Protective effect of black seed oil from Nigella sativa against murine cytomegalovirus infection

In this study, antiviral effect of black seed oil (BSO) from Nigella sativa was investigated using murine cytomegalovirus (MCMV) as a model. The viral load and innate immunity mediated by NK cells and Mφ during early stage of the infection were analyzed. Intraperitoneal (i.p.) administration of BSO to BALB/c mice, a susceptible strain of MCMV infection, strikingly inhibited the virus titers in spleen and liver on day 3 of infection with 1x10(5) PFU MCMV. This effect coincided with an increase in serum level of IFN-gamma. Although BSO treatment decreased both number and cytolytic function of NK cells on day 3 of infection, it increased numbers of Mφ and CD4(+) T cells. On day 10 of infection, the virus titer was undetectable in spleen and liver of BSO-treated mice, while it was detectable in control mice. Although spleen of both control and BSO-treated mice showed similar CTL activities on day 10 after infection, serum level of IFN-gamma in BSO-treated mice was higher. Furthermore, BSO treatment upregulated suppressor function of Mφ in spleen. These results show that BSO exhibited a striking antiviral effect against MCMV infection which may be mediated by increasing of Mφ number and function, and IFN-gamma production.     

The specific NK cell response in concert with perforin prevents CD8<Superscript>+</Superscript> T cell-mediated immunopathology after mouse cytomegalovirus infection

Natural killer (NK) and CD8⁺ T cells play a crucial role in the control of mouse cytomegalovirus (MCMV) infection. These effector cells exert their functions by releasing antiviral cytokines and by cytolytic mechanisms including perforin activation. In addition to their role in virus control, NK cells play an immunoregulatory role since they shape the CD8⁺ T cell response to MCMV. To investigate the role of perforin-dependent cytolytic mechanism in NK cell modulation of CD8⁺ T cell response during acute MCMV infection, we have used perforin-deficient C57BL/6 mice (Prf1^?/?) and have shown that virus control by CD8⁺ T cells in Prf1^?/? mice is more efficient if NK cells are activated by the engagement of the Ly49H receptor with the m157 MCMV protein. A lack of perforin results in severe liver inflammation after MCMV infection, which is characterized by immunopathological lesions that are more pronounced in Prf1^?/? mice infected with virus unable to activate NK cells. This immunopathology is caused by an abundant infiltration of activated CD8⁺ T cells. The depletion of CD8⁺ T cells has markedly reduced pathohistological lesions in the liver and improved the survival of Prf1^?/? mice in spite of an increased viral load. Altogether, the results of our study suggest that a lack of perforin and absence of the specific activation of NK cells during acute MCMV infection lead to an unleashed CD8⁺ T cell response that is detrimental for the host.     

Functional interactions between dendritic cells and NK cells during viral infection

Ly49H(+)NK1.1(+) natural killer (NK) cells are essential for the control of murine cytomegalovirus (MCMV) during the acute stage of infection. This cell subset expands at the later stages of infection in an MCMV-specific fashion. Here we demonstrate a critical interaction between Ly49H(+) NK cells and CD8alpha(+) dendritic cells (DCs) whereby the presence of Ly49H(+) NK cells results in maintenance of CD8alpha(+) DCs in the spleen during acute MCMV infection. Reciprocally, CD8alpha(+) DCs are essential for the expansion of Ly49H(+) NK cells by a mechanism involving interleukin 18 (IL-18) and IL-12. This study provides evidence for a functional interrelationship between DCs and NK cells during viral infection and defines some of the critical cytokines.     

