CD94/NKG2A expression is associated with proliferative potential of CD8 T cells during persistent polyoma virus infection

Memory CD8 T cells comprise a critical component of durable immunity because of their capacity to rapidly proliferate and exert effector activity upon Ag rechallenge. During persistent viral infection, memory CD8 T cells repetitively encounter viral Ag and must maintain a delicate balance between limiting viral replication and minimizing immunopathology. In mice infected by polyoma virus, a natural mouse pathogen that establishes long-term persistent infection, the majority of persistence-phase antiviral CD8 T cells express the inhibitory NK cell receptor CD94/NKG2A. In this study, we asked whether CD94/NKG2A expression is associated with Ag-specific recall of polyoma virus-specific CD8 T cells. During the persistent phase of infection, polyoma virus-specific CD8 T cells that express CD94/NKG2A were found to preferentially proliferate; this proliferation was dependent on cognate Ag both in vitro and in vivo. In addition, CD94/NKG2A(+) polyoma-specific CD8 T cells have a markedly enhanced capacity to produce IL-2 upon ex vivo Ag stimulation compared with CD94/NKG2A(-) polyoma-specific CD8 T cells. Importantly, CD94/NKG2A(+) anti-polyoma virus CD8 T cells appear to be essential for Ag-specific recall responses in mice persistently infected by polyoma virus. Because of its higher proliferative potential and capacity to produce IL-2, we propose that the CD94/NKG2A(+) subpopulation represents a less differentiated state than the CD94/NKG2A(-) subpopulation. Identification of proliferation-competent subpopulations of memory CD8 T cells should prove valuable in designing therapeutic vaccination strategies for persistent viral infections.     

Memory CD8+ T cells in HIV infection

Cytotoxic T lymphocytes (CTLs) play a central role in the control of persistent HIV infection in humans. The kinetics and general features of the CTL response are similar to those found during other persisting virus infections in humans. During chronic infection there are commonly between 0.1 and 1.0% of all CD8+ T cells in the blood that are specific for immunodominant virus epitopes, as measured by HLA class I peptide tetramers. These figures are greatly in excess of the numbers found by limiting dilution assays; the discrepancy may arise because in the latter assay, CTLs have to divide many times to be detected and many of the HIV-specific CD8+ T cells circulating in infected persons may be incapable of further division. Many tetramer-positive T cells make interferon-gamma, beta-chemokines and perforin, so are probably functional. It is not known how fast these T cells turn over, but in the absence of antigen they decay in number. Impairment of CTL replacement, because CD4+ T helper cells are depleted by HIV infection, may play a major role in the development of AIDS.     

NKG2D is a costimulatory receptor for human naive CD8+ T cells

In humans, all alpha beta CD8+ T cells express NKG2D, but in mouse, it is only expressed by activated and memory CD8+ T cells. We purified human naive CD8+ T cells to show that NKG2D serves as a costimulatory receptor for TCR induced Ca2+ mobilization and proliferation. The resulting effector cells are skewed toward a type 1 phenotype and produce high levels of IFN-gamma and TNF-alpha. NKG2D ligands, MHC class I chain-related (MIC)A, MICB, and UL16-binding proteins are expressed on the proliferating cells and NKG2D is down-regulated. The addition of the homeostatic cytokines IL-7 and IL-15 to the culture medium not only enhances proliferation but also counteracts the down-regulation of NKG2D, more so than the addition of IL-2. These results indicate that NKG2D can regulate the priming of human naive CD8+ T cells, which may provide an alternative mechanism for potentiating and channeling the immune response.     

