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The main purpose of this review is to present justification for the urgent need to implement specific prophylaxis of invasive Staphylococcus aureus infections. We emphasize the difficulties in achieving this goal due to numerous S. aureus virulence factors important for the process of infection and the remarkable ability of these bacteria to avoid host defense mechanisms. We precede these considerations with a brief overview of the global necessitiy to intensify the use of vaccines against other pathogens as well, particularly in light of an impasse in antibiotic therapy. Finally, we point out global trends in research into modern technologies used in the field of molecular microbiology to develop new vaccines. We focus on the vaccines designed to fight the infections caused by S. aureus , which are often resistant to the majority of available therapeutic options.

Staphylococcus aureus causes a wide range of diseases from skin infections to life threatening invasive diseases such as bacteremia, endocarditis, pneumonia, surgical site infections, and osteomyelitis. Skin infections such as furuncles, carbuncles, folliculitis, erysipelas, and cellulitis constitute a large majority of infections caused by S. aureus (SA). These infections cause significant morbidity, healthcare costs, and represent a breeding ground for antimicrobial resistance. Furthermore, skin infection with SA is a major risk factor for invasive disease. Here we describe the pre-clinical efficacy of a multicomponent toxoid vaccine (IBT-V02) for prevention of S. aureus acute skin infections and recurrence. IBT-V02 targets six SA toxins including the pore-forming toxins alpha hemolysin (Hla), Panton-Valentine leukocidin (PVL), leukocidin AB (LukAB), and the superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxins A and B. Immunization of mice and rabbits with IBT-V02 generated antibodies with strong neutralizing activity against toxins included in the vaccine, as well as cross-neutralizing activity against multiple related toxins, and protected against skin infections by several clinically relevant SA strains of USA100, USA300, and USA1000 clones. Efficacy of the vaccine was also shown in non-naïve mice pre-exposed to S. aureus . Furthermore, vaccination with IBT-V02 not only protected mice from a primary infection but also demonstrated lasting efficacy against a secondary infection, while prior challenge with the bacteria alone was unable to protect against recurrence. Serum transfer studies in a primary infection model showed that antibodies are primarily responsible for the protective response., Competing Interests: MA and HK have stocks and RA and FH have stock options in Integrated Biotherapeutics Inc. JL serves on the Scientific Advisory Board of Integrated BioTherapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Karauzum, Venkatasubramaniam, Adhikari, Kort, Holtsberg, Mukherjee, Mednikov, Ortines, Nguyen, Doan, Diep, Lee and Aman.)

Staphylococcus aureus remains a leading cause of human infection. These infections frequently recur when the skin is a primary site of infection, especially in infants and children. In contrast, invasive staphylococcal disease is less commonly associated with reinfection, suggesting that tissue-specific mechanisms govern the development of immunity. Knowledge of how S. aureus manipulates protective immunity has been hampered by a lack of antigen-specific models to interrogate the T cell response. Using a chicken egg OVA-expressing S. aureus strain to analyze OVA-specific T cell responses, we demonstrated that primary skin infection was associated with impaired development of T cell memory. Conversely, invasive infection induced antigen-specific memory and protected against reinfection. This defect in adaptive immunity following skin infection was associated with a loss of DCs, attributable to S. aureus α-toxin (Hla) expression. Gene- and immunization-based approaches to protect against Hla during skin infection restored the T cell response. Within the human population, exposure to α-toxin through skin infection may modulate the establishment of T cell-mediated immunity, adversely affecting long-term protection. These studies prompt consideration that vaccination targeting S. aureus may be most effective if delivered prior to initial contact with the organism.

