Correlation between natural killer cell activation in the bone marrow and haemopoietic dysfunction following cytomegalovirus infection of mice.

We describe here the activation of natural killer (NK) cells in the bone marrows and spleens of mice infected with murine cytomegalovirus (MCMV). NK activity at these sites peaked at day 2 to 3 post-infection (p.i.) and declined between days 6 and 10 p.i. in BALB/c and C57BL/6 mice. In BALB/c mice, the increases in NK activity coincided with depletion of colony-forming units of the granulocyte-monocyte lineage (CFU-GM) from the marrow. CFU-GM depletion in MCMV-infected C57BL/6 mice was less severe, despite the presence of activated NK cells in the marrow. Treatment of BALB/c mice with anti-asialo GM1 prior to MCMV infection resulted in less severe CFU-GM depletion at day 2 p.i. than infection with MCMV alone. When homozygous C57BL/6 or CBA/CaH bg/bg mice were infected with MCMV, depletion of marrow CFU-GM was more severe than in their heterozygous littermates. Finally, we observed some inhibition of colony formation when marrow cells from MCMV-infected and uninfected BALB/c donors were mixed and incubated prior to the CFU-GM assay. These results suggest that activated NK cells may contribute to depletion of haemopoietic cells soon after MCMV infection of BALB/c mice, but may limit the loss of these cells in C57BL/6 and CBA/CaH mice.     

The Genetic Background Modulates the Effect of the Beige Gene on Susceptibility to Cytomegalovirus Infection in Mice

Homozygous beige (bg/bg) mice were more susceptible to the development of fatal disease induced by murine cytomegalovirus (MCMV) than their bg/ + littermates. However, the increase in susceptibility depended on the genetic background of the strain carrying the bg gene. C57BL/6, SB/Le, DBA/2, and CBA bg/bg mice showed, respectively, 2.5-, 3.2-, 9.5-, and 18.6-fold increases in susceptibility compared with the corresponding bg/+ animals. Beige mice showed higher liver titres of MCMV than bg/ + by the 2nd or 3rd day after infection, and tissue damage was also greater. Splenic NK cells were not detected in uninfected bg/bg mice, and after virus inoculation the increment in cytotoxicity was greater in bg/ + than in bg/bg mice. However, cytotoxicity towards WEHI-164 cells was not impaired in bg/bg mice and was not augmented by MCMV. Interferon titres were also not impaired by the beige mutation. Of the strains examined, CBA had the highest endogenous levels of NK cells and were most genetically resistant to MCMV. Thus, our observation that the beige gene had the greatest effect on susceptibility in this strain suggests that NK cells are important mediators of genetically determined resistance to MCMV.     

Effect of a retroviral immunodeficiency syndrome on murine cytomegalovirus-induced hepatitis.

We have studied the effects of LP-BM5 MuLV-induced murine acquired immunodeficiency syndrome (MAIDS) on concomitant murine cytomegalovirus (MCMV) infection in the livers of C57BL mice. A delayed inflammatory response in livers of mice with MAIDS (M+) on day 4 was associated with impaired clearance of MCMV-infected cells 6 days after infection. This correlated with increased levels of inflammation and serum alanine transaminase. The latter reflects enhanced hepatic necrosis, which was evident histologically. Delayed-type hypersensitivity responses to MCMV antigen were unimpaired in M+ mice and were mediated by CD8+ cells. Depletion of NK1.1+ cells from M+ mice increased MCMV replication and associated liver damage on day 6, whereas CD8+ depletion had little effect. In contrast, in the presence of CD8+ cells M- C57BL mice did not require NK1.1+ cells for control of hepatic MCMV infection, but dual NK1.1+ and CD8+ depletion dramatically potentiated hepatic MCMV replication. Our results suggest that M+ mice may acquire non-NK1.1+ and non-CD8+ cells that are able to partially control hepatic MCMV infection. These findings are discussed with reference to mortality in M+ mice after high-dose MCMV infection, as this is initially delayed but ultimately higher than in M- controls.     

The association between murine cytomegalovirus induced hepatitis and the accummulation of oval cells

The accumulation of oval cells is an early event in the development of hepatocellular carcinoma induced by certain experimental regimes involving hepatocarcinogens. Oval cells have also been observed during chronic hepatitis induced by alcohol and iron overload. In this study, livers of murine cytomegalovirus (MCMV) infected mice were examined to determine whether hepatitis induced by this virus could initiate oval cell proliferation. BALB/c and C57BL/6 mice were infected with MCMV and studied 4, 8, 10 and 12 months later, alongside control (uninfected) mice. The livers were examined histochemically, immunocytochemically and by in situ hybridization to identify oval cells, inflammatory cells and proliferating cells. Oval cells were seen in the periportal regions of livers from MCMV infected BALB/c mice. These increased in number from 4 to 12 months after infection in parallel with increases in the numbers of inflammatory cells, even though cells expressing MCMV antigens were no longer evident in these samples. Proliferating oval cells and hepatocytes were identified by PCNA staining, indicating an increased level of liver regeneration in the infected livers. C57BL/6 mice are less susceptible to persistent MCMV hepatitis and had fewer oval cells than BALB/c mice. Thus the study demonstrates an association between MCMV induced hepatitis, inflammation, and presence of oval cells.     

Cytotoxic effects of natural killer cells have no significant role in controlling infection with the intracellular protozoon Eimeria vermiformis.

The course of infection with Eimeria vermiformis in C57BL/6J; NK cell-defective C57BL/6J bg/bg; BALB/c; T-cell-defective BALB/c nu/nu; and T-cell-, B-cell-, and NK cell-defective BALB/c x C57BL/6 scid/scid bg/bg mice was monitored. For young C57BL/6J mice, the bg/bg mutants consistently produced fewer oocysts than the controls; there were no differences between older mice of these strains. Wild-type BALB/c mice were more resistant to infection than the nu/nu and scid/scid bg/bg mutants, but there was no difference between the mutants. Treatment of BALB/c mice with poly(I.C) had no effect on the course of infection. These findings confirm the ineffectiveness of NK cells in this system.     

Contrasting phenotypes of liver-infiltrating leucocytes isolated from MCMV-infected BALB/c and C57 BL/6 mice

We characterized liver-infiltrating leucocytes (LIL) from BALB/c and C57BL/6 mice 0-56 days after murine cytomegalovirus (MCMV) infection. Inflammation clears from C57BL/6 mice 4-5 weeks post infection (pi), but persists for several months in BALB/c mice. The LIL obtained were 60-80% Thy 1.2+ by flow cytometry. The percentage of CD8+ cells rose sharply in all mice 7 days pi, with little decrease in BALB/c mice by day 56. CD4-CD8-Thy 1.2+/TCR alpha beta + cells were more prevalent in LIL than lymph node cells (LNC) irrespective of MCMV infection, whilst infection increased the proportion of CD8+ L-selectin- LIL (but not LNC). LIL from both mouse strains demonstrated cytotoxic activity against YAC-1 cells, but only LIL from BALB/c mice proliferated spontaneously ex vivo 21 days pi, as measured by tritiated thymidine incorporation. BALB/c LIL produced IFN gamma and IgG2a 7-21 days pi, whilst IL-10 secretion was similar in both strains. Thus, persistent hepatitis in BALB/c mice is associated with activation and proliferation of intrahepatic leucocytes with some bias towards a Th1 response.     

Quantitative measurement of infectious murine cytomegalovirus genomes in real-time PCR

Acute virus replication in murine (M) CMV infected C57BL/6 (Cmv1(r)) mice is severely limited by Ly49H+ NK cells, but not in MCMV infected BALB/c or BXD8 (Cmv1(s)) mice that lack Ly49H+ NK cells. Interestingly, other NK cell receptors may also play a role in MCMV immunity, as CMV encoded gp40 protein can diminish expression of protein ligands recognized by the NK cell receptor NKG2D. To determine whether other additional gene products might influence MCMV immunity, we designed an efficient, sensitive and reliable method for screening resistance or susceptibility phenotypes in mice. Although multiple methods are frequently used to detect and quantify infectious MCMV in mouse tissue samples collected during acute viral infection, these are not readily adaptable to high-throughput screening strategies. Hence, we utilized real-time PCR for detection and quantitative measurement of infectious MCMV genomes present in various tissues of infected mice. MCMV genomic sequence was accurately and reproducibly detected over the range 10(2)-10(8) molecules in mouse genomic DNA samples using this methodology. Importantly, it was found that quantitative real-time PCR and viral plaque assay measurements of MCMV in tissues collected from infected mice, including resistant and susceptible strains, were directly correlated. Moreover, quantitative real-time PCR results obtained during a 3-week time-course study of virus replication in spleens, livers and salivary glands of infected mice demonstrated sensitive, accurate and reproducible detection and measurement of infectious MCMV.     

Strain differences in mouse cellular responses to Mycobacterium lepraemurium and BCG subcutaneous infections. I. Analysis of cell surface phenotype in local granulomas.

C57BL/6, BALB/c and CBA mice were subcutaneously infected with either Mycobacterium lepraemurium (MLM) or BCG, and studied for bacillary growth, granuloma size of infected footpads and draining lymph nodes (DLN), and DLN cell surface phenotype. Whereas, BCG-infected mice controlled the infection and developed early and large granulomas, MLM-infected mice exhibited major strain variations in their resistance to the infection, as well as in the granuloma size and kinetics. C57BL/6 mice, highly resistant, displayed early and regressive granulomas; BALB/c mice showed lower resistance and early granulomas that grew continuously; CBA mice, highly susceptible, developed late, soft, phagocyte-rich granulomas. Important strain differences in lymph node lymphocyte subset distribution could be observed prior to any infection: C57BL/6 mice displayed higher B cell percentages than both of the other strains and BALB/c mice showed the highest CD4/CD8 ratios, followed by CBA and C57BL/6 mice. BCG and MLM infections both induced similar changes of these parameters in all three strains: that is a decrease of the B cell percentage and a decrease of the CD4/CD8 ratio, and the strain differences observed in uninfected mice persisted. On the other hand, DLN cells stimulated by the infecting bacillus and interleukin 2 also displayed an increase of the CD8 T cell percentage as compared with normal lymph node cells, but this phenomenon was much less pronounced in BALB/c mice, whether infected by MLM or BCG, and in MLM-infected CBA mice, than in BCG- or MLM-infected C57BL/6 (B6) mice. Thus the ability of C57BL/6 mice to generate an early and persistent CD8 T cell response to mycobacteria may contribute to their resistance to MLM.     

Bone marrow colony-formation in vitro after infection of genetically defined inbred mice with Candida albicans

The effect of C. albicans infection on the production of haematopoietic precursor cells in the bone marrow of CBA/CaH and BALB/c mice was evaluated by assay of colony formation in vitro. In immunocompetent mice, neither systemic nor oral infection induced significant alterations in colony formation by bone marrow from the two mouse strains, and Candida infection did not alter the proportion of morphological cell types in the colonies. However, the number of neutrophil-like was relatively greater in colonies derived from acutely infected CBA/CaH nude mice than in those from BALB/c nude mice, whereas small mononuclear cells were present in higher proportions in the latter strain. In both strains of nude mice, there was an increase in colony formation at 6 days after oral infection, but at 8 weeks, when the infection had become chronic, the production of bone marrow cells by CBA/CaH nude mice was significantly less than that by BALB/c nude mice. Reconstitution of nude mice with syngeneic lymphocytes enhanced the production of bone marrow precursor cells by BALB/c, but not by CBA/CaH mice, suggesting that T cells can enhance host resistance by promoting the colony-forming response of the bone marrow in BALB/c mice that are genetically resistant to tissue damage, but not in CBA/CaH that are prone to severe lesions. Finally, culture with Candida antigen in vitro decreased the number of colony-forming cells in cultures from CBA/CaH, but not from BALB/c mice.     

Role of Macrophages in Resistance to Murine Cytomegalovirus

The role of macrophages in protecting mice from murine cytomegalovirus (MCMV) was studied in Swiss, CBA/J, and C57BL/6J mice. CBA/J mice were more resistant to virus than were C57BL/6J mice at all ages tested. Prior treatment of adult Swiss mice with 60 mg of silica, a dose selectively toxic to macrophages, increased mortality due to MCMV infection. Transfer of syngeneic adult macrophages to suckling mice significantly increased their resistance to subsequent MCMV infection. Transfer of syngeneic, nonimmune adult lymphocytes to suckling mice also had a lesser but significant protective effect against subsequent MCMV challenge. In vitro infection of adult CBA/J and C57BL/6J macrophages with virulent and attenuated MCMV resulted in productive infection in only a small percentage of cells and recovery of very little virus from the extracellular fluid. Infection of CBA macrophages was no less productive than C57BL/6J nor was infection with virulent virus more productive than with attenuated virus. Histological examination of the livers of MCMV-infected CBA/J and C57BL/6J mice suggested that divergent cellular immune responses to infection might account for differences in susceptibility. It is postulated that the macrophage may facilitate the inductive phase of cellular immunity, one possible explanation for its demonstrated importance in host defenses against MCMV.     

