IL-18 time-dependently modulates Th1/Th2 cytokine production by ligand-activated NKT cells

While IL-18 synergizes with IL-12 to induce a Th1 immune response, it also promotes a Th2 response. Here we investigate the modulatory role of IL-18 on the Th1/Th2 cytokine response. The injection of alpha-galactosylceramide (alpha-GalCer), a ligand for NKT cells, elevated mouse serum levels of both IFN-gamma and IL-4. When the mice were treated 2 h before alpha-GalCer challenge with IL-18, IFN-gamma production but not IL-4 production was remarkably up-regulated. In contrast, pretreatment with IL-18 6 h before the challenge enhanced IL-4 production. However, this IL-18-enhanced IL-4 production was not elicited in mice injected with anti-CD3 Ab. Liver mononuclear cells (MNC) produced a similar cytokine production pattern when MNC from mice treated with IL-18 either 2 h or 6 h before challenge were stimulated with alpha-GalCer in vitro. Expression of SOCS1 and SOCS3 was notably up-regulated in the liver MNC from mice pretreated 6 h before with IL-18; in particular, SOCS3 expression was confined to the liver NKT cells. Inhibition of SOCS3 by RNA interference up-regulated the phosphorylation of STAT3 and suppressed in vitro IL-4 production by IL-18-primed liver MNC stimulated with alpha-GalCer, but it did not affect IFN-gamma production. These results suggest that IL-18 time-dependently modulates Th1/Th2 cytokine production in ligand-activated NKT cells by regulating/inducing SOCS3 expression.     

Increased Th1 and Th2 allergen-induced cytokine responses in children with atopic disease

BACKGROUND: Polyclonal cytokine responses following stimulation of T cells with mitogens or superantigens provides information on cytokine production from a wide range of T cells. Alternatively allergen-induced T cell responses can provide information on cytokine production by allergen-reactive T cells. While there is evidence of increased Th2 and reduced Th1 cytokine production following T cell stimulation with non-specific mitogens and superantigens, the evidence that Th1 cytokine production to allergens is decreased in line with a postulated imbalance in Th1/Th2 responses is unclear, with studies finding decreased, no difference or increased IFN-gamma responses to allergens in atopic subjects. OBJECTIVE: To examine childhood polyclonal and allergen-induced cytokine responses in parallel to evaluate cytokine imbalances in childhood atopic disease. METHODS: PBMC cytokine responses were examined in response to a polyclonal stimulus, staphylococcal superantigen (SEB), in parallel with two inhalant allergens, house dust mite (HDM) and rye grass pollen (RYE), and an ingested allergen, ovalbumin (OVA), in (a) 35 healthy children (non-atopic) and (b) 36 children with atopic disease (asthma, eczema and/or rhinitis) (atopic). RESULTS: Atopic children had significantly reduced IFN-gamma and increased IL-4 and IL-5 but not IL13 production to SEB superantigen stimulation when compared with non-atopic children. HDM and RYE allergens stimulated significantly increased IFN-gamma, IL-5 and IL-13, while OVA stimulated significantly increased IFN-gamma production in atopic children. CONCLUSION: We show that a polyclonal stimulus induces a reduced Th1 (IFN-gamma) and increased Th2 (IL-4 and IL-5) cytokine pattern. In contrast, the allergen-induced cytokine responses in atopic children were associated with both increased Th1 (INF-gamma) and Th2 (IL-5 and IL-13) cytokine production. The increased Th1 response to allergen is likely to reflect prior sensitization and indicates that increases in both Th1 and Th2 cytokine production to allergens exists concomitantly with a decreased Th1 response to a polyclonal stimulus in atopic children.     

Effect of exogenous leukotriene B4 (LTB4) on BALB/c mice splenocyte production of Th1 and Th2 lymphokines

The effect of exogenous leukotriene B₄ (LTB₄) on the production of cytokines typical of Th₁ (interleukin-2 and interferon-γ) and Th₂ (interleukin-4 and interleukin-10) lymphocytes was studied. Splenocytes were stimulated with concanavalin A (ConA) with or without different concentrations of LTB₄ (3 × 10⁻¹⁰ to 3 × 10⁻⁷M) for various times in the presence of BW 755C to inhibit the endogenous synthesis of eicosanoids. LTB₄ was not able to induce cytokine secretion by itself. However, LTB₄ augmented ConA spleen cell production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) from Th₁ cells and interleukin-4 (IL-4) and interleukin-10 (IL-10) from Th₂ cells more than the controls treated with ConA alone. The pre-exposition of splenocytes to LTB₄ for 3 h made these cells more sensitive to ConA in terms of IL-2 and IL-10 production than those treated with LTB₄ at the onset of the incubation and maintained during the whole culture period. The results suggest that LTB₄ may participate as a component of the signal transduction process for ConA-induced Th₁ and Th₂ cytokine production in a timedependent manner.     