Staphylococcus aureus is known to produce persistent and chronic infections in both humans and animals. It is recognized that small-colony variants (SCVs), which produce higher levels of biofilm and that are capable of intracellular persistence, contribute to the chronicity or recurrence of infections and that this phenotype is inherent to the pathogenesis process. Prevention of S. aureus infections through vaccination has not yet met with considerable success. Some of the current vaccine formulations for S. aureus bovine mastitis consist of inactivated S. aureus bacteria, sometimes combined to E. coli J5. As such, the stimulation of cell-mediated immunity by these vaccines might not be optimal. With this in mind, we recently engineered a genetically stable double mutant SCV (ΔvraGΔhemB), which was highly attenuated in a mastitis model of infection. The present work describes the immune responses elicited in mice by various experimental vaccine compositions including the live-attenuated SCV double mutant and its inactivated form, combined or not with inactivated E. coli J5. The live-attenuated SCV was found to provoke a strong and balanced humoral response in immunized mice, as well as strong proliferation of ex-vivo stimulated splenocytes isolated from these animals. These splenocytes were also found to release high concentration of IL-17 and IFN-γ when compared to every other vaccination formulation. Inversely, the inactivated whole-cell vaccine, alone or in combination with the E. coli J5 bacterin, elicited lower antibody titers and failed to induce Th1 or Th17 cell-mediated responses in the splenocyte proliferation assay. Our results suggest that live-attenuated SCVs can trigger host immunity differently than inactivated bacteria and could represent a suitable vector for inducing strong humoral and cell-mediated immune responses, which are crucial for protection. This could represent an important improvement over existing vaccine formulations for preventing S. aureus bovine mastitis and other infections caused by this pathogen., Competing Interests: The authors have declared that no competing interests exist.

Staphylococcus aureus wound infections delay healing and result in invasive complications such as osteomyelitis, especially in the setting of diabetic foot ulcers. In preclinical animal models of S. aureus skin infection, antibody neutralization of alpha-toxin (AT), an S. aureus -secreted pore-forming cytolytic toxin, reduces disease severity by inhibiting skin necrosis and restoring effective host immune responses. However, whether therapeutic neutralization of alpha-toxin is effective against S. aureus -infected wounds is unclear. Herein, the efficacy of prophylactic treatment with a human neutralizing anti-AT monoclonal antibody (MAb) was evaluated in an S. aureus skin wound infection model in nondiabetic and diabetic mice. In both nondiabetic and diabetic mice, anti-AT MAb treatment decreased wound size and bacterial burden and enhanced reepithelialization and wound resolution compared to control MAb treatment. Anti-AT MAb had distinctive effects on the host immune response, including decreased neutrophil and increased monocyte and macrophage infiltrates in nondiabetic mice and decreased neutrophil extracellular traps (NETs) in diabetic mice. Similar therapeutic efficacy was achieved with an active vaccine targeting AT. Taken together, neutralization of AT had a therapeutic effect against S. aureus -infected wounds in both nondiabetic and diabetic mice that was associated with differential effects on the host immune response., (Copyright © 2018 American Society for Microbiology.)

The aim of this work was the assessment of the "in vivo" immune response of a poly(lactide-co-glycolide)-based nanoparticulate adjuvant for a sub-unit vaccine, namely, a purified recombinant collagen-binding bacterial adhesion fragment (CNA19), against Staphylococcus aureus-mediated infections. "In vivo" immunogenicity studies were performed on mice: immunisation protocols encompassed subcutaneous and intranasal administration of CNA19 formulated as nanoparticles (NPs) and furthermore, CNA19-loaded NPs formulated in a set-up thermosetting chitosan-β-glycerolphosphate (chitosan-β-GP) solution for intranasal route in order to extend antigen exposure to nasal mucosa. CNA19 loaded NPs (mean size of about 195 nm, 9.04 ± 0.37μg/mg as CNA19 loading capacity) confirmed as suitable vaccine for subcutaneous administration with a more pronounced adjuvant effect (about 3-fold higher) with respect to aluminium, recognised as "reference" adjuvant. CNA19 loaded NPs formulated in an optimised thermogelling chitosan-β-GP solution showed promising results for eliciting an effective humoral response and a good chance as intranasal boosting dose.

