Dendritic cell targeting of survivin protein in a xenogeneic form elicits strong CD4+ T cell immunity to mouse survivin

To determine whether strong CD4+ T cell immunity could be induced to a nonmutated self protein that is important for tumorigenesis, we selectively targeted the xenogeneic form of survivin, a survival protein overexpressed in tumors, to maturing dendritic cells in lymphoid tissues. Dendritic cell targeting via the DEC205 receptor in the presence of anti-CD40 and poly(I:C) as maturation stimuli, induced strong human and mouse survivin-specific CD4+ T cell responses, as determined by IFN-gamma, TNF-alpha, and IL-2 production, as well as the development of lytic MHC class II-restricted T cells and memory. Immunity was enhanced further by depletion of CD25+foxp3+ cells before vaccination. anti-DEC205-human survivin was superior in inducing CD4+ T cell responses relative to other approaches involving survivin plasmid DNA or survivin peptides with adjuvants. However, we were unable to induce CD8+ T cell immunity to survivin by two doses of DEC205-targeted survivin or the other strategies. Therefore, significant CD4+ T cell immunity to a self protein that is overexpressed in most human cancers can be induced by DEC205 targeting of the Ag in its xenogeneic form to maturing DCs.     

Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity

There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.     

The instructive role of dendritic cells on T-cell responses

Immune responses are initiated in the T-cell areas of secondary lymphoid organs where na?ve T lymphocytes encounter dendritic cells (DCs) that present antigens taken up in peripheral tissues. DCs represent the interface between the universe of foreign and tissue-specific antigens and T lymphocytes, and they are the key players in the regulation of cell-mediated immunity. We discuss how the nature of the DC maturation stimuli and the density and quality of DCs present in the T-cell areas of secondary lymphoid organs determine the magnitude and class of the T-cell response.     

Novel exosome-targeted CD4+ T cell vaccine counteracting CD4+25+ regulatory T cell-mediated immune suppression and stimulating efficient central memory CD8+ CTL responses

T cell-to-T cell Ag presentation is increasingly attracting attention. In this study, we demonstrated that active CD4+ T (aT) cells with uptake of OVA-pulsed dendritic cell-derived exosome (EXO(OVA)) express exosomal peptide/MHC class I and costimulatory molecules. These EXO(OVA)-uptaken (targeted) CD4+ aT cells can stimulate CD8+ T cell proliferation and differentiation into central memory CD8+ CTLs and induce more efficient in vivo antitumor immunity and long-term CD8+ T cell memory responses than OVA-pulsed dendritic cells. They can also counteract CD4+25+ regulatory T cell-mediated suppression of in vitro CD8+ T cell proliferation and in vivo CD8+ CTL responses and antitumor immunity. We further elucidate that the EXO(OVA)-uptaken (targeted)CD4+ aT cell's stimulatory effect is mediated via its IL-2 secretion and acquired exosomal CD80 costimulation and is specifically delivered to CD8+ T cells in vivo via acquired exosomal peptide/MHC class I complexes. Therefore, EXO-targeted active CD4+ T cell vaccine may represent a novel and highly effective vaccine strategy for inducing immune responses against not only tumors, but also other infectious diseases.     

Diminished Memory T-Cell Expansion Due to Delayed Kinetics of Antigen Expression by Lentivectors

Memory CD8⁺ T lymphocytes play a central role in protective immunity. In attempt to increase the frequencies of memory CD8⁺ T cells, repeated immunizations with viral vectors are regularly explored. Lentivectors have emerged as a powerful vaccine modality with relatively low pre-existing and anti-vector immunity, thus, thought to be ideal for boosting memory T cells. Nevertheless, we found that lentivectors elicited diminished secondary T-cell responses that did not exceed those obtained by priming. This was not due to the presence of anti-vector immunity, as limited secondary responses were also observed following heterologous prime-boost immunizations. By dissecting the mechanisms involved in this process, we demonstrate that lentivectors trigger exceptionally slow kinetics of antigen expression, while optimal activation of lentivector-induced T cells relays on durable expression of the antigen. These qualities hamper secondary responses, since lentivector-encoded antigen is rapidly cleared by primary cytotoxic T cells that limit its presentation by dendritic cells. Indeed, blocking antigen clearance by cytotoxic T cells via FTY720 treatment, fully restored antigen presentation. Taken together, while low antigen expression is expected during secondary immunization with any vaccine vector, our results reveal that the intrinsic delayed expression kinetics of lentiviral-encoded antigen, further dampens secondary CD8⁺ T-cell expansion.     

CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes

The success of immunization with irradiated sporozoites is unparalleled among the current vaccination approaches against malaria, but its mechanistic underpinnings have yet to be fully elucidated. Using a model mimicking natural infection by Plasmodium yoelii, we delineated early events governing the development of protective CD8(+) T-cell responses to the circumsporozoite protein. We demonstrate that dendritic cells in cutaneous lymph nodes prime the first cohort of CD8(+) T cells after an infectious mosquito bite. Ablation of these lymphoid sites greatly impairs subsequent development of protective immunity. Activated CD8(+) T cells then travel to systemic sites, including the liver, in a sphingosine-1-phosphate (S1P)-dependent fashion. These effector cells, however, no longer require bone marrow-derived antigen-presenting cells for protection; instead, they recognize antigen on parenchymal cells-presumably parasitized hepatocytes. Therefore, we report an unexpected dichotomy in the tissue restriction of host responses during the development and execution of protective immunity to Plasmodium.     

CD8+ cytotoxic T-APC stimulate central memory CD8+ T cell responses via acquired peptide-MHC class I complexes and CD80 costimulation, and IL-2 secretion

We previously showed that naive CD4+ Th cells acquire peptide-MHC class I (pMHC I) and costimulatory molecules from OVA-pulsed dendritic cells (DC(OVA)), and act as Th-APCs in stimulation of CD8+ CTL responses. In this study, we further demonstrated that naive CD8+ cytotoxic T (Tc) cells also acquire pMHC I and costimulatory CD54 and CD80 molecules by DC(OVA) stimulation, and act as Tc-APC. These Tc-APC can play both negative and positive modulations in antitumor immune responses by eliminating DC(OVA) and neighboring Tc-APC, and stimulating OVA-specific CD8+ central memory T responses and antitumor immunity. Interestingly, the stimulatory effect of Tc-APC is mediated via its IL-2 secretion and acquired CD80 costimulation, and is specifically targeted to OVA-specific CD8+ T cells in vivo via its acquired pMHC I complexes. These principles could be applied to not only antitumor immunity, but also other immune disorders (e.g., autoimmunity).     

Continuous CD8⁺ T-cell priming by dendritic cell cross-presentation of persistent antigen following adeno-associated virus-mediated gene delivery

Recombinant adeno-associated virus (rAAV) vectors establish persistent transgene expression in the skeletal muscle of mice. How dendritic cells acquire encoded antigens for CD8(+) T-cell priming is unknown. Here we document CD8(+) T-cell priming after lethal irradiation and bone marrow reconstitution of mice treated with an AAV vector several weeks earlier. Temporal separation of vector delivery and successful class I antigen presentation indicated that T-cell priming does not necessarily require antigen synthesis in AAV-transduced dendritic cells. An apparent cross-presentation of antigen acquired from muscle suggests that strategies to limit transgene expression in dendritic cells will not prevent unwanted CD8(+) T-cell responses.     

Virus-specific CD4+ and CD8+ cytotoxic T-cell responses and long-term T-cell memory in individuals vaccinated against polio

The presence of poliovirus (PV)-specific CD4(+) T cells in individuals vaccinated against polio has been shown, but CD8(+) T-cell responses have not been described. Here, we functionally characterize the CD4(+) T-cell response and show for the first time that dendritic cells and macrophages can stimulate PV-specific CD8(+) T-cell responses in vitro from vaccinees. Both CD4(+) T and CD8(+) T cells secrete gamma interferon in response to PV antigens and are cytotoxic via the perforin/granzyme B-mediated pathway. Furthermore, the T cells also recognize and kill Sabin 1 vaccine-infected targets. The macrophage-stimulated CD4(+) T and CD8(+) T cells most likely represent memory T cells that persist for long periods in vaccinated individuals. Thus, immunity to PV vaccination involves not only an effective neutralizing antibody titer but also long-term CD4(+) and CD8(+) cytotoxic T-cell responses.     

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.     

Lack of intrinsic CTLA-4 expression has minimal effect on regulation of antiviral T-cell immunity

CTLA-4 is considered one of the most potent negative regulators of T-cell activation. To circumvent experimental limitations due to fatal lymphoproliferative disease associated with genetic ablation of CTLA-4, we have used radiation chimeras reconstituted with a mixture of CTLA-4+/+ and CTLA-4-/- bone marrow that retain a normal phenotype and allow the evaluation of long-term T-cell immunity under conditions of intrinsic CTLA-4 deficiency. Following virus infection, we profiled primary, memory, and secondary CD8+ and CD4+ T-cell responses directed against eight different viral epitopes. Our data demonstrate unaltered antigen-driven proliferation, acquisition of effector functions, distribution of epitope hierarchies, T-cell receptor repertoire selection, functional avidities, and long-term memory maintenance in the absence of CTLA-4. Moreover, regulation of memory T-cell survival and homeostatic proliferation, as well as secondary responses, was equivalent in virus-specific CTLA4+/+ and CTL-A-4-/- T-cell populations. Thus, lack of CTLA-4 expression by antigen-specific T cells can be compensated for by extrinsic factors in the presence of CTLA-4 expression by other cells. These findings have implications for the physiologic, pathological, and therapeutic regulation of costimulation.     

