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.     

CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection.

In this study, we have examined the relative contributions of CD4+ and CD8+ T cells in controlling an acute or chronic lymphocytic choriomeningitis virus (LCMV) infection. To study acute infection, we used the LCMV Armstrong strain, which is cleared by adult mice in 8 to 10 days, and to analyze chronic infection, we used a panel of lymphocyte-tropic and macrophage-tropic variants of LCMV that persist in adult mice for several months. We show that CD4+ T cells are not necessary for resolving an acute LCMV infection. CD4+ T-cell-depleted mice were capable of generating an LCMV-specific CD8+ cytotoxic T-lymphocyte (CTL) response and eliminated virus with kinetics similar to those for control mice. The CD8+ CTL response was critical for resolving this infection, since beta 2-microglobulin knockout (CD8-deficient) mice were unable to control the LCMV Armstrong infection and became persistently infected. In striking contrast to the acute infection, even a transient depletion of CD4+ T cells profoundly affected the outcome of infection with the macrophage- and lymphocyte-tropic LCMV variants. Adult mice given a single injection of anti-CD4 monoclonal antibody (GK1.5) at the time of virus challenge became lifelong carriers with high levels of virus in most tissues. Unmanipulated adult mice infected with the different LCMV variants contained virus for prolonged periods (> 3 months) but eventually eliminated infection from most tissues, and all of these mice had LCMV-specific CD8+ CTL responses. Although the level of CTL activity was quite low, it was consistently present in all of the chronically infected mice that eventually resolved the infection. These results clearly show that even in the presence of an overwhelming viral infection of the immune system, CD8+ CTL can remain active for long periods and eventually resolve and/or keep the virus infection in check. In contrast, LCMV-specific CTL responses were completely lost in chronically infected CD4-depleted mice. Taken together, these results show that CD4+ T cells are dispensable for short-term acute infection in which CD8+ CTL activity does not need to be sustained for more than 2 weeks. However, under conditions of chronic infection, in which CD8+ CTLs take several months or longer to clear the infection, CD4+ T-cell function is critical. Thus, CD4+ T cells play an important role in sustaining virus-specific CD8+ CTL during chronic LCMV infection. These findings have implications for chronic viral infections in general and may provide a possible explanation for the loss of human immunodeficiency virus-specific CD8+ CTL activity that is seen during the late stages of AIDS, when CD4+ T cells become limiting.     

Marginal zone B cells regulate antigen-specific T cell responses during infection

Marginal zone B cells (MZB) participate in the early immune response to several pathogens. In this study, we show that in μMT mice infected with Leishmania donovani, CD8 T cells displayed a greater cytotoxic potential and generated more effector memory cells compared with infected wild type mice. The frequency of parasite-specific, IFN-γ(+) CD4 T cells was also increased in μMT mice. B cells were able to capture parasites, which was associated with upregulation of surface IgM and MyD88-dependent IL-10 production. Moreover, MZB presented parasite Ags to CD4 T cells in vitro. Depletion of MZB also enhanced T cell responses and led to a decrease in the parasite burden but did not alter the generation of effector memory T cells. Thus, MZB appear to suppress protective T cell responses during the early stages of L. donovani infection.     

CD4 T cells are required for CD8 T cell survival during both primary and memory recall responses

The role of CD4 T cell help in primary and secondary CD8 T cell responses to infectious pathogens remains incompletely defined. The primary CD8 T response to infections was initially thought to be largely independent of CD4 T cells, but it is not clear why some primary, pathogen-specific CD8 T cell responses are CD4 T cell dependent. Furthermore, although the generation of functional memory CD8 T cells is CD4 T cell help dependent, it remains controversial when the "help" is needed. In this study, we demonstrated that CD4 T cell help was not needed for the activation and effector differentiation of CD8 T cells during the primary response to vaccinia virus infection. However, the activated CD8 T cells showed poor survival without CD4 T cell help, leading to a reduction in clonal expansion and a diminished, but stable CD8 memory pool. In addition, we observed that CD4 T cell help provided during both the primary and secondary responses was required for the survival of memory CD8 T cells during recall expansion. Our study indicates that CD4 T cells play a crucial role in multiple stages of CD8 T cell response to vaccinia virus infection and may help to design effective vaccine strategies.     

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.     