Staphylococcus aureus is the major cause of human septic arthritis and osteomyelitis, which deserve special attention due to their rapid evolution and resistance to treatment. The progression of the disease depends on both bacterial presence in situ and uncontrolled disruptive immune response, which is responsible for chronic disease. Articular and bone infections are often the result of blood bacteremia, with the knees and hips being the most frequently infected joints showing the worst clinical outcome. We report the development of a hematogenous model of septic arthritis in murine knees, which progresses from an acute to a chronic phase, similarly to what occurs in humans. Characterization of the local and systemic inflammatory and immune responses following bacterial infection brought to light specific signatures of disease. Immunization of mice with the vaccine formulation we have recently described (4C-Staph), induced a strong antibody response and specific CD4+ effector memory T cells, and resulted in reduced bacterial load in the knee joints, a milder general inflammatory state and protection against bacterial-mediated cellular toxicity. Possible correlates of protection are finally proposed, which might contribute to the development of an effective vaccine for human use., Competing Interests: This work was sponsored by Novartis Vaccines and Diagnostics Srl, now acquired by the GSK group of companies. All authors have declared the following interests: all authors were permanent employees of Novartis Vaccines at the time of the study. Following the acquisition of Novartis Vaccines by the GSK group of companies in March, 2015, all but A.C. and T.S. are now permanent employees of the GSK group of companies. E.D.G., F.B., A.S. and S.B. report ownership of GSK shares and/or restricted GSK shares. A.C., F.B., G.B. and E.C. are listed as inventors on patents owned by the GSK group of companies.

S. aureus vaccine development has proven particularly difficult. The conventional approach to achieve sterile immunity through opsonophagocytic killing has been largely unsuccessful. S. aureus is highly toxigenic and a great body of evidence suggests that a successful future vaccine for this organism should target extracellular toxins which are responsible for host tissue destruction and immunosuppression. Major staphylococcal toxins are alpha toxin (a single subunit hemolysin) along with a group of bicomponent pore-forming toxins (BCPFT), namely Panton-Valentine leukocidin (PVL), gamma hemolysins (HlgCB and AB), LukAB and LukED. In our previous report, an attenuated mutant of LukS-PV (PVL- S subunit) named as "LukS-mut9" elicited high immunogenic response as well as provided a significant protection in a mouse sepsis model. Recent discovery of PVL receptors shows that mice lack receptors for this toxin, thus the reported protection of mice with the PVL vaccine may relate to cross protective responses against other homologous toxins. This manuscript addresses this issue by demonstrating that polyclonal antibody generated by LukS-mut9 can neutralize other canonical and non-canonical leukotoxin pairs. In this report, we also demonstrated that several potent toxins can be created by non-canonical pairing of subunits. Out of 5 pairs of canonical and 8 pairs of non-canonical toxins tested, anti-LukS-mut9 polyclonal antibodies neutralized all except for LukAB. We also studied the potential hemolytic activities of canonical and noncanonical pairs among biocomponent toxins and discovered that a novel non-canonical pair consisting of HlgA and LukD is a highly toxic combination. This pair can lyse RBC from different species including human blood far better than alpha hemolysin. Moreover, to follow-up our last report, we explored the correlation between the levels of pre-existing antibodies to new sets of leukotoxins subunits and clinical outcomes in adult patients with S. aureus bacteremia. We found that there is an inversed correlation between the antibody titer to sepsis for leukotoxins LukS-mut9, LukF-PV, HlgC, LukE and LukAB, suggesting the risk of sepsis was significantly lower in the patients with higher antibody titer against those toxins.

Staphylococcus aureus is a fastidious pathogen of global concern. S. aureus can not only cause a wide range of serious infections, but can also colonize healthy individuals. Nosocomial- associated S. aureus infection is a major concern for healthcare facilities. Recent increases in antibiotic resistance and community-acquired infection highlight the need for improved understanding for prevention and treatment of this pathogen. This minireview addresses the current state of S. aureus prevalence, correlates of immunity, and vaccine development.

This study was undertaken to compare the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine for the prevention of mastitis using the mouse as a model system. Mice immunized with formalin-killed whole cell vaccine of S. aureus residing in a biofilm when delivered via an intramammary route produced a cell mediated immune response. Mice immunized with this biofilm vaccine showed significant reductions in colonization by S. aureus in mammary glands, severity of clinical symptoms and tissue damage in mammary glands in comparison with the mice immunized with formalin-killed whole cells of planktonic S. aureus. The planktonic vaccine administered by a subcutaneous route produced a significantly higher humoral immune response (IgG1 and IgG) than the biofilm vaccine. However, considering the host response, tissue damage, the clinical severity and colonization of S. aureus in mammary glands, the biofilm vaccine performed better in immunogenicity and protective potential when administered by the intramammary route.

