Activation of human dendritic cells following infection with Mycobacterium tuberculosis

Dendritic cells (DC) play an essential role in the initiation of primary T cell responses to foreign Ag. It is likely that these potent APC are critical in the initiation of immune responses to pathogens, such as bacteria or parasites. However, little is known about the interaction of these important APC with pathogens. To address this issue, the interaction of the bacterium Mycobacterium tuberculosis with human DC was studied. DC generated from human peripheral blood by short term culture in medium containing recombinant human cytokines granulocyte-macrophage-CSF and IL-4 were capable of phagocytosing M. tuberculosis. Infection of DC with live M. tuberculosis bacilli resulted in increased APC surface expression of the costimulatory molecules CD54, CD40, and B7.1, as well as MHC class I molecules. In addition, infected DC secreted elevated levels of inflammatory cytokines, including TNF-alpha, IL-1, and IL-12. M. tuberculosis-infected human monocytes also secreted inflammatory cytokines, but exhibited no enhancement of costimulatory or MHC class I molecule expression. These data indicate that infection with M. tuberculosis results in the direct activation and maturation of these DC. In vivo, such activation may facilitate migration to the lymph nodes, and enhance presentation of Ag to T cells, thereby facilitating the induction of the immune response against this pathogen.     

Complement opsonization is required for presentation of immune complexes by resting peripheral blood B cells

Complement receptor 2 (CD21, CR2) is a B cell receptor for complement degradation products bound to Ag or immune complexes. The role of CD21 in mediating Ag presentation of soluble immune complexes by resting B cells was studied. Complement-coated immune complexes were formed by the incubation of influenza virus with serum from immune donors. These complexes bound to peripheral blood B cells in a complement-dependent manner. The binding required CD21 or, to a lesser extent, complement receptor 1 (CR1, CD35). B cells pulsed with immune complexes containing complement elicited a response from a panel of influenza-specific T cell clones, while those pulsed with immune complexes formed in the absence of complement did not. The expression of the early activation marker CD69 and the costimulatory molecule CD86 were not induced by CD21 ligation alone, suggesting that CD21-mediated Ag presentation occurs independently of B cell activation. Up-regulation of these markers required exposure to T cell factors elicited by the recognition of Ag derived from complement-containing immune complexes. These findings suggest that binding of Ag to CD21 enables Ag-nonspecific B cells to participate in the activation of Ag-specific T cells in a process that occurs independently of well-characterized B cell activation events.     

Partial activation of naive CD4 T cells and tolerance induction in response to peptide presented by resting B cells

Tolerance is thought to occur when Ag is presented to T cells in the absence of costimulatory interactions from APC accessory molecules. Of the professional APC, the resting B cell may be the main tolerizing cell in vivo. We have analyzed several aspects of activation of naive transgenic CD4 cells stimulated with resting or activated B cells presenting peptide Ag. Similar results were obtained with stimulation from peptide presenting fibroblast APC lacking or expressing B7-1 with intracellular adhesion molecule-1. TCR ligation with little or no accessory molecule coreceptor engagement induced efficient blastogenesis; up-regulation of CD25, CD44, CD69, CD95 and CD71; and down-regulation of CD62L over a 48-h period. Accessory molecule help enhanced the expression of CD25, CD44, CD69, and CD71, but to very modest degrees. Only two molecules, CD40 ligand and IL-2, were found to be extremely dependent on accessory molecule help, with little or no expression evident with peptide presented on resting B cells or class II-positive fibroblasts. T cells induced on resting B cells expanded minimally over 3 days, and this was followed by extensive cell death and hyporesponsiveness of the resulting cells. These studies suggest that under tolerizing conditions, such as Ag presentation by resting B cells, much of the naive CD4 response is induced efficiently. Partial activation, however, may be the overall result due to the lack of CD40 ligand expression, which may regulate costimulatory activity in APC and, in turn, may contribute to limiting the production of IL-2 required for T cell expansion and survival.     