IL-21R expression on CD8+ T cells promotes CD8+ T cell activation in coxsackievirus B3 induced myocarditis

IL-21 is a multi-functional cytokine which can promote survival, proliferation and activation of T and B lymphocytes including CD8 T cells. Previous studies have shown that autoimmune CD8+ T cells are the primary pathogenic effector cell in coxsackievirus B3 (CVB3) induced myocarditis in C57Bl/6 mice. To evaluate the role of IL-21 in promoting CD8+ T cell mediated cardiac injury in myocarditis, C57Bl/6 and IL-21RKO mice were infected with CVB3. IL-21RKO mice developed significantly less myocarditis than C57Bl/6 animals although cardiac virus titers were equivalent between the mouse strains. Numbers of CD8+IFNγ+ cells were decreased in IL-21RKO mice but numbers of either CD4+IFNγ+ or CD4+IL-4+ cells were not significantly different from C57Bl/6 animals indicating a selective effect of IL-21 signaling on the CD8+ T cell response. To confirm that IL-21 signaling exclusively functions at the level of the CD8+ T cell in CVB3 induced myocarditis, purified CD8+ cells were isolated from either C57Bl/6 or IL-21RKO donors and adoptively transferred into CD8KO recipients prior to CVB3 infection. CD8KO recipients given either C57Bl/6 or IL-21RKO CD8+ cells showed equivalent reconstitution of the CD8+ cells in the spleen but the recipients given C57Bl/6 CD8+ cells showed significantly greater myocarditis than recipients of IL-21RKO CD8+ cells. These data demonstrate that IL-21 signaling directly in the CD8+ cell population is required for CVB3-induced myocarditis.     

Experimental histoplasmosis in the beige mouse

Mice carrying the beige mutation (bg/bg) on a C57Bl/6 background were challenged with Histoplasma capsulatum. bg/bg mice had higher mortality and higher lung tissue fungal counts in their lungs than either bg/+ or C57Bl/6 mice challenged with equal inocula. Immunologic studies showed that bg/bg mice developed normal delayed-type hypersensitivity (DTH) reactions to histoplasmin, but had deficient NK cell cytotoxic activity against YAC-1 target cells. Studies of macrophage killing of H. capsulatum in vitro showed that T lymphocytes of either bg/+ or bg/bg mice were able to activate fungal killing by bg/+ but not by bg/bg macrophages. These studies, while not excluding a role for the NK cell, suggest that macrophage dysfunction may be critical in the greater susceptibility of the bg/bg mouse and, by extension, that macrophage function is of major importance in host defense against H. capsulatum.     

Endothelial cells and renal epithelial cells do not express the Wegener''s autoantigen, proteinase 3.

In vitro and in vivo T cell responses were determined during the course of bronchopulmonary infection with mucoid Pseudomonas aeruginosa. T cell responses were compared in two inbred mouse strains, namely BALB/c mice, which are resistant to the establishment of chronic bronchopulmonary Ps. aeruginosa infection, and C57Bl/6 mice, which have high numbers of bacteria in the lungs through 14 days post-infection. Unseparated lung cells and lung T cells from BALB/c mice exhibited significantly higher in vitro proliferative responses to both heat-killed Ps. aeruginosa and concanavalin A (Con A) than cells from C57Bl/6 mice through 20 days post-intratracheal infection with 10(4) colony-forming units (CFU) Ps. aeruginosa. Proliferation of unseparated lung cells but not lung T cells from BALB/c mice infected 6 days previously with 10(5) CFU Ps. aeruginosa was suppressed in response to Con A; these cells were unresponsive to specific antigen. Suppression of lymphocyte proliferation in the lungs of C57Bl/6 mice infected with 10(4) CFU Ps. aeruginosa and in BALB/c mice infected with 10(5) CFU was found to be mediated by adherent lung cells via the production of nitric oxide and prostaglandins. Determination of in vivo T cell-mediated responses in infected mice demonstrated that resistant BALB/c mice had high DTH and low Pseudomonas-specific antibody responses, while C57Bl/6 mice had low DTH and high antibody levels, in particular, IgG2b and IgM.     

