Perforin and IFN-gamma are involved in the antitumor effects of antibody-targeted superantigens

The bacterial superantigen staphylococcal enterotoxin A (SEA) is a potent inducer of cytokine production and cytotoxic T cell responses. To target a T cell attack against tumor cells we have genetically engineered a fusion protein of SEA and the Fab part of the tumor-reactive mAb C215. Injection of this Fab-SEA fusion protein to mice carrying lung metastases of the poorly immunogenic B16 melanoma transfected with the C215 Ag resulted in infiltration of cytokine-producing T cells, perforin-containing CTL, and a marked tumor elimination. Fab-SEA therapy induced substantial levels of IFN-gamma and TNF-alpha in serum. In the present study we have characterized the molecular mechanisms of the antitumor effect induced by Fab-SEA treatment in vivo. Neutralization of cytokines by specific Abs demonstrated a major role for IFN-gamma in the suppression of tumor growth. In addition, a minor contribution of TNF-alpha was recorded. Injections of Fab-SEA into normal mice induced strong CTL activity but failed to promote cytotoxic function in perforin knockout mice. Also, a markedly reduced therapy was noted in perforin knockout mice, implicating a role for CTL in Fab-SEA-mediated tumor eradication. The data suggest that Fab-SEA-targeted T cells may suppress tumor growth by both perforin-dependent cytotoxicity and local release of cytokines such as IFN-gamma. The latter mechanism may have an important role in cytostatic effects against Ag-negative bystander tumor cells.     

Effects of bacterial DNA on cytokine production by (NZB/NZW)F1 mice

Microbial DNA has multiple immune effects including the capacity to induce polyclonal B cell activation and cytokine production in normal mice. We recently described the accelerated induction of anti-DNA Abs in NZB/NZW mice immunized with Escherichia coli (EC) dsDNA; paradoxically these mice developed less renal disease than unimmunized mice or mice immunized with calf thymus DNA. We postulated that alterations in cytokine production induced by bacterial DNA may play a key role in renal protection. To determine the effect of bacterial DNA on cytokine production in NZB/NZW mice, we measured the serum cytokine levels, cell culture supernatant cytokine levels, and number of cytokine-producing splenocytes in NZB/NZW mice injected with EC DNA, calf thymus DNA, or an immune active oligonucleotide. There was a 10- to 25-fold increase in the number of cells secreting IFN-gamma compared with IL-4 in mice immunized with EC DNA. IL-12-secreting cells were also increased by bacterial DNA immunization. In parallel with the increase in IFN-gamma secreting cells, there was a significant rise in serum IFN-gamma levels in mice receiving EC DNA. These results indicate that EC DNA modulates systemic cytokine levels in NZB/NZW mice, selectively increasing IL-12 and IFN-gamma while decreasing IL-4 production. The cytokine response of NZB/NZW mice to bacterial DNA may be of significance in disease pathogenesis and relevant to the treatment of lupus-like disease.     

The effect of LHRH and TRH on human interferon-gamma production in vivo and in vitro

Accumulating evidence suggests that hypothalamic luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH) are two hypophysiotropic factors which modulate the immune response. The aim of the present study was to determine the in vivo effects of an intravenous bolus of LHRH and TRH on plasma interferon (IFN)-gamma production in five normoprolactinemic women with irregular menstrual cycles. We also determined prolactin (PRL), thyrotropin (TSH), follicle stimulating hormone (FSH), and luteinizing hormone (LH) levels before and after intravenous administration of LHRH and TRH. The results demonstrate that intravenous bolus of LHRH/TRH increases plasma IFN-gamma levels, with the maximum response 45 min after in vivo administration of hypothalamic peptides and after peak levels of adenohypophyseal hormones (PRL: 15 min; TSH: 30 min; FSH: 30 min; LH: 30 min). In order to investigate a possible direct action of hypothalamic hormones on immune cells, we also evaluated, in the same subjects, the influence of LHRH and TRH on IFN-gamma production by human peripheral blood mononuclear cells (PBMCs), collected before the intravenous administration of the peptides and stimulated in vitro with bacterial superantigen staphylococcal enterotoxin A (SEA) and concanavalin A (Con A). LHRH and TRH, separately and together, significantly enhanced in vitro IFN-gamma production by SEA- and ConA-activated PBMCs. The present results suggest that hypothalamic peptides (LHRH and TRH) directly, and/or indirectly pituitary hormones (PRL, TSH, FSH, and LH) or IL-2, have stimulatory effect on IFN-gamma producing cells and are further evidence of interactions between the neuroendocrine and immune systems.     

