based multi-analyte technology (PMAT, Inova Diagnostics, US, research use only). The analysis focused on antibodies to EJ, SRP, Jo-1, NXP-2, MDA5, TIF1-γ, and Mi-2. RESULTS: Significant variations were observed among all methods. Overall, the novel PMAT assays showed slightly better correlation with IP, but the kappa agreement was strongly dependent on the antibody tested. When the results obtained from IP were used as reference for receiver operating characteristic (ROC) curve analysis, good discrimination and a high area under the curve (AUC) value were found for PMAT (AUC = 0.83, 95% confidence interval, CI 0.70-0.95) which was significantly higher (p = .0332) than the LIA method (AUC = 0.70, 95% CI 0.56-0.84). CONCLUSION: The novel PMAT used to detect a spectrum of MSA in IIM represents a potential alternative to IP and other diagnostic assays. Additional studies based on larger cohorts are needed to fully assess the performance of the novel PMAT system for the detection of autoantibodies in myositis.

Natural killer cell deficiencies in a consecutive series of children with herpetic encephalitis.

Natural killer (NK) cells play a fundamental role in innate and early phases of adaptive immunity against viral infections, both in humans and in animal models. To date, NK cell deficiencies in patients with severe herpetic infections have been reported in single cases, and their role as predisposing factor is still controversial. Five children affected by herpetic encephalitis were consecutively admitted to the Anna Meyer Children's Hospital in Florence (Italy) between 2003 and 2005. We therefore investigated the presence of NK cell deficiencies in a consecutive series of children with herpetic encephalitis. Five healthy children were included in the study as controls. Differential WBC counts, main Ig and IgE class serum analysis, cytofluorimetric analysis of circulating T, B and NK cells were performed on our study population. Sequencing of a selected region of CD16A gene transcript was carried out in two patients. All patients resulted to be affected by deficiencies related to NK cells in respect to controls. One patient was also affected by lymphopenia, while no other significant deficits of immunity were detected in the study population. To date, this is the first survey that demonstrates isolated NK cell deficiencies in a cohort of consecutive patients affected by severe herpes simplex infections. These findings suggest a role for NK cell deficiencies as a predisposing factor for increased susceptibility and severe course of disease in these patients.

Influence of impaired T- and B-cell compartments on efficacy of IVIg in dysimmune neuropathies.

Autoimmune mechanisms are postulated to play a role in the development and progression of dysimmune neuropathies (DN). We investigated the relation between lymphocyte number and marker expression, and disease activity in 20 patients with DN under intravenous immunoglobulins (IVIg) treatment. B- and T-lymphocyte markers were studied by flow cytometry of the expression of CD5, CD25, CD23 and CD38 markers on B cells and of CD3, CD4 and CD8 markers, respectively. These parameters were compared with those obtained from matched healthy volunteers. The proportions of CD38+ B cells were higher in patients compared with those of controls. Proportions of activated CD4+ and CD8+ T cells were comparable in peripheral blood mononuclear cells of patients and controls, but a significant reduction of the absolute numbers of CD3+, CD4+ and CD8+ cells were observed in DN patients. The percentages of CD25+ memory T cells were instead significantly increased in DN patients. Lastly, T-cell reduction and the CD19/CD38 ratio over total B (CD19+) cells directly correlated with a poor response to IVIg therapy. In DN, whereas T-cell number is reduced, activated T and B cells are increased, thus suggesting an intrinsic defect of the immune response.

Functional characterization and modulation of cytokine production by CD8+ T cells from human immunodeficiency virus-infected individuals.

