IL-2-activated CD8+CD44high cells express both adaptive and innate immune system receptors and demonstrate specificity for syngeneic tumor cells

CD8(+) T cells depend on the alphabeta TCR for Ag recognition and function. However, Ag-activated CD8(+) T cells can also express receptors of the innate immune system. In this study, we examined the expression of NK receptors on a population of CD8(+) T cells expressing high levels of CD44 (CD8(+)CD44(high) cells) from normal mice. These cells are distinct from conventional memory CD8(+) T cells and they proliferate and become activated in response to IL 2 via a CD48/CD2-dependent mechanism. Before activation, they express low or undetectable levels of NK receptors but upon activation with IL-2 they expressed significant levels of activating NK receptors including 2B4 and NKG2D. Interestingly, the IL-2-activated cells demonstrate a preference in the killing of syngeneic tumor cells. This killing of syngeneic tumor cells was greatly enhanced by the expression of the NKG2D ligand Rae-1 on the target cell. In contrast to conventional CD8(+) T cells, IL-2-activated CD8(+)CD44(high) cells express DAP12, an adaptor molecule that is normally expressed in activated NK cells. These observations indicate that activated CD8(+)CD44(high) cells express receptors of both the adaptive and innate immune system and may play a unique role in the surveillance of host cells that have been altered by infection or transformation.     

TRAIL deficiency does not rescue impaired CD8+ T cell memory generated in the absence of CD4+ T cell help

Ag-specific CD8(+) T cells immunized in the absence of CD4(+) T cell help, so-called "unhelped" CD8(+) T cells, are defective in function and survival. We investigated the role of the proapoptotic molecule TRAIL in this defect. We first demonstrate that TRAIL does not contribute to the CD8(+) T cell response to Listeria monocytogenes strain expressing OVA (LmOVA) in the presence of CD4(+) T cells. Secondly, we generated mice doubly deficient in CD4(+) T cells and TRAIL and analyzed their CD8(+) T cell response to LmOVA. Memory CD8(+) T cells in double-deficient mice waned over time and were not protective against rechallenge, similar to their TRAIL-sufficient unhelped counterparts. To avoid the effects of CD4(+) T cell deficiency during memory maintenance, and to address whether TRAIL plays a role in the early programming of the CD8(+) T cell response, we performed experiments using heterologous prime and early boost immunizations. We did not observe activation-induced cell death of unhelped CD8(+) T cells when mice were infected with followed vaccinia virus expressing OVA 9 days later by LmOVA infection. Furthermore, primary immunization of CD4(+) T cell-deficient mice with cell-associated Ag followed by LmOVA infection did not reveal a role for TRAIL-mediated activation-induced cell death. Overall, our results suggest that CD4(+) T cell help for the CD8(+) T cell response is not contingent on the silencing of TRAIL expression and prevention of TRAIL-mediated apoptosis.     

Tec kinases Itk and Rlk are required for CD8+ T cell responses to virus infection independent of their role in CD4+ T cell help

Itk and Rlk are members of the Tec kinase family of nonreceptor protein tyrosine kinases that are expressed in T cells, NK cells, and mast cells. These proteins are involved in the regulation of signaling processes downstream of the TCR in CD4(+) T cells, particularly in the phosphorylation of phospholipase C-gamma1 after TCR activation; furthermore, both Itk and Rlk are important in CD4(+) T cell development, differentiation, function, and homeostasis. However, few studies have addressed the roles of these kinases in CD8(+) T cell signaling and function. Using Itk(-/-) and Itk(-/-)Rlk(-/-) mice, we examined the roles of these Tec family kinases in CD8(+) T cells, both in vitro and in vivo. These studies demonstrate that the loss of Itk and Rlk impairs TCR-dependent signaling, causing defects in phospholipase C-gamma1, p38, and ERK activation as well as defects in calcium flux and cytokine production in vitro and expansion and effector cytokine production by CD8(+) T cells in response to viral infection. These defects cannot be rescued by providing virus-specific CD4(+) T cell help, thereby substantiating the important role of Tec kinases in CD8(+) T cell signaling.     

