Influence of drying methods over in vitro antitumoral effects of exopolysaccharides produced by Agaricus blazei LPB 03 on submerged fermentation.

Agaricus blazei is a mushroom that belongs to the Brazilian biodiversity and is considered as an important producer of bioactive compounds beneficial to human health. Studies have demonstrated that these compounds present immuno-modulatory, antioxidant and antitumor properties. In order to compare the most used method for fungal polysaccharide drying, lyophilization with other industrial-scale methods, the aim of this work was to submit A. blazei LPB 03 polysaccharide extracts to vaucum, spray and freeze drying, and evaluate the maintenance of its antitumoral effects in vitro. Exopolysaccharides produced by A. blazei LPB 03 on submerged fermentation were extracted with ethanol and submitted to drying processes. The efficiency represents the water content that was removed during the drying process. The resultant dried products showed water content around 3% and water activity less than 0.380, preventing therefore the growth of microorganisms and reactions of chemical degradation. Exopolysaccharide extracts dried by vacuum and spray dryer did not showed any significant cytotoxic effect on cell viability of Wistar mice macrophages. Content of total sugars and protein decrease after drying, nevertheless, 20 mg/ml of exopolysaccharides dried by spray dryer reached 33% of inhibition rate over Ehrlich tumor cells in vitro.

Distinct role of antigen-specific T helper type 1 (Th1) and Th2 cells in tumor eradication in vivo.

The role of T helper type 1 (Th1) and Th2 cells in tumor immunity was investigated using Th cells induced from ovalbumin (OVA)-specific T cell receptor transgenic mice. Although Th1 cells exhibited stronger cytotoxicity than Th2 cells, both cell types completely eradicated tumors when transferred into mice bearing A20 tumor cells transfected with the OVA gene (A20-OVA). Th1 cells eradicated the tumor mass by inducing cellular immunity, whereas Th2 cells destroyed the tumor by inducing tumor necrosis. Both Th1 and Th2 cells required CD8(+) T cells to eliminate tumors, and neither of these cells were able to completely eliminate A20-OVA tumors from T and B cell-deficient RAG2(-/-) mice. Mice cured from tumors by Th1 and Th2 cell therapy rejected A20-OVA upon rechallenge, but CD8(+) cytotoxic T lymphocytes were induced only from spleen cells prepared from cured mice by Th1 cell therapy. Moreover, we demonstrated that Th1 and Th2 cells used distinct adhesion mechanisms during tumor eradication: the leukocyte function-associated antigen (LFA)-1-dependent cell-cell adhesion step was essential for Th1 cell therapy, but not for Th2 cell therapy. These findings demonstrated for the first time the distinct role of antigen-specific Th1 and Th2 cells during eradication of established tumors in vivo.

Interleukin 7 transgenic mice develop chronic colitis with decreased interleukin 7 protein accumulation in the colonic mucosa.

We have demonstrated that intestinal epithelial cells produce interleukin 7 (IL-7), and IL-7 serves as a potent regulatory factor for proliferation of intestinal mucosal lymphocytes expressing functional IL-7 receptor. To clarify the mechanism by which locally produced IL-7 regulates the mucosal lymphocytes, we investigated IL-7 transgenic mice. Here we report that transgenic mice expressing murine IL-7 cDNA driver by the SRalpha promoter developed chronic colitis in concert with the expression of SRalpha/IL-7 transgene in the colonic mucosa. IL-7 transgenic but not littermate mice developed chronic colitis at 4-12 wk of age, with histopathological similarity to ulcerative colitis in humans. Southern blot hybridization and competitive PCR demonstrated that the expression of IL-7 messenger RNA was increased in the colonic mucosal lymphocytes but not in the colonic epithelial cells. IL-7 protein accumulation was decreased in the goblet cell-depleted colonic epithelium in the transgenic mice. Immunohistochemical and cytokine production analysis showed that lymphoid infiltrates in the lamina propria were dominated by T helper cell type 1 CD4+ T cells. Flow cytometric analysis demonstrated that CD4+ intraepithelial T cells were increased, but T cell receptor gamma/delta T cells and CD8alpha/alpha cells were not increased in the area of chronic inflammation. Increased IL-7 receptor expression in mucosal lymphocytes was demonstrated in the transgenic mice. These findings suggest that chronic inflammation in the colonic mucosa may be mediated by dysregulation of colonic epithelial cell-derived IL-7, and this murine model of chronic colitis may contribute to the understanding of the pathogenesis of human inflammatory bowel disease.