Relationship between Th1/Th2 cytokine patterns and the arthritogenic response in collagen-induced arthritis

It is hypothesized that the balance of cytokines produced by Th1/Th2 subsets of T helper cells plays an important role in the development of autoimmune diseases. Murine collagen-induced arthritis (CIA) is an example of an autoimmune disease in which immunization with cartilage-derived type II collagen induces, firstly, a T cell response to type II collagen and, secondly, the manifestation of a destructive inflammatory response in affected joints. We have investigated the role of Th1/Th2 responses in the development of CIA by monitoring levels of interferon (IFN)-γ (a Th1 cytokine) and interleukin (IL)-4 and IL-10 (Th2 cytokines), and IL-1β and tumor necrosis factor (TNF) (pro-inflammatory cytokines) produced by cultured draining lymph node cells (LNC) from collagen-immunized DBA/1 mice during the induction phase of arthritis and throughout the time of clinical manifestation and subsequent remission of the disease. Although a transient increase in IL-10 was detected 3 days after immunization, Th2 cytokine production was found to be almost completely suppressed 6 days after immunization. In contrast, IFN-γ was detected in LNC cultures as early as 6 days after immunization and the addition of type II collagen to the culture medium resulted in an approximately 10-fold increase in IFN-γ production, indicating that a predominantly Th1 response had become established by this time. IFN-γ production by LNC was found to be further increased at the time of clinical manifestation of arthritis and could be up-regulated by co-culture with type II collagen. IL-10 was not detected in LNC cultures at the onset of arthritis and IL-4, although present, was found to be markedly suppressed in LNC cultures containing type II collagen. These findings indicate that Th1 responses are predominant at the time of onset of arthritis and that the activation of collagen-specific Th1 cells may result in suppression of Th2 activity. IFN-γ production declined progressively during the progression and subsequent remission of arthritis whereas levels of IL-10 increased and low, though persistent, levels of IL-4 were detected throughout this period. High levels of IL-1β and TNF-α production were detected at the onset of the disease. The role of Th1 responses in the development of CIA was further emphasized by the observation that immunization of mice with type II collagen in incomplete Freund's adjuvant, which normally fails to induce arthritis, resulted in a predominantly Th2 cytokine profile.     

TCF1 deficiency ameliorates autoimmune lymphoproliferative syndrome (ALPS)‐like phenotypes of lpr/lpr mice

Autoimmune lymphoproliferative syndrome (ALPS) is an incurable disease, which is characterized by non‐malignant autoimmune lymphoproliferation. TCF1 is a key effector in the canonical Wnt/β‐catenin pathway, regulating the development, activation and function of T cells. In this study, we aimed to explore the potential role of TCF1 in the development of ALPS‐like phenotypes of lpr/lpr mice. We acquired TCF1^−/−lpr/lpr double mutant mice by crossing TCF1 deficiency mice with lpr/lpr mice. Splenocyte compositions, serum cytokines levels, antidsDNA antibody production and kidney pathology were examined in the TCF1^−/−lpr/lpr mice. With these examinations, we revealed that TCF1 deficiency relieved most manifestations of ALPS‐like phenotype, which were caused by Fas mutation in TCF1^−/−lpr/lpr mice. Splenocyte total numbers and compositions were downregulated to the similar levels with wildtype mice. T_E and T_EM cells were decreased in TCF1^−/−lpr/lpr compared with lpr/lpr mice. The levels of autoantibodies and proinflammatory factors in serum, and the histopathology changes and the relative mRNA levels of proinflammatory factors in kidney all displayed parallel tendency in TCF1^−/−lpr/lpr mice. Our study demonstrated that TCF1 deficiency ameliorated the ALPS‐like phenotypes of TCF1^−/−lpr/lpr mice, which might indicate a potential therapeutic direction for ALPS.     