Development of intestinal intraepithelial lymphocytes, NK cells, and NK 1.1+ T cells in CD45-deficient mice

The transmembrane protein tyrosine phosphatase CD45 is differentially required for the development and function of B, T, and NK cells, with mice partially deficient for CD45 having a significant inhibition of T cell, but not NK or B cell, development. CD45-mediated signaling has also been implicated in the development of intrathymic, but not extrathymic, intestinal intraepithelial T lymphocytes (iIELs) in the CD45ex6(-/-) mouse. As NK1.1(+) CD3(+) (NK-T) cells can also develop through extrathymic pathways, we have investigated the role of CD45 in NK-T cell development. In mice with a complete absence of CD45 expression (CD45ex9(-/-)) the NK-T cell population was maintained in the iIEL compartment, but not in the spleen. Functionally, CD45-deficient NK-T cells were unable to secrete IL-4 in response to TCR-mediated signals, a phenotype similar to that of CD45-deficient iIELs, in which in vitro cytokine production was dramatically reduced. Using the CD45ex9(-/-) mouse strain, we have also demonstrated that only one distinct population of NK-T cells (CD8(+)) appears to develop normally in the absence of CD45. Interestingly, although an increase in cytotoxic NK cells is seen in the absence of CD45, these NK calls are functionally unable to secrete IFN-gamma. In the absence of CD45, a significant population of extrathymically derived CD8alphaalpha(+) iIELs is also maintained. These results demonstrate that in contrast to conventional T cells, CD45 is not required during the development of CD8(+) NK-T cells, NK cells, or CD8alphaalpha(+) iIELs, but is essential for TCR-mediated function and cytokine production.     

The NK cell response to mouse cytomegalovirus infection affects the level and kinetics of the early CD8(+) T-cell response

Natural killer (NK) cells and CD8(+) T cells play a prominent role in the clearance of mouse cytomegalovirus (MCMV) infection. The role of NK cells in modulating the CD8(+) T-cell response to MCMV infection is still the subject of intensive research. For analyzing the impact of NK cells on mounting of a CD8(+) T-cell response and the contribution of these cells to virus control during the first days postinfection (p.i.), we used C57BL/6 mice in which NK cells are specifically activated through the Ly49H receptor engaged by the MCMV-encoded ligand m157. Our results indicate that the requirement for CD8(+) T cells in early MCMV control inversely correlates with the engagement of Ly49H. While depletion of CD8(+) T cells has only a minor effect on the early control of wild-type MCMV, CD8(+) T cells are essential in the control of Δm157 virus. The frequencies of virus epitope-specific CD8(+) T cells and their activation status were higher in mice infected with Δm157 virus. In addition, these mice showed elevated levels of alpha interferon (IFN-α) and several other proinflammatory cytokines as early as 1.5 days p.i. Although the numbers of conventional dendritic cells (cDCs) were reduced later during infection, particularly in Δm157-infected mice, they were not significantly affected at the peak of the cytokine response. Altogether, we concluded that increased antigen load, preservation of early cDCs' function, and higher levels of innate cytokines collectively account for an enhanced CD8(+) T-cell response in C57BL/6 mice infected with a virus unable to activate NK cells via the Ly49H-m157 interaction.     

Innate NK cells and macrophages recognize and reject allogeneic nonself in vivo via different mechanisms

Both innate and adaptive immune cells are involved in the allograft response. But how the innate immune cells respond to allotransplants remains poorly defined. In the current study, we examined the roles of NK cells and macrophages in recognizing and rejecting allogeneic cells in vivo. We found that in naive mice NK cells are the primary effector cells in the killing of allogeneic cells via "missing self" recognition. However, in alloantigen-presensitized mice, NK cells are dispensable. Instead, macrophages become alloreactive and readily recognize and reject allogeneic nonself. This effect requires help from activated CD4(+) T cells and involves CD40/CD40L engagement, because blocking CD40/CD40L interactions prevents macrophage-mediated rejection of allogeneic cells. Conversely, actively stimulating CD40 triggers macrophage-mediated rejection in the absence of CD4(+) T cells. Importantly, alloantigen-primed and CD4(+) T cell-helped macrophages (licensed macrophages) exhibit potent regulatory function in vivo in an acute graft-versus-host disease model. Together, our data uncover an important role for macrophages in the alloimmune response and may have important clinical implications.     

Multiple dendritic cell populations activate CD4+ T cells after viral stimulation

Dendritic cells (DC) are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8alpha DC play a prominent, and sometimes exclusive, role in driving amplification of CD8(+) T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4(+) T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4(+) and CD8(+) T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8alpha DC populations in the amplification of CD8(+) and CD4(+) T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8(+) T cells are dominated by presentation of antigen by CD8alpha DC but can involve non-CD8alpha DC. In contrast, CD4(+) T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4(+) T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity.     

NK cells regulate CD4 responses prior to antigen encounter

NK cells not only respond rapidly to infection, shaping subsequent adaptive immunity, but also play a role in regulating autoimmune disease. The ability of NK cells to influence adaptive immunity before Ag exposure was examined in a gender-dependent model of preferential Th1 and Th2 activation. The inability of young adult male SJL mice to activate Th1 cells was reversed via depletion of NK1.1(+) cells, whereas the presence or the absence of NK1.1(+) cells did not alter responses in age-matched females. Consistent with a gender-dependent role in regulating adaptive immunity, significantly more NK1.1(+) cells were present in males compared with females, and this difference was reversed by castration. In contrast to NK1.1(+) cells derived from C57BL/6 mice, no spontaneous cytokine secretion was detected in NK1.1(+) cells derived from either male or female SJL mice, although an increased frequency of IL-10-secreting NK1.1(+) cells was observed in males vs females following in vitro stimulation. Direct evidence that NK1.1(+) cells in males influence CD4(+) T cell activation before Ag exposure was demonstrated via the adoptive transfer of APC from control and NK1.1-depleted males. The absence of a functional NK T cell population in SJL mice suggests that NK cells influence adaptive immunity before Ag exposure via alterations in APC activity.     

Multiple CD4+ T cell subsets produce immunomodulatory IL-10 during respiratory syncytial virus infection

The host immune response is believed to contribute to the severity of pulmonary disease induced by acute respiratory syncytial virus (RSV) infection. Because RSV-induced pulmonary disease is associated with immunopathology, we evaluated the role of IL-10 in modulating the RSV-specific immune response. We found that IL-10 protein levels in the lung were increased following acute RSV infection, with maximum production corresponding to the peak of the virus-specific T cell response. The majority of IL-10-producing cells in the lung during acute RSV infection were CD4(+) T cells. The IL-10-producing CD4(+) T cells included Foxp3(+) regulatory T cells, Foxp3(-) CD4(+) T cells that coproduce IFN-γ, and Foxp3(-) CD4(+) T cells that do not coproduce IFN-γ. RSV infection of IL-10-deficient mice resulted in more severe disease, as measured by increased weight loss and airway resistance, as compared with control mice. We also observed an increase in the magnitude of the RSV-induced CD8(+) and CD4(+) T cell response that correlated with increased disease severity in the absence of IL-10 or following IL-10R blockade. Interestingly, IL-10R blockade during acute RSV infection altered CD4(+) T cell subset distribution, resulting in a significant increase in IL-17A-producing CD4(+) T cells and a concomitant decrease in Foxp3(+) regulatory T cells. These results demonstrate that IL-10 plays a critical role in modulating the adaptive immune response to RSV by limiting T-cell-mediated pulmonary inflammation and injury.     

CD4+ T-cell responses are required for clearance of West Nile virus from the central nervous system

Although studies have established that innate and adaptive immune responses are important in controlling West Nile virus (WNV) infection, the function of CD4(+) T lymphocytes in modulating viral pathogenesis is less well characterized. Using a mouse model, we examined the role of CD4(+) T cells in coordinating protection against WNV infection. A genetic or acquired deficiency of CD4(+) T cells resulted in a protracted WNV infection in the central nervous system (CNS) that culminated in uniform lethality by 50 days after infection. Mice surviving past day 10 had high-level persistent WNV infection in the CNS compared to wild-type mice, even 45 days following infection. The absence of CD4(+) T-cell help did not affect the kinetics of WNV infection in the spleen and serum, suggesting a role for CD4-independent clearance mechanisms in peripheral tissues. WNV-specific immunoglobulin M (IgM) levels were similar to those of wild-type mice in CD4-deficient mice early during infection but dropped approximately 20-fold at day 15 postinfection, whereas IgG levels in CD4-deficient mice were approximately 100- to 1,000-fold lower than in wild-type mice throughout the course of infection. WNV-specific CD8(+) T-cell activation and trafficking to the CNS were unaffected by the absence of CD4(+) T cells at day 9 postinfection but were markedly compromised at day 15. Our experiments suggest that the dominant protective role of CD4(+) T cells during primary WNV infection is to provide help for antibody responses and sustain WNV-specific CD8(+) T-cell responses in the CNS that enable viral clearance.     

Characterization of CD4(+) CTLs ex vivo

The cytotoxic potential of CD8(+) T cells and NK cells plays a crucial role in the immune response to pathogens. Although in vitro studies have reported that CD4(+) T cells are also able to mediate perforin-mediated killing, the in vivo existence and relevance of cytotoxic CD4(+) T cells have been the subject of debate. Here we show that a population of CD4(+) perforin(+) T cells is present in the circulation at low numbers in healthy donors and is markedly expanded in donors with chronic viral infections, in particular HIV infection, at all stages of the disease, including early primary infection. Ex vivo analysis shows that these cells have cytotoxic potential mediated through the release of perforin. In comparison with more classical CD4(+) T cells, this subset displays a distinct surface phenotype and functional profile most consistent with end-stage differentiated T cells and include Ag experienced CD4(+) T cells. The existence of CD4(+) cytotoxic T cells in vivo at relatively high levels in chronic viral infection suggests a role in the immune response.     

Role of CD8+ T cells in control of West Nile virus infection

Infection with West Nile virus (WNV) causes fatal encephalitis more frequently in immunocompromised humans than in those with a healthy immune system. Although a complete understanding of this increased risk remains unclear, experiments with mice have begun to define how different components of the adaptive and innate immune response function to limit infection. Previously, we demonstrated that components of humoral immunity, particularly immunoglobulin M (IgM) and IgG, have critical roles in preventing dissemination of WNV infection to the central nervous system. In this study, we addressed the function of CD8(+) T cells in controlling WNV infection. Mice that lacked CD8(+) T cells or classical class Ia major histocompatibility complex (MHC) antigens had higher central nervous system viral burdens and increased mortality rates after infection with a low-passage-number WNV isolate. In contrast, an absence of CD8(+) T cells had no effect on the qualitative or quantitative antibody response and did not alter the kinetics or magnitude of viremia. In the subset of CD8(+)-T-cell-deficient mice that survived initial WNV challenge, infectious virus was recovered from central nervous system compartments for several weeks. Primary or memory CD8(+) T cells that were generated in vivo efficiently killed target cells that displayed WNV antigens in a class I MHC-restricted manner. Collectively, our experiments suggest that, while specific antibody is responsible for terminating viremia, CD8(+) T cells have an important function in clearing infection from tissues and preventing viral persistence.     

CD40 on APCs is needed for optimal programming, maintenance, and recall of CD8+ T cell memory even in the absence of CD4+ T cell help

CD40 stimulation is one of the many signals that can activate APCs and we have recently shown it to have a unique function in generating maximum primary CD8(+) T cell responses. However, whether CD40 signaling plays a role in memory CD8(+) T cell responses is still not completely understood. In this study, we show that in the absence of CD40 on all APCs or specifically on dendritic cells, memory CD8(+) T cells are generated but at significantly reduced levels. This reduction is due to a contribution of CD40 at several different steps in the generation of CD8(+) memory. In the initial T cell response, CD40 contributes to maximizing not only the number of effector cells that are generated but also the programming of ones that will differentiate into memory. Subsequently, CD40 is needed to maintain maximal numbers of the committed memory cells in a manner that is independent of the immunizing Ag. Finally, when memory CD8(+) T cells are reactivated there is a variable requirement for CD40 depending on whether CD40 or CD4(+) Th cells were present during the primary response. Therefore, CD40 signaling on APCs plays an important role in all phases of a memory CD8(+) T cell response.     