The use of plasma lipid and lipoprotein ratios in interpreting the hyperlipidaemia of pregnancy.

The aim of this study was to determine the lipid and lipoproteins ratio in pregnant mothers and to evaluate their role in the interpretation of hyperlipidaemias. A total of 222 pregnant women who registered for ANC and 222 non-pregnant healthy women of the sameage and parity as control were recruited for the study. A sample of venous blood after an overnight fast was collected for analysis and interpretation. The mean ± SD age (years) of pregnant women, 27.317 ± 7.283 years and that of the non-pregnant women, 26.234 ± 6.234 years are not significantly different, p = 0.429. Total cholesterol, HDL-c and TGs were significantly higher in pregnant women (5.29 ± 1.04 mmol/l, 1.64 ± 0.42 mmol/l and 1.74 ± 0.42 mmol/l) compared with that of non-pregnant women (4.64 ± 0.92 mmol/l, 1.25 ± 0.35 mmol/l and 1.37 ± 0.45 mmol/l, respectively) All showed p < 0.000. The frequencies of hypercholesterolaemia, 96(43.2%) and hypertriglyceridaemia, 82 (36.9%), are significantly higher in the pregnant women than in the non-pregnant women, 58 (26.1%) and 26 (11.7%), respectively. TC/HDL-C ratio, 3.33 ± 1.01 and LDL/HDL-C ratio, 1.91 ± 0.85 are significantly lower in pregnant women compared with non-pregnant women counterparts, 3.89 ± 0.97 and 2.35 ± 0.84, respectively. Similarly the frequencies of increased TC/HDL-C ratio, 22 (9.9%) and LDL/HDL-C ratio, 16 (7.2%) are significantly less in the pregnant compared with the non-pregnant women, 54 (24.3%) and 28 (12.6%), respectively.

Antigen-specific antibody production of human B cells in NOG mice reconstituted with the human immune system.

Passive antibody administration shows strong potential as a new therapeutic method. In clinical applications, human-derived antibodies with antigen specificity are more useful without putting individuals at risk. Production of human-derived antibodies against given antigens can be obtained from animal models if the human immune system is established in the animals. In fact, past reports revealed that human T and B cells develop from hematopoietic progenitor cells in immunodeficient mice. However, there have been few reports on sufficient induction of antigen-specific antibodies, particularly IgG, in immunodeficient mice reconstituted with human immune cells. In this chapter, we discuss a major shortcoming of induction of antigen-specific IgG antibodies in human immune cells developed in the murine environment based on our data. We demonstrated that human T cell development is restricted by the murine MHC and consequently T cells may not achieve cognate interaction with human B cells. Human B cells developed in the mouse are mainly CD5+B1 cells that preferentially produce IgM. At the same time, human LN transplantation on the spleen enabled NOG mice to produce antigen-specific IgG antibody. These results suggest that if efficient cognate interaction mediated by a certain antigen on MHC class II between human T and B-2 cells occurs, human B cells can produce IgG antibody against a given antigen in the murine environment.

Morphology and function of ganglio-N-tetraosylceramide-positive lymphocyte mediators of natural killer activity.

More than 90% of nylon wool-passed spleen cells from nude mice reacted with ganglio-N-tetraosylceramide (asialo GM1) which eliminated mouse natural killer (NK) cell activity in the presence of complement. The successful enrichment of asialo GM1-positive (asialo GM1+) cells made possible the demonstration that the number of asialo GM1+ cells was correlated with NK cell activity as well as with binding frequency to YAC-1 target cells. Morphologically, by light and immunoelectron microscopic examinations, the enriched asialo GM1+ cells were composed of 90% lymphocytes and 10% large cells with the characteristics of monocytes. However, the asialo GM1+ lymphocytes (but not the monocytes) bound to the target cells and gave rise to the asialo GM1+ monocytes were sometimes observed to bind to target cells, cytolytic features of the target cells were not demonstrated. These results strongly suggest that asialo GM1+ lymphocytes are essential for NK cytolysis by directly binding to target cells.

Chemical composition of urinary calculi in Maiduguri, Nigeria.