NK cell CD94/NKG2A inhibitory receptors are internalized and recycle independently of inhibitory signaling processes

Human CD94/NKG2A is an inhibitory receptor that recognizes HLA-E and is expressed by NK cells and a subset of T cells. We have analyzed the cellular trafficking of the CD94/NKG2A receptor using the NKL cell line and peripheral blood NK cells. Flow cytometric, confocal microscopic, and biochemical analyses show that CD94/NKG2A continuously recycles in an active process that requires the cytoskeleton between the cell surface and intracellular compartments that are distinguishable from recycling compartments used by well-characterized receptors, such as transferrin receptor (CD71). CD94/NKG2A, an inhibitory receptor, traffics differently from the closely related CD94/NKG2C molecule, an activating receptor. Using transfection/expression analyses of wild-type and mutant CD94/NKG2A molecules in the HLA-E negative rat basophilic cell line RBL-2H3, we demonstrate that CD94/NKG2A internalization is independent of ligand cross-linking or the presence of functional immunoreceptor tyrosine-based inhibition motifs. Thus, the mechanisms that control cell surface homeostasis of CD94/NKG2A are independent of functional signaling.     

Gene expression in antigen-specific CD8+ T cells during viral infection

Following infection with intracellular pathogens, Ag-specific CD8(+) T cells become activated and begin to proliferate. As these cells become activated, they elaborate effector functions including cytokine production and cytolysis. After the infection has been cleared, the immune system returns to homeostasis through apoptosis of the majority of the Ag-specific effector cells. The surviving memory cells can persist for extended periods and provide protection against reinfection. Little is known about the changes in gene expression as Ag-specific cells progress through these stages of development, i.e., naive to effector to memory. Using recombinant MHC class I tetramers, we isolated Ag-specific CD8(+) T cells from mice infected with lymphocytic choriomeningitis virus at various time points and performed semiquantitative RT-PCR. We examined expression of: 1) genes involved in cell cycle control, 2) effector and regulatory functions, and 3) susceptibility to apoptosis. We found that Ag-specific CD8(+) memory T cells contain high steady-state levels of Bcl-2, BAX:, IFN-gamma, and lung Kruppel-like factor (LKLF), and decreased levels of p21 and p27 mRNA. Moreover, the pattern of gene expression between naive and memory cells is distinct and suggests that these two cell types control susceptibility to apoptosis through different mechanisms.     

2B4+ CD8+ T cells play an inhibitory role against constrained HIV epitopes

Cytotoxic T cells play a critical role in the control of HIV and the progression of infected individuals to AIDS. 2B4 (CD244) is a member of the SLAM family of receptors that regulate lymphocyte development and function. The expression of 2B4 on CD8⁺ T cells was shown to increase during AIDS disease progression. However, the functional role of 2B4⁺ CD8⁺ T cells against HIV infection is not known. Here, we have examined the functional role of 2B4⁺ CD8⁺ T cells during and after stimulation with HLA B14 or B27 restricted HIV epitopes. Interestingly, IFN-γ secretion and cytotoxic activity of 2B4⁺ CD8⁺ T cells stimulated with HIV peptides were significantly decreased when compared to influenza peptide stimulated 2B4⁺ CD8⁺ T cells. The expression of the signaling adaptor molecule SAP was downregulated in 2B4⁺ CD8⁺ T cells upon HIV peptide stimulation. These results suggest that 2B4⁺ CD8⁺ T cells play an inhibitory role against constrained HIV epitopes underlying the inability to control the virus during disease progression.     

HLA-E is the ligand for the natural killer cell CD94/NKG2 receptors

CD94/NKG2 is a recently described receptor present on natural killer (NK) cells and certain T cells that is composed of the CD94 chain covalently associated with a member of the NKG2 family of molecules. Both chains are glycosylated members of the C-type lectin superfamily. The CD94/NKG2 receptors are functionally heterogenous depending on which NKG2 family member is associated with CD94. Initially, it was thought that CD94/NKG2 receptors recognized a broad array of HLA-A, -B and -C (classical), as well as the nonclassical HLA-G, MHC class I molecules. Instead, recent data have suggested that this receptor is specific for HLA-E complexed with a peptide derived from the signal sequence (residues 3–11) of certain classical MHC class I molecules. Position 2 (residue 4) in the signal sequence derived peptides appears pivotal in determining whether the HLA-E/peptide complex confers resistance to NK-mediated lysis. The potential roles that the CD94/NKG2-HLA-E receptor ligand interaction might play in infection and tumor development are discussed.     