Th1/Th2 cytokine gene expression after mercuric chloride in susceptible and resistant rat strains

Mercuric chloride (HgCl₂) has contrasting effects on different rat strains: susceptible strains, e.g. Brown Norway (BN) develop polyclonal B cell activation, multiple autoantibodies and widespread tissue injury. Lewis (LEW) rats are resistant: no autoimmune response occurs after HgCl₂; instead, there is immunosuppression. We have previously shown, by fully quantitative polymerase chain reaction (PCR), up-regulation of interleukin-4 (IL-4) gene expression in HgCl₂-treated BN rats, implicating Th2 cells in the autoimmune syndrome. Involvement of the reciprocal Th1 subset, producing interferon-γ (IFN-γ), in resistance of LEW rats to HgCl₂ has been suggested. We now report extensive analysis of Th1 and Th2 cytokine gene expression in spleen and lymph nodes of susceptible (BN) and resistant (LEW) rats after HgCl₂. IL-4 and IFN-γ were analyzed by quantitative PCR, other cytokines were assessed using semiquantitative PCR: the relative merits of these two techniques are discussed. We show pronounced up-regulation of IL-4 and more modest up-regulation of IFN-γ in BN rats, but no up-regulation of either in LEW rats. Baseline levels of IFN-γ were higher in LEW rats. Semi-quantitative PCR showed increased expression of IL-2, IL-6 and IL-10 in BN; in LEW rats only IL-10 was increased. There was no marked change in IL-5, IL-13 or transforming growth factor-β (TGF-β) in either strain. These data further support the key role of IL-4 in HgCl₂-induced autoimmunity, and suggest that failure of up-regulation of IL-4, together with higher baseline IFN-γ expression, accounts for resistance of LEW rats to HgCl₂. However, neither IFN-γ nor TGF-β can be implicated in HgCl₂-induced immunosuppression in the LEW rat in vivo: our data suggest a role for IL-10 in this phenomenon.     

Cell‐Mediated Immune Response and Th1/Th2 Cytokine Profile of B‐T Constructs of F1 and V Antigen of Yersinia pestis

Yersinia pestis, a Gram‐negative bacterium, is the etiological agent of pneumonic and bubonic plague and still active in various regions of the world. Because plague is highly infectious and can readily spread by aerosolization, it poses a bioterrorism threat. The effective induction of mucosal as well as systemic immunity is an important attribute of an improved vaccine for plague. An alternative approach described here is the use of protective epitopes derived from immunodominant antigens (F1 and V) of Yersinia pestis. As T‐cell immunity is also a major contributor of protection, microencapsulated B‐T constructs of F1 and V antigen were used to immunize outbred and inbred mice through intranasal route, and lympho‐proliferative response and cytokine profile of both Th₁ and Th₂ arms were measured in spleen, lamina propria and Peyer’s patches. Three B‐T constructs of F1 antigen and seven of V antigen showed significantly high T‐cell response in terms of inducing systemic as well as mucosal response when compared to constituent peptides. These ten conjugates showed Th₁ cytokine profile whereas rest of the conjugates showed mixed Th₁/Th₂ response. Four conjugates of V antigen showed high level of IL‐10 production. In present study, microencapsulated B‐T constructs after intranasal immunization generated systemic as well as mucosal immune response in all three sites, which offers an alternative approach for plague vaccine.     

Vaccination with autoreactive CD4Th1 clones in lupus-prone MRL/Mp-Fas mice

We studied the efficacy of T cell vaccination with CD4⁺αβTh1 clones in lupus-prone MRL/Mp-Fas^lpr/lpr (MRL/lpr) mice. CD4⁺αβ Th1 clones, dna51 (Vβ8.3) and rnp2 (Vβ14), which stimulated anti-dsDNA or U1 ribonucleoprotein (U1RNP) antibody (Ab) production respectively, were isolated from splenocytes of MRL/lpr mice. Antinuclear Ab kinetics, renal function, renal histology, survival rate, and lymphocyte subpopulations in the spleen were monitored after intravenous adoptive transfer of IL-2-stimulated (s-) or irradiated (i-) clones to 3 week-old female MRL/lpr mice. Anti-idiotypic humoral and T cell responses against the transferred autoreactive Th1 clones were determined in parallel. Compared with PBS-treated MRL/lpr mice, anti-dsDNA Ab titers, and the activity index for lupus nephritis were all decreased in MRL/lpr mice vaccinated with i-dna51 cells, whereas survival rate was not improved. The numbers of CD4⁺Vβ8.3 T cells in the spleen were also significantly decreased in these mice. Anti-idiotypic Abs recognizing a 12 amino acid sequence of clone dna51 T cell receptor Vβ8.3-complementarity-determining region (CDR) 3 were detected in the MRL/lpr mice that received i-dna51 or s-dna51 cells. These Abs suppressed dna51 cell proliferation, as well as cytotoxicity of CD8⁺T cells against dna51. The present study suggests that vaccination with CD4⁺αβTh1 clone, dna51, elicits anti-idiotypic T cell and humoral responses against dna51 in MRL/lpr mice, although the immunoregulatory effects on lupus may be limited.     

