Possible role of LECT2 as an intrinsic regulatory factor in SEA-induced toxicity in d-galactosamine-sensitized mice.

To elucidate whether leukocyte cell-derived chemotaxin 2 (LECT2) controls the progression of staphylococcal enterotoxin A (SEA)-induced toxicity, we examined the role of LECT2 in a mouse model. Almost all the C57BL/6J (B6) mice survived for 72 h after the injection of 0.1 µg of SEA and 20 mg of d-galactosamine (d-GalN). However, the same treatment protocol in LECT2(-/-) mice produced a high lethality (~90%), severe hepatic apoptosis, and massive hepatic and pulmonary hemorrhage, similar to the situation observed in B6 mice treated with 1.0 µg SEA/d-GalN. The plasma LECT2 levels in B6 mice treated with 1.0 µg SEA/d-GalN were inversely correlated with the plasma cytokine levels and were associated with prognosis. LECT2 administration increased the survival of B6 mice and down-regulated TNF-α and IL-6. These results suggest the involvement of LECT2 in the regulation of fatal SEA-induced toxicity in d-GalN-sensitized mice.

Long-term bacterial exposure can trigger nonsuppurative destructive cholangitis associated with multifocal epithelial inflammation.

Bacterial infection has become a focus of attention in the pathogenesis of primary biliary cirrhosis (PBC). We earlier reported that the bacterial lipoteichoic acid was detected at the sites of inflammation around damaged bile ducts in the livers of PBC, and PBC patients' sera showed high titers against streptococcal histone-like protein. Here, we investigated whether chronic bacterial exposure could trigger PBC-like epithelial cell damage in normal mouse. BALB/c mice were repeatedly inoculated with various bacteria for 8 weeks. At 1 week (Group 1) and 3, 4, or 20 months (long term; Group 2) after the final inoculation, mice were killed to obtain samples. In the livers of the Streptococcus intermedius (S.i.)-inoculated mice in Group 1, cellular infiltration was predominantly observed around the bile ducts over the hepatic parenchyma. In the S.i.-inoculated mice in Group 2, portal but not parenchymal inflammation was observed in the livers, and periductal cellular infiltrates were detected in the salivary glands. Both S.i.-inoculated Groups 1 and 2 BALB/c mice sera had antibodies against HuCCT1 biliary epithelial cells, anti-nuclear antibodies, and anti-gp210 antibodies, but not anti-mitochondrial antibodies. Immunoreactivity to histone-like DNA-binding protein of S.i. (S.i.-HLP) was detectable around the sites of chronic nonsuppurative destructive cholangitis in the portal area in the livers of both S.i.-inoculated Groups 1 and 2 BALB/c mice. Furthermore, anti-S.i.-HLP antibody bound to synthetic gp210 peptide, as well. Bacteria triggered PBC-like cholangitis, multifocal epithelial inflammation, and autoantibody production. Bacteria are likely involved in the pathogenesis of PBC and of associated multifocal epithelial inflammation.

T cell activation in abnormal perinatal events.

The aim of this study was to determine the percentage of CD45RO(+) T cells in umbilical cord blood from neonates born at less than 37 weeks of gestation. Fifty-nine patients were enrolled in this study, including 49 with preterm and 10 with term deliveries. Preterm deliveries were divided into two categories; spontaneous (Group A, n= 31) and indicated (Group B, n= 18). Perinatal infection was categorized as C-CAM, H-CAM and neonatal infection. The percentage of CD45RO(+) T cells in the umbilical cord was assessed using flow cytometry. IL-6 was measured using ELISA. In Group A, the percentage of CD45RO(+) T cells and concentrations of IL-6 in patients with perinatal infection (n= 18) were significantly higher than in those without perinatal infection (n= 13). A significant correlation between percentage of CD45RO(+) T cells and IL-6 concentrations was observed in the cord blood (r= 0.62, P= 0.001). In Group B, pink-tinged amniotic fluid was observed in seven cases. In these cases, an increase in the percentage of CD45RO(+) T cells (>10%) was noted. In the cases without perinatal infection, which included all those delivered at term (n= 32), no correlation was observed between the percentage of CD45RO(+) T cells and gestational age at delivery (r=-0.139, P= 0.448). We concluded that a high percentage of CD45RO(+) cord blood T cells is observed not only in perinatal infection, but also in the presence of abnormal perinatal events such as maternal bleeding in preterm gestation.