CD8+ T-cell clones were generated from peripheral blood mononuclear cells (PBMC) of three human immunodeficiency virus (HIV)-seronegative individuals and six HIV-seropositive individuals and assessed for their cytokine secretion profile, cytolytic potential, and chemokine production. While the great majority of CD8+ T-cell clones generated from HIV-seronegative individuals produced interferon (IFN)-gamma, but not interleukin-4 (IL-4), that is a type 1 cytotoxic (Tc1) profile, high numbers of CD8+ T-cell clones generated from HIV-seropositive individuals produced IL-4 in addition to IFN-gamma or IL-4 alone, thus showing a type 0 cytotoxic (Tc0)- or a type 2 cytotoxic (Tc2) profile, respectively. Tc0/Tc2 cells displayed lower cytolytic activity than Tc1 cells, including a reduced ability to lyse autologous targets pulsed with HIV or HIV peptides. By contrast, the production of chemokines RANTES and macrophage inflammatory protein-1alpha was comparable in Tc1, Tc0, and Tc2 clones irrespective of whether they were derived from HIV-seronegative or HIV-seropositive individuals. When CD8+ T-cell clones were generated from PBMC cultures of HIV-seronegative individuals conditioned with IL-4 plus an anti-IL-12 antibody (Ab), a shift towards the Tc0/Tc2-like profile was observed. Conversely, the addition to PBMC cultures of IL-12 plus an anti-IL-4 Ab shifted the differentiation of CD8+ T cells from HIV-infected individuals towards the Tc1-like profile, whereas IL-12 or anti-IL-4 Ab alone had a lower Tc1-promoting effect. Irradiated PBMC from HIV-infected individuals, used as feeder cells, shifted the differentiation of CD8+ T cells from a healthy HIV-seronegative individual towards the Tc0/Tc2-like profile. On the other hand, a shift towards the Tcl-like profile was noted in CD8+ T-cell clones generated from the skin specimens of two HIV-seropositive patients with Kaposi's sarcoma, successfully treated with IFN-alpha, in comparison to CD8+ clones generated from the same skin areas before treatment. The IFN-alpha-induced Tc1 shift could be prevented by the incubation of skin-infiltrating CD8+ T cells with IL-4 before cloning. Taken together, these data indicate that both defective production of IL-12 and abnormal IL-4 production in bulk PBMC populations of HIV-infected individuals may contribute to the development of high numbers of CD8+ T-cell clones showing a Tc0/Tc2-like phenotype and reduced cytolytic potential against HIV itself. They also suggest that the cytokine profile of CD8+ T-cell clones can be modulated by cytokines (or anticytokine Ab) both in vitro and in vivo.

Role for CD30 in HIV expression.

CD30 is a member of the tumor necrosis factor (TNF)-receptor superfamily, whose ligand (CD30L) has been identified on B cells, activated macrophages and a subset of activated T cells. We show here that infection in vitro with human immunodeficiency virus (HIV) of CD4+ T-cell clones generated from HIV-seronegative individuals can enhance the expression of CD30, which often preceeds and is associated with the death of clonal T cells. Furthermore, cross-linking CD30 with an agonistic CD30-specific monoclonal antibody potentiated HIV replication induced by an insolubilized anti-CD3 antibody in T-cell lines generated from HIV-infected individuals. More importantly, paraformaldehyde-fixed CD8+ T-cell clones expressing CD30L enhanced HIV replication in anti-CD3-stimulated allogeneic or autologous HIV-infected CD4+ T-cell lines and such a potentiating effect was inhibited by an anti-CD30L antibody. The anti-CD30L antibody also exerted a suppressive effect on the spontaneous HIV replication occurring in lymph node cells, freshly derived from an HIV-seropositive patient showing CD30 expression in B cells and in a proportion of CD8+ T lymphocytes. Thus, CD30 triggering may play an important role in both HIV replication and the death of HIV-infected CD4+ T cells.

Activation of HIV expression by CD30 triggering in CD4+ T cells from HIV-infected individuals.

CD30 is a member of the tumor necrosis factor receptor superfamily, preferentially expressed by T cells producing type 2 helper (Th2) cytokines, whose ligand (CD30L) has been identified on B cells, activated macrophages, and a subset of activated T cells. We show here that cross-linking CD30 with an agonistic CD30-specific monoclonal antibody, as well as with CD30L+ CD8+ T cell clones or CD30L+ B cells, enhanced HIV replication in CD4+ T cells from HIV-infected individuals, and such a potentiating effect was inhibited by anti-CD30L antibody. The anti-CD30L antibody also exerted a suppressive effect on spontaneous HIV replication occurring in lymph node cells from an HIV-sero-positive patient, showing CD30L expression by both B and CD8+ T lymphocytes. Thus, CD30 triggering by CD30L-expressing cells may plan an important role in the activation of HIV expression from latently infected CD4+ T cells.

Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane CD30 expression in established Th1 cell clones.