Urolithiasis is a common disorder in Maiduguri and constitutes a significant proportion of surgical diseases in Nigerian Hospitals today. Although, the analysis of the stones is an integral part of the assessment of stone formers, earlier report in Maiduguri did not dwell well on it. We therefore, carry out this study to report on the composition of urinary tract calculi removed during surgery at the University of Maiduguri Teaching Hospital and to find out if stone composition in the area changes with time. Fourty-nine urinary tract calculi removed in the surgery unit of the Hospital in 2003 were chemically analyzed in the Department of Chemical Pathology of the same Hospital. We also retrieved results of stones analyzed in 1989 (41) and 1999 (21) and compared the results with the 49 analyzed in 2003. The results showed a male preponderance with male: female ratio 12:1, and the calculi occurred more in the upper part of the tract (70.9%) than the lower part of the tract (29.1%). Calcium containing stones constituted the majority; 76.9%, uric acid/urate was associated with 16.3% of the stones while struvite constitutes 4.3%, xanthine 1.7% and cystine 0.9%. There was subsequent reduction of struvite stones with time. Urinary tract stone is common in Maiduguri. There is need for identification of risk factors of calculi formation in the environment to enable the health care providers plan preventive measures in order to reduce the high incidence in the area.

Primary response of naive CD4(+) T cells to amino acid-substituted analogs of an antigenic peptide can show distinct activation patterns: Th1- and Th2-type cytokine secretion, and helper activity for antibody production without apparent cytokine secretion.

Naive CD4(+) T cells differentiate into two types of helper T cells showing an interferon-gamma-predominant (Th1) or an interleukin-4-predominant (Th2) cytokine secretion profile after repeated antigenic stimulation. Their differentiation can be influenced by slight differences in the interaction between the T cell receptor (TCR) and its ligand at the time of primary activation. However, the primary response of freshly isolated naive CD4(+) T cells to altered TCR ligands is still unclear. Here, we investigated the primary response of splenic naive CD4(+) T cells derived from transgenic mice expressing TCR specific for residues 323-339 of ovalbumin (OVA323-339) bound to I-A(d) molecules. Naive CD4(+) T cells secreted either Th1- or Th2-type cytokines immediately after stimulation with OVA323-339 or its single amino acid-substituted analogs. Helper activity for antibody secretion by co-cultured resting B cells was also found in the primary response, accompanied by either low-level Th2-type cytokine secretion or no apparent cytokine secretion. Our results clearly indicate that dichotomy of the Th1/Th2 cytokine secretion profile can be elicited upon primary activation of naive CD4(+) T cells. We also demonstrate that the helper activity of naive CD4(+) T cells for antibody production does not correspond to the amounts of the relevant cytokines secreted.

Establishment of a T cell-dependent nude mouse liver injury model induced by Propionibacterium acnes and LPS.

Normal ICR mice developed severe liver injury when they were given intravenous injections of Propionibacterium acnes and lipopolysaccharide (LPS) with a 7 day interval. In contrast, T cell-deficient ICR nude mice were resistant to P. acnes and LPS-induced liver injury. However, athymic ICR nude mice, which were treated with cell transfer of normal ICR mouse spleen cells (10(8) cells) or ICR mouse nylon-wool passed splenic T-enriched cells (over 10(7) cells), showed severe liver injury as assessed by elevation of serum transaminase activities. Histological analyses also demonstrated that the transferred cells migrated into the liver of nude mice to induce liver injury. However, depletion of both CD4+ T cells and CD8+ T cells from transferred cell populations caused a marked decrease in the elevation of serum transaminase, indicating the actual involvement of T cells in liver injury. Moreover, in vivo administration of anti-LFA-1 mAb blocked P. acnes and LPS-induced liver injury in nude mice following T cell transfer. Thus, this model will provide a new strategy to investigate T cell-dependent cell-cell interaction during the induction of liver damage.

Manipulation of Th1/Th2 balance in vivo by adoptive transfer of antigen-specific Th1 or Th2 cells.

We have investigated the possibility that the Th1/Th2 balance in vivo may be modulated by adoptive transfer of Th1 or Th2 cells induced in vitro. Thl cells were induced from I-Ad-binding OVA323-339-specific T-cell receptor-transgenic (TCR-Tg) mouse spleen cells by culturing with OVA323-339 peptide and antigen presenting cells (APC) in the presence of IL-2, IL-12 and anti-IL-4 mAb. Th2 cells were induced from TCR-Tg mouse spleen cells by culturing with IL-2, IL-4 and anti-IL-12 mAb in addition to OVA323-339 plus APC. Immunomodulating activities of both Th1 and Th2 cells were determined by their effect on delayed type hypersensitivity (DTH) responses or cytokine production. No significant DTH responses (footpad swelling) were observed in untreated BALB/c mice following a single injection of OVA323-339-pulsed syngeneic spleen cells. However, adoptive transfer of Th1 cells into BALB/c mice induced strong dose dependent DTH responses in response to I-Ad-bound OVA323-339 but not unrelated peptide. In contrast, only slight DTH responses were detected in BALB/c mice transferred with Th2 cells. In parallel with the DTH responses, increased levels of serum IFN-gamma were demonstrated in mice adoptively transferred with Th1, while no significant increase was observed in Th2-transferred mice. In vitro analysis also demonstrated that both spleen cells and popliteal lymph node cells prepared from Th1-transferred mice showed Th1-type cytokine production, while cells obtained from Th2-transferred mice revealed Th2-dominant cytokine production. Such immune deviation induced by antigen-specific Th1 cells was demonstrated up to three months after cell transfer. Therefore, it may be possible to manipulate the Th1/Th2 balance in vivo by adoptive transfer of antigen-specific Th1 or Th2 cells.

Interleukin-4-dependent induction of preproenkephalin in antigen-specific T helper-type 2 (Th2) cells.

Naive Th cells obtained from OVA(323-339)-specific DO11.10 TCR-Tg mice did not express preproenkephalin (PPE) mRNA. However, culture of naive Th cells with OVA(323-339) peptide (OVA-pep) plus IL-2 under Th2-inducing conditions for 7 days resulted in an induction of PPE mRNA. The PPE mRNA was also induced by culturing with OVA-pep plus IL-2 (neutral condition). However, PPE mRNA induction under neutral conditions was totally abrogated by addition of anti-IL-4 mAb. The existence of methionine-enkephalin was also demonstrated in peptidase-digested peptides derived from Th2 cell lysate. These results demonstrate that IL-4 is a critical factor for the induction of PPE mRNA in freshly expanded antigen-specific Th2 cells.

CD4+CD8+ cells lacking self-Mls reactive T cells are induced in mesenteric lymph nodes of Salmonella enteritidis-infected mice.

The percentage of mesenteric lymph node (MLN) cells that co-expressed both CD4 and CD8 was found to be from 5 to 7% in BALB/c and AKR/N mice bred under conventional conditions. In mice maintained under specific pathogen-free (SPF) conditions, the percentage fell below 2%. When mice were infected with an attenuated strain of Salmonella enteritidis (SER), the percentage of CD4+CD8+ cells in MLN rose to 20-30% transiently. In these mice, the total cell number and the percentage of CD8+ cells were not changed, but the CD4+ cell percentage was decreased. The expression intensity of TCR-alpha beta on CD4+CD8+ cells in the infected mice was higher in the MLN than in the thymus, but was similar to that of mature peripheral T cells. Among the CD4+CD8+ cell population in MLN, TCR-V beta 3+ cells were deleted but V beta 6+ cells were present in BALB/c mice which possess endogenous superantigen Mls-2a, but lack Mls-la. In AKR mice with the inverse of the occurrence of the superantigens, TCR-V beta 3+ cells were present and V beta 6+ cells were absent. These data suggest that CD4+CD8+ cells in the MLN of SER-infected mice may belong to thymus-derived mature T cells undergoing negative selection and that they may appear following exogenous stimulation.

Characterization of the T cells in aged rat bone marrow.

In this study, we determined the characteristics of CD3-positive (CD3+) T cells existing in rat bone marrow (BM). In contrast to splenic T cells, BM CD3+ T cells are composed of a higher proportion of CD8+ T cells, and the number of both cell types increased with age. Such CD3+ T cells in aged rats showed a similar usage of TCR V beta as splenic T cells, suggesting that BM CD3+ T cells are thymus-dependent and composed of an ordinary population in view of the expression of the TCR beta-chain. Purified T cells obtained from aged rat BM showed a markedly proliferative response by stimulation with immobilized anti-CD3 mAb, as did splenic T cells. However, the addition of BM non-T cells completely inhibited the response of both BM and splenic T cells in vitro. These results suggest that T cells in rat BM are negatively regulated by BM non-T cells in their response to the TCR-mediated signal not to disrupt the microenvironment of the BM.

Beta-selection of immature thymocytes is less dependent on CD45 tyrosinephosphatase.

Tyrosine kinase p56lck plays a pivotal role in beta-selection from CD4-8- (DN) to CD4+8+ (DP) developing pathway, but it is unclear how CD45 transmembrane tyrosinephosphatase is involved in this process although CD45 activates p56lck by dephosphorylating its tyrosine-505. To analyze this issue, we produced double mutant mice of T-cell receptor transgenic mice (TCR-Tg) or RAG-2 knock out mice backcrossed with either p56lck or CD45 knock out mice. In TCR-Tg, CD25+DN thymocytes almost disappeared and CD25-44-DN cells of further developing stage increased, implying that all DN thymocytes can undergo beta-selection due to the expression of functionally rearranged TCR-beta on CD25+ DN thymocytes. However, CD25+ thymocytes increased in DN stage when TCR-Tg were backcrossed with p56lck deficient mice but not with CD45 deficient mice. Similarly, DP thymocyte induction with CD25+ cell reduction in RAG-2 knock out mice by injection of anti-CD3 mAb was inhibited in p56lck deficient but not in CD45 deficient mice. This suggests that CD45 is dispensable for beta-selection though p56lck is required.

Thymic nurse cell clone supports the differentiation of CD4-8- thymocytes into CD4+8+ thymocytes in vitro.

A previously reported thymic nurse cell clone, TNC-R3.1 could form a unique complex with isolated adult mouse CD4-8- (DN) thymocytes and greatly sustained the cell viability of DN thymocytes in suspension culture. In addition, the TNC-R3.1 clone supported the differentiation of DN thymocytes into CD4+8+ (DP) thymocytes in a short-term culture. Addition of IL-7 into the coculture markedly enhanced DN thymocyte-TNC interaction and induced the proliferation and differentiation of DN thymocytes, though IL-7 alone did not induce the differentiation of DN thymocytes. Separation of DN thymocytes from TNC-R3.1 monolayer using a Millicell caused a great inhibition of the DN thymocyte differentiation, suggesting that direct contact between TNC-R3.1 cells and immature thymocytes was required for the differentiation of DN thymocytes. The kinetics study demonstrated that DN thymocytes started to differentiate into DP thymocytes through CD3-CD4+J11d+ intermediate cells 8-12 h after the initiation of the culture with TNC-R3.1 plus IL-7. The generation of DP thymocytes became maximal 20 h after coculture and gradually decreased thereafter. Furthermore, we demonstrated that TNC-R3.1 could support the differentiation of CD3+CD4+CD8- or CD3+CD4-CD8+ thymocytes from CD3-CD4-CD8- thymocytes in the presence of IL-7 and IL-2. These data indicate that our established in vitro culture system mimics the early stage of the intrathymic T cell developing pathway.

Natural cytotoxic T cells responsible for anti-CD3-induced cytotoxicity in mice.

T cells obtained from normal mouse spleen cells showed significant cytotoxic activity against Fc receptor positive tumor cells in the presence of anti-CD3 monoclonal antibody (mAb). This activity was designated as natural cytotoxic T cell (NCT) activity and compared with natural killer (NK) activity. Considerable levels of NCT activity were detected in mouse strains with both high and low NK activity. NCT cells were distributed in both lower and higher density fractions of Percoll discontinuous density gradients, while NK cells were enriched in the lower density fraction of Percoll gradients. Moreover, NCT activity was resistant to in vivo anti-asialo GM1 treatment, in contrast to NK cells. These results indicate that NCT cells, which have different characteristics from NK cells, are present in normal, nonimmunized mouse spleen cells. Unexpectedly, CD4+ T cells sorted from normal mouse spleen T cells revealed significant NCT activity, as did CD8+ T cells. It was also demonstrated that NCT cells require the LFA-1 molecule to lyse tumor cells in the presence of anti-CD3 mAb.

Tumor-associated glycoantigen, sialyl Lewis(a) as a target for bispecific antibody-directed adoptive tumor immunotherapy.

The KM231 mAb recognizing sialyl Lewis(a) (sLe(a)) epitope of glycoprotein or glycolipid expressed on various human cancers was used to prepare bispecific antibody (BSAb) containing anti-CD3 x anti-sLe(a) mAb. The effect of anti-CD3 x anti-sLe(a) BSAb on the induction of cytotoxicity by activated T cells was investigated. The activated CD3+ T cells expressing CD8 or CD4 were induced from human peripheral blood mononuclear cells by culture with recombinant IL-2 plus immobilized anti-CD3 mAb. The activated CD8+ and CD4+ T cells showed marginal cytotoxicity against tumor cells by themselves. However, addition of anti-CD3 x anti-sLe(a) BSAb resulted in a great augmentation of their cytotoxicity against gastrointestinal tumor cells. The BSAb also triggered IL-2 production of CD4+ helper/killer T cells during lysis of tumor cells. Moreover, the BSAb was demonstrated to have a potent in vivo antitumor activity against human colon cancer implanted in nude mice by combination with CD4+ helper/killer cells. These results demonstrated that sLe(a) antigen might be a good target molecule for BSAb-directed adoptive tumor immunotherapy.

Phenotypic and functional characteristics of in vivo-induced interleukin-12-activated killer cells.

A single i.p. administration of IL-12 (2000 U/mouse) into the mice caused the elevation of serum IFN-gamma activity and the generation of killer cells which can lyse various kinds of tumor cells including both NK-sensitive and -resistant tumor cells. Such in vivo induced killer cells were not detected in the mice treated with the same dose of IL-2. The generation of IL-12-activated killer cells (IL-12AK) peaked at day 1 and sustained their cytotoxicity until day 3 after IL-12 administration. The generation of IL-12AK was inhibited by in vivo administration of anti-asialo GM1 (ASGM1) Ab but not anti-CD4 or anti-CD8 mAbs, suggesting that the precursor cells for IL-12AK were ASGM1+CD4-CD8- NK cells. The phenotypic characterization of in vivo induced effector cells with IL-12AK activity was carried out by separating the cells with FACStar. The IL-12AK activity was highly enriched in ASGM1+CD4-8- or NK1.1+CD4-8- NK cells, but not in CD8+ T cells and CD4+ T cells. The IL-12AK cells were also generated in tumor-inoculated mice. In parallel with the in vivo generation of IL-12AK generation, the growth of i.p. inoculated MBL-2 lymphoma cells was markedly inhibited by the administration with IL-12. The in vivo antitumor activity of IL-12 was blocked by the administration of anti-ASGM1 but not anti-CD4 or anti-CD8 mAbs in concomitant with the decrease of IL-12AK generation. From these results, it was indicated that ASGM1+NK1.1+CD4-8- NK type IL-12AK cells might play an important role in IL-12-induced local therapy of tumor in vivo.

The restraint stress drives a shift in Th1/Th2 balance toward Th2-dominant immunity in mice.

When mice were physically restrained in 50-ml tubes for 24 h, a marked decrease of NK activity was demonstrated in parallel with the elevation of serum corticosterone levels. The release of mice from restraint stress resulted in the recovery of NK activity, with a decrease of serum corticosterone levels within 48 h. Using this stress model, we also investigated the influence of restraint stress on mouse Th1/Th2 balance. Consistent with the decrease of NK activity, IFN-gamma production of mouse spleen cells greatly reduced after suffering from restraint stress. In contrast, the IL-4 producing ability of spleen cells was not so much affected by restraint stress. These results initially indicated that stress may induce the skewing of the Th1/Th2 balance toward Th2-dominant immunity, which stimulates the occurrence of infectious diseases and allergic disorders.

Functional characterization of NK1.1 + Ly-6C+ cells.

It was found that NK1.1+ cells were subdivided by their different expression pattern of Ly-6C antigen. To characterize their functional significance in immunoregulation, we separated NK1.1 + Ly6C+ cells and NK1.1 + Ly-6C- cells from C57BL/6 mouse nylon-passed spleen cells by FACStar. Both NK1.1 + Ly-6C+ and NK1.1 + Ly-6C- cells responded to the stimulation with IL-2 plus IL-12 and showed strong cytotoxicity against YAC-1 cells. However, these cells revealed different ability in terms of IFN-gamma production. Only NK1.1 + Ly-6C+ cells, but not NK1.1 + Ly-6C- cells, cultured with IL-12 alone or IL-2 plus IL-12, produced high levels of IFN-gamma. Flow cytometric analysis demonstrated that NK1.1 + Ly-6C+ cells consisted of NK1.1 + CD3-Ly-6C+ NK cells and NK1.1 + CD3 + Ly-6C+ NKT cells. Therefore, we further separated these two populations from NK1.1 + Ly-6C+ cells to define their functions. Although, both NK1.1 + CD3-Ly-6C+ NK cells and NK1.1 + CD3+ NKT cells showed the same level of cytotoxicity. It was clearly demonstrated that NK1.1 + CD3+Ly-6C+ NKT cells were major immunoregulatory cells to produce IFN-gamma in respond to IL-12 alone or IL-2 plus IL-12.