Superantigen-presentation by rat major histocompatibility complex class II molecules RT1.Bl and RT1.Dl.

Rat major histocompatibility complex (MHC) class II molecules RT1.B(l) (DQ-like) and RT1.D(l) (DR-like) were cloned from the LEW strain using reverse transcription-polymerase chain reaction and expressed in mouse L929 cells. The transduced lines bound MHC class II-specific monoclonal antibodies in an MHC-isotype-specific manner and presented peptide antigens and superantigens to T-cell hybridomas. The T-cell-hybridomas responded well to all superantigens presented by human MHC class II, whereas the response varied considerably with rat MHC class II-transduced lines as presenters. The T-cell hybridomas responded to the pyrogenic superantigens Staphylococcus enterotoxin B (SEB), SEC1, SEC2 and SEC3 only at high concentrations with RT1.B(l)-transduced and RT1.D(l)-transduced cells as presenters. The same was true for streptococcal pyrogenic exotoxin A (SPEA), but this was presented only by RT1.B(l) and not by RT1.D(l). SPEC was recognized only if presented by human MHC class II. Presentation of Yersinia pseudotuberculosis superantigen (YPM) showed no MHC isotype preference, while Mycoplasma arthritidis superantigen (MAS or MAM) was presented by RT1.D(l) but not by RT1.B(l). Interestingly, and in contrast to RT1.B(l), the RT1.D(l) completely failed to present SEA and toxic shock syndrome toxin 1 even after transduction of invariant chain (CD74) or expression in other cell types such as the surface MHC class II-negative mouse B-cell lymphoma (M12.4.1.C3). We discuss the idea that a lack of SEA presentation may not be a general feature of RT1.D molecules but could be a consequence of RT1.D(l)beta-chain allele-specific substitutions (arginine 80 to lysine, asparagine 82 to aspartic acid) in the extremely conserved region flanking the Zn(2+)-binding histidine 81, which is crucial for high-affinity SEA-binding.

Antitumor activity and some immunological properties of gammadelta T-cells from patients with gastrointestinal carcinomas.

OBJECTIVES: Human gammadelta T-cells expressing Vgamma2Jgamma1.2Vdelta2-TCR recognize microbial pyrophosphomonoesters in an MHC-independent manner and exert cytotoxic activity on a wide variety of tumor cells. In the present study, the immunological properties of gammadelta T-cells derived from patients with gastrointestinal carcinomas were examined and compared with those from healthy adult individuals, aiming to develop a novel cancer immunotherapy using gammadelta T-cells stimulated with one of the nonpeptide antigens, 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP). MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMs) and tumor-associated lymphocytes (TAL) were obtained from patients with gastrointestinal carcinomas. The mononuclear cells were stimulated with 2M3B1PP for 2 weeks and the expanded gammadelta T cells were examined for cytokine production upon T-cell receptor (TCR) engagement and cytotoxic activity against allogeneic tumors and autologous tumor cells. For comparison, PBMCs derived from healthy adult volunteers were similarly stimulated with 2M3B1PP and the resulting gammadelta T-cells were analyzed for effector functions. RESULTS: All the peripheral blood- and tumor-associated gammadelta T-cell preparations from patients with gastrointestinal carcinomas proliferated vigorously in response to 2M3B1PP to comparable levels to those from healthy donors. When challenged with CD3 monoclonal antibodies, the carcinoma patient-derived gammadelta T-cells secreted a large amount of inflammatory cytokine, IFN-gamma, and exhibited a potent cytotoxic activity against allogeneic tumor cell lines as well as autologous tumor cells. CONCLUSION: Both peripheral blood- and tumor-associated gammadelta T-cells derived from patients with gastrointestinal carcinomas were as immunologically active as those from healthy individuals and could be utilized for a novel cancer immunotherapy for gastrointestinal malignancies.

A possible role of histone-like DNA-binding protein of Streptococcus intermedius in the pathogenesis of bile duct damage in primary biliary cirrhosis.

