Quantitative assessment of the functional plasticity of memory CD8(+) T cells.

While the functional plasticity of memory CD4(+) T cells has been studied extensively, less is known about this property in memory CD8(+) T cells. Here, we report the direct measurement of plasticity by paired daughter analysis of effector and memory OT-I CD8(+) T cells primed in vivo with ovalbumin. Naïve, effector, and memory OT-I cells were isolated and activated in single-cell culture; then, after the first division, their daughter cells were transferred to new cultures with and without IL-4; expression of IFN-γ and IL-4 mRNAs was measured 5 days later in the resultant subclones. Approximately 40% of clonogenic memory CD8(+) T cells were bipotential in this assay, giving rise to an IL-4(-) subclone in the absence of IL-4 and an IL-4(+) subclone in the presence of IL-4. The frequency of bipotential cells was lower among memory cells than naïve cells but markedly higher than among 8-day effectors. Separation based on high or low expression of CD62L, CD122, CD127, or Ly6C did not identify a phenotypic marker of the bipotential cells. Functional plasticity in memory CD8(+) T-cell populations can therefore reflect modulation at the level of a single memory cell and its progeny.

Interleukin-4-induced loss of CD8 expression and cytolytic function in effector CD8 T cells persists long term in vivo.

Activation of naive CD8(+) T cells in the presence of interleukin-4 modulates their CD8 co-receptor expression and functional differentiation, resulting in the generation of CD8(low) cells that produce type 2 cytokines and display poor cytolytic and anti-tumour activity. Although this CD8(low) phenotype becomes stable after about a week and can persist with further stimulation in vitro, it is not known whether it can be maintained long term in vivo. Here we report that CD8(low) cells derived from oval-bumin(257-264) -specific T-cell receptor-transgenic CD8(+) T cells activated in the presence of interleukin-4 could be detected in the spleen for at least 4 months after adoptive transfer into normal mice. A significant proportion of the long-term surviving cells retained their CD8(low) phenotype in vivo and after clonal re-activation in vitro. Although long-term surviving CD8(low) cells lacked detectable cytolytic activity or perforin expression, they showed some anti-tumour function in vivo. The persistence of functional cells with a CD8(low) phenotype in vivo raises the possibility that such cells can contribute to effector or regulatory responses to tumours or pathogens.

IFN-gamma inhibits IL-4-induced type 2 cytokine expression by CD8 T cells in vivo and modulates the anti-tumor response.

Activation of naive CD8 T cells in vitro in the presence of IL-4 induces type 2 cytokine expression, loss of CD8 expression, and reduced cytolytic potential. This represents a major shift from the canonical phenotype of effector CD8 T cells. It has not been established, however, whether IL-4 can induce comprehensive type 2 cytokine expression by CD8 T cells in vivo, nor whether the effects of IL-4 on type 2 cytokine production by CD8 T cells can be inhibited by IFN-gamma. Furthermore, disparate results have been reported regarding the anti-tumor ability of type 2 polarized effector CD8 T cells, and the effects of IFN-gamma in this respect remain unknown. To address these questions, wild-type or IFN-gamma-deficient OVA-specific CD8(+) T cells were activated in RAG-2(-/-) gamma c(-/-) recipients with control or IL-4-expressing OVA(+) tumor cells, and then transferred to secondary recipients for tumor challenge. Tumor-derived IL-4 induced the expression of type 2 cytokines and the transcription factor GATA-3 by responding CD8 T cells while reducing their CD8 coreceptor expression and ability to eliminate a secondary tumor challenge. Each of these effects of IL-4 was exaggerated in IFN-gamma-deficient, compared with wild-type, CD8 T cells. The results demonstrate that endogenous IFN-gamma counteracts the induction of type 2 cytokines and the downregulation of both CD8 coreceptor levels and the anti-tumor response in CD8 T cells exposed to IL-4 during activation in vivo. These findings may explain the anomalies in the reported functional phenotype of type 2 polarized CD8 T cells.

Interferon-gamma and interleukin-4 reciprocally regulate CD8 expression in CD8+ T cells.