Staphylococcus aureus (S. aureus) is one of the most widespread agent of diseases in humans and animals. In dairy cows, S. aureus is the most frequently isolated contagious pathogens in mastitis cases and vaccines are one of the potential tools to control the infections, thus decreasing the use of antibiotics. Among all the virulence factors produced by S. aureus, extra cellular fibrinogen binding protein (Efb) is an important one in the pathogenesis of mastitis. Plants are useful bioreactors to produce antigens and the aim of the study was the production of Efb in two cultivars of Nicotiana tabacum as a mean to produce vaccine against S. aureus in plants. A matrix attachment region (MAR) sequence was inserted near the two borders of transfer-DNA in the transformation vector in the two possible orientations. The presence of MAR elements in the transformation system significantly improved transformation efficiency and Efb protein yield up to a 2% level on total soluble protein (TSP). Mice orally immunized with transgenic lyophilized leaves produced an antigen-specific immune response., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

To develop a safer, more immunogenic and efficacious vaccine against Streptococcus equi ssp. zooepidemicus (SEZ) infections, the gene of M-like protein (SzP) was placed under the strong vaccinia virus promoter P28 and then inserted into swinepox virus (SPV) genome. The recombinant swinepox virus (rSPV-szp) was isolated in a non-selective medium by the co-expression of Escherichia coli LacZ gene and verified by PCR, western blotting and immunofluorescence assays. To evaluate the immunogenicity of this rSPV-szp, ICR mice were immunized with the rSPV-szp, inactivated SEZ vaccine (positive control), wild type SPV (negative control), or PBS (challenge control). All mice were intraperitoneally challenged with 5 LD(50) of homogenous ATCC 35246 strain 14 days post-vaccination. The results showed that at least 70% mice in rSPV-szp-vaccinated group were protected against homogenous ATCC 35246 challenge, the survival rate was significantly higher compared with mice in the negative control group and the challenge control group (P<0.001). The antibody titers of the rSPV-szp-vaccinated group were significantly higher (P<0.05) than the other three groups. Passive immune protection assays showed that the hyperimmune sera against M-like protein could provide mice with complete protection against challenge of ATCC 35246. Semi-quantitative RT-PCR analysis showed a marked increased in levels of IL-4 and IFN-γ mRNA in immunized mice. The results suggested that the recombinant rSPV-szp provided mice with significant protection from the SEZ infections. It is a promising candidate for the vaccine development against SEZ infections., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The objective of the present study was to analyze an extracellular component from Staphylococcus aureus (S. aureus), which we refer to as slime associated antigenic complex (SAAC), and to investigate the role of SAAC-specific antibody production in protection from S. aureus bovine mastitis. Twelve primiparous gestating cows were randomly assigned to one of the three groups: Group 1 was vaccinated with a S. aureus bacterin with very limited SAAC content; Group 2 received a S. aureus bacterin with high SAAC content and Group 3 served as unvaccinated controls. Animals were vaccinated at 45 days before the expected parturition date and revaccinated 35 days later. All groups were challenged by intramammary infusion with a virulent heterologous strain of S. aureus 23 days after calving. Antibody response against SAAC in serum and in milk, general clinical signs, mastitis score, somatic cell count (SCC) and count of S. aureus in milk were evaluated before and after challenge. Immunization with a high SAAC content in the S. aureus bacterin (Group 2) significantly enhanced antibody titers against SAAC (in serum and milk) and reduced the S. aureus concentration in milk during the post-challenge period compared to Group 1 and Group 3. Moreover, a significant negative correlation was observed between SAAC antibody production on the day of the challenge and the S. aureus count in milk by 1 day after challenge. However, there was no evidence of a difference between vaccinated and control groups with regard to clinical signs of mastitis following the challenge. Nevertheless, the SAAC antibody concentration on the day of the challenge negatively correlated with the mastitis score in quarters infected with S. aureus at 2 days post-challenge. These results indicate that the vaccines did not prevent S. aureus intramammary infection (IMI) after the experimental challenge, but immunization with a S. aureus bacterin with high SAAC content was able to reduce S. aureus multiplication in the mammary gland after challenge and suggests that the SAAC-specific antibody response could be involved in the protection against S. aureus intramammary infection. Although further studies should be performed to confirm the efficacy (under experimental conditions and in field trials), we propose bacterins from strong biofilm-producing bacteria and with high SAAC content, rather than with limited SAAC content, as a cost-efficient vaccine design against S. aureus bovine mastitis., (Copyright 2009 Elsevier B.V. All rights reserved.)

Staphylococci and most other Gram-positive bacteria incorporate complex teichoic acid (TA) polymers into their cell envelopes. Several crucial roles in Staphylococcus aureus fitness and cell wall maintenance have been assigned to these polymers, which are either covalently linked to peptidoglycan (wall teichoic acid, WTA) or to the cytoplasmic membrane (lipoteichoic acid, LTA). However, the exact TA structures, functions, and biosynthetic pathways are only superficially understood. Recently, most of the enzymes mediating TA biosynthesis have been identified and mutants lacking or with defined changes in WTA or LTA have become available. Their characterization has revealed crucial roles of TAs in protection against harmful molecules and environmental stresses; in control of enzymes directing cell division or morphogenesis and of cation homeostasis; and in interaction with host or bacteriophage receptors and biomaterials. Accordingly, several in vivo studies have demonstrated the importance of WTA and LTA in S. aureus colonization, infection, and immune evasion. TAs and enzymes required for TA biosynthesis represent attractive candidates for novel vaccines and antibiotics and are targeted by recently developed antibacterial therapeutics., (Copyright 2009 Elsevier GmbH. All rights reserved.)

Effect of Staphylococcus aureus vaccine on metabolic and functional activities of polymorphonuclear leucocytes and guinea pig macrophages was observed. The tested vaccine following a short contact with human polymorphonuclear leucocytes isolated from peripheral blood inhibited candidacidal and phagocytic activities, whereas the phagocytic index stayed unchanged. The tested vaccine stimulated the INT-reductase activity and superoxide production, as well as extracellular liberation of lysosome enzymes from polymorphonuclear leucocytes. Application of the Staphylococcus aureus vaccine to guinea pigs 35, 30, 28, and 23 days prior to the acquirement of peritoneal macrophages, augmented, as compared to the control group, the amount of peritoneal macrophages and their phagocytic activity. Candidacidal and metabolic activities stayed unchanged. The results imply that the contact of phagocytic cells with the antigen, affects their biochemical and functional activities. Activities are dependent of the length of the period during which the antigen stimulation takes place, and of its quantity. Examination of phagocytes can be recommended for adjustment of both, vaccine doses and intervals between their individual applications. (Tab. 5, Ref. 19.).

Anti-CD45RA antibody defined by anti-2H4 monoclonal antibody has been reported to split CD4+T cells into two distinct subpopulations. CD45RA antigen is present on the surface of virtually more than 95% B lymphocytes in the purified tonsillar B-cell preparations. We examined the role of CD45RA antigen on human B-cell function using this antibody. The addition to anti-2H4 to tonsillar B cells inhibited the proliferative response induced by Staphylococcus aureus Cowan strain I(SAC) in a dose-dependent manner. Kinetic analysis indicated that anti-2H4 exerted its inhibitory effect when added within the first 24 h of culture initiation during a 72-h culture period. Anti-2H4 inhibited the transferrin receptor expression without interfering with the expression of the IL-2 receptor on SAC-stimulated B cells in a short-term culture. Anti-2H4 blocked the progress of SAC-stimulated B cells from the G1 to S phase of the cell cycle. These events suggested that anti-CD45RA MoAb inhibited the proliferative response by directly acting on B cells in the G1 phase. In addition, anti-CD45RA antibody also had a suppressive effect on early phase of B-cell differentiation. This effect appeared to be independent of its suppressive effect on proliferation, because anti-CD45RA did not inhibit the proliferative response of preactivated B cells with lymphokines. These studies suggested that the restricted epitope recognized by anti-2H4 antibody may be directly involved in regulatory function on B cells.

In this investigation lymphocytes sensitized with Corynebacterium diphtheriae antigens obtained from carriers and convalescents were used. The new diphtheria bacterial vaccine Codivac, in contrast to other comparable preparations (diphtheria toxoid, staphylococcal toxoid, staphylococcal vaccine, levamisole), was found to produce a more direct effect by modulating the levels of T-lymphocytes, depending on their initial levels in the patient. Codivac, together with other preparations, can be used for the study of the problems of immunostimulation and immunocorrective therapy.

The role of prostaglandin E2 (PGE2) in the regulation of human B cell proliferation in vitro was examined. Initial studies demonstrated that monocyte (M phi)-mediated suppression of B cell proliferation in Staphylococcus aureus (SA)-stimulated cultures was diminished by indomethacin, and thus it was suggested that cyclooxygenase pathway products of arachidonic acid played a role in the regulation of B cell activation. The possibility that PGE2, one of the major products of this pathway generated by M phi, affected human B cell responses was therefore investigated. PGE2 was found to suppress B cell DNA synthesis and proliferation stimulated by SA in the presence or absence of B cell growth factor (BCGF)-containing T cell supernatants. Suppression was concentration-dependent; significant inhibition was observed in indomethacin-blocked cultures with 10(-10) to 10(-9) M PGE2. In contrast to the effect of PGE2 on SA-induced B cell proliferation, it had minimal effect on pokeweed mitogen (PWM)-stimulated B cell or T cell DNA synthesis. The effect of PGE2 on the generation of BCGF activity from mitogen-stimulated T cells was also examined. PGE2 (10(-7) M) did not inhibit the production of BCGF activity from PWM-stimulated T cells, or from T cells stimulated with the combination of phytohemagglutinin and phorbol myristate acetate. Thus, PGE2 inhibits B cell proliferation in response to SA but not PWM, and has little effect on the production of BCGF by T cells. These results indicate that PGE2 at physiologically relevant concentrations exerts selective regulatory effects on human B cell responses.

The responses of resting human B lymphocytes to a variety of activation signals were studied. Human tonsillar B lymphocytes were separated according to size by countercurrent elutriation. The small B lymphocytes were then stimulated in vitro with various concentrations of anti-mu antibody in the presence or absence of B cell growth factor (BCGF) or with Staphylococcus aureus Cowan strain I (SAC). Cellular volume changes and RNA synthesis were measured over the first 24 h of stimulation and were similar with either 15 micrograms/ml of anti-mu, 100 micrograms/ml of anti-mu, or SAC. In the subsequent 24 h, however, substantial increases occurred in the amount of RNA synthesis and cell enlargement only in those cultures stimulated with 100 micrograms/ml of anti-mu or SAC, but not in the cultures stimulated with 15 micrograms/ml of anti-mu. The addition of BCGF to those cultures stimulated with 15 micrograms/ml of anti-mu did not alter the increases in cellular volume and RNA synthesis found 24 h after stimulation with anti-mu alone. However, over the subsequent 24 h, the presence of BCGF in culture enhanced both B cell volume changes and RNA synthesis, when compared to cultures stimulated with 15 micrograms/ml of anti-mu alone. In addition, BCGF enhanced DNA synthesis in cultures stimulated with low and high concentrations of anti-mu. DNA content changes following stimulation with anti-mu, anti-mu plus BCGF, and SAC were also measured using propidium iodide staining and flow cytometry. Optimal concentrations of anti-mu induced 20% of the resting B cells to enter S phase, while optimal concentrations of anti-mu plus BCGF or SAC induced approximately 40%. Finally, prestimulation of resting B cells for 24 h with a low concentration of anti-mu, sufficient for cell enlargement but not S phase progression, allowed for rapid entrance of the prestimulated B cells into S phase when a high concentration of anti-mu or SAC was added. These findings suggest the existence of a control point in the progression of human B cells through the cell cycle. This control point is located in the G1 phase of the cycle and is reached 24 to 36 h after a surface immunoglobulin-mediated stimulus.

In certain human IgM and IgG cell lines, immunoglobulin (Ig) secretion is highly stimulated by a B cell inducing factor (BIF) that is free of interleukin 2 (IL 2). BIF also induces Ig secretion in purified peripheral blood B cell populations that have been mitogenically stimulated by Staphylococcus aureus bacteria. Low concentrations of IL 2 (less than 20 U/ml) are not active in these systems. We now show that IL 2 at concentrations above 100 U/ml can induce Ig secretion in these blood B cells and B cell lines. Both conventional IL 2, purified from the human JURKAT and gibbon MLA-144 cell lines, and recombinant IL 2 are active. Very high concentrations approaching 10(4) U/ml are optimal for Ig secretion. Antibody to the T cell IL 2 receptor, anti-Tac, did not inhibit stimulation of the IgM cell line SKW6.4 by IL 2, and no Tac antigen was detected on the cells. The 9B11 monoclonal anti-IL 2 antibody that neutralizes T cell growth activity also abrogates stimulation of Ig secretion by conventional and recombinant IL 2 in the SKW6.4 cell line. However, the 1H11 monoclonal anti-(conventional thr3-glycosylated IL 2), which does not neutralize T cell growth activity, does inhibit induction of Ig secretion by the corresponding IL 2 in the B cell line. These results suggest that IL 2 stimulates B cells via a low-affinity interaction with a receptor different from the Tac receptor identified on T cells, and that the active site on the IL 2 molecule for B cells differs from that for T cell targets. If IL 2 promotes Ig secretion by binding with a low affinity to the B cell BIF receptor, IL 2 and BIF could be homologous proteins.

Transferrin receptors are expressed on proliferating cells and are required for their growth. Transferrin receptors can be detected after, but not before, mitogenic stimulation of normal peripheral blood T and B cells. T cells demonstrate a functional requirement for transferrin receptors in the activation process. These receptors, in turn, are induced to appear by T cell growth factor (interleukin 2). In the experiments reported here, we examined the regulation of transferrin receptor expression on activated human B cells and whether these receptors are necessary for activation to occur. Activation was assessed by studying both proliferation and immunoglobulin secretion. We determined that transferrin receptor expression on B cells is regulated by a factor contained in supernatants of mitogen-stimulated T cells (probably B cell growth factor). This expression is required for proliferation to occur, because antibody to transferrin receptor (42/6) blocks B cell proliferation. Induction of immunoglobulin secretion, however, although dependent on phytohemagglutinin-treated T cell supernatant, is not dependent on transferrin receptor expression and can occur in mitogen-stimulated cells whose proliferation has been blocked by anti-transferrin receptor antibody. These findings support a model for B cell activation in which mitogen (or antigen) delivers two concurrent but distinct signals to B cells: one, dependent on B cell growth factor and transferrin receptor expression, for proliferation; and a second, dependent on T cell-derived factors and not requiring transferrin receptors, which leads to immunoglobulin secretion.

The role of IL-4 in human B cell activation, proliferation, and differentiation was examined. rIL-2, but not rIL-4, was able to promote maximum proliferation and generation of Ig-secreting cells in cultures of highly purified B cells stimulated with Cowan I Staphylococcus aureus (SA). Addition of rIL-4 to rIL-2-supported cultures of SA-stimulated peripheral blood, spleen, or lymph node B cells dramatically suppressed both proliferation and differentiation. Results from experiments in which rIL-4 was added to culture at progressively later times indicated a requirement for rIL-4 to be present during the first 2 days of a 5-day incubation to cause inhibition of responsiveness. When a two-stage culture system was utilized, rIL-4 was found to support proliferation or differentiation of B cells initially activated with SA for 2 days only minimally. However, rIL-4 did not inhibit responses of SA preactivated B cells supported by IL-2. The presence of rIL-4 during the initial 48-h activation of B cells with SA and rIL-2 resulted in a profound inhibition of the ability of the activated B cells to respond subsequently to rIL-2 or lymphokine-rich T cell supernatants. A similar 48-h incubation with rIL-4 alone without SA had no effect on subsequent B cell responsiveness. The presence of rIFN-gamma during B cell activation decreased the inhibitory effect of IL-4. Other cytokines including IFN-alpha, IL-1, and commercially available low m.w. B cell growth factor also diminished the inhibitory effect of IL-4. These results indicate that IL-4 inhibits the capacity of human B cells to be activated maximally by SA and rIL-2 and therefore suggest a new immunomodulatory role for this cytokine.