Normal human CD4+ memory T cells display broad heterogeneity in their activation threshold for cytokine synthesis

CD4+ memory T cells coordinate immune responses against viruses and other pathogens via the Ag-induced secretion of potent effector cytokines. The efficacy of these responses depends on both the overall number of pathogen-specific memory T cells and the particular array of cytokines that these cells are programmed to secrete. Here, we provide evidence that heterogeneity in Ag triggering thresholds constitutes an additional critical determinant of memory T cell function. Using a novel assay that allows single-cell detection of Ag-specific T cell cytokine production, we demonstrate that CMV-specific CD4+ memory cells from human peripheral blood display pronounced differences in their costimulatory requirements for Ag-induced triggering of IFN-gamma and IL-2 secretion, ranging from cells that trigger with little costimulation (e.g., resting APC alone) to cells requiring potent costimulation through multiple pathways (resting APC plus multiple costimulatory mAbs, or activated APC). These differences in costimulatory requirements are independent of clonal differences in TCR signaling intensity, consistent with an intrinsic activation-threshold heterogeneity that is "downstream" from the TCR. Thus, "effective" frequencies of Ag-specific CD4+ memory T cells appear to depend on the activation status of available APC, a dependence that would allow the immune system to rapidly adjust the number of functional Ag-specific memory T cells in a particular effector site according to local conditions.     

Activation of human CD8+ alpha beta TCR+ cells by Mycobacterium tuberculosis via an alternate class I MHC antigen-processing pathway

Human immune responses to M. tuberculosis are characterized by activation of multiple T cell subsets including CD4+, CD8+, and gammadelta T cells, and the role of CD8+ alphabeta TCR+ T cells in this response is poorly understood. Stimulation of T cells from healthy tuberculin skin test-positive persons with live M. tuberculosis-H37Ra or soluble M. tuberculosis Ags readily up-regulated IL-2Ralpha (CD25) expression on CD8+ T cells. Purified resting and activated CD8+ T cells produced IFN-gamma and proliferated to both M. tuberculosis bacilli and soluble mycobacterial Ags with monocytes as APC. Precursor frequency of mycobacterial Ag-specific CD8+ T cells by IFN-gamma enzyme-linked immunospot was 5-10-fold lower than the precursor frequency of CD4+ T cells, and IFN-gamma secretion by CD8+ T cells was 50-100-fold lower. CD8+ T cells secreted approximately 10-fold less IFN-gamma per cell than CD4+ T cells in response to mycobacterial Ags. CD8+ T cell responses to M. tuberculosis bacilli were blocked by anti-MHC class I antibody and required Ag processing. Processing of M. tuberculosis bacilli by monocytes for presentation to MHC class I-restricted CD8+ T cells was insensitive to brefeldin A treatment, which blocks the conventional MHC class I Ag-processing pathway. These results represent the first demonstration that human cells can process pathogen Ags via an alternate Ag-processing pathway for MHC class I and suggest a mechanism for participation of IFN-gamma-secreting CD8+ T cells in the human immune responses to M. tuberculosis.     

Modulation of T cell proliferative response by accessory cell interactions

Antigen-specific activation of the T cell is accomplished by engagement of the T cell receptor (TCR) by an antigen (Ag)/MHC complex presented on the surface of an antigen- presenting cell (APC). However, it has been demonstrated that engagement of the TCR by Ag/HC complexes alone is normally insufficient to lead to a proliferative response and the development of effector function. Thus it has been proposed that the APC also provides additional signals which serve to modulate the T cell's response. These second or costimulatory signals are thought to be critical in the generation of a T cell-driven immune response. Several receptors have been proposed to be capable of serving as costimulatory receptors. Candidate molecules include CD28 and LFA-1 as well as other receptors. In this review the studies that we have performed to clarify the role of both LFA-1 and CD28 in providing costimulatory activity for T cell activation are discussed. In addition, we present evidence that under certain conditions, TCR signalling alone can be sufficient to lead to T cell proliferation.     

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Nonionic triblock copolymers are relatively nontoxic adjuvants that induce high-titer, long-lasting antibody responses. We have previously shown that these adjuvants also induce cell-mediated immunity including lymphokine production by CD4(+) T cells and cytolytic responses by CD8(+) T cells. These copolymers are thought to modulate hydrophobic adhesive interactions between antigens (Ag) and lymphoid cells. We sought to test the hypothesis that copolymers facilitate uptake of exogenous Ag by antigen-presenting cells (APC) using an in vitro model system. Our data show that nonionic triblock copolymers enhanced presentation of soluble ovalbumin (OVA) to the major histocompatibility complex (MHC) class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells, respectively. Presentation of OVA via the class I pathway was enhanced by copolymers in both phagocytic and nonphagocytic APC. However, copolymers did not enhance binding of peptides to the MHC molecules on APC, presentation of endogenously synthesized Ag, or presentation of exogenous Ag delivered by electroporation. These results provide additional evidence that these nonionic triblock copolymers can serve as powerful adjuvants for augmenting both humoral and cell-mediated immunity to protein Ag.     

A mutant antigen-presenting cell defective in antigen presentation expresses class II MHC molecules with an altered conformation

Ag presentation by APC to class II MHC-restricted T cells involves a sequence of events: 1) intracellular processing of protein Ag into immunogenic peptides, 2) specific binding of peptides to class II MHC molecules, and then 3) transport of the MHC-peptide complexes to the plasma membrane. The critical event in the activation of T cells by APC is the recognition of MHC-associated antigenic determinants by the TCR/CD3 complex. In this report we describe the isolation and characterization of a mutant APC with a defect in an intracellular process that results in its inability to form MHC-peptide complexes for recognition by T cells. The mutant APC cannot present many different protein Ag with both I-A and I-E molecules but is able to present processing-independent peptides. The functional defect in the mutant APC is not caused by either a decrease in expression or a structural mutation in class II MHC molecules. Further, there is no mutation in the invariant chain (li) and it displays a normal kinetics of association and dissociation from the class II MHC molecules during biosynthesis. Although the mutation is not in the genes encoding for the class II MHC molecules or li, the mutant APC expresses class II MHC molecules with distinct serological epitopes suggestive of an altered conformation. Pulse-chase experiments suggest that a conformational difference between I-Ad molecules of wild-type and mutant cells occurs after the class II molecules exit from the endoplasmic reticulum but while they are still associated with li. The mutant cell produces few compact (SDS-resistant) class II heterodimers. This mutant APC provides a tool for studying the cell biology of Ag processing and presentation.     

MHC class II-transfected tumor cells directly present antigen to tumor-specific CD4+ T lymphocytes

We have developed and shown to be efficacious an immunotherapeutic strategy to enhance the generation of tumor-specific CD4+ T helper lymphocytes. The approach uses autologous tumor cells genetically modified to express syngeneic MHC class II genes as cell-based immunogens and is based on the hypothesis that tumor cells directly present tumor Ags to CD4+ T cells. Since the conventional pathway for CD4+ T cell activation is indirect via professional APC, induction of immunity following immunization with class II-transfected tumor cells was examined in bone marrow chimeric mice. Both tumor and host-derived cells are APC for tumor Ags, suggesting that the efficacy of tumor cell vaccines can be significantly improved by genetic modifications that enhance tumor cell Ag presentation.     

Engineering DNA vaccines via co-delivery of co-stimulatory molecule genes

DNA immunization has been investigated as a potential immunization strategy against infectious diseases and cancer. To enhance a DNA vaccine's ability to induce CTL response in vivo, we co-administered CD80 and CD86 expression cassettes along with HIV-1 immunogens. This manipulation resulted in a dramatic increase in MHC class I-restricted and CD8+ T-cell-dependent CTL responses in both mice and chimpanzees. This strategy of engineering vaccine producing cells to be more efficient T-cell activators could be an important tool for optimizing antigen-specific T-cell-mediated immune responses in the pursuit of more rationally designed vaccines and immune therapies.     

Molecular and immunological analysis of genetic prostate specific antigen (PSA) vaccine

Nucleic acid immunization has been investigated as immunotherapy for infectious diseases as well as for treating specific types of cancers. In this approach, nucleic acid expression cassettes are directly inoculated into the host, whose transfected cells become the production source of novel and possibly immunologically foreign protein. We have developed a DNA vaccine construct which encodes for PSA by cloning a cDNA for PSA into a mammalian expression vector under control of a CMV promoter. We investigated and characterized the immunogenicity of PSA DNA expression cassettes in mice. PSA-specific immune responses induced in vivo by immunization were characterized by enzyme-linked immunosorbent assay (ELISA), T helper proliferation cytotoxic T lymphocyte (CTL), and flow cytometry assays. We observed a strong and persistent antibody response against PSA for at least 180 days following immunization. In addition, a significant T helper cell proliferation was observed against PSA protein. Using synthetic peptides spanning the PSA open frame, we identified four dominant T helper epitopes of PSA. Furthermore, immunization with PSA plasmid induced MHC Class I CD8+ T cell-restricted cytotoxic T lymphocyte response against tumor cell targets expressing PSA. The prostate represents a very specific functional organ critical for reproduction but not for the health and survival of the individual. Understanding the immunogenicity of PSA DNA immunization cassettes offers insight into the possible use of this tumor-associated antigen as a target for immunotherapy. These results demonstrate the ability of the genetic PSA to serve as a specific immune target capable of generating both humoral and cellular immune responses in vivo.     

IL-3 enhances both presentation of exogenous particulate antigen in association with class I major histocompatibility antigen and generation of primary tumor-specific cytolytic T lymphocytes

Recent studies have reported that APC can present particulate exogenous Ag in the context of class I MHC to CD8+ CTL, and our laboratory demonstrated that IL-3 could enhance CTL generation to exogenous Ag. In this paper, we wished to determine whether presentation of particulate Ag could be enhanced by IL-3. A T cell hybridoma, B3Z86/90.14 (B3Z) restricted to Ova/Kb, was used as an indicator for presentation of particulate Ag with class I MHC. When activated, this hybridoma expresses lacZ, allowing a simple colorimetric measurement of Ag-specific T cell stimulation. We demonstrated that bone marrow cells stimulated by IL-3 in vivo and in vitro exhibited significantly increased presentation of exogenous OVA linked to beads. Lysate from OVA-transfected line 1 murine lung adenocarcinoma cells (line 1/OVA) was also presented by IL-3-stimulated bone marrow cells, suggesting that these APC can process tumor fragments or debris. Studies using TAP1/2-deficient mice and Ag presentation inhibitors indicate that this exogenous Ag presentation is mediated via the conventional class I MHC pathway. Adoptive transfer of IL-3-stimulated bone marrow cells pulsed with lysate from line 1/OVA tumor cells into naive recipient mice led to the generation of a potent CTL response. These observations indicate that use of such cells may provide a new avenue for development of tumor vaccines.     

Inhibitory and stimulatory effects of IL-10 on human CD8+ T cells

IL-10 is a well-documented immunosuppressant that inhibits macrophage-dependent Ag presentation and CD4+ T cell proliferation in vitro. We report that IL-10 inhibits alloantigen-specific proliferative responses and induces a long lasting anergic state in human purified CD8+ T cells when added concomitantly with the Ag in the presence of APC. Moreover, the generation of allospecific cytotoxic activity is inhibited by IL-10. These effects are indirect and are mediated through inhibition of the costimulatory functions of APC. In contrast, IL-10 has no direct inhibitory effects on the proliferation of purified CD8+ T cells activated by anti-CD3 mAb and promotes the growth of activated CD8+ T cells in combination with low doses of IL-2. Taken together, these results indicate that IL-10 has differential effects on CD8+ T cells depending on their state of activation, which may explain both the enhancing and inhibitory effects observed after IL-10 treatment in different in vivo experimental models.     

Antigen specificity of dual reactive T hybridomas determines the requirement for CD40 ligand-CD40 interactions

CD40 ligand (CD40L) expression on T cells is known to play a crucial role in B cell responses. Some evidence also supports a role for CD40L-CD40 interactions in T cell responses, at least in vivo. Whether the T cell requirement for these interactions is an invariable finding, however, is less clear. Here, we provide evidence that the Ag specificity of T cells influences the requirement for CD40L. T cell hybridomas with dual reactivity for two different Ags, allo-H2-Ap and Mls(a) superantigens, display a differential requirement for CD40L expression. Whereas the response to splenic APC expressing Mls(a) Ags requires CD40L expression, the response to alloantigen-bearing APC does not. The requirement for CD40L expression for the Mls(a) response appears to reflect a strong dependence of this response on ICAM-1 (intercellular adhesion molecule-1) and the ability of CD40-mediated signals to regulate ICAM-1 expression. These findings demonstrate that CD40L-CD40-mediated cross-talk is important for some but not all T cell responses and is influenced by both the type of Ag recognized and the type of APC.     

CD40 activation boosts T cell immunity in vivo by enhancing T cell clonal expansion and delaying peripheral T cell deletion

In this report we show that activation of APC with an agonist anti-CD40 mAb profoundly alters the behavior of CD4 T cells in vivo. Stimulation of mice with anti-CD40 2 days before, but not 1 day after, administration of superantigen (SAg) enhanced CD4 and CD8 T cell clonal expansion by approximately threefold. Further, CD40 activation also delayed peripheral T cell deletion after activation. Dying, activated T cells were quantitated by detecting extracellular phosphatidylserine with concomitant staining for SAg-reactive T cells using a TCR Vbeta-specific mAb. Upon close examination, it was shown that CD40 activation delayed the death of the activated T cells. Additionally, it was found that enhanced survival of CD4 T cells was equally dependent on APC expression of B7-1 and B7-2. This is in contrast to CD8 T cells, which did not depend as much on B7-1 as B7-2. Thus, CD40 activation indirectly promotes T cell growth and delays the death of SAg-stimulated CD4 T cells in vivo. These data suggest that one way CD40 activation promotes a more robust immune response is by indirectly increasing the production of effector T cells and by keeping them alive for longer periods of time.     

Dose-dependent induction of distinct anergic phenotypes: multiple levels of T cell anergy

T cell anergy has been proposed as one of the mechanisms underlying peripheral T cell tolerance. In recent years, the functional relevance of T cell anergy has been studied extensively in vitro and in vivo, using different species, cell systems, and ways to induce anergy. Although these studies concurred about the induction of unresponsiveness, conflicting findings were obtained with respect to the function of anergic T cells and to the persistence of T cell anergy. In the present study, T cell anergy was induced through T-T presentation of the specific Ag by rat MHC class II+ T cells in the absence of professional APC. We show that, depending on the Ag dose with which T cells were incubated, distinct anergic phenotypes were induced. Incubation of T cell clones with a low (suboptimal) Ag dose induced hyporesponsiveness. Incubation with a higher (optimal) Ag dose induced an anergic state capable of exerting immunoregulatory effects. Incubation with a high (supraoptimal) Ag dose led to an anergic suppressive phenotype that was persistent and was not reversed by APC, Ag, and rIL-2. These findings demonstrate that T cell anergy is not confined to a single state of functional inactivation. Instead, multiple levels of T cell anergy exist. Thus, anergic T cells can contribute to the regulation of the immune response either in a persistent and active manner or in a passive manner, depending on their level of T cell anergy.     

Naive versus memory CD4 T cell response to antigen. Memory cells are less dependent on accessory cell costimulation and can respond to many antigen-presenting cell types including resting B cells

Secondary responses to Ag in vivo are characterized by more rapid kinetics and greatly enhanced magnitude compared with primary responses. For CD4+ T cells, this is in part due to a greater frequency of Ag-specific memory cells, and may also reflect differences in responsiveness of memory vs naive cells to stimulation. To compare activation requirements and the role of accessory cells, naive and memory cells were stimulated with immobilized anti-CD3 in the presence or absence of APC. With anti-CD3 alone, naive cells proliferated slightly but produced no detectable IL-2, whereas memory cells proliferated well with significant IL-2 production. Increasing numbers of T-depleted APC greatly enhanced responses of naive cells to levels equivalent to those of memory cells, whereas for memory cells only IL-2 production increased slightly. The response of naive cells was equivalent in magnitude and kinetics to that of memory cells when low density APC, enriched in dendritic cells and depleted of resting B cells, were used with anti-CD3. To directly compare naive and memory responses in an Ag-specific model, we examined CD4+ cells specific for a peptide of pigeon cytochrome c fragment isolated from TCR-alpha beta transgenic mice. Naive cells were compared with 4-day activated blasts (effectors) and memory cells generated by adoptive transfer of effectors to adult thymectomized bone marrow reconstituted mice, in which the cells return to a resting state but still respond to recall Ag. Naive cells responded to Ag on dendritic cells and activated B cells but not on resting B cells or macrophages. In contrast, both memory cells and effectors were stimulated by all APCs, including resting B cells and macrophage to a limited extent. The ability of memory cells to respond to all APC types was confirmed using Ag-specific cells generated by in vivo priming with keyhole limpet hemocyanin. These results suggest that memory cells are considerably less dependent on accessory cell costimulation than naive cells, but that naive cells can respond equivalently in both magnitude and kinetics if Ag is presented on costimulatory APCs such as dendritic cells. In addition, these studies suggest that the enhanced secondary T cell response is due to a combination of the increased frequency of Ag-specific cells and their ability to react to Ag presented on a wider range of APC types, rather than an inherent capacity of memory T cells to respond better and faster.     

Hepatocytes induce functional activation of naive CD8+ T lymphocytes but fail to promote survival

Intraperitoneal peptide injection of TCR-transgenic mice or expression of antigen in hepatocytes leads to an accumulation in the liver of specific apoptotic CD8+ T cells expressing activation markers. To determine whether liver cells are capable of directly activating naive CD8+ T cells, we have studied the ability of purified hepatocytes to activate TCR-transgenic CD8+ T cells in vitro. We show that hepatocytes which do not express CD80 and CD86 co-stimulatory molecules are able to induce activation and effective proliferation of specific naive CD8+ T cells in the absence of exogenously added cytokines, a property only shared by professional antigen-presenting cells (APC). Specific T cell proliferation induced by hepatocytes was comparable in magnitude to that seen in response to dendritic cells and was independent of CD4+ T cell help or bystander professional APC co-stimulation. During the first 3 days, the same number of divisions was observed in co-cultures of CD8+ T cells with either hepatocytes or splenocytes. Both APC populations induced expression of early T cell activation markers and specific cytotoxic T lymphocyte (CTL) activity. However, in contrast to T cells activated by splenocytes, T cells activated by hepatocytes lost their cytolytic function after 3 days of co-culture. This correlated with death of activated T cells, suggesting that despite efficient activation, proliferation and transient CTL function, T cells activated by hepatocytes did not survive. Death could be prevented by adding antigen-expressing splenocytes or exogenous IL-2 to the co-culture, indicating that hepatocytes are not involved in direct killing of CD8+ T cells but rather fail to promote survival. Dying cells acquired a CD8(low) TCR(low) B220+ phenotype similar to the one described for apoptotic intrahepatic T cells, suggesting an alternative model to account for the origin of these cells in the liver. The importance of these findings for the understanding of peripheral tolerance and the ability of liver grafts to be accepted is discussed.     

Targeting a polyepitope protein incorporating multiple class II-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by CD4+ cytotoxic T lymphocytes: a novel approach to vaccine design

The role of CD4+ and CD8+ cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4+- and CD8+-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8+- or CD4+-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4+ T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4+ CTLs. In the present study we attempted to enhance the efficiency of class II-restricted endogenous presentation of minimal class II-restricted CTL epitopes by specifically targeting a polyepitope protein to class II processing compartments through the endosomal and/or lysosomal pathway. A significantly enhanced stimulation of virus-specific CD4+-T-cell clones by antigen-presenting cells (APC) expressing the recombinant polyepitope protein targeted to the endocytic/secretory pathway was readily demonstrated in cytotoxicity assays. In addition, in vitro activation of Epstein-Barr virus- and influenza virus-specific CD4+ memory CTLs by the recombinant constructs encoding the polyepitope protein, specifically targeted to the lysosomal compartment, was also demonstrated. The enhanced stimulatory capacity of APC expressing a lysosome-targeted polyepitope protein has important implications for vaccine design.