Sequential production of IL-2, IFN-gamma and IL-10 by individual staphylococcal enterotoxin B-activated T helper lymphocytes

Upon primary activation, T helper (Th) cell populations express different cytokines transiently and with different kinetics. Stimulation of naive murine splenic Th cells with the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) in vitro results in expression of IL-2, IFN-gamma and IL-10 with fast, intermediate and slow kinetics, respectively. This first report of a functional analysis of cells separated alive according to cytokine expression shows that these cytokines are not produced by different Th cell subpopulations, but can be expressed sequentially by individual Th cells. Th cells, activated with SEB for 1 day and isolated according to expression of IL-2, using the cellular affinity matrix technology, upon continued stimulation with SEB later secrete most of the IFN-gamma and IL-10. Likewise, after 2 days of SEB culture, cells expressing IFN-gamma, separated according to specific surface-associated IFN-gamma as detected by magnetofluorescent liposomes, 1 day later secrete IL-10. Thus, individual Th1 cells can contribute to the control of their own IFN-gamma expression by sequential expression of first IL-2, supporting their proliferation, and later IL-10, down-regulating the production of IFN-gamma-inducing monokines and limiting the pro-inflammatory effects of IFN-gamma.     

Phospholipid vesicle binding and aggregation by four novel fish annexins are differently regulated by Ca2+

The aim of this study was to further assess the role of pooled human immunoglobulin (PHIG) on cytokine production from PBMC stimulated with a bacterial superantigen. Human PBMC were cultured with Streptococcus pyrogenic exotoxin A (SPE-A) with or without PHIG and several proinflammatory cytokine levels of culture supernatants were measured. Serum cytokine levels of KD patients before and after PHIG therapy were also examined. PHIG greatly reduced the production of IL-12, interferon-gamma (IFN-gamma) and other cytokines from SPE-A-stimulated PBMC, while exogenous IL-12, but neither IL-1 nor tumour necrosis factor-alpha (TNF-alpha), restored IFN-gamma production inhibited by PHIG. Although PHIG partially adsorbed SPE-A, its inhibitory effect on cytokine production was not played by anti-SPE-A antibody. Although purified CD4+ T cells cultured with human HLA-DR-transfected mouse L cells and SPE-A could not effectively produce IFN-gamma, they produced large amounts of IFN-gamma if exogenous IL-12 was introduced. KD patients in the acute phase had higher levels of serum IFN-gamma than did controls and patients with bacterial infection. Although IL-12 levels of children with or without KD were not significantly different, IL-12 levels of children were much higher than those of adults. However, serum levels of IL-12 of KD patients were transiently but significantly decreased by PHIG therapy and IFN-gamma amounts subsequently reverted to basal levels thereafter. These findings indicate that PHIG inhibits IL-12 production of SPE-A-activated monocytes and thereby decreases IFN-gamma synthesis by T cells and suggest that inhibition of IL-12 and IFN-gamma production is an important part of the mechanisms underlying PHIG therapy on KD.     

Evidence for Clostridium perfringens enterotoxin (CPE) inducing a mitogenic and cytokine response in vitro and a cytokine response in vivo

We investigated some immunogenic properties of Clostridium perfringens enterotoxin (CPE) in vitro using murine J774A macrophages (MPhi) and in vivo using Swiss Webster (SW) mice. CPE was a potent mitogen in vitro, where cell proliferation increased with CPE concentration. CPE was nonmitogenic when MPhi were concurrently incubated with CPE and interferon gamma (IFN-gamma). MPhi incubated in the presence of CPE induced the synthesis of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma), but not interleukin-2 (IL-2). In vivo, CPE induced a pro-inflammatory cytokine response with striking production of IFN-gamma, IL-1, and IL-6. Regardless of route of CPE entry, serum cytokine levels generally peaked within 1 h of administration and were maintained for 4-8 h. Although CPE engenders an intense immune response during toxicosis, the toxin does not appear to be a superantigen. Death from CPE-induced shock appears to result from various interrelating immunological mechanisms.     