Differential regulation of GM1 and asialo-GM1 expression by T cells and natural killer (NK) cells in respiratory syncytial virus infection

We previously reported that respiratory syncytial virus (RSV) infection increases lung CD8(+) T cell GM1 expression. The related lipid asialo-GM1 (ASGM1) is expressed by T cells in viral infection and by natural killer (NK) cells. The in vivo co-expression of GM1 and ASGM1 by immune cells is not defined. Here we analyzed lung lymphocyte GM1 and ASGM1 expression in RSV-infected mice. GM1 and ASGM1 were coordinately upregulated by activated CD8(+) T cells in RSV-infected BALB/c and C57BL/6 mice. In contrast, RSV infection had no effect on constitutively high NK cell GM1 expression, while increasing NK cell ASGM1 expression. GM1 and ASGM1 co-localized in lipid raft structures in NK and CD8(+) T cells sorted from the lungs of RSV-infected mice. Anti-ASGM1 Ab treatment of RSV-infected BALB/c mice depleted GM1/ASGM1-expressing NK cells and GM1/ASGM1-expressing T cells, reduced lung IFN-gamma levels, increased viral load, delayed viral clearance, and reduced illness. STAT1(-/-) mice are more susceptible to RSV replication and disease than wild-type mice. In RSV-infected STAT1(-/-) mice, anti-ASGM1 Ab altered cytokine levels, but in contrast to BALB/c mice, antibody treatment had no effect on viral load or illness. Taken together, GM1 and ASGM1 expression are differentially regulated by T and NK cells in RSV infection. Also, GM1/ASGM1-expressing cells are important for control of RSV in BALB/c mice, whereas STAT1(-/-) mice clear RSV by an alternative pathway.     

A GRA1 DNA vaccine primes cytolytic CD8(+) T cells to control acute Toxoplasma gondii infection

Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma interferon (IFN-gamma). The contribution of CD4(+) and CD8(+) T-lymphocyte subsets to protective immune responses against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4(+) and CD8(+) T-cell subsets produced IFN-gamma upon restimulation with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8(+) T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that CD8(+) T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii infection, while depletion of CD4(+) T cells led to an increase in brain cyst burden during the chronic phase of infection.     

NK cell functions restrain T cell responses during viral infections

NK cell functions for regulation of T cell responses were evaluated during acute viral infections. In vivo depletion studies established that the presence of NK cells in murine cytomegalovirus (MCMV)-infected immunocompetent mice negatively affected CD4 and CD8 T cell IFN-gamma expression, bromodeoxyuridine (BrdU) incorporation, and expansion. To evaluate NK cell effects, under conditions when NK cells do not control viral replication, experiments were performed using lymphocytic choriomeningitis virus (LCMV). Depletion of NK cells did not affect LCMV-elicited T cell responses in immunocompetent mice; however, the presence of NK cells did inhibit CD4 T cell IFN-gamma production, BrdU incorporation, and expansion in infected MHC class I- and CD8 T cell-deficient beta2M-/- mice. Together, the results reveal a previously unappreciated immunoregulatory role of NK cells for downstream T cell responses.     

Depletion of CD8(+) T cells in vivo impairs host defense of mice resistant and susceptible to pulmonary paracoccidioidomycosis

Using a pulmonary model of infection, we demonstrated previously that A/Sn and B10.A mice are, respectively, resistant and susceptible to Paracoccidioides brasiliensis infection. Employing the same experimental model, we examined herein the role of CD8(+) T cells in the course of paracoccidioidomycosis. Treatment with anti-CD8 monoclonal antibodies caused a selective depletion of pulmonary and splenic CD8(+) T cells in both mouse strains. The number of pulmonary CD4(+) T cells and immunoglobulin-positive cells was independent of the number of CD8(+) T cells. In susceptible mice, the loss of CD8(+) T cells by in vivo treatment with anti-CD8 monoclonal antibodies impaired the clearance of yeasts from the lungs and increased the fungal dissemination to the liver and spleen. The same treatment in resistant mice increased fungal dissemination to extrapulmonary tissues but did not alter the pulmonary fungal load. Furthermore, CD8(+) T-cell depletion did not modify delayed-type hypersensitivity reactions of A/Sn mice but increased these reactions in B10.A mice. The production of P. brasiliensis-specific antibodies by resistant and susceptible mice depleted of CD8(+) T cells was similar to that of mice given control antibody. Histopathologically, depletion of CD8(+) T cells did not disorganize the focal granulomatous lesions developed by both mouse strains. These results indicate that CD8(+) T cells are necessary for optimal clearance of the fungus from tissues of mice infected with P. brasiliensis and demonstrate more prominent protective activity by those cells in the immune responses mounted by susceptible animals.     

NK cells help to induce CD8(+)-T-cell immunity against Toxoplasma gondii in the absence of CD4(+) T cells

CD8(+) T-cell immunity plays an important role in protection against intracellular infections. Earlier studies have shown that CD4(+) T-cell help was needed for launching in vivo CD8(+) T-cell activity against these pathogens and tumors. However, recently CD4(+) T-cell-independent CD8 responses during several microbial infections including those with Toxoplasma gondii have been described, although the mechanism is not understood. We now demonstrate that, in the absence of CD4(+) T cells, T. gondii-infected mice exhibit an extended NK cell response, which is mediated by continued interleukin-12 (IL-12) secretion. This prolonged NK cell response is critical for priming parasite-specific CD8(+) T-cell immunity. Depletion of NK cells inhibited the generation of CD8(+) T-cell immunity in CD4(-/-) mice. Similarly neutralization of IL-12 reduces NK cell numbers in infected animals and leads to the down-regulation of CD8(+) T-cell immunity against T. gondii. Adoptive transfer of NK cells into the IL-12-depleted animals restored their CD8(+) T-cell immune response, and animals exhibited reduced mortality. NK cell gamma interferon was essential for cytotoxic T-lymphocyte priming. Our studies for the first time demonstrate that, in the absence of CD4(+) T cells, NK cells can play an important role in induction of primary CD8(+) T-cell immunity against an intracellular infection. These observations have therapeutic implications for immunocompromised individuals, including those with human immunodeficiency virus infection.     

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.     

T-cell help permits memory CD8(+) T-cell inflation during cytomegalovirus latency

CD4(+) T cells are implied to sustain CD8(+) T-cell responses during persistent infections. As CD4(+) T cells are often themselves antiviral effectors, they might shape CD8(+) T-cell responses via help or via controlling antigen load. We used persistent murine CMV (MCMV) infection to dissect the impact of CD4(+) T cells on virus-specific CD8(+) T cells, distinguishing between increased viral load in the absence of CD4(+) T cells and CD4(+) T-cell-mediated helper mechanisms. Absence of T-helper cells was associated with sustained lytic MCMV replication and led to a slow and gradual reduction of the size and function of the MCMV-specific CD8(+) T-cell pool. However, when virus replication was controlled in the absence of CD4(+) T cells, CD8(+) T-cell function was comparably impaired, but in addition CD8(+) T-cell inflation, a hallmark of CMV infection, was completely abolished. Thus, CD8(+) T-cell inflation during latent CMV infection is strongly dependent on CD4(+) T-cell helper functions, which can partially be compensated by ongoing lytic viral replication in the absence of CD4(+) T cells.     

CD8+ T cells responding to influenza infection reach and persist at higher numbers than CD4+ T cells independently of precursor frequency

The activation, localization, phenotypic changes, and function of CFSE-labeled naive influenza-specific CD8(+) and CD4(+) T cells following influenza infection were examined. Response of adoptively transferred CD8(+) T cells was seen earliest in draining lymph node. Highly activated cells were found later in the lung, airways, and spleen, were cytolytic, and expressed IFN-gamma upon restimulation. Similar amounts of division at early time points, but higher numbers of CD8(+) T cells, were detected at 9 and 30 days postinfection after cotransfer of CD4(+) and CD8(+) T cells followed by infection. Transfer of much smaller numbers of CD4(+) and CD8(+) T cells led to more extensive expansion but the same difference in final number between the two cell types. These studies demonstrate how CD8(+) and CD4(+) T cells respond to influenza at early time points postinfection and the differential kinetics of antigen-specific CD4(+) and CD8(+) T cells.     

Plasmacytoid dendritic cell ablation impacts early interferon responses and antiviral NK and CD8(+) T cell accrual

Plasmacytoid dendritic cells (pDCs) mediate type I interferon (IFN-I) responses to viruses that are recognized through the Toll-like receptor 7 (TLR7) or TLR9 signaling pathway. However, it is unclear how pDCs regulate the antiviral responses via innate and adaptive immune cells. We generated diphtheria toxin receptor transgenic mice to selectively deplete pDCs by administration of diphtheria toxin. pDC-depleted mice were challenged with viruses known to activate pDCs. In murine cytomegalovirus (MCMV) infection, pDC depletion reduced early IFN-I production and augmented viral burden facilitating the expansion of natural killer (NK) cells expressing the MCMV-specific receptor Ly49H. During vesicular stomatitis virus (VSV) infection, pDC depletion enhanced early viral replication and impaired the survival and accumulation of virus-specific cytotoxic T lymphocytes. We conclude that pDCs mediate early antiviral IFN-I responses and influence the accrual of virus-specific NK or CD8(+) T?cells in?a virus-dependent manner.     

HIV-specific cytolytic CD4 T cell responses during acute HIV infection predict disease outcome

Early immunological events during acute HIV infection are thought to fundamentally influence long-term disease outcome. Whereas the contribution of HIV-specific CD8 T cell responses to early viral control is well established, the role of HIV-specific CD4 T cell responses in the control of viral replication after acute infection is unknown. A growing body of evidence suggests that CD4 T cells-besides their helper function-have the capacity to directly recognize and kill virally infected cells. In a longitudinal study of a cohort of individuals acutely infected with HIV, we observed that subjects able to spontaneously control HIV replication in the absence of antiretroviral therapy showed a significant expansion of HIV-specific CD4 T cell responses-but not CD8 T cell responses-compared to subjects who progressed to a high viral set point (P = 0.038). Markedly, this expansion occurred before differences in viral load or CD4 T cell count and was characterized by robust cytolytic activity and expression of a distinct profile of perforin and granzymes at the earliest time point. Kaplan-Meier analysis revealed that the emergence of granzyme A(+) HIV-specific CD4 T cell responses at baseline was highly predictive of slower disease progression and clinical outcome (average days to CD4 T cell count <350/μl was 575 versus 306, P = 0.001). These data demonstrate that HIV-specific CD4 T cell responses can be used during the earliest phase of HIV infection as an immunological predictor of subsequent viral set point and disease outcome. Moreover, these data suggest that expansion of granzyme A(+) HIV-specific cytolytic CD4 T cell responses early during acute HIV infection contributes substantially to the control of viral replication.     

Interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective antiviral immunity

Dendritic cells (DCs) regulate various aspects of innate immunity, including natural killer (NK) cell function. Here we define the mechanisms involved in DC-NK cell interactions during viral infection. NK cells were efficiently activated by murine cytomegalovirus (MCMV)-infected CD11b(+) DCs. NK cell cytotoxicity required interferon-alpha and interactions between the NKG2D activating receptor and NKG2D ligand, whereas the production of interferon-gamma by NK cells relied mainly on DC-derived interleukin 18. Although Toll-like receptor 9 contributes to antiviral immunity, we found that signaling pathways independent of Toll-like receptor 9 were important in generating immune responses to MCMV, including the production of interferon-alpha and the induction of NK cell cytotoxicity. Notably, adoptive transfer of MCMV-activated CD11b(+) DCs resulted in improved control of MCMV infection, indicating that these cells participate in controlling viral replication in vivo.     

Involvement of both donor cytotoxic T lymphocytes and host NK1.1+ T cells in the thymic atrophy of mice suffering from acute graft-versus-host disease.

It has been reported that a dramatic decrease in the number of thymocytes (thymic atrophy) in mice suffering from acute graft-versus-host disease (GVHD) is ascribed to glucocorticoids. In this study, we examined the possibility that cellular immune responses may thus be involved in thymic atrophy. In contrast to chronic GVHD mice, acute GVHD (C57BL/6 X DBA/2) F1 (BDF1) hybrid mice, which were injected intravenously with both spleen and lymph node cells from C57BL/6 mice, showed a dramatic decrease in the number of CD4 CD8 double-positive thymocytes 2 or 3 weeks after the induction of GVHD. A flow cytometric analysis revealed the donor-derived T cells with either CD4 or CD8 molecules to infiltrate the thymus of the mice undergoing acute GVHD for 10 days. In a cytolytic assay, such thymus-containing cells exhibited a cytolytic activity specific to the host cells. In addition, anti-H-2d cytolytic T cells showed a high level of cytolytic activity against BDF1 (H-2bXd) thymocytes, whereas they also showed a low level of cytolytic activity against C57BL/6 (H-2b) thymocytes, thus suggesting that the thymus-infiltrating donor-derived T cells killed the host thymocytes through both anti-H-2d-specific and non-specific mechanisms. Interestingly, a flow cytometric analysis revealed both the percentage and the absolute cell number of host-derived NK1.1+ CD3+ cells to increase in the thymus of mice suffering from acute GVHD for 10 days. In addition, they also showed the cytolytic activity against YAC-1 cells and the mRNA expression of interleukin-12 (IL-12) in the thymus to be also significantly augmented on day 7 after the induction of acute GVHD. Collectively, our results indicate that the cellular immune responses such as donor cytotoxic T lymphocytes and host NK1.1+ T cells are therefore involved in the thymic atrophy of mice suffering from acute GVHD.     

Characterization of early gamma interferon (IFN-gamma) expression during murine listeriosis: identification of NK1.1+ CD11c+ cells as the primary IFN-gamma-expressing cells

Though it is well established that gamma interferon (IFN-gamma) is crucial to the early innate defense of murine listeriosis, its sources remain controversial. In this study, intracellular cytokine staining of IFN-gamma-expressing splenocytes early after Listeria monocytogenes infection revealed that NK1.1(+), CD11c(+), CD8(+) T, and CD4(+) T cells expressed IFN-gamma 24 h after infection. Contrary to the previous report, most IFN-gamma(+) dendritic cells (DC) were CD8alpha(-) DC. Unexpectedly, almost all CD11c(+) IFN-gamma-expressing cells also expressed NK1.1. These NK1.1(+) CD11c(+) cells represented primary IFN-gamma-expressing cells after infection. In situ studies showed these NK1.1(+) CD11c(+) cells were recruited to the borders of infectious foci and expressed IFN-gamma. A significant NK1.1(+) CD11c(+) population was found in uninfected spleen, lymph node, blood, and bone marrow cells. And its number increased significantly in spleen, lymph node, and bone marrow after L. monocytogenes infection. Using interleukin-12 (IL-12) p40(-/-) mice, IFN-gamma expression was found to be largely IL-12 p40 dependent, and the number of IFN-gamma-expressing cells was only about one-third of that of wild-type mice. Moreover, the IFN-gamma expression was absolutely dependent on live L. monocytogenes infection, as no IFN-gamma was detected after inoculation of heat-killed L. monocytogenes. Our findings not only provide an insight into IFN-gamma expression after in vivo infection but may also change the current perceptions of DC and natural killer cells.     

Lactoferrin-mediated protection of the host from murine cytomegalovirus infection by a T-cell-dependent augmentation of natural killer cell activity

Summary The administration of bovine lactoferrin (LF) with 1 mg/g body weight before the murine cytomegalovirus (MCMV) infection completely protected the BALB/c mice from death due to the infection. In these LF-treated mice, a significant increase in the activity was found in the NK cells but not in the cytolytic T lymphocytes which recognized an MCMV-derived peptide. Moreover, the elimination of the NK cell activity by an injection with anti-asialo GM1 antibody abrogated such augmented resistance, thus supporting the hypothesis that the LF-mediated antiviral effect in vivo is performed through the augmentation of NK cell activity. No such LF-mediated antiviral effect in vivo with the increased NK cell activity was found in athymic nude mice, whereas it was restored completely by the transfer of splenic T cells from LF-treated donors. These findings therefore suggest that T lymphocytes induce both the augmentation of NK cell activity and the resultant antiviral effect in the LF-treated hosts.     

Phenotypic and functional characterization of CD8(+) T cell clones specific for a mouse cytomegalovirus epitope

A series of CD8(+) T cell clones, specific for the IE1 epitope YPHFMPTNL, of the immediate-early protein 1 of the murine cytomegalovirus (MCMV) were generated in order to determine their protective activity against this infection and correlate their phenotypic markers with antiviral activity. We found that the adoptive transfer of three of these anti-MCMV CD8(+) T cell clones into irradiated naive mice resulted in protection against challenge, while another CD8(+) T cell clone, of the same specificity, failed to confer protection. The clones that conferred protection against lethal challenge reduced greatly viral replication in the lung and other organs of the mice. Using one of the protective anti-MCMV CD8(+) T cell clones we found that in order to be fully protective the cells had to be transferred to recipient mice no later than 1 day after MCMV challenge. The adoptive transfer of these CD8(+) T cell clones also protected CD4(+) T-cell-depleted mice. Phenotypic characterization of the anti-MCMV clones revealed that the nonprotective clone expressed very low levels of CD8 molecules and produced only small amounts of TNF-alpha upon antigenic stimulation. Most importantly, our current study demonstrates that this MHC class I-restricted IE1 epitope of MCMV is efficiently presented to CD8(+) T cell clones in vivo and further strengthens the possibility of the potential use of CD8(+) T cell clones as immunotherapeutic tools against cytomegalovirus-induced disease.     

NK cell-mediated immunopathology during an acute viral infection of the CNS

Natural killer (NK) and cytotoxic T (Tc) cells are prime effector populations in the antiviral response of the host. Tc cells are essential for recovery from many viral diseases but may also be responsible for immunopathology. The role of NK cells in recovery from viral infections is less well established. We have studied acute virulent Semliki Forest virus (vSFV) infection of the central nervous system in C57BL/6J mice, which was mainly controlled by NK cells without marked Tc cell involvement. We show that mice with defects in the Fas and/or granule exocytosis pathways of cytotoxicity are more resistant to lethal vSFV infection than wild-type mice. On the other hand, mice defective in the IFN-gamma response are more sensitive than wild-type mice, whereas mice lacking the Tc cell compartment (beta-2 microglobulin-deficient mice) exhibit susceptibility similar to wild-type mice. The additional finding that depletion of NK cells significantly delayed the mean time to death but did not prevent mortality in SFV-infected B6 mice suggests that cytolytic activity of NK cells is detrimental, while IFN-gamma production is beneficial for recovery from SFV infection. This is the first study illustrating an NK cell-mediated immunopathological outcome to an acute viral infection.     

NK and T cells constitute two major, functionally distinct intestinal epithelial lymphocyte subsets in the chicken

Non-mammalian NK cells have not been characterized in detail; however, their analysis is essential for the understanding of the NK cell receptor phylogeny. As a first step towards defining chicken NK cells, several tissues were screened for the presence of NK cells, phenotypically defined as CD8(+) cells lacking T- or B-lineage specific markers. By this criteria, approximately 30% of CD8(+) intestinal intraepithelial lymphocytes (IEL), but <1% of splenocytes or peripheral blood lymphocytes were defined as NK cells. These CD8(+)CD3(-) IEL were used for the generation of the 28-4 mAb, immunoprecipitating a 35-kDa glycoprotein with a 28-kDa protein core. The CD3 and 28-4 mAb were used to separate IEL into CD3(+) IEL T cells and 28-4(+) cells, both co-expressing the CD8 antigen. During ontogeny, 28-4(+) cells were abundant in the IEL and in the embryonic spleen, where two subsets could be distinguished according to their CD8 and c-kit expression. Most importantly, 28-4(+) IEL lysed NK-sensitive targets, whereas intestinal T cells did not have any spontaneous cytolytic activity. These results define two major, phenotypically and functionally distinct IEL subpopulations, and imply an important role of NK cells in the mucosal immune system.