The effect of progesterone (P) on the cytokine production profile of Ag-specific human CD4+ T cell lines and clones was investigated. T cell lines specific for purified protein derivative or streptokinase (SK) derived in the presence of P exhibited significant increased ability to produce IL-5 in comparison with T cell lines derived in the absence of P. Moreover, IL-4 was significantly increased in SK-specific T cell lines derived in the presence of P in comparison with SK-specific T cell lines derived in the absence of this hormone. In addition, SK-specific T cell lines generated in the presence of P developed into T cell clones showing a Th0-, instead of Th1-like, cytokine profile. Furthermore, SK-specific T cell clones with an established Th1 profile of cytokine secretion did express mRNA for, and produced detectable amounts of, IL-4 when stimulated with P in combination with insoluble anti-CD3 mAb. Combined stimulation with P and insoluble anti-CD3 mAb also enabled Th1 clones to express CD30 on their surface membrane. These results indicate that P can favor the development of Th cells producing Th2-type cytokines and is an inducer of both transient IL-4 production and CD30 expression in established Th1 cells. Thus, P production at the placental level may be responsible, at least in part, for increased production of Th2-type cytokines which have been implied in fetal allograft survival and maintenance of successful pregnancy.

CD30 expression by CD8+ T cells producing type 2 helper cytokines. Evidence for large numbers of CD8+CD30+ T cell clones in human immunodeficiency virus infection.

A large panel of CD8+ T cell clones generated from peripheral blood lymphocytes (PBL) of healthy donors or human immunodeficiency virus (HIV)-infected individuals were assessed for both cytokine secretion profile and CD30 expression and release. The great majority of CD8+ T cell clones generated from healthy individuals showed the ability to produce interferon gamma (IFN-gamma), but not interleukin 4 (IL-4), and none of them either expressed membrane CD30 or released substantial amounts of soluble CD30 (sCD30) in their supernatant. In contrast, high numbers of CD8+ T cell clones generated from HIV-infected individuals, which produced IL-4 (and IL-5) in addition to IFN-gamma or IL-4 (and IL-5) alone, expressed membrane CD30 and released detectable amounts of sCD30 in their supernatants. Indeed, CD30 expression appeared to be positively correlated with the ability of CD8+ T cell clones to produce IL-4 and IL-5 and inversely correlated with their ability to produce IFN-gamma, whereas no correlation between CD30 expression and production of IL-10 was observed. These data suggest that CD30 is a marker for CD8+ T cells that have switched to the production of type 2 helper cytokines.

Th2-like CD8+ T cells showing B cell helper function and reduced cytolytic activity in human immunodeficiency virus type 1 infection.

We analyzed at clonal level the functional profile of circulating or skin-infiltrating T lymphocytes from two individuals infected with the human immunodeficiency virus type 1 (HIV-1), suffering from a Job's-like syndrome (eczematous dermatitis, recurrent skin and sinopulmonary infections, and hypergammaglobulinemia E) and showing virtually no circulating CD4+ T cells. Most of the CD3+ T cell clones generated from both patients were CD4- CD8+ TCR alpha beta +. The others were CD4- CD8- TCR alpha beta + which exhibited reduced mRNA expression for the CD8 molecule or no mRNA expression for either CD4 or CD8 molecules. The great majority of both CD4- CD8+ and CD4- CD8- did not produce interferon (IFN) gamma and exhibited reduced cytolytic activity. Rather, most of them produced large amounts of both interleukin (IL) 4 and IL-5 and provided B cell helper function for IgE synthesis. These data suggest that a switch of cytolytic CD8+ T cells showing a Th1-like cytokine secretion profile to cells that make Th2-type cytokines, exhibit reduced cytolytic potential, and provide B cell helper function can occur in the course of HIV-1 infection. These cells may contribute to the reduced defense against viral infections and intracellular parasites and account for the elevated IgE serum levels, eosinophilia, and the allergic-like clinical manifestations seen in a proportion of HIV-1-infected individuals.

Spontaneous in vitro differentiation of a myoepithelial cell line (PA 16/23) from a pleomorphic adenoma of the parotid gland is associated with reduced production of the autocrine growth factor interleukin 6.