Differential kinetics of antigen-specific CD4+ and CD8+ T cell responses in the regression of retrovirus-induced sarcomas

Despite the accepted role for CD4+ T cells in immune control, little is known about the development of Ag-specific CD4+ T cell immunity upon primary infection. Here we use MHC class II tetramer technology to directly visualize the Ag-specific CD4+ T cell response upon infection of mice with Moloney murine sarcoma and leukemia virus complex (MoMSV). Significant numbers of Ag-specific CD4+ T cells are detected both in lymphoid organs and in retrovirus-induced lesions early during infection, and they express the 1B11-reactive activation-induced isoform of CD43 that was recently shown to define effector CD8+ T cell populations. Comparison of the kinetics of the MoMSV-specific CD4+ and CD8+ T cell responses reveals a pronounced shift toward CD8+ T cell immunity at the site of MoMSV infection during progression of the immune response. Consistent with an important early role of Ag-specific CD4+ T cell immunity during MoMSV infection, CD4+ T cells contribute to the generation of virus-specific CD8+ T cell immunity within the lymphoid organs and are required to promote an inflammatory environment within the virus-infected tissue.     

Anatomical and cellular requirements for the activation and migration of virus-specific CD8+ T cells to the brain during Theiler's virus infection

Theiler's murine encephalomyelitis virus (TMEV) infection of the brain induces a virus-specific CD8(+) T-cell response in genetically resistant mice. The peak of the immune response to the virus occurs 7 days after infection, with an immunodominant CD8(+) T-cell response against a VP2-derived capsid peptide in the context of the D(b) molecule. The process of activation of antigen-specific T cells that migrate to the brain in the TMEV model has not been defined. The site of antigenic challenge in the TMEV model is directly into the brain parenchyma, a site that is considered immune privileged. We investigated the hypothesis that antiviral CD8(+) T-cell responses are initiated in situ upon intracranial inoculation with TMEV. To determine whether a brain parenchymal antigen-presenting cell is responsible for the activation of virus-specific CD8(+) T cells, we evaluated the CD8(+) T-cell response to the VP2 peptide in bone marrow chimeras and mutant mice lacking peripheral lymphoid organs. The generation of the anti-TMEV CD8(+) T-cell response in the brain requires priming by a bone marrow-derived antigen-presenting cell and the presence of peripheral lymphoid organs. Although our results show that activation of TMEV-specific CD8(+) T cells occurs in the peripheral lymphoid compartment, they do not exclude the possibility that the immune response to TMEV is initiated by a brain-resident, bone marrow-derived, antigen-presenting cell.     

Helper T cells, dendritic cells and CTL Immunity

In this review, we examine the emerging view that all CTL responses depend on CD4 T-cell help for the generation of efficient memory. We further review the evidence that CD4 and CD8 T cells must recognize antigen on the same dendritic cell, and examine why this corecognition is required. Earlier studies have suggested that CD4 T cells must activate the dendritic cell via CD40 to license it for the capacity to prime CTL immunity. More recently, however, CD40 signalling of the CTL has been reported. Here, we argue that the main reason for corecognition of antigen on the dendritic cell may be related to the time taken to activate and release CD4 and CD8 T cells from their priming dendritic cell. CD4 T cells may only be capable of activating one dendritic cell during the period that CD8 T cells are primed. In this case, corecognition of this same dendritic cell would be essential.     

The CC chemokine receptor-7 ligands 6Ckine and macrophage inflammatory protein-3 beta are potent chemoattractants for in vitro- and in vivo-derived dendritic cells

Dendritic cell migration to secondary lymphoid tissues is critical for Ag presentation to T cells necessary to elicit an immune response. Despite the importance of dendritic cell trafficking in immunity, at present little is understood about the mechanisms that underlie this phenomenon. Using a novel transwell chemotaxis assay system, we demonstrate that the CC chemokine receptor-7 (CCR7) ligands 6Ckine and macrophage inflammatory protein (MIP)-3 beta are selective chemoattractants for MHC class IIhigh B7-2high bone marrow-derived dendritic cells at a potency 1000-fold higher than their known activity on naive T cells. Furthermore, these chemokines stimulate the chemotaxis of freshly isolated lymph node dendritic cells, as well as the egress of skin dendritic cells ex vivo. Because these chemokines are expressed in lymphoid organs and 6Ckine has been localized to high endothelial venules and lymphatic endothelium, we propose that they may play an important role in the homing of dendritic cells to lymphoid tissues.     

Immunotherapy of established tumors using bone marrow transplantation with antigen gene--modified hematopoietic stem cells

A major focus of cancer immunotherapy is to develop strategies to induce T-cell responses through presentation of tumor antigens by dendritic cells (DCs). Current vaccines are limited in their ability to efficiently transfer antigens to DCs in vivo. Ex vivo-generated DCs can be efficiently loaded with antigen but after reinjection, few DCs traffic to secondary lymphoid organs, the critical sites for antigen presentation. To enhance efficiency and durability of antigen presentation by DCs, we transduced hematopoietic stem-progenitor cells (HSCs) with a model tumor antigen and then transplanted the gene-modified cells into irradiated recipient mice, which resulted in efficient expression of the transgene in a large proportion of donor derived DCs in lymphoid organs. The combination of bone marrow transplantation (BMT) using transduced HSCs, systemic agents that generate and activate DCs, and mature T-cell infusion resulted in substantial expansion and activation of antigen-specific T cells. This tripartite strategy provided potent antigen-specific immunotherapy for an aggressive established tumor.     

Functional expression of human CD8 in fully reconstituted mice after retroviral-mediated gene transfer of hemopoietic stem cells.

Retroviral-mediated gene transfer has been used in an attempt to efficiently and stably express functional cell-surface molecules in lymphoid and myeloid cells. The human CD8 molecule is a T cell-specific surface receptor that is intimately involved in class I MHC-restricted Ag recognition and subsequent T cell activation. After infection with a recombinant, replication-defective retrovirus containing the human CD8 alpha cDNA, bone marrow cells were transplanted into lethally irradiated recipients. The majority of lymphoid and myeloid cells of reconstituted animals expressed high levels of human CD8 for at least 8 months after transplantation. Transfer of bone marrow and spleen cells from these recipients 100 days after transplantation into secondary recipients also resulted in long term expression of CD8 in lymphoid and myeloid cells. CD8 expressed in splenic T cells associated with the lymphoid-specific tyrosine protein kinase p56lck, participated in T cell activation and conferred an increased xenogeneic response to human MHC class I Ag. Thus, retroviral-mediated gene transfer allows the long term, functional expression of cell-surface molecules in normal murine lymphoid and myeloid cells.     

Unusual T cell populations in adult murine bone marrow. Prevalence of CD3+CD4-CD8- and alpha beta TCR+NK1.1+ cells

The T cell populations present in normal murine bone marrow have not been previously analyzed in detail, mainly because of their relative rarity. In order to permit such analyses, bone marrow T cells were enriched by depleting Mac1-positive cells, which constitute 65 to 90% of bone marrow cells (BMC), and then studied by two-color flow cytometry. Analysis of the remaining cells revealed that the T cell profile of adult murine bone marrow is markedly different from that of other lymphoid organs. A very high proportion of bone marrow CD3+ cells (approximately one-third) are CD4-CD8-. CD3+CD4-CD8- cells are much more concentrated among BMC T cells than among thymocytes or splenic T cells, suggesting that bone marrow may be either a site of extrathymic TCR gene rearrangement, or a major site to which such cells home from the thymus. The expression of NK1.1 was also evaluated on Mac1-depleted BMC populations. Surprisingly, up to 39% of alpha beta TCR+ BMC were found to express NK1.1. Most alpha beta TCR+NK1.1+ BMC also expressed CD4 or CD8. NK1.1+ alpha beta TCR+ cells represented a much greater proportion of BMC T cells than of other lymphoid (splenocyte or thymocyte) T cell populations. Mac1-depleted BMC of nude mice contained very few cells with this phenotype. These results are consistent with the hypothesis that NK1.1+ alpha beta TCR+ cells are generated primarily in the thymus of normal animals and migrate preferentially to bone marrow, where they may function as regulatory elements in hematopoiesis.     

Dendritic cells: driving the differentiation programme of T cells in viral infections

Protective immunity against viral pathogens depends on the generation and maintenance of a small population of memory CD8(+) T cells. Successful memory cell generation begins with early interactions between na?ve T cell and dendritic cells (DCs) within the inflammatory milieu of the secondary lymphoid tissues. Recent insights into the role of different populations of DCs, and kinetics of antigen presentation, during viral infections have helped to understand how DCs can shape the immune response. Here, we review the recent progress that has been made towards defining how specific DC subsets drive effector CD8(+) T-cell expansion and differentiation into memory cells. Further, we endeavour to examine how the molecular signals imparted by DCs coordinate to generate protective CD8(+) T-cell immunity.