免疫治疗对母胎交界面NK细胞表面CD69表达水平的影响及其与鼠胚和鼠仔预后的关系

目的:研究反复自然流产(recurrent spontaneous abortion,RSA)模型CBA/J×DBA/2小鼠母胎交界面NK细胞表面CD69分子的表达水平,并评价淋巴细胞免疫治疗(lymphocyteimmunotherapy,LIT)对CD69表达水平的影响及其与鼠胚和鼠仔预后的关系。方法:评价CBA/J×DBA/2、C57BL/6×DBA/2和BALB/c×DBA/2小鼠胚胎和鼠仔的预后,绘制出生后1-21 d鼠仔离乳前生长曲线和Kaplan-Meier生存分析曲线。并且采用PE-CD69和FITC-DX5双色流式细胞术检测在有或无LIT的情况下,母胎交界面NK细胞表面CD69分子的表达水平。此外,对CD16/CD32⁺NK细胞亚群等也进行检测。结果:在孕期母鼠体重平均增长幅度、平均每窝产仔数、新生鼠仔出生第1 d平均体重和胚胎吸收率等方面,CBA/J×DBA/2小鼠与生殖力正常的对照组相比均有显著差异。在CBA/J×DBA/2小鼠中,代表活化型NK细胞的CD69⁺DX5⁺细胞比率也显著高于对照组。而有效的LIT能够显著降低CD69在母胎交界面NK细胞表面的表达水平,并相应地显著降低胚胎吸收率。结论:CD69⁺DX5⁺细胞在胚胎被排斥的机制中可能具有重要作用。有效的LIT通过降低CD69分子在母胎交界面NK细胞表面的表达水平而显著降低流产率。     

T Cells Augment Monocyte and Neutrophil Function in Host Resistance against Oropharyngeal Candidiasis

The purpose of this study was to identify the cell populations involved in recovery from oral infections with Candida albicans. Monoclonal antibodies specific for CD4+ cells, CD8+ cells, and polymorphonuclear leukocytes were used to deplete BALB/c and CBA/CaH mice of the relevant cell populations in systemic circulation. Monocytes were inactivated with the cytotoxic chemical carrageenan. Mice were infected with 10(8) C. albicans yeast cells and monitored for 21 days. Systemic depletion of CD4+ and CD8+ T lymphocytes alone did not increase the severity of oral infection compared to that of controls. Oral colonization persisted in animals treated with head and neck irradiation and depleted of CD4+ T cells, whereas infections in animals that received head and neck irradiation alone or irradiation and anti-CD8 antibody cleared the infection in a comparable fashion. The depletion of polymorphonuclear cells and the cytotoxic inactivation of mononuclear phagocytes significantly increased the severity of oral infection in both BALB/c and CBA/CaH mice. High levels of interleukin 12 (IL-12) and gamma interferon (IFN-gamma) were produced by lymphocytes from the draining lymph nodes of recovering animals, whereas IL-6, tumor necrosis factor alpha, and IFN-gamma were detected in the oral mucosae of both na?ve and infected mice. The results indicate that recovery from oropharyngeal candidiasis in this model is dependent on CD4+-T-cell augmentation of monocyte and neutrophil functions exerted by Th1-type cytokines such as IL-12 and IFN-gamma.     

C57BL/6小鼠不同组织器官中NKT细胞的比较

目的比较C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结中NKT细胞的含量、亚型和功能的特点。方法分离正常C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结的淋巴细胞,利用细胞表面分子染色的方法,观察不同组织器官中CD3+NK1.1+NKT细胞及其亚型的含量;淋巴细胞经过PMA和离子霉素刺激后,应用细胞内细胞因子染色的方法,通过流式细胞仪观察NKT细胞IFN-γ、IL-4、IL-9和IL-17的产生情况。结果肝脏中NKT细胞的含量为(25.2±12)%,显著高于肺脏、脾脏和肠系膜淋巴结。肝脏、脾脏和肠系膜淋巴结中NKT细胞以CD4+细胞亚群为主,而肺脏中NKT细胞以CD4-CD8-亚群为主,同时肠系膜淋巴结的NKT细胞中存在CD4+CD8+亚群。不同组织器官中NKT细胞IFN-γ、IL-4、IL-9和IL-17产生的能力有差别。结论 C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结的NKT细胞在含量、表型和功能方面可能存在明显的差异。     

Immune reactions in different mouse strains

The responses of immunocompetent cells to thymus-dependent antigen differ in mice of different strains. Immunization stimulated phagocytic activity of peritoneal macrophages in CBA/CaLac, DBA/2, and BALB/c mice and suppressed it in CC57W mice. By the formation of antibody-producing cells in the spleen in response to thymus-dependent antigen DBA/2 and CBA/CaLac mice can be classified as high responders, BALB/c mice as medium-responders, and C57Bl/6 and CC57W mice as low responders.     

A flow cytometry-based method for detecting antibody responses to murine cytomegalovirus infection

An assay based on target cells infected with green fluorescent protein labeled murine cytomegalovirus (GFP-MCMV) and dual color flow cytometry for detecting antibody to MCMV is described. After optimizing conditions for this technique, kinetics of anti-MCMV IgG antibody response was tested in susceptible (BALB/c) and resistant (C57BL/6) mouse strains following primary MCMV infection. Previously published antibody kinssetics were confirmed in susceptible mice, with peak IgG response seen approximately 8 weeks after primary infection, decreasing by 20 weeks after infection. In contrast, MCMV resistant C57BL/6 mice showed significantly lower IgG antibody responses than susceptible mice. Although several techniques have been previously described to detect murine antibody responses to MCMV, including nuclear anti-complement immunofluorescence, viral immunoblotting, complement fixation, indirect immunofluorescence, indirect hemagglutination, and enzyme-liked immunosorbent assay techniques, these techniques are all time consuming and laborious. The technique presented is a simple time efficient alternative to detect previous MCMV antibody responses in experimentally infected mice.     

Experimental African trypanosomiasis: differences in cytokine and nitric oxide production by macrophages from resistant and susceptible mice.

Immunosuppression in experimental infections with Trypanosoma congolense is mediated by the synergistic action of macrophages and a novel lymphocyte(s), which involves the activity of IFN-gamma as well as IL-10. BALB/c mice are highly susceptible while C57Bl/6 mice are relatively resistant to T. congolense infections. Plasma and/or supernatants of spleen cell cultures of infected susceptible BALB/c mice have more IL-10 but less IL-12 than those of infected relatively resistant C57Bl/6 mice. Cells of a BALB/c macrophage cell line, when pulsed with T. congolense, produce more IL-10 and IL-6, but have less TNF-alpha mRNA, than equally treated cells of a C57Bl/6 cell line. Peritoneal and/or bone marrow-derived macrophages obtained from BALB/c mice, pulsed with T. congolense in culture, produce less nitric oxide, TNF-alpha and IL-12, but more IL-6 and IL-10 than equally treated macrophages isolated from C57Bl/6 mice. We suggest that genetic resistance to African trypanosomiasis is expressed at the level of the macrophage.     

Characterization of CD4- CD8- CD3+ T-cell receptor-alphabeta+ T cells in murine cytomegalovirus infection

In this study, we have investigated that after the intraperitoneal infection with murine cytomegalovirus (MCMV), the CD3+ CD4- CD8-(double negative; DN) T-cell receptor (TCR)alphabeta+ T cells increased in peritoneal cavity, liver and spleen in both resistant C57BL/6 and susceptible BALB/c mice. The total cellular population of these cells showed peak levels around day 5 after infection in all the three investigated organs and the following phenotypical and functional characteristics emerged. The peritoneal DN TCRalphabeta+ T cells expressed highly skewed TCRVbeta8 on day 5 after infection compared with the uninfected mice, but those in spleen and liver showed moderate and low skewed TCRVbeta8, respectively. The percentages of NK1.1+ DN TCRalphabeta+ T cells gradually decreased as did modulation of some of their activation markers consistent with an activated cell phenotype. The peritoneal DN TCRalphabeta+ T cells on day 5 after infection expressed the genes of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha, Eta-1 (early T-cell activation-1) and MCP-1 (monocyte chemoattractant protein 1) but lacked expression of interleukin-4 (IL-4). After in vitro stimulation with phorbol 12-myristate 13-acetate and calcium ionophore in the presence of Brefeldin A, higher frequencies of intracellular IFN-gamma+ DN TCRalphabeta+ T cells were detected in all three investigated organs of infected mice compared with those of uninfected mice. Stimulation of peritoneal DN TCRalphabeta+ T cells with plate-bound anti-TCRbeta monoclonal antibodies showed proliferation and also produced IFN-gamma but not IL-4. These results suggest that DN TCRalphabeta+ T cells were activated and may have an antiviral effect through producing IFN-gamma and some macrophage-activating factors during an early phase of MCMV infection.