CD4+CD25+ regulatory T cells in HIV infection

The immune system faces the difficult task of discerning between foreign, potentially pathogen-derived antigens and self-antigens. Several mechanisms, including deletion of self-reactive T cells in the thymus, have been shown to contribute to the acceptance of self-antigens and the reciprocal reactivity to foreign antigens. Over the last decade it has become increasingly clear that CD4(+)CD25(+) T(Reg) cells are crucial for maintenance of T cell tolerance to self-antigens in the periphery, and to avoid development of autoimmune disorders. Recently, evidence has also emerged that demonstrates that CD4(+)CD25(+) T(Reg) cells can also suppress T cell responses to foreign pathogens, including viruses such as HIV. In this article we review the current knowledge and potential role of CD4(+)CD25(+) T(Reg) cells in HIV infection.     

Role that each NKG2A immunoreceptor tyrosine-based inhibitory motif plays in mediating the human CD94/NKG2A inhibitory signal

The human NKG2A chain of the CD94/NKG2A receptor contains two immunoreceptor Tyr-based inhibitory motifs (ITIMs) in its cytoplasmic tail. To determine the relative importance of membrane-distal (residues 6-11) and membrane-proximal (residues 38-43) ITIMs in mediating the inhibitory signal, we made site-directed mutants of NKG2A at the Y (Y8F, Y40F, Y8F/Y40F) and the residues two positions N-terminal (Y-2) of Y (V6A, I38A, V6A/I38A) in each motif. Wild-type (wt) and mutated NKG2A were then cotransfected with CD94 into rat basophilic leukemia 2H3 cells. Immunochemical analyses after pervanadate treatment showed that each of the mutant molecules could be phosphorylated to expected levels relative to wt NKG2A and that all the mutations significantly reduced the avidity of SH2 domain-bearing tyrosine phosphatase-1 for NKG2A. Confocal microscopy was used to determine whether SH2 domain-bearing tyrosine phosphatase-1 and CD94/NKG2A colocalized intracellularly after receptor ligation. Only the Y8F/Y40F and Y8F mutant NKG2A molecules failed to show a dramatic colocalization. In agreement with this result, the Y8F/Y40F mutant was unable to inhibit FcepsilonRI-mediated serotonin release and the Y8F mutant was relatively ineffective compared with wt NKG2A. In contrast, the Y40F mutant was 70% as effective as wt in mediating inhibition, and the Y-2 mutations did not remarkably affect inhibitory function. These results show that, like KIR, both NKG2A ITIMs are required for mediating the maximal inhibitory signal, but opposite to KIR, the membrane-distal ITIM is of primary importance rather than the membrane-proximal ITIM. This probably reflects the opposite orientation of the ITIMs in type II vs type I proteins.     

Regulation of antiviral CD8+ T cells by inhibitory natural killer cell receptors

Recent evidence indicates that CD8(+) T cells express natural killer cell receptors that constrain the range and magnitude of their activities. For virus-specific CD8(+) T cells, upregulation of these receptors serves to control infection, while concurrently minimizing bystander pathology. Dysregulated expression of these receptors, however, may foster the establishment of persistent virus infection.     

Cytotoxicity of CD56(bright) NK cells towards autologous activated CD4+ T cells is mediated through NKG2D, LFA-1 and TRAIL and dampened via CD94/NKG2A

In mouse models of chronic inflammatory diseases, Natural Killer (NK) cells can play an immunoregulatory role by eliminating chronically activated leukocytes. Indirect evidence suggests that NK cells may also be immunoregulatory in humans. Two subsets of human NK cells can be phenotypically distinguished as CD16(+)CD56(dim) and CD16(dim/-)CD56(bright). An expansion in the CD56(bright) NK cell subset has been associated with clinical responses to therapy in various autoimmune diseases, suggesting an immunoregulatory role for this subset in vivo. Here we compared the regulation of activated human CD4(+) T cells by CD56(dim) and CD56(bright) autologous NK cells in vitro. Both subsets efficiently killed activated, but not resting, CD4(+) T cells. The activating receptor NKG2D, as well as the integrin LFA-1 and the TRAIL pathway, played important roles in this process. Degranulation by NK cells towards activated CD4(+) T cells was enhanced by IL-2, IL-15, IL-12+IL-18 and IFN-α. Interestingly, IL-7 and IL-21 stimulated degranulation by CD56(bright) NK cells but not by CD56(dim) NK cells. NK cell killing of activated CD4(+) T cells was suppressed by HLA-E on CD4(+) T cells, as blocking the interaction between HLA-E and the inhibitory CD94/NKG2A NK cell receptor enhanced NK cell degranulation. This study provides new insight into CD56(dim) and CD56(bright) NK cell-mediated elimination of activated autologous CD4(+) T cells, which potentially may provide an opportunity for therapeutic treatment of chronic inflammation.     

Massive load of functional effector CD4+ and CD8+ T cells against cytomegalovirus in very old subjects

A progressive, systemic, and low-grade proinflammatory status is one of the major characteristics of immunosenescence. Emerging data suggest a possible contribution of CMV, known to chronically infect a large proportion of humans, lifelong from newborns to centenarians. To test this hypothesis, we evaluated functional T cell responses to two CMV immunogenic proteins, pp65 and IE-1, in 65 chronically infected subjects aged 25-100 years. PBMC were stimulated with mixtures of peptides spanning the entire sequence of both proteins, and Ag specificity and magnitude of intracellular IFN-gamma- and TNF-alpha-positive cells were then analyzed within both CD4+ and CD8+ T cells. Results indicate that pp65 and, to a lesser extent, IE-1 constitute major Ags against which aged people target functionally efficient T cell effector responses with massive production of Th1 cytokines and exhibition of CD107a degranulation marker. As a result, the production of IFN-gamma induced in T cells by both Ags was seven to eight times greater in very old than in young subjects. The comparative analysis of pp65-specific responses in these very long-term carriers revealed a reciprocal relationship between CD4+ and CD8+ producing IFN-gamma in the same individuals. These results indicate that CMV represents an important pathogen responsible for a strong immune activation in human aging. Such a remarkable burden of effector CD4+ and CD8+ T cells may be necessary to protect the elderly from CMV endogenous reactivation, but can turn detrimental by giving a substantial contribution to the proinflammatory status that accompanies the main age-related diseases.     

Activated iNKT cells promote memory CD8+ T cell differentiation during viral infection

α-Galactosylceramide (α-GalCer) is the prototypical lipid ligand for invariant NKT cells. Recent studies have proposed that α-GalCer is an effective adjuvant in vaccination against a range of immune challenges, however its mechanism of action has not been completely elucidated. A variety of delivery methods have been examined including pulsing dendritic cells with α-GalCer to optimize the potential of α-GalCer. These methods are currently being used in a variety of clinical trials in patients with advanced cancer but cannot be used in the context of vaccine development against pathogens due to their complexity. Using a simple delivery method, we evaluated α-GalCer adjuvant properties, using the mouse model for cytomegalovirus (MCMV). We measured several key parameters of the immune response to MCMV, including inflammation, effector, and central memory CD8(+) T cell responses. We found that α-GalCer injection at the time of the infection decreases viral titers, alters the kinetics of the inflammatory response, and promotes both increased frequencies and numbers of virus-specific memory CD8(+) T cells. Overall, our data suggest that iNKT cell activation by α-GalCer promotes the development of long-term protective immunity through increased fitness of central memory CD8(+) T cells, as a consequence of reduced inflammation.     