Role of CD4 T cell help and costimulation in CD8 T cell responses during Listeria monocytogenes infection

CD4 T cells are known to assist the CD8 T cell response by activating APC via CD40-CD40 ligand (L) interactions. However, recent data have shown that bacterial products can directly activate APC through Toll-like receptors, resulting in up-regulation of costimulatory molecules necessary for the efficient priming of naive T cells. It remains unclear what role CD4 T cell help and various costimulation pathways play in the development of CD8 T cell responses during bacterial infection. In this study, we examined these questions using an intracellular bacterium, Listeria monocytogenes, as a model of infection. In CD4 T cell-depleted, CD4(-/-), and MHC class II(-/-) mice, L. monocytogenes infection induced CD8 T cell activation and primed epitope-specific CD8 T cells to levels commensurate with those in normal C57BL/6 mice. Furthermore, these epitope-specific CD8 T cells established long-term memory in CD4(-/-) mice that was capable of mounting a protective recall response. In vitro analysis showed that L. monocytogenes directly stimulated the activation and maturation of murine dendritic cells. The CD8 T cell response to L. monocytogenes was normal in CD40L(-/-) mice but defective in CD28(-/-) and CD137L(-/-) mice. These data show that in situations where infectious agents or immunogens can directly activate APC, CD8 T cell responses are less dependent on CD4 T cell help via the CD40-CD40L pathway but involve costimulation through CD137-CD137L and B7-CD28 interactions.     

Persistence and turnover of antigen-specific CD4 T cells during chronic tuberculosis infection in the mouse

CD4 T cells are critical for resistance to Mycobacterium tuberculosis infection, but how effective T cell responses are maintained during chronic infection is not well understood. To address this question we examined the CD4 T cell response to a peptide from ESAT-6 during tuberculosis infection in the mouse. The ESAT-6(1-20)/IA(b)-specific CD4 T cell response in the lungs, mediastinal lymph nodes, and spleen reached maxima 3-4 wk postinfection, when the bacteria came under the control of the immune response. Once chronic infection was established, the relative frequencies of Ag-specific CD4 T cells were maintained at nearly constant levels for at least 160 days. ESAT-6(1-20)/IA(b)-specific CD4 T cells that responded in vitro expressed activation markers characteristic of chronically activated effector cells and used a limited Vbeta repertoire that was clonally stable in vivo for at least 12 wk. 5-Bromo-2-deoxyuridine incorporation studies indicated a relatively high rate of cell division among both total CD4 and ESAT-6(1-20)/IA(b)-specific CD4 T cells during acute infection, but the degree of 5-bromo-2-deoxyuridine incorporation by both the CD4 T cells and the Ag-specific cells declined at least 3-fold during chronic infection. The data indicate that the peripheral ESAT-6(1-20)/IA(b)-specific CD4 T cell response to M. tuberculosis is characterized during the acute phase of infection by a period of extensive proliferation, but once bacterial control is achieved, this is followed during chronic infection by an extended containment phase that is associated with a persistent response of activated, yet more slowly proliferating, T cells.     

Lung neutrophils facilitate activation of naive antigen-specific CD4+ T cells during Mycobacterium tuberculosis infection

Initiation of the adaptive immune response to Mycobacterium tuberculosis occurs in the lung-draining mediastinal lymph node and requires transport of M. tuberculosis by migratory dendritic cells (DCs) to the local lymph node. The previously published observations that 1) neutrophils are a transiently prominent population of M. tuberculosis-infected cells in the lungs early in infection and 2) that the peak of infected neutrophils immediately precedes the peak of infected DCs in the lungs prompted us to characterize the role of neutrophils in the initiation of adaptive immune responses to M. tuberculosis. We found that, although depletion of neutrophils in vivo increased the frequency of M. tuberculosis-infected DCs in the lungs, it decreased trafficking of DCs to the mediastinal lymph node. This resulted in delayed activation (CD69 expression) and proliferation of naive M. tuberculosis Ag85B-specific CD4 T cells in the mediastinal lymph node. To further characterize the role of neutrophils in DC migration, we used a Transwell chemotaxis system and found that DCs that were directly infected by M. tuberculosis migrated poorly in response to CCL19, an agonist for the chemokine receptor CCR7. In contrast, DCs that had acquired M. tuberculosis through uptake of infected neutrophils exhibited unimpaired migration. These results revealed a mechanism wherein neutrophils promote adaptive immune responses to M. tuberculosis by delivering M. tuberculosis to DCs in a form that makes DCs more effective initiators of naive CD4 T cell activation. These observations provide insight into a mechanism for neutrophils to facilitate initiation of adaptive immune responses in tuberculosis.     