Bacterial infection has become a focus of attention in the pathogenesis of primary biliary cirrhosis (PBC). It was reported that anti-histone autoantibody was detected in PBC, suggesting that bacterial histone-like DNA-binding protein (HLP) may be involved in the pathogenesis of PBC. To identify bacterial species in PBC to confirm this possibility, serum reactivity to bacterial cells was studied by ELISA. The IgM class Streptococcus intermedius titers were significantly higher in PBC than chronic hepatitis due to hepatitis C virus (CH-C) and healthy subjects. Among the streptococci, S. intermedius was selected for further study. The antigenic peptide of S. intermedius of HLP was synthesized to examine the serum reactivity to Si-HLP. IgM class anti-Si-HLP peptide titers were significantly higher in PBC. Immunoreactivity to anti-Si-HLP was detected in the cytoplasm of biliary epithelial cells and inflammatory cells in the portal area in PBC patients' livers. Streptococci, especially S. intermedius, might play a key role in the pathogenesis of PBC, possibly involving HLP.

Long-term exposure to superantigen induces p27Kip1 and Bcl-2 expression in effector memory CD4+ T cells.

The long-term exposure of mice to superantigen SEA using a mini-osmotic pump (SEA pump) induced a long-lasting expansion of Vbeta3+ CD4+ T cells with T helper (Th) 2 cell-type properties. Removal of the SEA pump 10 days after pump implantation did not significantly alter the level of Vbeta3+ CD4+ T cell expansion/maintenance. Furthermore, CFSE-labeled CD4+ T cells failed to divide when transferred to post-implantation day 15 mice. Thus, CD4+ T cells appeared to survive for at least 30 days in the absence of a sufficient amount of antigen to trigger cell division. STAT6 deficient mice, in which Th2 cell development is largely impaired, also exhibited a protracted cell expansion, similar to that observed in normal mice, suggesting that the Th2 cell property is dispensable for the maintenance of Vbeta3+ CD4+ T cell expansion. The expanded CD4+ T cells on post-implantation day 26 were arrested in the G0/G1 phase of the cell cycle and showed a lower level of cell division upon restimulation. The Cdk inhibitor p27(Kip1) was highly expressed, and Cdk2 was downregulated. Moreover, the CD4+ T cells were resistant to in vitro apoptosis induction in parallel with their level of Bcl-2 expression. Collectively, the Vbeta3+ CD4+ T cells appeared to develop into long-lived memory T cells with cell cycle arrest upon long-term exposure to SEA.

Molecular and supra-molecular structure related differences in toxicity and granulomatogenic activity of mycobacterial cord factor in mice.

To establish the structure biological activity relationship of cord factor (trehalose 6,6'-dimycolate, TDM), we compared the molecular or supra-molecular structure of TDM micelles with toxicity, thymic atrophy and granulomatogenicity in lungs and spleen of BALB/c mice. According to the difference in the mycolyl subclass composition, TDM was divided into two groups, one possessing alpha-, methoxy- and keto-mycolates in M. tuberculosis H37Rv, M. bovis BCG and M. kansasii (group A) and the other having alpha-, keto- and wax ester-mycolates in M. avium serotype 4, M. phlei and M. flavescens (group B), although mycolic acid molecular species composition differed in each group considerably. Supra-molecular structure of TDM micelle differed species to species substantially and the micelle size of TDM from M. bovis BCG Connaught was the largest. The highest toxicity was shown with TDM from M. tuberculosis H37Rv which possessed the highest amount of alpha- (47.3%) and methoxy-mycolates (40.8%), while TDM from M. phlei having the low amount of alpha-mycolate (11.6%) showed almost no toxicity with the given doses. The thymic atrophy was observed with TDM from group A, but not with TDM from group B. On the other hand, TDM from group B showed massive lung granulomatogenic activity based on the histological observations and organ indices. Taken together, group A TDM showed a wide variety of micelle sizes and specific surface areas, high to low toxicity and marked to moderate granulomatogenicity, while group B TDM showed smaller sizes of micelles and larger specific surface areas, lower toxicity but higher granulomatogenicity in lungs. Existence of higher amount of longer chain alpha-mycolates in TDM appeared to be essential for high toxicity and thymic apoptotic activity, whereas TDM possessing wax ester-mycolate with smaller sized micelles seemed to be less toxic, but more granulomatogenic in lungs in mice. Thus, the mycolic acid subclass and molecular species composition of TDM affect critically the micelle forms, toxicity and granulomatogenicity in mice, while the relative abundances and carbon chain length of alpha-mycolate affected the toxicity in mice.