A myoepithelial cell line (PA 16/23) was derived from a pleomorphic adenoma of the parotid gland. PA 16/23 cells have light microscopic, immunophenotypical and ultrastructural features of immature myoepithelial cells, i.e. they are of fusiform or stellate shape and show keratin and actin cytofilaments located mainly in the perinuclear cytoplasm, desmosomes and tracts of basal lamina. The PA 16/23 cells grew actively and expressed mRNA for and produced interleukin 6 (IL-6) which was released into the culture medium. This cytokine, in turn, acted as an autocrine growth factor on the cells. PA 16/23 cells also expressed high-affinity IL-6 receptors. In these cells, both IL-6 production and proliferation could be modulated by exogenous stimulants, such as IL-6 itself, IL-1, IL-4, tumour necrosis factor alpha, interferon gamma and lipopolysaccharide. From the 40th culture passage onwards, the PA 16/23 cells ceased to grow, either spontaneously or in response to exogenous stimulants. Moreover, they strongly reduced IL-6 production, and underwent morphological differentiation into more mature myoepithelial cells, with an increased amount and a different arrangement of the keratin and actin cytofilaments, which formed thick bundles in the peripheral cytoplasm. These findings suggest a role for IL-6 in modulating the proliferation and, possibly, the differentiation of the PA 16/23 cells.

Interleukin 12 induces stable priming for interferon gamma (IFN-gamma) production during differentiation of human T helper (Th) cells and transient IFN-gamma production in established Th2 cell clones.

Interleukin 12 (IL-12) facilitates the generation of a T helper type 1 (Th1) response, with high interferon gamma (IFN-gamma) production, while inhibiting the generation of IL-4-producing Th2 cells in polyclonal cultures of both human and murine T cells and in vivo in the mouse. In this study, we analyzed the effect of IL-12, present during cloning of human T cells, on the cytokine profile of the clones. The culture system used allows growth of clones from virtually every T cell, and thus excludes the possibility that selection of precommitted Th cell precursors plays a role in determining characteristics of the clones. IL-12 present during the cloning procedures endowed both CD4+ and CD8+ clones with the ability to produce IFN-gamma at levels severalfold higher than those observed in clones generated in the absence of IL-12. This priming was stable because the high levels of IFN-gamma production were maintained when the clones were cultured in the absence of IL-12 for 11 d. The CD4+ and some of the CD8+ clones produced variable amounts of IL-4. Unlike IFN-gamma, IL-4 production was not significantly different in clones generated in the presence or absence of IL-12. These data suggest that IL-12 primes the clone progenitors, inducing their differentiation to high IFN-gamma-producing clones. The suppression of IL-4-producing cells observed in polyclonally generated T cells in vivo and in vitro in the presence of IL-12 is not observed in this clonal model, suggesting that the suppression depends more on positive selection of non-IL-4-producing cells than on differentiation of individual clones. However, antigen-specific established Th2 clones that were unable to produce IFN-gamma with any other inducer did produce IFN-gamma at low but significant levels when stimulated with IL-12 in combination with specific antigen or insoluble anti-CD3 antibodies. This induction of IFN-gamma gene expression was transient, because culture of the established clones with IL-12 for up to 1 wk did not convert them into IFN-gamma producers when stimulated in the absence of IL-12. These results suggest that Th clones respond to IL-12 treatment either with a stable priming for IFN-gamma production or with only a transient low level expression of the IFN-gamma gene, depending on their stage of differentiation.

Immortalization with herpesvirus saimiri modulates the cytokine secretion profile of established Th1 and Th2 human T cell clones.

T blasts of six established human CD4+ T cell clones with defined Ag specificity and cytokine secretion profile (3 Th1 and 3 Th2) were immortalized with Herpesvirus saimiri (HVS) and compared with their uninfected counterparts for their ability to proliferate, produce cytokines, and express cytolytic activity. HVS-transformed Th1 and Th2 clones neither substantially changed their original surface markers nor lose their ability to proliferate in response to their specific Ag but did acquire the ability to proliferate in response to contact signals delivered by SRBC or autologous APC alone. In addition, transformation by HVS substantially enhanced the lectin-dependent cytolytic activity of Th1 clones and enabled noncytolytic Th2 clones to exert cytolytic activity. HVS-transformed Th1 clones but not their uninfected counterparts spontaneously transcribed and secreted Th1-type cytokines (IL-2, IFN-gamma, and TNF-beta) and such a production was further enhanced by stimulation with either SRBC or PMA plus anti-CD3 mAb. HVS transformed but not uninfected Th2 clones constitutively expressed both IL-4 and IL-2 mRNA and secreted IFN-gamma. Stimulation with PMA plus anti-CD3 mAb induced uninfected Th2 clones to secrete high amounts of IL-4 and IL-5 but not Th1-type cytokines, whereas the same HVS-transformed Th2 showed minimal IL-4 and IL-5 secretion with concomitant high production of IL-2, IFN-gamma, and TNF-beta. Transformation by HVS also resulted in up-regulation of TNF-alpha and IL-3 production by both Th1 and Th2 clones. The ongoing proliferation of HVS-transformed clones was partially inhibited by either anti-IL-2 or anti-IL-3 antibodies and virtually abolished by the combined addition of the two anticytokine antibodies, suggesting that both IL-2 and IL-3 can function as autocrine growth factors for HVS-transformed Th1 and Th2 clones.

Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells.

The effects exerted on the in vitro development of antigen-specific T cell lines and T cell clones by addition or neutralization of interleukin 12 (IL-12) in lymphocyte bulk culture were examined. T cell lines specific for Dermatophagoides pteronyssinus group I (Der p I) derived in the presence of IL-12 exhibited reduced ability to produce IL-4 and increased ability to produce interferon gamma (IFN-gamma), and developed into Der p I-specific CD4+ T cell clones showing a T helper type 0 (Th0)- or Th1-, instead of Th2-, like cytokine profile. In contrast, purified protein derivative (PPD)-specific T cell lines derived in the presence of anti-IL-12 antibody exhibited an increased ability to produce IL-4 and developed into PPD-specific CD4+ T cell clones showing a Th0-, instead of Th1-, like profile. The influence of IL-12 on the cytokine secretion profile of Der p I-specific T cell lines was not prevented by addition to lymphocyte bulk cultures of anti-IFN-gamma antibody, but could be at least partially inhibited by the removal from bulk cultures of CD16+ cells. Thus, IL-12 and CD16+ cells appear to have inhibitory effects on the development of IL-4-producing cells and to play an inductive role in promoting Th1-like responses.

Human IL-10 is produced by both type 1 helper (Th1) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production.

IL-10 gene transcription and IL-10 protein production was assessed in both type 1 (Th1) and type 2 (Th2) CD4+ human T cell clones by polymerase chain reaction and ELISA, respectively. Although Th2 clones apparently showed higher IL-10 mRNA levels, IL-10 mRNA expression was consistently found in Th1 clones, as well. Likewise, measurable IL-10 levels were found in the supernatants of both Th1 and Th2 clones. The effect of human IL-10 (h-IL-10) and viral IL-10 (v-IL-10) on the proliferative response and cytokine production by Th1 and Th2 human clones was also investigated. Addition in culture of h-IL-10 and v-IL-10 significantly reduced the proliferation of both Th1 and Th2 clones in response to the specific Ag and to PHA, but it had no inhibitory effect on the proliferative response of Th1 and Th2 clones to IL-2. h-IL-10 and v-IL-10 also inhibited the Ag-induced production of gamma-interferon (IFN-gamma) by Th1 clones and the production of IL-4 and IL-5 by Th2 clones, whereas they had no effect on the cytokine synthesis by the same clones stimulated with PMA plus anti-CD3 antibody. Preincubation of APC, but not of clonal T blasts, with h-IL-10 resulted in the inhibition of Ag-induced proliferation of both Th1 and Th2 clones, supporting the view that h-IL-10 primarily affects APC. These data demonstrate that, unlike the murine system where IL-10 is a product of Th2 (but not Th1) cells and seems to mainly down-regulate the Th1 response, in the human system, IL-10 is produced by, and down-regulates the function of, both Th1 and Th2 cells.

Human bone marrow non-B, non-T cells produce interleukin 4 in response to cross-linkage of Fc epsilon and Fc gamma receptors.