TGF-beta 1 regulates antigen-specific CD4+ T cell responses in the periphery

T cell expansion typically is due to cognate interactions with specific Ag, although T cells can be experimentally activated through bystander mechanisms not involving specific Ag. TGF-beta1 knockout mice exhibit a striking expansion of CD4+ T cells in the liver by 11 days of age, accompanied by CD4+T cell-dependent necroinflammatory liver disease. To examine whether hepatic CD4+T cell expansion in TGF-beta1(-/-) mice is due to cognate TCR-peptide interactions, we used spectratype analysis to examine the diversity in TCR Vbeta repertoires in peripheral CD4+T cells. We reasoned that Ag-nonspecific T cell responses would yield spectratype profiles similar to those derived from control polyclonal T cell populations, whereas Ag-specific T cell responses would yield perturbed spectratype profiles. Spleen and liver CD4+T cells from 11-day-old TGF-beta1(-/-) mice characteristically exhibited highly perturbed nonpolyclonal distributions of TCR Vbeta CDR3 lengths, indicative of Ag-driven T cell responses. We quantitatively assessed spectratype perturbation to derive a spectratype complexity score. Spectratype complexity scores were considerably higher for TGF-beta1(-/-) CD4+ T cells than for TGF-beta1(+/-) CD4+T cells. TCR repertoire perturbations were apparent as early as postnatal day 3 and preceded both hepatic T cell expansion and liver damage. By contrast, TGF-beta1(-/-) CD4+ single-positive thymocytes from 11-day-old mice exhibited normal unbiased spectratype profiles. These results indicate that CD4+ T cells in TGF-beta1(-/-) mice are activated by and respond to self-Ags present in the periphery, and define a key role for TGF-beta1 in the peripheral regulation of Ag-specific CD4+ T cell responses.     

NKT cells inhibit antigen-specific effector CD8 T cell induction to skin viral proteins

We recently demonstrated that CD1d-restricted NKT cells resident in skin can inhibit CD8 T cell-mediated graft rejection of human papillomavirus E7-expressing skin through an IFN-γ-dependent mechanism. In this study, we examined the role of systemically derived NKT cells in regulating the rejection of skin grafts expressing viral proteins. In lymph nodes draining transplanted skin, Ag-specific CD8 T cell proliferation, cytokine production, and cytotoxic activity were impaired by NKT cells. NKT cell suppression was mediated via CD11c(+) dendritic cells. Inhibition of CD8 T cell function did not require Foxp3(+) regulatory T cells or NKT cell-secreted IFN-γ, IL-10, or IL-17. Thus, following skin grafting or immunization with human papillomavirus-E7 oncoprotein, NKT cells reduce the capacity of draining lymph node-resident APCs to cross-present Ag to CD8 T cell precursors, as evidenced by impaired expansion and differentiation to Ag-specific CD8 T effector cells. Therefore, in the context of viral Ag challenge in the skin, systemic NKT cells limit the capacity for effective priming of adaptive immunity.     

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.     

Regulatory NK cells suppress antigen-specific T cell responses

The immune system has a variety of regulatory/suppressive processes, which are decisive for the development of a healthy or an allergic immune response to allergens. NK1 and NK2 subsets have been demonstrated to display counterregulatory and provocative roles in immune responses, similar to Th1 and Th2 cells. T regulatory cells suppressing both Th1 and Th2 responses have been the focus of intensive research during the last decade. In this study, we aimed to investigate regulatory NK cells in humans, by characterization of NK cell subsets according to their IL-10 secretion property. Freshly purified IL-10-secreting NK cells expressed up to 40-fold increase in IL-10, but not in the FoxP3 and TGF-beta mRNAs. PHA and IL-2 stimulation as well as vitamin D3/dexamethasone and anti-CD2/CD16 mAbs are demonstrated to induce IL-10 expression in NK cells. The effect of IL-10+ NK cells on Ag-specific T cell proliferation has been examined in bee venom major allergen, phospholipase A2- and purified protein derivative of Mycobecterium bovis-induced T cell proliferation. IL-10+ NK cells significantly suppressed both allergen/Ag-induced T cell proliferation and secretion of IL-13 and IFN-gamma, particularly due to secreted IL-10 as demonstrated by blocking of the IL-10 receptor. These results demonstrate that a distinct small fraction of NK cells display regulatory functions in humans.     

Characterization of CD4+ T cell responses during natural infection with Salmonella typhimurium

CD4+ T cells are important for resistance to infection with Salmonella typhimurium. However, the Ag specificity of this T cell response is unknown. Here, we demonstrate that a significant fraction of Salmonella-specific CD4+ T cells respond to the flagellar filament protein, FliC, and that this Ag has the capacity to protect naive mice from lethal Salmonella infection. To characterize this Ag-specific response further, we generated FliC-specific CD4+ T cell clones from mice that had resolved infection with an attenuated strain of Salmonella. These clones were found to respond to an epitope from a constant region of FliC, enabling them to cross-react with flagellar proteins expressed by a number of distinct Salmonella serovars.     