Safety profile and anti-tumor effects of adoptive immunotherapy using gamma-delta T cells against advanced renal cell carcinoma: a pilot study.

PURPOSE: Although various types of immunotherapy have been used to improve the prognosis of patients with advanced renal cell carcinoma (RCC), adoptive immunotherapy using gamma-delta (gammadelta) T cells has not yet been tried. In this study, we designed a pilot study of adoptive immunotherapy using in vitro activated gammadelta T cells against advanced RCC to evaluate the safety profile and possible anti-tumor effects of this study. EXPERIMENTAL DESIGN: Patients with advanced RCC after radical nephrectomy were administered via intravenous infusion in vitro-activated autologous gammadelta T cells every week or every 2 weeks, 6-12 times, with 70 JRU of teceleukin. Adverse events, anti-tumor effects and immunomonitoring were assessed. The anti-tumor effects were evaluated according to tumor doubling time (DT) by computed tomography (CT) and immunomonitoring was performed by flow cytometric analysis. RESULTS: Seven advanced RCC patients were entered in this study. The most common adverse events were fever, general fatigue and elevation of hepatobiliary enzymes, but no severe adverse events were seen. Prolongation of tumor DT was seen in three out of five patients; these three patients showed an increase in the number of gammadelta T cells in peripheral blood and also a high response to the antigen in vitro. CONCLUSIONS: The results indicated that adoptive immunotherapy using in vitro-activated autologous gammadelta T cells was well tolerated and induced anti-tumor effects.

Inhibitory effect of antimicrobial agents and anisodamine on the staphylococcal superantigenic toxin-induced overproduction of proinflammatory cytokines by human peripheral blood mononuclear cells.

Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), produces superantigenictoxins, such as toxic shock syndrome toxin-1 (TSST-1). TSST-1 abnormally activates T cells to overproduce inflammatory cytokines (such as tumor necrosis factor-alpha, interleukin-2, and interferon-gamma) leading to shock. In this study, we investigated the inhibitory effect of antimicrobial agents and anisodamine (a Chinese herbal extract) on TSST-1-induced cytokine production. Among the macrolides and related agents examined, azithromycin and rokitamycin showed the greatest inhibitory activity against the TSST-1-induced cytokine production. This inhibitory effect was similar to that of anisodamine, which, however, had no inhibitory activity against bacterial growth. Vancomycin, teicoplanin, arbekacin, and linezolid (anti-MRSA and related agents) had no significant inhibitory effect on cytokine production. The inhibitory effect of the drugs on cell proliferation was not significant. These data indicate that some antimicrobial agents, e.g., azithromycin and rokitamycin, manifest anti-superantigenic toxin activity through the inhibition of cytokine production, just like anisodamine.

Characterization of novel staphylococcal enterotoxin-like toxin type P.

We investigated the biological properties of a novel staphylococcal enterotoxin (SE)-like toxin type P (SElP). SElP induced a substantial proliferative response and the production of cytokines interleukin-2, gamma interferon, tumor necrosis factor alpha, and interleukin-4 from human T cells when administered at a concentration of 0.4 pM (0.01 ng/ml) or more. The expression of major histocompatibility complex class II molecules on accessory cells was required for T-cell stimulation by SElP. SElP selectively stimulated a vast number of human T cells bearing receptors Vbeta 5.1, 6, 8, 16, 18, and 21.3. These results indicated that SElP acts as a superantigen. SElP proved to be emetic in the house musk shrew emetic assay, although at a relatively high dose (50 to 150 mug/animal). A quantitative assay of SElP production with 30 Staphylococcus aureus strains harboring selp showed that 60% of these strains produced significant amounts of SElP in vitro. All 10 strains carrying seb and selp produced SEB but not SElP, suggesting the inactivation of the selp locus in S. aureus strains with a particular se gene constitution.

Studies of recombinant streptococcal pyrogenic exotoxin B/cysteine protease (rSPE B/SCP) in the skin of guinea pigs & the release of histamine from cultured mast cells & basophilic leukocytes.