Tim-3 negatively regulates cytotoxicity in exhausted CD8+ T cells in HIV infection

Cytotoxic CD8(+) T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell 'exhaustion' is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection. These Tim-3 expressing T cells are dysfunctional in terms of their capacities to proliferate or to produce cytokines. In this study, we evaluated the effect of Tim-3 expression on the cytotoxic capabilities of CD8(+) T cells in the context of HIV infection. We investigated the cytotoxic capacity of Tim-3 expressing T cells by examining 1) the ability of Tim-3(+) CD8(+) T cells to make perforin and 2) the direct ability of Tim-3(+) CD8(+) T cells to kill autologous HIV infected CD4(+) target cells. Surprisingly, Tim-3(+) CD8(+) T cells maintain higher levels of perforin, which was mainly in a granule-associated (stored) conformation, as well as express high levels of T-bet. However, these cells were also defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8(+) T cells from chronic progressors by increasing; a) their degranulation capacity, b) their ability to release perforin, c) their ability to target activated granzyme B to HIV antigen expressing CD4(+) T cells and d) their ability to suppress HIV infection of CD4(+) T cells. In this latter effect, blocking the Tim-3 pathway enhances the cytotoxcity of CD8(+) T cells from chronic progressors to the level very close to that of T cells from viral controllers. Thus, the Tim-3 receptor, in addition to acting as a terminator for cytokine producing and proliferative functions of CTLs, can also down-regulate the CD8(+) T cell cytotoxic function through inhibition of degranulation and perforin and granzyme secretion.     

Exclusion of lipid rafts and decreased mobility of CD94/NKG2A receptors at the inhibitory NK cell synapse

CD94/NKG2A is an inhibitory receptor expressed by most human natural killer (NK) cells and a subset of T cells that recognizes human leukocyte antigen E (HLA-E) on potential target cells. To elucidate the cell surface dynamics of CD94/NKG2A receptors, we have expressed CD94/NKG2A-EGFP receptors in the rat basophilic leukemia (RBL) cell line. Photobleaching experiments revealed that CD94/NKG2A-EGFP receptors move freely within the plasma membrane and accumulate at the site of contact with ligand. The enriched CD94/NKG2A-EGFP is markedly less mobile than the nonligated receptor. We observed that not only are lipid rafts not required for receptor polarization, they are excluded from the site of receptor contact with the ligand. Furthermore, the lipid raft patches normally observed at the sites where FcepsilonR1 activation receptors are cross-linked were not observed when CD94/NKG2A was coengaged along with the activation receptor. These results suggest that immobilization of the CD94/NKG2A receptors at ligation sites not only promote sustenance of the inhibitory signal, but by lipid rafts exclusion prevent formation of activation signaling complexes.     

Mitochondrial potential and reactive oxygen intermediates in antigen-specific CD8+ T cells during viral infection

Following many viral infections, there are large expansions of Ag-specific CD8(+) T cells. After viral clearance, mechanisms exist to ensure that the vast majority of effector cells undergo apoptosis. In studies of thymocyte apoptosis, loss of mitochondrial potential (deltapsi(m)) and excess production of reactive oxygen intermediates have been implicated as key events in cellular apoptosis. The purpose of the experiments presented in this work was to determine these parameters in Ag-specific CD8(+) T cells during a physiological response such as viral infection. Using lymphocytic choriomeningitis virus infection of mice, we found that Ag-specific CD8(+) effector T cells that had undergone recent TCR stimulation had an increased deltapsi(m). These cells also had increased levels of superoxide. As these cells progressed through the contraction of the immune response, their potential decreased, but superoxide levels remained similar to naive cells. One of the consequences of reduced mitochondrial potential is membrane permeability and subsequent caspase activation. We examined both the enzymatic activity and levels of cleaved caspase 3, an effector caspase, and could only detect increased levels in Ag-specific CD8(+) T cells on day 5 postinfection, a time point in which virus was still present. This contrasts with Ag-specific effector cells examined during the contraction phase that had no detectable caspase activity directly ex vivo. These data suggest that the apoptotic program begins earlier than previously expected on day 5, during the expansion phase.     

Interferon-alpha administration enhances CD8+ T cell activation in HIV infection

Background

Type I interferons play important roles in innate immune defense. In HIV infection, type I interferons may delay disease progression by inhibiting viral replication while at the same time accelerating disease progression by contributing to chronic immune activation.