Ablation of CD8 and CD4 T cell responses by high viral loads

To evaluate the impact of sustained viral loads on anti-viral T cell responses we compared responses that cleared acute lymphocytic choriomeningitis virus infection with those that were elicited but could not resolve chronic infection. During acute infection, as replicating virus was cleared, CD8 T cell responses were down-regulated, and a pool of resting memory cells developed. In chronically infected hosts, the failure to control the infection was associated with pronounced and prolonged activation of virus-specific CD8 T cells. Nevertheless, there was a progressive diminution of their effector activities as their capacity to produce first IL-2, then TNF-alpha, and finally IFN-gamma was lost. Chronic lymphocytic choriomeningitis virus infection was also associated with differential contraction of certain CD8 T cell responses, resulting in altered immunodominance. However, this altered immunodominance was not due to selective expansion of T cells expressing particular TCR Vbeta segments during chronic infection. High viral loads were not only associated with the ablation of CD8 T cell responses, but also with impaired production of IL-2 by virus-specific CD4 T cells. Taken together, our data show that sustained exposure to high viral loads results in the progressive functional inactivation of virus-specific T cell responses, which may further promote virus persistence.     

CD4 T cells promote CD8 T cell immunity at the priming and effector site during viral encephalitis

CD4 T cell activation during peripheral infections not only is essential in inducing protective CD8 T cell memory but also promotes CD8 T cell function and survival. However, the contributions of CD4 T cell help to antiviral CD8 T cell immunity during central nervous system (CNS) infection are not well established. Encephalitis induced by the sublethal coronavirus JHMV was used to identify when CD4 T cells regulate CD8 T cell responses following CNS infection. Peripheral expansion of virus-specific CD8 T cells was impaired when CD4 T cells were ablated prior to infection but not at 4 days postinfection. Delayed CD4 T cell depletion abrogated CD4 T cell recruitment to the CNS but only slightly diminished CD8 T cell recruitment. Nevertheless, the absence of CNS CD4 T cells was associated with reduced gamma interferon (IFN-γ) and granzyme B expression by infiltrating CD8 T cells, increased CD8 T cell apoptosis, and impaired control of infectious virus. CD4 T cell depletion subsequent to CD4 T cell CNS migration restored CD8 T cell activity and virus control. Analysis of γc-dependent cytokine expression indicated interleukin-21 (IL-21) as a primary candidate optimizing CD8 T cell activity within the CNS. These results demonstrate that CD4 T cells play critical roles in both enhancing peripheral activation of CD8 T cells and prolonging their antiviral function within the CNS. The data highlight the necessity for temporally and spatially distinct CD4 T cell helper functions in sustaining CD8 T cell activity during CNS infection.     

MHC-based detection of antigen-specific CD8+ T cell responses

The hallmark of adaptive immunity is its ability to recognise a wide range of antigens and technologies that capture this diversity are therefore of substantial interest. New methods have recently been developed that allow the parallel analysis of T cell reactivity against vast numbers of different epitopes in limited biological material. These technologies are based on the joint binding of differentially labelled MHC multimers on the T cell surface, thereby providing each antigen-specific T cell population with a unique multicolour code. This strategy of ‘combinatorial encoding’ enables detection of many (at least 25) different T cell populations per sample and should be of broad value for both T cell epitope identification and immunomonitoring.     

IL-17 production is dominated by gammadelta T cells rather than CD4 T cells during Mycobacterium tuberculosis infection

IL-17 is a cytokine produced by T cells in response to IL-23. Recent data support a new subset of CD4 Th cells distinct from Th1 or Th2 cells that produce IL-17 and may contribute to inflammation. In this study, we demonstrate that, in naive mice, as well as during Mycobacterium tuberculosis infection, IL-17 production is primarily from gammadelta T cells and other non-CD4(+)CD8(+) cells, rather than CD4 T cells. The production of IL-17 by these cells is stimulated by IL-23 alone, and strongly induced by the cytokines, including IL-23, produced by M. tuberculosis-infected dendritic cells. IL-23 is present in the lungs early in infection and the IL-17-producing cells, such as gammadelta T cells, may represent a central innate protective response to pulmonary infection.     

CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection

Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4(+) T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4(+) T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4(+) T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4(+) T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4(+) T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis.