Memory CD8+ T Cells Specific for a Single Immunodominant or Subdominant Determinant Induced by Peptide-Dendritic Cell Immunization Protect from an Acute Lethal Viral Disease

The antigens recognized by individual CD8(+) T cells are small peptides bound to major histocompatibility complex (MHC) class I molecules. The CD8(+) T cell response to a virus is restricted to several peptides, and the magnitudes of the effector as well as memory phases of the response to the individual peptides are generally hierarchical. The peptide eliciting a stronger response is called immunodominant (ID), and those with smaller-magnitude responses are termed subdominant (SD). The relative importance of ID and SD determinants in protective immunity remains to be fully elucidated. We previously showed that multispecific memory CD8(+) T cells can protect susceptible mice from mousepox, an acute lethal viral disease. It remained unknown, however, whether CD8(+) T cells specific for single ID or SD peptides could be protective. Here, we demonstrate that immunization with dendritic cells pulsed with ID and some but not all SD peptides induces memory CD8(+) T cells that are fully capable of protecting susceptible mice from mousepox. Additionally, while natural killer (NK) cells are essential for the natural resistance of nonimmune C57BL/6 (B6) to mousepox, we show that memory CD8(+) T cells of single specificity also protect B6 mice depleted of NK cells. This suggests it is feasible to produce effective antiviral CD8(+) T cell vaccines using single CD8(+) T cell determinants and that NK cells are no longer essential when memory CD8(+) T cells are present.     

CD27 stimulation promotes the frequency of IL-7 receptor-expressing memory precursors and prevents IL-12-mediated loss of CD8(+) T cell memory in the absence of CD4(+) T cell help

Fully functional CD8(+) T cell memory is highly dependent upon CD4(+) T cell support. CD4(+) T cells play a critical role in inducing the expression of CD70, the ligand for CD27, on dendritic cells. In this study, we demonstrate that CD27 stimulation during primary CD8(+) T cell responses regulates the ability to mount secondary CD8(+) T cell responses. CD27 stimulation during vaccinia and dendritic cell immunization controls the expression of the IL-7R (CD127), which has been shown to be necessary for memory CD8(+) T cell survival. Furthermore, CD27 stimulation during primary CD8(+) T cell responses to vaccinia virus restrained the late expression on memory precursor cells of cytokine receptors that support terminal differentiation. The formation of CD8(+) T cell memory precursors and secondary CD8(+) T cell responses was restored in the absence of CD27 costimulation when endogenous IL-12 was not available. Similarly, the lesion in CD8(+) T cell memory that occurs in the absence of CD4(+) T cells did not occur in mice lacking IL-12. These data indicate that CD4(+) T cell help and, by extension, CD27 stimulation support CD8(+) T cell memory by modulating the expression of cytokine receptors that influence the differentiation and survival of memory CD8(+) T cells.     

CD8(+) T cells sabotage their own memory potential through IFN-γ-dependent modification of the IL-12/IL-15 receptor α axis on dendritic cells

CD8(+) T cell responses have been shown to be regulated by dendritic cells (DCs) and CD4(+) T cells, leading to the tenet that CD8(+) T cells play a passive role in their own differentiation. In contrast, by using a DNA vaccination model, to separate the events of vaccination from those of CD8(+) T cell priming, we demonstrate that CD8(+) T cells, themselves, actively limit their own memory potential through CD8(+) T cell-derived IFN-γ-dependent modification of the IL-12/IL-15Rα axis on DCs. Such CD8(+) T cell-driven cytokine alterations result in increased T-bet and decreased Bcl-2 expression, and thus decreased memory progenitor formation. These results identify an unrecognized role for CD8(+) T cells in the regulation of their own effector differentiation fate and a previously uncharacterized relationship between the balance of inflammation and memory formation.     

Cognate memory CD4+ T cells generated with dendritic cell priming influence the expansion, trafficking, and differentiation of secondary CD8+ T cells and enhance tumor control

CD4(+) T cells are known to provide support for the activation and expansion of primary CD8(+) T cells, their subsequent differentiation, and ultimately their survival as memory cells. However, the importance of cognate memory CD4(+) T cells in the expansion of memory CD8(+) T cells after re-exposure to Ag has been not been examined in detail. Using bone marrow-derived dendritic cells pulsed with cognate or noncognate MHC class I- and class II-restricted peptides, we examined whether the presence of memory CD4(+) T cells with the same Ag specificity as memory CD8(+) T cells influenced the quantity and quality of the secondary CD8(+) T cell response. After recombinant vaccinia virus-mediated challenge, we demonstrate that, although cognate memory CD4(+) T cells are not required for activation of secondary CD8(+) T cells, their presence enhances the expansion of cognate memory CD8(+) T cells. Cognate CD4(+) T cell help results in an approximate 2-fold increase in the frequency of secondary CD8(+) T cells in secondary lymphoid tissues, and can be accounted for by enhanced proliferation in the secondary CD8(+) T cell population. In addition, cognate memory CD4(+) T cells further selectively enhance secondary CD8(+) T cell infiltration of tumor-associated peripheral tissue, and this is accompanied by increased differentiation into effector phenotype within the secondary CD8(+) T cell population. The consequence of these improvements to the magnitude and phenotype of the secondary CD8(+) T cell response is substantial increase in control of tumor outgrowth.     

Chemokine-guided CD4+ T cell help enhances generation of IL-6RalphahighIL-7Ralpha high prememory CD8+ T cells

CD4(+) T cells promote effective CD8(+) T cell-mediated immunity, but the timing and mechanistic details of such help remain controversial. Furthermore, the extent to which innate stimuli act independently of help in enhancing CD8(+) T cell responses is also unresolved. Using a noninfectious vaccine model in immunocompetent mice, we show that even in the presence of innate stimuli, CD4(+) T cell help early after priming is required for generating an optimal pool of functional memory CD8(+) T cells. CD4(+) T cell help increased the size of a previously unreported population of IL-6Ralpha(high)IL-7Ralpha(high) prememory CD8(+) T cells shortly after priming that showed a survival advantage in vivo and contributed to the majority of functional memory CD8(+) T cells after the contraction phase. In accord with our recent demonstration of chemokine-guided recruitment of naive CD8(+) T cells to sites of CD4(+) T cell-dendritic cell interactions, the generation of IL-6Ralpha(high)IL-7Ralpha(high) prememory as well as functional memory CD8(+) T cells depended on the early postvaccination action of the inflammatory chemokines CCL3 and CCL4. Together, these findings support a model of CD8(+) T cell memory cell differentiation involving the delivery of key signals early in the priming process based on chemokine-guided attraction of naive CD8(+) T cells to sites of Ag-driven interactions between TLR-activated dendritic cells and CD4(+) T cells. They also reveal that elevated IL-6Ralpha expression by a subset of CD8(+) T cells represents an early imprint of CD4(+) T cell helper function that actively contributes to the survival of activated CD8(+) T cells.     

CD154 blockade alters innate immune cell recruitment and programs alloreactive CD8+ T cells into KLRG-1(high) short-lived effector T cells

CD154/CD40 blockade combined with donor specific transfusion remains one of the most effective therapies in prolonging allograft survival. Despite this, the mechanisms by which these pathways synergize to prevent rejection are not completely understood. Utilizing a BALB/c (H2-K(d)) to B6 (H2-K(b)) fully allogeneic skin transplant model system, we performed a detailed longitudinal analysis of the kinetics and magnitude of CD8(+) T cell expansion and differentiation in the presence of CD154/CD40 pathway blockade. Results demonstrated that treatment with anti-CD154 vs. DST had distinct and opposing effects on activated CD44(high) CD62L(low) CD8(+) T cells in skin graft recipients. Specifically, CD154 blockade delayed alloreactive CD8(+) T cell responses, while DST accelerated them. DST inhibited the differentiation of alloreactive CD8(+) T cells into multi-cytokine producing effectors, while CD40/CD154 blockade led to the diminution of the KLRG-1(low) long-lived memory precursor population compared with either untreated or DST treated animals. Moreover, only CD154 blockade effectively inhibited CXCL1 expression and neutrophil recruitment into the graft. When combined, anti-CD154 and DST acted synergistically to profoundly diminish the absolute number of IFN-γ producing alloreactive CD8(+) T cells, and intra-graft expression of inflammatory chemokines. These findings demonstrate that the previously described ability of anti-CD154 and DST to result in alloreactive T cell deletion involves both delayed kinetics of T cell expansion and differentiation and inhibited development of KLRG-1(low) memory precursor cells.     

Discriminating between different pathways of memory CD8+ T cell differentiation

Despite the rapid accumulation of quantitative data on the dynamics of CD8(+) T cell responses following acute viral or bacterial infections of mice, the pathways of differentiation of naive CD8(+) T cells into memory during an immune response remain controversial. Currently, three models have been proposed. In the "stem cell-associated differentiation" model, following activation, naive T cells differentiate into stem cell-like memory cells, which then convert into terminally differentiated short-lived effector cells. In the "linear differentiation" model, following activation, naive T cells first differentiate into effectors, and after Ag clearance, effectors convert into memory cells. Finally, in the "progressive differentiation" model, naive T cells differentiate into memory or effector cells depending on the amount of specific stimulation received, with weaker stimulation resulting in formation of memory cells. This study investigates whether the mathematical models formulated from these hypotheses are consistent with the data on the dynamics of the CD8(+) T cell response to lymphocytic choriomeningitis virus during acute infection of mice. Findings indicate that two models, the stem cell-associated differentiation model and the progressive differentiation model, in which differentiation of cells is strongly linked to the number of cell divisions, fail to describe the data at biologically reasonable parameter values. This work suggests additional experimental tests that may allow for further discrimination between different models of CD8(+) T cell differentiation in acute infections.     

IL-15 expands unconventional CD8alphaalphaNK1.1+ T cells but not Valpha14Jalpha18+ NKT cells

Despite recent gains in knowledge regarding CD1d-restricted NKT cells, very little is understood of non-CD1d-restricted NKT cells such as CD8(+)NK1.1(+) T cells, in part because of the very small proportion of these cells in the periphery. In this study we took advantage of the high number of CD8(+)NK1.1(+) T cells in IL-15-transgenic mice to characterize this T cell population. In the IL-15-transgenic mice, the absolute number of CD1d-tetramer(+) NKT cells did not increase, although IL-15 has been shown to play a critical role in the development and expansion of these cells. The CD8(+)NK1.1(+) T cells in the IL-15-transgenic mice did not react with CD1d-tetramer. Approximately 50% of CD8(+)NK1.1(+) T cells were CD8alphaalpha. In contrast to CD4(+)NK1.1(+) T cells, which were mostly CD1d-restricted NKT cells and of which approximately 70% were CD69(+)CD44(+), approximately 70% of CD8(+)NK1.1(+) T cells were CD69(-)CD44(+). We could also expand similar CD8alphaalphaNK1.1(+) T cells but not CD4(+) NKT cells from CD8alpha(+)beta(-) bone marrow cells cultured ex vivo with IL-15. These results indicate that the increased CD8alphaalphaNK1.1(+) T cells are not activated conventional CD8(+) T cells and do not arise from conventional CD8alphabeta precursors. CD8alphaalphaNK1.1(+) T cells produced very large amounts of IFN-gamma and degranulated upon TCR activation. These results suggest that high levels of IL-15 induce expansion or differentiation of a novel NK1.1(+) T cell subset, CD8alphaalphaNK1.1(+) T cells, and that IL-15-transgenic mice may be a useful resource for studying the functional relevance of CD8(+)NK1.1(+) T cells.     

Pathogen-induced inflammatory environment controls effector and memory CD8+ T cell differentiation

In response to infection, CD8(+) T cells integrate multiple signals and undergo an exponential increase in cell numbers. Simultaneously, a dynamic differentiation process occurs, resulting in the formation of short-lived effector cells (SLECs; CD127(low)KLRG1(high)) and memory precursor effector cells (CD127(high)KLRG1(low)) from an early effector cell that is CD127(low)KLRG1(low) in phenotype. CD8(+) T cell differentiation during vesicular stomatitis virus infection differed significantly than during Listeria monocytogenes infection with a substantial reduction in early effector cell differentiation into SLECs. SLEC generation was dependent on Ebi3 expression. Furthermore, SLEC differentiation during vesicular stomatitis virus infection was enhanced by administration of CpG-DNA, through an IL-12-dependent mechanism. Moreover, CpG-DNA treatment enhanced effector CD8(+) T cell functionality and memory subset distribution, but in an IL-12-independent manner. Population dynamics were dramatically different during secondary CD8(+) T cell responses, with a much greater accumulation of SLECs and the appearance of a significant number of CD127(high)KLRG1(high) memory cells, both of which were intrinsic to the memory CD8(+) T cell. These subsets persisted for several months but were less effective in recall than memory precursor effector cells. Thus, our data shed light on how varying the context of T cell priming alters downstream effector and memory CD8(+) T cell differentiation.     

Involvement of CD1 in peripheral deletion of T lymphocytes is independent of NK T cells

During peripheral T cell deletion, lymphocytes accumulate in nonlymphoid organs including the liver, a tissue that expresses the nonclassical, MHC-like molecule, CD1. Injection of anti-CD3 Ab results in T cell activation, which in normal mice is followed by peripheral T cell deletion. However, in CD1-deficient mice, the deletion of the activated T cells from the lymph nodes was impaired. This defect in peripheral T cell deletion was accompanied by attenuated accumulation of CD8(+) T cells in the liver. In tetra-parental bone marrow chimeras, expression of CD1 on the T cells themselves was not required for T cell deletion, suggesting a role for CD1 on other cells with which the T cells interact. We tested whether this role was dependent on the Ag receptor-invariant, CD1-reactive subset of NK T cells using two other mutant mouse lines that lack most NK T cells, due to deletion of the genes encoding either beta(2)-microglobulin or the TCR element J alpha 281. However, these mice had no abnormality of peripheral T cell deletion. These findings indicate a novel role for CD1 in T cell deletion, and show that CD1 functions in this process through mechanisms that does not involve the major, TCR-invariant set of NK T cells.     

Viral and bacterial infections induce expression of multiple NK cell receptors in responding CD8(+) T cells

NK cells express several families of receptors that play central roles in target cell recognition. These NK cell receptors are also expressed by certain memory phenotype CD8(+) T cells, and in some cases are up-regulated in T cells responding to viral infection. To determine how the profile of NK receptor expression changes in murine CD8(+) T cells as they respond to intracellular pathogens, we used class I tetramer reagents to directly examine Ag-specific T cells during lymphocytic choriomeningitis virus and Listeria monocytogenes infections. We found that the majority of pathogen-specific CD8(+) T cells initiated expression of the inhibitory CD94/NKG2A heterodimer, the KLRG1 receptor, and a novel murine NK cell marker (10D7); conversely, very few Ag-specific T cells expressed Ly49 family members. The up-regulation of these receptors was independent of IL-15 and persisted long after clearance of the pathogen. The expression of CD94/NKG2A was rapidly initiated in naive CD8(+) T cells responding to peptide Ags in vitro and on many of the naive T cells that proliferate when transferred into lymphopenic (Rag-1(-/-)) hosts. Thus, CD94/NKG2A expression is a common consequence of CD8(+) T cell activation. Binding of the CD94/NKG2A receptor by its ligand (Qa-1(b)) did not significantly inhibit CD8(+) T cell effector functions. However, expression of CD94 and NKG2A transgenes partially inhibited early events of T cell activation. These subtle effects suggest that CD94/NKG2A-mediated inhibition of T cells may be limited to particular circumstances or may synergize with other receptors that are similarly up-regulated.     