The CD8 co-receptor can modulate CD8(+) T cell function through its contributions to T cell receptor (TCR) binding and signaling. Here we show that IFN-gamma and IL-4 exert opposing effects on the expression of CD8alpha mRNA and surface CD8 protein during CD8(+) T cell activation. IL-4 caused down-regulation of surface CD8 on ovalbumin (OVA)(257-264)-specific TCR-transgenic OT-I CD8(+) T cells activated with OVA(257-264)-coated antigen presenting cells or polyclonal stimuli, and on wild type CD8(+) T cells activated with polyclonal stimuli. This effect was enhanced in each case when the cells lacked a functional IFN-gamma or IFN-gamma R gene. When WT or IFN-gamma-deficient OT-I CD8(+) T cells were analyzed 9 days after co-injection with control or IL-4-expressing OVA(+) tumor cells into RAG-2(-/-)gamma c(-/-) mice, CD8 levels were highest on WT donor cells from mice that received the control tumor and lowest on IFN-gamma-deficient donor cells from mice that received the IL-4-expressing tumor. The latter CD8(low) cells displayed markedly impaired binding of OVA(257-264)/MHC tetramers and peptide/MHC-dependent degranulation. The data reveal an unexpected role for IFN-gamma in tuning the CD8 co-receptor during primary CD8(+) T cell activation both in vitro and in vivo.

Echinococcus granulosus: induction of T-independent antibody response against protoscolex glycoconjugates in early experimental infection.

In this work CD4-knockout mice were used as a model to analyse the role of CD4+ T cells in the antibody response against Echinococcus granulosus immunization or experimental infection. Results obtained with mice immunized with protoscolex antigens indicated that these contain T-independent antigens. After infection, CD4-knockout mice and C57Bl/6 mice showed similar titres of specific antibodies indicating that T-independent antibody production was quantitatively important in early infection. We have also identified an antigenic fraction from protoscoleces (E4+) which induces CD4 T cell independent antibody response in early stages of infection. In conclusion, the results presented here directly support the existence of T-independent immunogens in E. granulosus protoscoleces and suggest that T-independent antibody response may be quantitatively important in early infection.

Branched and linear lipopeptide vaccines have different effects on primary CD4+ and CD8+ T-cell activation but induce similar tumor-protective memory CD8+ T-cell responses.

We compared murine T-cell responses to synthetic lipopeptide vaccines in which the TLR2 ligand Pam(2)Cys was attached to co-linear CD4+ and CD8+ T-cell epitopes of ovalbumin (OVA) in a linear or branched configuration. Mice received OVA-specific transgenic CD8+ and CD4+ T-cells followed by one injection of vaccine. Although the branched lipopeptide was more potent in activating OVA-specific CD4+ and CD8+ T-cells in the primary response, both vaccines induced cytolytic T lymphocytes (CTL) that expressed perforin, granzyme A-C, and IFN-gamma mRNAs and conferred long-term protection of most mice against challenge with OVA-expressing tumor cells. OVA epitope display was reduced in tumors that developed in some mice, suggesting CD8+ T-cell dependent selection.

The relevance of cytokines for development of protective immunity and rational design of vaccines.

Cytokines are key regulators of the immune system that shape innate and adaptive immune responses. An adequate balance of the cytokine environment is critical to achieve protective immunity and to avoid immunopathology. Present knowledge allows a deeper understanding of the cytokine network and their sometimes conflicting roles in the development of immune responses, as well as their relevance in the establishment and maintenance of immunological memory. New insights have been gained into the role of different T cell subsets for protection against infection or tumor growth. The incorporation of cytokines as molecular adjuvants in vaccines has been attempted to strengthen vaccine-induced immune responses, and as a rational approach to modulate cytokine milieu in vivo and tailor host immunity for specific situations. These approaches have been tried in experimental models and veterinary species, and a few of them have entered into clinical trials. However, manipulating the cytokine network to modulate immune responses is not a simple task, because cytokine functions are complex and the final effects on the immune response will depend on timing and length of exposure, cell(s) targeted and other cytokines present in the same microenvironment. Here, we will review our present understanding on the role of cytokines in the development of effector and memory T cell responses. Also the potential use of cytokines as molecular adjuvant for vaccines against infectious diseases and cancer will be revised.

Complexity and function of cytokine responses in experimental infection by Echinococcus granulosus.