BACKGROUND & OBJECTIVES: Streptococcal pyrogenic exotoxin B/streptococcal cysteine protease (SPE B/SCP) is considered to be one of the virulence factors of Streptococcus pyogenes (S. pyogenes) which causes serious diseases such as severe invasive infections and streptococcal toxic shock syndrome (STSS). There are no reports on the histamine releasing activity of SPE B/SCP from mast cells, although several biological activities have been studied. It is not clear whether SPE B/SCP have the superantigenic activity. We studied whether SPE B/SCP plays as a pathogenic factor in streptococcal infections and STSS through a histamine releasing activity. METHODS: Human mast cells and basophils were generated from CD34 positive cells isolated from cord blood and cultured in the presence of rIL-6, stem cell factor and/or rIL-3. The capacity of increasing capillary permeability of recombinant SPE B/SCP (rSPE B/SCP) was studied by using the skin of guinea pigs. Mitogenic activity to human T-cells of rSPE B/SCP was studied by incorporation of (3)Hthymidine. The levels of histamine in the plasma of patients with STSS and controls were measured by ELISA kit. RESULTS: rSPE B/SCP induced increased capillary permeability in the skin of guinea pigs, but both SPE A and SPE C did not exhibit such activity. Histamine was released from cultured human mast cells stimulated with rSPE B/SCP. The rSPE B/SCP did not exhibit mitogenic activity to human T-cells. Three of the 7 patients with STSS showed higher levels of plasma histamine than those of normal subjects. INTERPRETATION & CONCLUSION: The results suggested that increased capillary permeability and histamine release from mast cells induced by rSPE B/SCP might be involved in STSS and/or streptococcal infection of skin and mucous membrane.

Akt is a neutral amplifier for Th cell differentiation.

Both CD28 and its relative, inducible costimulator (ICOS), have a binding motif for phosphatidylinositol 3-kinase (PI3K) in their cytoplasmic tail, and the binding of PI3K leads to activation of a serine/threonine kinase, Akt. The role of Akt in cytokine production and helper T (Th) cell differentiation remains obscure. In this study, we found that enforced expression of the constitutively active form (E40K) of Akt rendered CD4(+) T cells activated. Wild-type of Akt and E40K promoted Th1 cell differentiation in C57BL/6-derived and Th1-polarized BALB/c-derived CD4(+) T cells, while both promoted Th2 cell differentiation in BALB/c-derived and Th2-polarized C57BL/6 CD4(+) T cells. E40K also facilitated Th1 differentiation in CD4(+) T cells from IL-4-deficient mice with the BALB/c background. E40K up-regulated expression of NF-AT and c-Myb, which may be related to the augmentation of cytokine production by E40K. These findings indicate that the mechanism by which Akt augments cytokine production via CD28 and ICOS is Th cell type-specific and reflects the intracellular status affected by the cytokine milieu. We conclude that Akt is a neutral amplifier of T cell activation and Th differentiation.

Regulatory roles of IL-2 and IL-4 in H4/inducible costimulator expression on activated CD4+ T cells during Th cell development.

We found a tight correlation among the levels of H4/inducible costimulator (ICOS) expression, IL-4 production, and GATA-3 induction, using activated CD4(+) T cells obtained from six different murine strains. BALB/c-activated CD4(+) T cells expressed approximately 10-fold more H4/ICOS on their surfaces and produced approximately 10-fold more IL-4 upon restimulation than C57BL/6-activated CD4(+) T cells. BALB/c naive CD4(+) T cells were shown to produce much higher amounts of IL-2 and IL-4 upon primary stimulation than C57BL/6 naive CD4(+) T cells. Neutralization of IL-4 with mAbs in culture of BALB/c naive CD4(+) T cells strongly down-regulated both H4/ICOS expression on activated CD4(+) T cells and IL-4 production upon subsequent restimulation. Conversely, exogenous IL-4 added to the culture of BALB/c or C57BL/6 naive CD4(+) T cells up-regulated H4/ICOS expression and IL-4 production upon restimulation. In addition, retroviral expression of GATA-3 during the stimulation of naive CD4(+) T cells from C57BL/6 or IL-4(-/-) mice increased H4/ICOS expression on activated CD4(+) T cells. A similar effect of IL-2 in the primary culture of BALB/c naive CD4(+) T cells appeared to be mediated by IL-4, the production of which was regulated by IL-2. These data suggest that IL-4 induced by IL-2 is critical to the maintenance of high H4/ICOS expression on BALB/c-activated CD4(+) T cells.

Maturation of adult peripheral blood CD38(+)CD4(+) T cells demonstrated by cytokine production in response to a superantigen, TSST-1.

This study looks at immunoincompetent CD4(+) T cells in adult peripheral blood (APB) using cytokine production in response to a superantigen as a measure of function. We compared the function of APB CD38(+)CD4(+) and CD38(-/low)CD4(+) T cells to that of cord blood (CB) CD4(+) T cells. APB CD4(+) T cell blasts produce substantial amounts of IL-2 in response to TSST-1 restimulation, while CB CD4(+) T cell blasts produce less. APB CD38(+)CD4(+) T cells produce low levels of IL-4 and IFN-gamma in response to TSST-1, even after activation, while APB CD38(-/low)CD4(+) T cells retain their ability to produce high levels of these cytokines despite high CD38 expression. These results suggest that the developmental stage of APB CD38(+)CD4(+) T cells lies between that of CB CD4(+) T cells and APB CD38(-/low)CD4(+) T cells and that APB CD38(+)CD45RO(-)CD4(+) T cells gradually cease to express CD38 as they acquire full function. We reconsider CD4(+) cell maturation and response to TSST-1 and discuss the implications of T cell maturity on infectious diseases.

Preparation of a superantigen-adsorbing device and its superantigen removal efficacies in vitro and in vivo.

OBJECTIVE: A new superantigen-adsorbing device (SAAD) was developed, and its characteristics and efficacy in septic animals were evaluated. METHODS: The SAAD was prepared by stepwise chemical modification of a polystyrene-based composite fiber reinforced with polypropylene. Adsorption affinities for several factors and the biological effect of superantigen (SAg) removal were measured in vitro. Also, superantigen-infused rabbits were treated with SAAD, and the efficacy was evaluated in vivo. RESULTS: When the SAAD was evaluated for its ability to adsorb SAg in human plasma (1 ng/mL each), the adsorption rates were 74%, 76% and 85% for staphylococcal enterotoxins A, B and C, respectively, and 80% and 72% for toxic shock syndrome toxin-1 (TSST-1) and streptococcal pyrogenic exotoxin A, respectively. In addition, the SAAD showed some affinity towards other molecules, such as streptococcal pyrogenic exotoxin B, beta2-microglobulin, and vancomycin. Residual activities in whole blood samples containing TSST-1 (1 ng/mL) after incubation with the SAAD were 125 pg/mL for tumor necrosis factor alpha (TNF-alpha) production, and 359 pg/mL for interleukin-8 (IL-8) production (the initial activities: 194 pg/mL for TNF-alpha production, and 1029 pg/mL for IL-8 production). When TSST-1/lipopolysaccharide (LPS)-infused rabbits were subjected to extracorporeal blood purification with a SAAD column, 50% of the animals survived for a 14-day period after the infusion. In contrast, all control animals died within 3 days after the infusion. CONCLUSION: These results indicate that the SAg-adsorbing device may be useful in treating SAg-related diseases.

A co-stimulatory molecule on activated T cells, H4/ICOS, delivers specific signals in T(h) cells and regulates their responses.

We examined the co-stimulatory activity of H4/ICOS on murine activated CD4(+) T cells and found that the cross-linking of H4/ICOS enhanced their proliferation, in addition to raising IFN-gamma, IL-4 and IL-10 production to levels comparable to those induced by CD28. However, IL-2 production was only marginally co-stimulated by H4/ICOS. This distinct pattern of lymphokine production appears to be induced by a specific intracellular signaling event. Compared with CD28, H4/ICOS dominantly elicited the Akt pathway via phosphatidylinositol 3-kinase. In addition, mitogen-activated protein kinase family kinases were activated in different ways by CD28 and H4/ICOS. The strong phosphorylation of p46 c-Jun N-terminal kinase was observed upon CD28 co-stimulation, but was less potently induced by H4/ICOS. The strain diversity in the induction of H4/ICOS was recognized. The expression of H4/ICOS on BALB/c activated CD4(+) T cells was >6-fold higher compared with C57BL/6 activated CD4(+) T cells. Furthermore, BALB/c activated CD4(+) T cells exhibited more T(h)2-deviated lymphokine production as compared with C57BL/6 activated CD4(+) T cells and signaling through H4/ICOS during the primary stimulation of naive CD4(+) T cells promoted the generation of T(h)2 cells. Thus, the difference in H4/ICOS expression on activated CD4(+) T cells, which is regulated among the mouse strains, may also regulate the polarization of T(h) cells.