Signaling through NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity

NK cells possess both effector and regulatory activities that may be important during the antitumor immune response. In fact, the generation of antitumor immunity by the administration of an agonistic mAb against CD137 is NK cell-dependent. In this study, we report that NK cells could be induced by IL-2 and IL-15 to express CD137 and ligation of CD137-stimulated NK cell proliferation and IFN-gamma secretion, but not their cytolytic activity. Importantly, CD137-stimulated NK cells promoted the expansion of activated T cells in vitro, demonstrating immunoregulatory or "helper" activity for CD8(+)CTL. Furthermore, tumor-specific CTL activity against P815 tumor Ags was abrogated following anti-CD137 treatment in NK-depleted mice. We further demonstrate that CD137-stimulated helper NK cells expressed the high-affinity IL-2R and were hyperresponsive to IL-2. Taken together with previous findings that CD137 is a critical receptor for costimulation of T cells, our findings suggest that CD137 is a stimulatory receptor for NK cells involved in the crosstalk between innate and adaptive immunity.     

Essential role for CD40 ligand interactions in T lymphocyte-mediated modulation of the murine immune response to pneumococcal capsular polysaccharides

Protection against infection with pneumococci is provided by anti-capsular polysaccharide (caps-PS) Abs. We investigated whether CD40 ligand (CD40L) plays a role in T lymphocyte-mediated regulation of the immune response to caps-PS, which are considered thymus-independent Ags. Administration of MR1, an antagonist mAb against murine CD40L, in BALB/c mice immunized with Pneumovax resulted in an inhibition of the IgM and IgG Ab response for various caps-PS serotypes. Evidence for the involvement of CD4(+) T lymphocytes in the Ab response to caps-PS was obtained in SCID/SCID mice that, when reconstituted with B lymphocytes and CD4(+) T lymphocytes, mounted a higher specific IgM response compared with SCID/SCID mice reconstituted with only B lymphocytes. This helper effect of CD4(+) T lymphocytes was abrogated by MR1. Blocking CD40L in vitro decreased the IgM response to caps-PS and abolished the helper effect of CD4(+) T lymphocytes. CD8(+) T lymphocyte-depleted murine spleen cells mounted a higher in vivo immune response than total murine spleen cells, which provided evidence for a suppressive role of CD8(+) T lymphocytes on the anti-caps-PS immune response. CD4(+) T lymphocyte-depleted murine spleen cells, leaving a B and CD8(+) T lymphocyte fraction, elicited only a weak in vivo and in vitro Ab response, which was enhanced after MR1 administration. In summary, our data provide evidence that T lymphocytes contribute to the regulation of the anti-caps-PS immune response in a CD40L-dependent manner.     

Vgamma4(+) T cells promote autoimmune CD8(+) cytolytic T-lymphocyte activation in coxsackievirus B3-induced myocarditis in mice: role for CD4(+) Th1 cells

T cells expressing the Vgamma4 T-cell receptor (TCR) promote myocarditis in coxsackievirus B3 (CVB3)-infected BALB/c mice. CD1, a major histocompatibility complex (MHC) class I-like molecule, is required for activation of Vgamma4(+) cells. Once activated, Vgamma4(+) cells initiate myocarditis through gamma interferon (IFN-gamma)-mediated induction of CD4(+) T helper type 1 (Th1) cells in the infected animal. These CD4(+) Th1 cells are required for activation of an autoimmune CD8(+) alphabeta TCR(+) effector, which is the predominant pathogenic agent in this model of CVB3-induced myocarditis. Activated Vgamma4(+) cells can adoptively transfer myocarditis into BALB/c mice infected with a nonmyocarditic variant of CVB3 (H310A1) but cannot transfer myocarditis into either uninfected or CD1(-/-) recipients, demonstrating the need for both infection and CD1 expression for Vgamma4(+) cell function. In contrast, CD8(+) alphabeta TCR(+) cells transfer myocarditis into either infected CD1(-/-) or uninfected recipients, showing that once activated, the CD8(+) alphabeta TCR(+) effectors function independently of both virus and CD1. Vgamma4(+) cells given to mice lacking CD4(+) T cells minimally activate the CD8(+) alphabeta TCR(+) cells. These studies show that Vgamma4(+) cells determine CVB3 pathogenicity by their ability to influence both the CD4(+) and CD8(+) adaptive immune response. Vgamma4(+) cells enhance CD4(+) Th1 (IFN-gamma(+)) cell activation through IFN-gamma- and CD1-dependent mechanisms. CD4(+) Th1 cells promote activation of the autoimmune CD8(+) alphabeta TCR(+) effectors.     