Cytokines are important in the regulation of the immune system and are secreted by a variety of cells in response to self and non-self stimuli. Communication within cells, in the same or distant anatomical sites, occurs via cytokines which determine the quality and intensity of inflammatory and adaptive immune responses. Infection by helminths is characterized by a dominant secretion of type-2 cytokines; IL-4, IL-5, IL-10 (among others), which down-regulates the induction and functions of type-1 cytokines. The molecular mechanisms involved in the polarization of type-2 responses and their biological significance in helminthic infections are unknown, and probably depends on each host-parasite system. Understanding these issues may contribute to immune therapy against parasitic infections. Here we summarize our data obtained in Echinococcus granulosus experimental infection regarding type-2 cytokine induction and its putative role in the host-parasite interaction. Results suggest that induction of cytokine responses at different stages of infection is complex and depends on several parameters. In addition, they support the hypothesis that early IL-10, secreted by B cells in response to non-proteic antigens, may favour parasite survival and the establishment of a polarized type-2 cytokine response.

Memory cytolytic T-lymphocytes: induction, regulation and implications for vaccine design.

The design of vaccines that protect against intracellular infections or cancer remains a challenge. In many cases, immunity depends on the development of antigen-specific memory CD8+ T-cells that can express cytokines and kill antigen-bearing cells when they encounter the pathogen or tumor. Here, the authors review current understanding of the signals and cells that lead to memory CD8+ T-cell differentiation, the relationship between the primary CD8+ T-cell response and the memory response and the regulation of memory CD8+ T-cell survival and function. The implications of this new knowledge for vaccine design are discussed, and recent progress in the development of lipidated peptide vaccines as a promising approach for vaccination against intracellular infections and cancer is reviewed.

Progressive differentiation and commitment of CD8+ T cells to a poorly cytolytic CD8low phenotype in the presence of IL-4.

Exposure to IL-4 during activation of naive murine CD8+ T cells leads to generation of IL-4-producing effector cells with reduced surface CD8, low perforin, granzyme B and granzyme C mRNA, and poor cytolytic function. We show in this study that maximal development of these cells depended on exposure to IL-4 for the first 5 days of activation. Although IL-4 was not required at later times, CD8 T cell clones continued to lose surface CD8 expression with prolonged culture, suggesting commitment to the CD8low phenotype. This state was reversible in early differentiation. When single CD8low cells from 4-day cultures were cultured without IL-4, 65% gave rise to clones that partly or wholly comprised CD8high cells; the proportion of reverted clones was reduced or increased when the cells were cloned in the presence of IL-4 or anti-IL-4 Ab, respectively. CD8 expression positively correlated with perforin and granzyme A, B, and C mRNA, and negatively correlated with IL-4 mRNA levels among these clones. By contrast, most CD8low cells isolated at later time points maintained their phenotype, produced IL-4, and exhibited poor cytolytic function after many weeks in the absence of exogenous IL-4. We conclude that IL-4-dependent down-regulation of CD8 is associated with progressive differentiation and commitment to yield IL-4-producing cells with little cytolytic activity. These data suggest that the CD4-CD8- cells identified in some disease states may be the product of a previously unrecognized pathway of effector differentiation from conventional CD8+ T cells.

Profiling the CD8low phenotype, an alternative career choice for CD8 T cells during primary differentiation.

A CD8+ T cell of naive phenotype has multiple career choices during its primary differentiation into an effector cell population. One of these career options is becoming a CD8low T cell. We have previously shown by in vitro studies that CD8low T cells have lost expression of CD8 surface protein and mRNA and are poorly cytolytic. In line with poor cytolytic function, CD8low T cells express low levels of perforin and granzyme B and C, mediators of the granule-exocytosis machinery. However, CD8low T cells express IFN-gamma and substantial amounts of IL-4, the signature cytokines of type 1 and type 2 T-cell polarization, respectively. Here, we argue that the CD8low phenotype is an alternative career choice for any naive CD8+ T cell during primary activation but that the probability of choosing this option is greatly enhanced by both IL-4 and strong activation conditions. CD8low T cells have downregulated CD8 alpha/beta heterodimers and no preferential CD8 alpha/alpha homodimer expression. As shown by anti-CD8 Ab blocking experiments, surface CD8 substantially contributes to the CD8 T cell's effector function (i.e. cytokine expression and cytolytic activity). The distinct effector profile of CD8low T cells gives an example of the complexity of different CD8 T cell careers during primary effector differentiation.

Isolation and characterization of a secretory component of Echinococcus multilocularis metacestodes potentially involved in modulating the host-parasite interface.

Echinococcus multilocularis metacestodes are fluid-filled, vesicle-like organisms, which are characterized by continuous asexual proliferation via external budding of daughter vesicles, predominantly in the livers of infected individuals. Tumor-like growth eventually leads to the disease alveolar echinococcosis (AE). We employed the monoclonal antibody (MAb) E492/G1, previously shown to be directed against a carbohydrate-rich, immunomodulatory fraction of Echinococcus granulosus, to characterize potentially related components in E. multilocularis. Immunofluorescence studies demonstrated that MAb E492/G1-reactive epitopes were found predominantly on the laminated layer and in the periphery of developing brood capsules. The respective molecules were continuously released into the exterior medium and were also found in the parasite vesicle fluid. The MAb E492/G1-reactive fraction in E. multilocularis, named Em492 antigen, was isolated by immunoaffinity chromatography. Em492 antigen had a protein/carbohydrate ratio of 0.25, reacted with a series of lectins, and is related to the laminated layer-associated Em2(G11) antigen. The epitope recognized by MAb E492/G1 was sensitive to sodium periodate but was not affected by protease treatment. Anti-Em492 immunoglobulin G1 (IgG1) and IgG2 and, at lower levels, IgG3 were found in sera of mice suffering from experimentally induced secondary, but not primary, AE. However, with regard to cellular immunity, a suppressive effect on concanavalin A- or crude parasite extract-induced splenocyte proliferation in these mice was observed upon addition of Em492 antigen, but trypan blue exclusion tests and transmission electron microscopy failed to reveal any cytotoxic effect in Em492 antigen-treated spleen cells. This indicated that Em492 antigen could be modulating the periparasitic cellular environment during E. multilocularis infection through as yet unidentified mechanisms and could be one of the factors contributing to immunosuppressive events that occur at the host-parasite interface.

Cytokine response and outcome of infection depends on the infective dose of parasites in experimental infection by Echinococcus granulosus.

We here analysed whether the cytokine responses in early and late experimental infection with Echinococcus granulosus depend on the dose of parasites to which the host is exposed. To this purpose Balb/c mice were inoculated intraperitoneally (i.p.) with either 500 or 2000 protoscoleces. Splenocytes of mice were obtained at days 3, 7, 14 and 21 and also on week 37 post-infection and cultured in vitro with protoscolex antigens. Type-1 and type-2 cytokines were analysed in supernatants by ELISA. Results showed that the inoculation of 500 protoscoleces induced an early type-0 and a late type-2 cytokine response, whereas the inoculation of 2000 protoscoleces induced an early type-2 and a late type-0 cytokine response. Parasite growth was lower in the group inoculated with the low infective dose. These results indicate that the cytokine response during the infection by the helminth E. granulosus depends on the dose of parasites to which the host has been exposed.

Towards new immunotherapies: targeting recombinant cytokines to the immune system using live attenuated Salmonella.

We have used Salmonella as a delivery system for eukaryotic expression plasmids encoding cytokines, and assessed its capacity to modulate immune responses in different experimental models. Plasmids encoding mouse IL-4 and IL-18 under cytomegalovirus promoter were constructed and transformed into live attenuated Salmonella enterica serovar Typhi strain CVD 908-htrA, and Salmonella enterica serovar Typhimurium strain SL3261. We have shown that systemic as well as mucosal immunization with such constructs can influence the antibody and cytokine responses to the Salmonella carrier and to co-administered bystander antigens, as well as the specific immune response elicited during a parasitic infection. Further, we have shown that oral cytokine-therapy using Salmonella as gene vector induce antitumoral effect as demonstrated by extended survival time in melanoma-bearing mice. This approach may be particularly suited for the development of new immunotherapies with applications in parasitic infections and cancer, were alterations of the host's immune responses are usually found, and therapy-induced modulation of the immune response is likely to be required.