Human CD8⁺ and CD4⁺ T cell memory to lymphocytic choriomeningitis virus infection

Although cellular immunity to acute lymphocytic choriomeningitis virus (LCMV) infection has been well characterized in experimental studies in mice, the T cell response to this virus in humans is incompletely understood. Thus, we analyzed the breadths, magnitudes, and differentiation phenotypes of memory LCMV-specific CD8(+) and CD4(+) T cells in three human donors displaying a variety of disease outcomes after accidental needle stick injury or exposure to LCMV. Although only a small cohort of donors was analyzed at a single time point postinfection, several interesting observations were made. First, we were able to detect LCMV-specific CD8(+) and CD4(+) T cell responses directly ex vivo at 4 to 8 years after exposure, demonstrating the longevity of T cell memory in humans. Second, unlike in murine models of LCMV infection, we found that the breadths of memory CD8(+) and CD4(+) T cell responses were not significantly different from one another. Third, it seemed that the overall CD8(+) T cell response was augmented with increasing severity of disease, while the LCMV-specific CD4(+) T cell response magnitude was highly variable between the three different donors. Next, we found that LCMV-specific CD8(+) T cells in the three donors analyzed seemed to undergo an effector memory differentiation program distinct from that of CD4(+) T cells. Finally, the levels of expression of memory, costimulatory, and inhibitory receptors on CD8(+) and CD4(+) T cell subsets, in some instances, correlated with disease outcome. These data demonstrate for the first time LCMV-specific CD8(+) and CD4(+) T cells in infected humans and begin to provide new insights into memory T cell responses following an acute virus infection.     

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.     

Role of cross-talk between IFN-alpha-induced monocyte-derived dendritic cells and NK cells in priming CD8+ T cell responses against human tumor antigens

In the present study we evaluated the role of IFN-alpha in the generation of dendritic cells (IFN-DCs) with priming activity on CD8(+) T lymphocytes directed against human tumor Ags. A 3-day treatment of monocytes, obtained as adherent PBMCs from HLA-A*0201(+) healthy donors, with IFN-alpha and GM-CSF led to the differentiation of DCs displaying a semimature phenotype, but promptly inducing CD8(+) T cell responses after one in vitro sensitization with peptides derived from melanoma (gp100(209-217) and MART-1/Melan-A(27-35)) and adenocarcinoma (CEA(605-613)) Ags. However, these features were lost when IFN-DCs were generated from immunosorted CD14(+) monocytes. The ability of adherent PBMCs to differentiate into IFN-DCs expressing higher levels of costimulatory molecules and exerting efficient T cell priming capacity was associated with the presence of contaminating NK cells, which underwent phenotypic and functional activation upon IFN-alpha treatment. NK cell boost appeared to be mediated by both direct and indirect (i.e., mediated by IFN-DCs) mechanisms. Experiments performed to prove the role of contaminating NK cells in DC differentiation showed that IFN-DCs generated in the absence of NK were phenotypically less mature and could not efficiently prime antitumor CD8(+) lymphocytes. Reciprocally, IFN-DCs raised from immunosorted CD14(+) monocytes regained their T cell priming activity when NK cells were added to the culture before IFN-alpha and GM-CSF treatment. Together, our data suggest that the ability of IFN-DCs to efficiently prime anti-tumor CD8(+) T lymphocytes relied mostly on the positive cross-talk occurring between DCs and NK cells upon stimulation with IFN-alpha.     

TNF-alpha controls intrahepatic T cell apoptosis and peripheral T cell numbers

At the end of an immune response, activated lymphocyte populations contract, leaving only a small memory population. The deletion of CD8(+) T cells from the periphery is associated with an accumulation of CD8(+) T cells in the liver, resulting in both CD8(+) T cell apoptosis and liver damage. After adoptive transfer and in vivo activation of TCR transgenic CD8(+) T cells, an increased number of activated CD8(+) T cells was observed in the lymph nodes, spleen, and liver of mice treated with anti-TNF-alpha. However, caspase activity was decreased only in CD8(+) T cells in the liver, not in those in the lymphoid organs. These results indicate that TNF-alpha is responsible for inducing apoptosis in the liver and suggest that CD8(+) T cells escaping this mechanism of deletion can recirculate into the periphery.     

Memory CD8+ T cells provide an early source of IFN-gamma

During the non-Ag-specific early phase of infection, IFN-gamma is believed to be primarily provided by NK and NKT cells in response to pathogen-derived inflammatory mediators. To test whether other cell types were involved in early IFN-gamma release, IFN-gamma-producing cells were visualized in spleens and lymph nodes of LPS-injected mice. In addition to NK and NKT cells, IFN-gamma was also detected in a significant fraction of CD8(+) T cells. CD8(+) T cells represented the second major population of IFN-gamma-producing cells in the spleen ( approximately 30%) and the majority of IFN-gamma(+) cells in the lymph nodes ( approximately 70%). LPS-induced IFN-gamma production by CD8(+) T cells was MHC class I independent and was restricted to CD44(high) (memory phenotype) cells. Experiments performed with C3H/HeJ (LPS-nonresponder) mice suggested that CD8(+) T cells responded to LPS indirectly through macrophage/dendritic cell-derived IFN-alpha/beta, IL-12, and IL-18. IFN-gamma was also detected in memory CD8(+) T cells from mice injected with type I IFN or with poly(I:C), a synthetic dsRNA that mimics early activation by RNA viruses. Taken together, these results suggest that in response to bacterial and viral products, memory T cells may contribute to innate immunity by providing an early non-Ag-specific source of IFN-gamma.     

Blocking intrahepatic deletion of activated CD8+ T cells by an altered peptide ligand

BACKGROUND: Activated CD8(+) T cells are retained by the healthy liver where the majority undergo apoptosis. The intrahepatic apoptosis of activated CD8(+) T cells is enhanced by the presence of SIINFEKL peptide. It is of great interest to identify strategies for maintaining intrahepatic T cell number and function in the presence of SIINFEKL peptides. AIM: Our aim was to test if low affinity peptides can block SIINFEKL peptide induced T cell deletion. METHODS: We used an in vivo model of intrahepatic CD8(+) T cell deletion with peptides of different affinities. RESULTS AND DISCUSSION: We show that the intrahepatic deletion of CD8(+) T cells by SIINFEKL peptide results in loss of in vivo cytotoxic T lymphocyte function. In contrast we show that a low affinity peptide (G4) does not result in intrahepatic deletion of CD8(+) T cells. High concentrations G4 peptide can however block intrahepatic deletion of activated CD8(+) T cells, and prevent loss of in vivo cytotoxicity due to SIINFEKL peptide. This is the first demonstration of blocking of SIINFEKL peptide induced CD8(+) T cell deletion in the liver, with enhancement of in vivo cytotoxicity.     

Contribution of CD8+ T cells to control of Mycobacterium tuberculosis infection

Tuberculosis is the number one cause of death due to infectious disease in the world today. Understanding the dynamics of the immune response is crucial to elaborating differences between individuals who contain infection vs those who suffer active disease. Key cells in an adaptive immune response to intracellular pathogens include CD8(+) T cells. Once stimulated, these cells provide a number of different effector functions, each aimed at clearing or containing the pathogen. To explore the role of CD8(+) T cells in an integrative way, we synthesize both published and unpublished data to build and test a mathematical model of the immune response to Mycobacterium tuberculosis in the lung. The model is then used to perform a series of simulations mimicking experimental situations. Selective deletion of CD8(+) T cell subsets suggests a differential contribution for CD8(+) T cell effectors that are cytotoxic as compared with those that produce IFN-gamma. We also determined the minimum levels of effector memory cells of each T cell subset (CD4(+) and CD8(+)) in providing effective protection following vaccination.