Methods

To investigate the effects of type I interferons in HIV-infection, we obtained cryopreserved peripheral blood mononuclear cell samples from 10 subjects who participated in AIDS Clinical Trials Group Study 5192, a trial investigating the activity of systemic administration of IFNα for twelve weeks to patients with untreated HIV infection. Using flow cytometry, we examined changes in cell cycle status and expression of activation antigens by circulating T cells and their maturation subsets before, during and after IFNα treatment.

Results

The proportion of CD38+HLA-DR+CD8+ T cells increased from a mean of 11.7% at baseline to 24.1% after twelve weeks of interferon treatment (p = 0.006). These frequencies dropped to an average of 20.1% six weeks after the end of treatment. In contrast to CD8+ T cells, the frequencies of activated CD4+ T cells did not change with administration of type I interferon (mean percentage of CD38+DR+ cells = 2.62% at baseline and 2.17% after 12 weeks of interferon therapy). As plasma HIV levels fell with interferon therapy, this was correlated with a “paradoxical” increase in CD8+ T cell activation (p<0.001).

Conclusion

Administration of type I interferon increased expression of the activation markers CD38 and HLA DR on CD8+ T cells but not on CD4+ T cells of HIV+ persons. These observations suggest that type I interferons may contribute to the high levels of CD8+ T cell activation that occur during HIV infection.     

Antiviral protection by CD8+ versus CD4+ T cells. CD8+ T cells correlating with cytotoxic activity in vitro are more efficient in antivaccinia virus protection than CD4-dependent IL.

T cell-mediated protection against a recombinant vaccinia virus was evaluated in mice with respect to the relative contributions of CTL vs that of T cell-dependent IL and of CD4+ cells. H-2b mice primed with the wildtype of vesicular stomatitis virus serotype Indiana (VSV-IND wt) mount an in vitro measurable cytotoxic response against the nucleoprotein (NP) of VSV-IND and are protected against a challenge infection with a vaccinia-VSV recombinant virus expressing the NP of VSV-IND (vacc-IND-NP). Their protective mechanism was highly susceptible to in vivo depletion of CD8+ T cells, but resistant to CD4+ depletion or treatment with anti-IFN-gamma and anti-TNF-alpha. Surprisingly, also VSV-CTL nonresponder H-2k mice were protected against a challenging infection with vacc-IND-NP when primed with VSV-IND wt. In contrast to the CTL responder H-2b mice, this protection was highly susceptible to CD4+ T cell depletion and to anti-IFN-gamma or anti-TNF-alpha treatment, but resistant to CD8+ T cell depletion. Antibodies were not responsible because they failed to transfer protection; in contrast CD4+ T cells conferred significant protection. VSV-CTL responder H-2b and nonresponder H-2k mice were protected almost equally well against a challenge dose of 10(3) pfu vacc-IND-NP inoculated intracerebrally. However, after intracerebral challenge with 5 x 10(6) pfu vacc-IND-NP, the CTL nonresponder mice died, whereas the CTL responder mice eliminated the virus by day 5. These results collectively show that CD4+ T cell-dependent IL may mediate antiviral protection, but their efficiency is relatively weak compared with CD8-mediated protection correlating with cytotoxic activity in vitro.     

A viral long terminal repeat expressed in CD4+CD8+ precursors is downregulated in mature peripheral CD4-CD8+ or CD4+CD8- T cells.

The long terminal repeat from a thymotropic mouse mammary tumor virus variant, DMBA-LV, was used to drive the expression of two reporter genes, murine c-myc and human CD4, in transgenic mice. Expression was observed specifically in thymic immature cells. Expression of c-myc in these cells induced oligoclonal CD4+ CD8+ T-cell thymomas. Expression of human CD4 was restricted to thymic progenitor CD4- CD8- and CD4+ CD8+ T cells and was shut off in mature CD4+ CD8- and CD4- CD8+ T cells, known to be derived from the progenitor double-positive T cells. These results suggest the existence of similar and common factors in CD4+ CD8- and CD4- CD8+ T cells and support a model of differentiation of CD4+ CD8+ T cells through common signal(s) involved in turning off the expression of the CD4 or CD8 gene.