Comparing the kinetics of NK cells, CD4, and CD8 T cells in murine cytomegalovirus infection

NK cells recognize virus-infected cells with germline-encoded activating and inhibitory receptors that do not undergo genetic recombination or mutation. Accordingly, NK cells are often considered part of the innate immune response. The innate response comprises rapid early defenders that do not form immune memory. However, there is increasing evidence that experienced NK cells provide increased protection to secondary infection, a hallmark of the adaptive response. In this study, we compare the dynamics of the innate and adaptive immune responses by examining the kinetic profiles of the NK and T cell response to murine CMV infection. We find that, unexpectedly, the kinetics of NK cell proliferation is neither earlier nor faster than the CD4 or CD8 T cell response. Furthermore, early NK cell contraction after the peak of the response is slower than that of T cells. Finally, unlike T cells, experienced NK cells do not experience biphasic decay after the response peak, a trait associated with memory formation. Rather, NK cell contraction is continuous, constant, and returns to below endogenous preinfection levels. This indicates that the reason why Ag-experienced NK cells remain detectable for a prolonged period after adoptive transfer and infection is in part due to the high precursor frequency, slow decay rate, and low background levels of Ly49H(+) NK cells in recipient DAP12-deficient mice. Thus, the quantitative contribution of Ag-experienced NK cells in an endogenous secondary response, with higher background levels of Ly49H(+) NK cells, may be not be as robust as the secondary response observed in T cells.     

Conventional alpha beta T cells are sufficient for innate and adaptive immunity against enteric Listeria monocytogenes

We have begun to dissect the cellular requirements for generation of immunity against enteric infection by Listeria monocytogenes using a novel T(-) B(-) NK(-) mouse strain (mice double deficient for the common cytokine receptor gamma-chain (gamma(c)) and the recombinase-activating gene-2 (RAG2/gamma(c) mice). Initial experiments showed that C57BL/6 mice and alymphoid RAG2/gamma(c) mice had similar kinetics of bacterial accumulation in the spleen, liver, and brain early after intragastric L. monocytogenes infection (up to day 3), calling into question the physiologic role of gut-associated lymphoid cells during the passage of this enterobacterium into the host. However, in contrast to C57BL/6 mice, RAG2/gamma(c) mice rapidly succumbed to disseminated infection by day 7. Polyclonal lymph node CD4(+) and CD8(+) alphabeta T cells were able to confer RAG2/gamma(c) mice with long-lasting protection against enteric L. monocytogenes infection in the absence of gammadelta T, NK, and NK-T cells. Moreover, these alphabeta T-reconstituted RAG2/gamma(c) mice produced IFN-gamma at levels comparable to C57BL/6 mice in response to L. monocytogenes both in vitro and in vivo. Protection was IFN-gamma dependent, as RAG2/gamma(c) mice reconstituted with IFN-gamma-deficient alphabeta T cells were unable to control enteric L. monocytogenes infection. Furthermore, alphabeta T cell-reconstituted RAG2/gamma(c) mice were able to mount memory responses when challenged with lethal doses of L. monocytogenes. These data suggest that NK, NK-T, gammadelta T, and B cells are functionally redundant in the immunity against oral L. monocytogenes infection, and that in their absence alphabeta T cells are able to mediate the early IFN-gamma production required for both innate and adaptive immunity.     

Emergence of CD8+ T cells expressing NK cell receptors in influenza A virus-infected mice

Both innate and adaptive immune responses play an important role in the recovery of the host from viral infections. In the present report, a subset of cells coexpressing CD8 and NKR-P1C (NK1.1) was found in the lungs of mice infected with influenza A virus. These cells were detected at low numbers in the lungs of uninfected mice, but represented up to 10% of the total CD8(+) T cell population at day 10 postinfection. Almost all of the CD8(+)NK1.1(+) cells were CD8alphabeta(+)CD3(+)TCRalphabeta(+) and a proportion of these cells also expressed the NK cell-associated Ly49 receptors. Interestingly, up to 30% of these cells were virus-specific T cells as determined by MHC class I tetramer staining and by intracellular staining of IFN-gamma after viral peptide stimulation. Moreover, these cells were distinct from conventional NKT cells as they were also found at increased numbers in influenza-infected CD1(-/-) mice. These results demonstrate that a significant proportion of CD8(+) T cells acquire NK1.1 and other NK cell-associated molecules, and suggests that these receptors may possibly regulate CD8(+) T cell effector functions during viral infection.