Differential modulation of T helper type 1 (Th1) and T helper type 2 (Th2) cytokine secretion by prostaglandin E2 critically depends on interleukin-2

Prostaglandin E₂ (PGE₂) favors T helper type 2 (Th2)-like cytokine secretion profiles in murine and human CD4⁺ T cells by inhibiting the production of the Th1-associated cytokines interleukin-2 (IL-2) and interferon-γ (IFN-γ) and up-regulating the production of the Th2-associated cytokines IL-4 and IL-5 in a dose-dependent way. However, the potent inhibition of IL-2 production by PGE₂ seems to be in contrast with the simultaneous up-regulation of IL-4 and IL-5 production, because the induction of these cytokines requires IL-2. We, therefore, investigated to which extent the net modulatory effect of PGE₂ is determined by the availability of IL-2. To this aim, we examined the effects of PGE₂ on the cytokine secretion profiles of a panel of human Th0 clones upon stimulation via different activation pathways, resulting either in high or low IL-2 production. The differential modulation of Th1 and Th2 cytokines by PGE₂ was observed only upon modes of stimulation resulting in high IL-2 production. When IL-2 production was low, PGE₂ inhibited the secretion of all four cytokines. These different modulation patterns were directly related to the IL-2 availability, because (i) neutralizing antibody to IL-2 abrogated the up-regulatory effect of PGE₂ on IL-4 and IL-5 secretion in experiments with high endogenous IL-2 levels, (ii) lack of differential cytokine modulation by PGE₂ in conditions with low levels of endogenous IL-2 could be restored with exogenous IL-2, and (iii) cell viability was comparable in all conditions. These results demonstrate that the net modulatory effect of PGE₂ on the cytokine secretion profile of T cells critically depends on the availability of IL-2. Since this parameter varies with the experimental conditions and the T cell population studied, this finding may explain why certain immune responses may be either up- or down-regulated by PGE₂ under different conditions.     

Infection of non-encapsulated species of Trichinella ameliorates experimental autoimmune encephalomyelitis involving suppression of Th17 and Th1 response

Epidemiological and experimental studies have indicated that helminth infections can ameliorate autoimmune diseases. The present study investigated the amelioration effect of the Trichinella pseudospiralis infection on experimental autoimmune encephalomyelitis (EAE), a T-cell-mediated autoimmune disease of central nervous system (CNS), and expression kinetics of Th17 and Th1 cytokine which play a crucial role in the pathogenesis of EAE. The results indicated that the infection of helminth T. pseudospiralis obviously ameliorated clinical severity and greatly delayed the onset of EAE induced by myelin oligodendrocyte glycoprotein (MOG) immunization. Infection caused much lesser inflammatory infiltration and demyilination in the CNS of infected EAE mice than uninfected EAE mice. The reduced infiltration was also suggested by the expressions of the inflammation cytokines, IL-17, IL-6, IL-1β, IFN-γ, and TNF-α, which were high in the spinal cords of the uninfected EAE mice, but was nearly normal or low in the infected EAE mice. The increased production of MOG-induced IL-17 and IFN-γ and the expression of IL-6, IL-1β, TGF-β in splenocytes after restimulation with MOG was inhibited in the infected EAE mice. On the other hand, the greatly induced Th2 response was observed in the splenocytes of the infected EAE mice. The present study showed that T. pseudospiralis infection can suppresses EAE by reducing the inflammatory infiltration in CNS, likely associated with the suppression of Th17 and Th1 responses by the infection.     

Selective Effect of Mercury on Th2-Type Cytokine Production in Humans

Mercury induces autoimmune disease and increases IL-4 production and IgE levels in certain rodent strains. The object of this study was to determine if mercury was capable of inducing Th2 cytokine production in human leucocytes. Human peripheral blood mononuclear cells (PBMC) were incubated with PMA/ionomycin or Con A in the presence or absence of methyl mercury (CH₃Hg) or mercuric chloride (HgCl₂). IL-4 and gamma-IFN were measured by ELISA. RESULTS: IL-4 production significantly increased at low concentrations of CH₃Hg (0.5 uM, p < 0.01), while gamma-IFN production was suppressed starting at CH₃Hg 2 uM (p = 0.004). Inorganic mercury (HgCl₂) increased IL-4 only at concentrations 10–20 times higher than CH₃Hg. These findings suggest a mechanism by which mercury could trigger or potentiate TH2 cytokine production in humans.     

Effect of cyclophosphamide on lymphokine production in MRL/lpr.Yaa mice

The Y chromosome (Yaa gene) from autoimmune BXSB mice has been shown to be responsible for the acceleration of autoimmune symptoms when transferred to MRL/lpr mice. We examined the pathological, serological and immunological characteristics of MRL/lpr.Yaa mice and the suppressive effect of cyclophosphamide (CP) on the mice. MRL/lpr.Yaa mice spontaneously developed a massive lymphadenopathy characterized by hypergammaglobulinemia, the presence of several autoantibodies, and autoimmune disease. In MRL/lpr.Yaa mice, IL-2, IL-4 and IL-5 production in concanavalin A (Con A)-stimulated splenocytes were about 10-fold lower than in BALB/c mice at 5 weeks of age.The concentrations of these lymphokines remained low until the mice were 16 weeks of age. The production of IFN- and IL-6 in 16 week old MRL/lpr.Yaa mice was about 4- and 2-fold lower, respectively, though these levels were similar in both strains at 8 weeks of age. It was found that this dysregulation of T cell function was almost identical to that in MRL/lpr mice. Administration of CP to MRL/lpr.Yaa mice ameliorated nephritis, and suppressed production of autoantibodies and the accumulation of abnormal T cells. CP also significantly elevated the production of lymphokines. These findings suggest that an abnormality of T cell function may contribute to the autoimmune pathogenesis of MRL/lpr.Yaa mice and that CP probably ameliorates autoimmune disease by improving the T cell functions.     

Th1 and Th2 cytokine modulation by IL-10/IL-12 imbalance in autoimmune haemolytic anaemia (AIHA)

Recent studies about autoimmune diseases in animal models and in humans focused their attention on lymphocyte activation and in vitro cytokine production. The respective contribution of the Th1 and Th2 cytokines to the pathogenesis of autoimmune diseases is still a matter of debate. In this study the role of IL-2, IL-4, IFN-gamma, IL-10 and IL-12 cytokines were investigated by examining their spontaneous and mitogen-induced (OKT3 and PHA or LPS) synthesis and T-cells proliferative response by peripheral blood mononuclear cells to determine their role in the pathogenesis of AIHA. Thirteen patients affected by AIHA, idiopathic or associated with other diseases, and 13 healthy subjects, randomly selected from a group of blood donors, were investigated. This study indicated that AIHA is characterised by increased basal synthesis of IL-4 and decreased levels of IFN-gamma compared with healthy controls (p < 0,01). These results suggest that there is a basal decrease of Th1 cytokine and an increase of the Th2 ones. Enhanced IL-2 levels in AIHA patients are likely due to the necessity of a T-cell proliferation stimulus rather than produced as Th1 prevalent stimulation. Furthermore, it has been observed a significant increase in IL-12 production in LPS stimulated cultures from healthy controls, but not in AIHA patients, that shows IL-10 increased levels, which could cause a secondary decrease in IFN-gamma production and a stimulation of Th2 differentiation. These observations indicate that decreased production of Th1-type cytokines and prevalent Th2 ones leading to autoantibodies production in AIHA may be secondary to the imbalance between IL-10 and IL-12. These results strongly suggest that manipulation of the cytokine network, i.e. IL-10/IL-12 balance, maintained by cells of the innate immune system, can have a strong effect on the incidence of AIHA and their modulation might be useful for a therapeutic control of the disorder.     

Th1 (IL-2, interferon-gamma (IFN-γ)) and Th2 (IL-10, IL-4) cytokine production by peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE)

We investigated the production of IL-2, IFN-γ, IL-10 and IL-4 by PBMC from 24 patients with SLE and 10 healthy individuals. Basal and mitogen-stimulated (lipopolysaccharide and phytohaemagglutinin (LPS + PHA)) cytokine production was determined in a whole blood assay (WBA). Supernatants were collected and assayed with specific ELISAs. Although the IL-2 and IFN-γ contents did not differ significantly between patients and controls under both conditions, statistically significant correlations were found between each cytokine and disease activity (SLAM index) after stimulation (respectively, r= 0.501, P = 0.01 and r = 0.631, P = 0.001). PBMC IL-10 production was significantly higher for patients than controls (P = 0.05), but no correlation between IL-10 levels and the SLAM index was obtained. IL-4 production was not statistically different between SLE patients and controls. For stimulated WBAs, the IL-10/IL-2 and IL-10/IFN-γ ratios were significantly correlated with disease severity (P = 0.02; P = 0.001, respectively). Overall, our data suggest that SLE is characterized by an elevated production of IL-10, reflecting the basal state of activation of the immune system. During exacerbation of SLE, IL-2 and IFN-γ are synthesized in larger amounts and may cause the tissue damage observed.     

Ontogenic development of Th1 and Th2 cytokine capabilities in random bred mice

Neonatal mouse Th1 capabilities mature by postnatal day 5. Neonatal T cells have been reported to exhibit a bias towards Th2 cytokine production when co-cultured with adult antigen presenting cells (APC). We studied mouse T cells co-cultured with contemporary APC to evaluate neonatal cytokine production capabilities. In response to allogeneic stimulation, T cells co-cultured with contemporary APC from day 5 pups produced 37-fold greater IFNgamma and 1.4-fold greater IL-2 levels than day 20 weanling mice. After CD3 ligation, cells from day 5 pups produced 4- (IL-2) and 10-fold (IFNgamma) greater levels than adults (day 45), and concentrations were 27- (IL-2) and 18-fold (IFNgamma) higher than with allogeneic stimulation alone. On average, the percent difference in concentrations was 418 (IL-4), 286 (IL-2) and 1140% (IFNgamma) higher in unseparated spleen cells than in isolated splenic CD4 cells and APC. These results demonstrate that, in response to allogeneic stimulation with or without CD3 ligation, lymphocytes of neonatal mice (day 5) have the capacity to produce equivalent or greater TcR-dependent Th1 cytokine (IL-2 and IFNgamma) levels than adult mice. Findings also support the idea that the reported Th2 bias of neonatal T cells may be the result of in vitro manipulation and choice of mouse strain, not of an inherent bias.     

Assessment of Th1 and Th2 cytokine modulatory activity of an edible fern,Diplazium esculentum

The present study was conducted to determine the effect of Diplazium esculentum on Th1 and Th2 cytokine modulation in Swiss albino mice that were administered orally with different doses of boiled D. esculentum (BDE), daily within a span of 180 days. After the treatment, serum was collected. Splenocytes were also cultured in vitro with different concentrations of BDE, and culture supernatant was collected. Both serum and culture supernatant were used for cytokine determination by enzyme-linked immunosorbent assay (ELISA) for different Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-10) cytokines. Results indicated significant decreases (p < 0.05, p < 0.01, and p < 0.001) in both Th1 and Th2 cytokine concentrations when compared with their respective controls. These results suggest that the intake of D. esculentum, even after cooking, may evoke immune dysfunction by altering Th1 and Th2 cytokine balance, may induce severe immunosuppression, and may be considered as alarming.     

Th1/Th2 cytokine responses following HIV-1 immunization in seronegative volunteers

The Th1/Th2 profile that follows human vaccination may profoundly influence the subsequent course of disease after infection. However, the ability to detect IL-4 has been limited outside trials of live vaccination. By using methods in which memory effector cells are allowed to antigenically expand by short term culture, followed by low-dose mitogenic stimulation, we have been able to follow the Th1/Th2 profile in HIV-1^?volunteers enrolled in two phase I studies of HIV immunogens (a recombinant gp120 and a multivalent, octomeric V3 loop peptide). Antigen-specific interferon-gamma (IFN-γ) could be detected in primary stimulation, but IL-4 was observed only after antigenic expansion and restimulation. In both of these studies the responses after initial immunizations were dominated by IFN-γ, with IL-4 appearing only after multiple rounds of immunization, and IL-4 was temporally related to antibody production. Concomitant with the IL-4 production, the amount of supernatant IFN-γ declined. Antigen-specific IL-10 was not detected in either study. Such techniques, which have been shown to correlate with outcomes in immunotherapy, may prove useful as future surrogates of human vaccine response.     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.