Interferon beta, a cofactor in the interferon gamma production induced by gram-negative bacteria in mice

The interferon (IFN) gamma production of splenocytes from closely related C57BL/10ScSn (Sn) and C57BL/10ScCr (Cr) mice was compared. Concanavalin A and CD3 monoclonal antibodies induced high levels of IFN-gamma in both Sn and Cr splenocytes. By contrast, treatment with gram-negative bacteria induced IFN-gamma only in Sn splenocytes; in Cr splenocytes, the IFN-gamma response was heavily impaired. The IFN-gamma induction by bacteria requires the cooperation of IFN-gamma-producing cells with macrophages. Depletion of macrophages from Sn splenocytes resulted in the loss of ability to produce IFN-gamma after bacterial stimulation. Reconstitution with new Sn macrophages restored the IFN-gamma responsiveness, whereas reconstitution with Cr macrophages failed to do so. Normal function of IFN-gamma-producing cells and a defective function of macrophages of Cr mice was demonstrated by evidence showing that whole or macrophage-depleted Cr splenocytes, when supplemented with Sn macrophages, acquire the ability to produce IFN-gamma in response to bacteria. A similar effect was achieved by supplementing Cr splenocytes with supernatants of bacteria-stimulated Sn macrophages or with recombinant murine IFN-beta or IFN-alpha. Preincubation of active macrophage supernatants with antibodies to IFN-beta suppressed the helper activity for Cr splenocytes. Moreover, the bacteria-induced production of IFN-gamma by Sn splenocytes could be inhibited by antibodies to murine IFN-beta. The results provide evidence that IFN-beta is an important cofactor of IFN-gamma induction, which is not induced in Cr mice by gram-negative bacteria.     

Leishmania donovani infection of a susceptible host results in apoptosis of Th1-like cells: rescue of anti-leishmanial CMI by providing Th1-specific bystander costimulation

A protective immune response against Leishmania donovani infection is mediated by T-helper type 1 (Th1) cells. Th1 induced cell-mediated immunity (CMI), as assessed by anti-leishmanial DTH response, is lost in a susceptible host such as BALB/c mice. Although the impaired Th1 function eventuates in unhindered parasite growth and in manifestation of the susceptible phenotype, the mechanism of down-regulation of the Th1 function is yet to be elucidated. Here, we provide evidence that the parasite down-regulates the expression of a Th1-specific costimulatory molecule, M150, on the surface of infected BALB/c mice-derived macrophages. Th cells are rendered unresponsive to anti-CD3 Ab-mediated stimulation after interaction with infected macrophages. The anergized T cells produce much less IL-2, IL-4 and IFN-gamma compared to those T cells which were costimulated using normal macrophages. The defect in proliferation, anti-CD3 Ab induced unresponsiveness and IFN-gamma but not IL-4 production can be restored by providing bystander costimulation through M150. These results not only unfold a novel immune evasion strategy used by the parasite but also clarify the mechanism of Th1 cell debilitation during the disease. Recovery of Th1 cytokine production by bystander costimulation through M150 may help in formulating a new strategy for the elimination of intracellular parasites.     

Murine immune responses to Schistosoma haematobium and the vaccine candidate rSh28GST

Longitudinal studies of Schistosoma haematobium infection in CBA mice revealed a progressive down-regulation of cellular immune responses, as measured by mitogenic and antigenic stimulation of in vitro lymphocyte cultures. Antigen-stimulated production of the Th1 cytokine IFN-gamma by splenocytes increased progressively up to 14 weeks post infection, (four weeks after the onset of parasite egg production), before declining swiftly. Levels of the Th2 cytokine IL-4 in the same cultures remained low until 14 weeks, after which they rose rapidly as IFN-gamma declined. High levels of IL-10 coincided with the peak in IFN-gamma production, suggesting a non Th2-restricted role for this cytokine. Both total and antigen-specific immunoglobulin production confirmed parasite egg deposition as being a major stimulus for host humoral responses. The S. haematobium infection failed to elicit detectable T cell responses to the antifecundity vaccine candidate rSh28GST. However, low levels of antibody were detectable in infected mouse serum and strong IgG and IgA production was induced by vaccination with rSh28GST plus adjuvant.     

Endothelial cells expressing an inflammatory phenotype are lysed by superantigen-targeted cytotoxic T cells

The objective of this study was to investigate whether the superantigen staphylococcal enterotoxin A (SEA), which binds to HLA class II and T-cell receptor Vbeta chains, can direct cytotoxic T cells to lyse cytokine-stimulated endothelial cells (EC). In addition, we wanted to determine whether SEA-primed cytotoxic T cells could be targeted to EC surface molecules as a means of a novel cancer immunotherapy. Human umbilical vein EC (HUVEC), dermal microvascular EC (HMVEC), or the EC line EA.hy926 stimulated with gamma interferon (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) displayed upregulated HLA class II and adhesion molecule (CD54 and CD106) expression, respectively. SEA-primed T cells induced a strong cytotoxicity against IFN-gamma- and TNF-alpha-activated EA.hy926 which had been preincubated with SEA. Blocking of CD54 completely abrogated the T-cell attack. SEA-D227A, which has a mutated class II binding site, did not promote any cytotoxicity. A strong lysis was observed when a fusion protein consisting of protein A and SEA-D227A was added together with T cells to TNF-alpha-induced EA.hy926 and HUVEC precoated with monoclonal antibodies (MAb) directed against HLA class I, CD54, or CD106 molecules. Finally, an scFv antibody fragment reactive with an unknown EC antigen was fused with SEA-D227A. Both EA.hy926 and HMVEC were efficiently lysed by scFv-SEA-D227A-triggered cytotoxic T cells. Taken together, superantigen-activated T-cell-dependent EC killing was induced when EC expressed an inflammatory phenotype. Moreover, specific MAb targeting of the superantigen to surface antigens induced EC lysis. Our data suggest that directed T-cell-mediated lysis of unwanted proliferating EC, such as those in the tumor microvasculature, can be clinically useful.     

Stimulation of leukaemic cells from adult T-cell leukaemia patients with bacterial superantigens

Bacterial superantigens stimulate T cells in a manner that is restricted to the Vbeta of the T-cell receptor. We examined the ability of adult T-cell leukaemia (ATL) cells to respond to these superantigens. Mononuclear cells from 10 patients were cultured with staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin-1 (TSST-1), and their response was determined by MTT assay and 3H-thymidine incorporation assay. Cells from six patients showed a specific response to a single superantigen. In two cases the cells responded to TSST-1 and bore Vbeta2, the known target of TSST-1. In three cases the cells responded to SEA with one bearing Vbeta9, a target of SEA, and one bearing Vbeta16. In one case the cells responded to SEB. Most of the cells which proliferated in response to superantigens were determined genetically to be leukaemic. The response to TSST-1 was inhibited by anti-Vbeta2 antibody. The responding cells showed a strongly enhancement expression of interleukin-2 receptor. These findings indicate that leukaemic cells from a proportion of ATL patients have an ability to respond to T-cell receptor-dependent superantigens. This suggests that bacterial infection in such patients may contribute to the expansion of ATL cells.     

Psychosocial influences on immune responses to HSV-1 infection in BALB/c mice

The effects of differential housing (one or four mice/cage) on T-helper (Th) cell markers of cellular and humoral immune responses were examined. Differentially housed male BALB/cJ mice were infected with herpes simplex virus (HSV)-1 (Patton strain), and in vitro cytokine production [interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-gamma] by splenocytes and popliteal lymph node cells and serum antibody titers (IgM and IgG) were evaluated. Differential housing of male BALB/c mice influenced the magnitude, but not the kinetics, of some, but not all, immune responses to HSV-1. Splenocytes from individually housed mice produced more IL-2, IFN-gamma, IL-4, and IL-10 than splenocytes from group-housed mice; in popliteal lymph node cells, only IFN-gamma and IL-10 production was influenced by housing. Although the social environment influenced cytokine production, there were no concomitant changes in circulating IgM or IgG antibody titers. These results do not support the hypothesis that dominant Th cell responses are the primary targets of this psychosocial manipulation, or that a reciprocal relationship exists between Th1 and Th2 cell-derived cytokines.     

Evidence that induction of tolerance in vivo involves active signaling via a B7 ligand-dependent mechanism: CTLA4-Ig protects V beta 8+ T cells from tolerance induction by the superantigen staphylococcal enterotoxin B

Co-stimulation through CD28 is thought to be necessary for the activation of unprimed CD4+ T cells, which are otherwise rendered tolerant. However, we previously found that CD4+ T cell priming was normal or augmented in mice which overexpressed a soluble form of CTLA4 where co-stimulation through CD28 was abrogated. To investigate this CD4+ T cell response, we exploited the capacity of the superantigen staphylococcal enterotoxin B to stimulate T lymphocytes bearing V beta 8+, which represent approximately 30% of all CD4+ T cells. In litter-mate controls of CTLA4-Ig transgenic mice, immunization with staphylococcal enterotoxin B leads to expansion, followed by deletion of V beta 8+ T cells, and the remaining cells are tolerant when stimulated in vitro. Comparable expansion and deletion of V beta 8 T cells occurs in CTLA4-Ig transgenic mice. However, in contrast to normal mice, the remaining V beta 8+ T cells from CTLA4-Ig transgenic mice are not anergic and remain responsive to superantigen in vitro.     

Ceramide-independent CD28 and TCR signaling but reduced IL-2 secretion in T cells of acid sphingomyelinase-deficient mice

Ceramide generated by lysosomal acid sphingomyelinase (aSMase) has been proposed to contribute to CD28 co-stimulatory signaling pathways. We used an aSMase-deficient mouse line (asmase-/-) to elucidate the role of the aSMase in splenocytes stimulated with either a combination of anti-CD3 and anti-CD28 antibodies, the lectin concanavalin A (Con A) or the superantigen staphylococcal enterotoxin B. All stimuli were shown to induce IL-2 expression, Con A additionally triggered the expression of high-affinity IL-2 receptor. However, in asmase-/- mice secretion of IL-2 was significantly reduced, whereas the intracellular IL-2 levels were elevated. Proliferation of anti-CD3/anti-CD28 or Con A-stimulated aSMase-deficient splenocytes was reduced up to 50% after 72 h in comparison to wild-type cells. We conclude that ceramide generated by aSMase is not involved in CD28 signal transduction, but rather a perturbation of the secretory system is responsible for the impaired proliferation of aSMase-deficient splenocytes.     

The cellular immune response to Bordetella pertussis in two children with whooping cough

There is increasing evidence that the cellular immune response to Bordetella pertussis plays an important role in the immune protection. Particularly in animal models, Bordetella pertussis-specific T-cells have been shown to confer immunity. In this case report, we therefore investigated the cellular immune response to whole cell Bordetella pertussis bacteria, to the pertussis antigens filamentous hemagglutinin and pertussis toxoid defined by lymphoproliferation and cytokine secretion. Two children with whooping cough were compared to three individuals vaccinated against whooping cough with a whole cell pertussis vaccine. In contrast to the vaccinated controls, the cellular immune response to Bordetella pertussis in children with whooping cough was characterized by a strong proliferation of T cells to whole pertussis bacteria as well as to filamentous hemagglutinin and pertussis toxoid. This response was defined by a marked Th-1 type T cell response with IFN-gamma secretion to all Bordetella pertussis antigens. However, in the control individuals IFN-gamma was secreted only to whole cell Bordetella pertussis bacteria and filamentous hemagglutinin but not to pertussis toxoid. A Th-2 type cytokine response could not be detected in any condition. Our observations suggest that in the immune defense of a natural Bordetella pertussis infection, the Th-1 specific T cell response to filamentous hemagglutinin and particularly to pertussis toxoid may play a major role.     

Use of a whole blood assay to monitor the immune response to mycobacterial antigens in leprosy patients: a predictor for type 1 reaction onset?

In this study, we investigated tick saliva effects on T cell proliferation, antigen presentation and IFN-gamma-induced macrophage activation, events which are associated with host immune defense mechanisms. Mice repeatedly infested with Rhipicephalus sanguineus ticks, similarly to dogs, did not develop resistance to further infestations. We determined that R. sanguineus tick saliva inhibited, in a dose-dependent manner, both Con-A and specific antigen-induced splenic T cell proliferation. Tick saliva diluted twenty times (64 microg/ml) inhibited Con-A-induced and antigen-specific T cell proliferation in 83% and 69%, respectively. In addition, the inhibition of cell proliferation correlated with a decrease in IL-2 production. Microconcentrator fractionated saliva was tested on a Con-A-induced cell proliferation assay, and showed that one fraction between 3 and 10 kDa and another smaller than 3 kDa can be responsible for the inhibition of T cell proliferation. Although saliva inhibited cell proliferation, it did not impair antigen presentation. Tick saliva further abrogated the killing of intracellular forms of Trypanosoma cruzi by IFN-gamma-activated macrophages. Moreover, saliva-induced macrophage inhibition of IFN-gamma-induced-trypanocidal activity was paralleled with 69% less nitric oxide (NO) production. Finally, tick saliva doubled the production of IL-10 and reduced 84.6% production of IFN-gamma by splenocytes cultured with T. cruzi, suggesting that decreased macrophage NO production may be due to a saliva-induced cytokine imbalance, leading to decreased NO synthase activity. Together, these data indicate that tick saliva can modulate host immune response, thus, contributing to its feeding success and favoring the transmission of tick-borne pathogens.     

Modulation of endotoxin- and enterotoxin-induced cytokine release by in vivo treatment with beta-(1,6)-branched beta-(1,3)-glucan

Leukocytes activated by endotoxin or enterotoxins release proinflammatory cytokines, thereby contributing to the cascade of events leading to septic shock. In the present studies, we analyzed the effects of in vivo administration of a soluble immunomodulator, beta-(1,6)-branched beta-(1,3)-glucan (soluble beta-glucan), on toxin-stimulated cytokine production in monocytes and lymphocytes isolated from treated mice. In vitro stimulation of lymphocytes isolated from soluble beta-glucan-treated mice with lipopolysaccharide (LPS) resulted in enhanced production of interleukin-6 (IL-6) and suppressed production of tumor necrosis factor alpha (TNF-alpha), while stimulation of these cells with staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin 1 (TSST-1) resulted in enhanced production of gamma interferon (IFN-gamma) and suppressed production of IL-2 and TNF-alpha compared to that in cells isolated from untreated mice. In vitro stimulation of monocytes isolated from soluble beta-glucan-treated mice with LPS also resulted in suppressed TNF-alpha production, while stimulation of these cells with SEB or TSST-1 resulted in suppressed IL-6 and TNF-alpha production compared to that in cells isolated from untreated mice. Thus, the overall cytokine pattern of leukocytes from soluble beta-glucan-treated mice reflects suppressed production of proinflammatory cytokines, especially TNF-alpha. Taken together, our results suggest that treatment with soluble beta-glucan can modulate the induction cytokines during sepsis, resulting in an overall decrease in host mortality.     

In vivo tumor transfection with superantigen plus cytokine genes induces tumor regression and prolongs survival in dogs with malignant melanoma

In vivo transfection of established tumors with immunostimulatory genes can elicit antitumor immunity. Therefore, we evaluated the safety and efficacy of intratumoral injections of a bacterial superantigen with a cytokine gene in dogs with malignant melanoma, a spontaneous and highly malignant canine tumor. 26 dogs with melanoma were treated with lipid-complexed plasmid DNA encoding staphylococcal enterotoxin B and either GM-CSF or IL-2. Dogs were evaluated for treatment-associated toxicity, tumor responses, immunologic responses, and survival times. The overall response rate (complete or partial remissions) for all 26 dogs was 46% (12 of 26), and was highest in patients with smaller tumors. Toxicity was minimal or absent in all dogs. Injected tumors developed marked infiltrates of CD4+ and CD8+ T cells and macrophages, and tumor regression was associated with development of high levels of antitumor cytotoxic T lymphocyte activity in peripheral blood lymphocytes. Survival times for animals with stage III melanomas treated by intratumoral gene therapy were prolonged significantly compared with animals treated with surgical tumor excision only. Thus, local tumor transfection with superantigen and cytokine genes was capable of inducing both local and systemic antitumor immunity in an outbred animal with a spontaneously developing malignant tumor.     

The dual phases of the response to neonatal exposure to a VH family-restricted staphylococcal B cell superantigen

In vitro studies of several naturally occurring proteins have characterized VH family-specific B lymphocyte binding and stimulatory properties that appear analogous to those of T cell superantigens. To examine the in vivo consequences of exposure to a putative B cell superantigen, we treated neonatal BALB/c mice with a form of staphylococcal protein A (MS) devoid of Fcgamma binding activity, which retains the clan VHIII Fab binding specificity. In naive adults, about 5% of peripheral B cells and >13% of splenic IgM-secreting cells display MS binding activity, in association with high IgM and low IgG circulating anti-MS Ab titers. Neonatal exposure to MS elicited two distinct temporal phases of immune responsiveness. The early phase, representing the first approximately 5 wk of life, was associated with MS-specific B cell and T cell tolerance. Microfluorometric assays revealed that exposure caused a dramatic MS-specific B cell clonal loss in bone marrow and spleen, but levels normalized by about 3 wk of life. The late phase (>6 wk of age) was associated with spontaneous priming for MS-specific T cell responses and production of MS-specific IgG1 Abs despite long term persistently depressed in vivo and in vitro MS-specific IgM responses. In vivo challenge during the late phase induced high frequencies of MS-specific IgG-secreting cells, indicating recruitment of highly focused Ab responses that were predominantly encoded by rearrangements of the S107 family, a member of the VHIII clan. These studies document the immunodominance of the VH-restricted Fab binding site on staphylococcal protein A and demonstrate the diverse effects of a B cell superantigen on the emerging peripheral B cell compartment.