Human bone marrow (BM) cells lacking T- and B-cell markers expressed RNA encoding interleukin (IL) 4 and secreted detectable amounts of IL-4 in supernatants in response to Fc epsilon or Fc gamma receptor (Fc epsilon R or Fc gamma R) cross-linking. In some experiments, IL-5 RNA expression in response to Fc epsilon R cross-linkage could also be detected. In contrast, RNA transcripts for, and secretion of, IL-2, IL-6, and interferon gamma were never observed. The presence of IL-3 in the cultures was essential for IL-4 production by non-B, non-T BM cells in response to Fc gamma R cross-linking and enhanced IL-4 RNA expression in response to Fc epsilon R cross-linking. Under the same experimental conditions, BM T and B lymphocytes, as well as peripheral blood T, B, and non-B, non-T cells, did not express IL-4 RNA. Prolonged incubation of non-B, non-T cells in IgE-free medium followed by extensive washing did not inhibit IL-4 production induced by anti-IgE antibodies, suggesting that the Fc epsilon R involved in the response has the characteristics of a high-affinity receptor. The Fc epsilon R+ cells were separated from the Fc epsilon R- cells by sorting non-B, non-T BM cell suspensions with fluorescein isothiocyanate-conjugated IgE and then assessed for both IL-4 RNA expression and alcian blue staining. Both IL-4-producing and alcian blue-positive cells segregated with the Fc epsilon R+ fraction. These data suggest that human BM cells, probably belonging to the mast cell and/or basophil lineage, are capable of producing IL-4 in response to Fc epsilon R or Fc gamma R cross-linkage.

A novel pathway of human B cell activation initiated by CK226 surface antigen.

In this study we analyzed the effect of CK226 monoclonal antibody (mAb) on human B cell activation and proliferation. This mAb was shown to recognize a 75-kDa surface molecule expressed on both T and B lymphocytes and to mediate T lymphocyte activation and proliferation. Flow cytometry analysis of B cell populations isolated from peripheral blood, tonsil and spleen showed that CK226 surface antigen is highly expressed on 40-80% of surface Ig+ cells. When purified B cells were cultured in the presence of CK226 mAb, up-regulation of major histocompatibility complex class II and CD23 surface structures and the de novo expression of CD25 antigen could be detected within 48 h. In addition, B cells underwent proliferation ([3H] thymidine uptake) in the absence of either T cells or exogenous lymphokines. Proliferation was potentiated by the addition of suboptimal concentrations (0.5 ng/ml) of phorbol 12-myristate 13-acetate (PMA). Cells recovered at day 5 were surface Ig+ and no CD3+ cells could be detected. CK226-induced proliferation (either in the presence or in the absence of PMA) was not inhibited by anti-CD25 mAb. Addition of exogenous interleukin 2 to CK226-stimulated B cells resulted in further increase of B cell proliferation. On the other hand, CK226 mAb did not display a co-stimulatory effect with submitogenic concentrations of either anti-Ig antibody or Staphylococcus aureus Cowan strain I bacteria. In addition proliferation induced by mitogenic concentrations of the above stimuli was inhibited in a dose-dependent fashion by CK226 mAb.

Analysis of the B-cell growth-promoting activity of human IL-4, the co-stimulatory assay with anti-immunoglobulin antibodies. Comparison with the B-cell growth-promoting activity of other lymphokines.

Human recombinant interleukin-4 (rIL-4) was assessed for its ability to promote the proliferative response of purified human B cells co-stimulated with submitogenic concentrations of soluble F(ab')2 fragments of anti-immunoglobulin (Ig) antibodies. The growth-promoting activity of rIL-4 was usually as potent as, or even more potent than, that of recombinant interleukin-2 (rIL-2), and more potent than that of recombinant interferon-gamma (rIFN-gamma). Preincubation with rIL-4 did not cause enhancement of the proliferative response of B cells to the subsequent addition of rIL-4 and anti-IgM antibody. In contrast, the proliferative response of B cells preincubated with anti-IgM antibody and rIL-4 was potentiated by the subsequent addition of rIL-4. The simultaneous addition of rIFN-gamma and rIL-2 or rIFN-gamma and rIL-4 had an additive effect in comparison with the response induced by rIL-2 or rIL-4 alone, respectively, whereas simultaneous addition of rIL-2 and rIL-4 induced a response equal or lower than that stimulated by rIL-2 or rIL-4 alone. The addition of rIFN-gamma at the beginning of culture or preincubation of B cells with rIFN-gamma and anti-IgM antibody potentiated the proliferative response of B cells to the subsequent addition of either rIL-2 or rIL-4. Taken together, these data suggest that rIL-4 acts as a growth factor for activated human B cells and displays on such cells a growth-promoting activity similar to that of rIL-2.