CD8 kinetically promotes ligand binding to the T-cell antigen receptor

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.     

Pre-existing anti-Salmonella vector immunity prevents the development of protective antigen-specific CD8 T-cell frequencies against murine listeriosis

Our laboratory has focused its research on the use of the type III secretion system of Salmonella enterica serovar Typhimurium to translocate heterologous antigens directly into the cytosol of antigen-presenting cells. We have previously reported that the single oral immunization of mice with a recombinant Salmonella aroA/sptP mutant strain expressing the translocated Yersinia outer protein E fused to the immunodominant antigen p60 from Listeria monocytogenes in a type III-mediated fashion results in the efficient induction of p60-specific CD8 T cells and confers protection against a lethal Listeria challenge infection. In the present study, we determined whether pre-existing anti-Salmonella vector immunity influences the induction of p60-specific CD8 T cells and modulates protective immunity against listeriosis after oral vaccination with recombinant Salmonella. After single oral immunization, the Salmonella aroA/sptP double mutant strain was found to colonize spleens of mice for 21days. In contrast, the period of colonization was significantly shortened to 6days due to anti-Salmonella vector immunity after second oral immunization. The latter scenario led to the induction of low-level frequencies of antigen-specific CD8 T cells. Compared to the significantly higher numbers of p60-specific T lymphocytes elicited after single oral immunization, the low amount of Listeria-specific CD8 T cells did not confer protection against listeriosis.     

Termination of antigen-specific immunity by CD95 ligand (Fas ligand) and IL-10

Following elimination of a foreign invader, the immune system must return to its normal quiescent levels. This process requires removal of reactive immune cells when they are no longer needed. We have explored the role of Fas/Fas ligand (FasL) in terminating immunity and demonstrate that mice defective in these proteins have prolonged immune responses. Studies demonstrate that termination of immunity occurs via the interaction of Fas(+) lymphoid cells with FasL(+) nonlymphoid cells at the site of Ag challenge. Our results also show that FasL is absent in quiescent tissue but is rapidly up-regulated during the local immune reaction. This occurs through the production of IL-10. Thus, FasL and IL-10 work in concert to eliminate inflammatory cells and control the duration of an immune response.     

Insights into human CD8(+) T-cell memory using the yellow fever and smallpox vaccines

Live virus vaccines provide a unique opportunity to study human CD8(+) T-cell memory in the context of a controlled, primary acute viral infection. Yellow fever virus-17D and Dryvax are two such live-virus vaccines that are highly efficacious, used worldwide and provide long-term immunity against yellow fever and smallpox respectively. In this review, we describe the properties of virus-specific memory CD8(+) T cells generated in smallpox and yellow fever vaccinees. We address fundamental questions regarding magnitude, functional quality and longevity of the CD8(+) T-cell response, which are otherwise challenging to address in humans. These findings provide insights into the attributes of the human immune system as well as provide a benchmark for the optimal quality of a CD8(+) T-cell response that can be used to evaluate novel candidate vaccines.     

Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory

Emerging evidence indicates that CD8+ and CD4+ T-cell immunity is differentially regulated. Here we have delineated differences and commonalities among antiviral T-cell responses by enumeration and functional profiling of eight specific CD8+ and CD4+ T-cell populations during primary, memory and recall responses. A high degree of coordinate regulation among all specific T-cell populations stood out against an approximately 20-fold lower peak expansion and prolonged contraction phase of specific CD4+ T-cell populations. Surprisingly, although CD8+ T-cell memory was stably maintained for life, levels of specific CD4+ memory T cells gradually declined. However, this decay, which seemed to result from less efficient rescue from apoptosis, did not affect functionality of surviving virus-specific CD4+ T cells. Our results indicate that CD4+ T-cell memory might become limiting under physiological conditions and that conditions precipitating CD4+ T-cell loss might compromise protective immunity even in the presence of unimpaired CD8+ T-cell responses.     

Cutting edge: cognate CD4 help promotes recruitment of antigen-specific CD8 T cells around dendritic cells

The cellular orchestration underlying help provided by CD4 T lymphocytes to CD8 T cell responses is not fully understood. We documented that the formation of three-cell clusters occurred as soon as day 1 and relied on long-lasting CD4 and CD8 T cell interactions with dendritic cells (DCs). The influence of CD4 help on CD8 T cell differentiation could be observed as early as the second round of cell division. Importantly, our results identify a new facet to the phenomenon of CD4 help in which DCs, upon cognate interactions with CD4 T cells, increase their ability to attract/retain Ag-specific CD8 T cells. Our results support a model in which CD4 help operates rapidly, in part by favoring CD8 T cells recruitment around those DCs that are the most competent for priming.     

IFN-alpha beta promote priming of antigen-specific CD8+ and CD4+ T lymphocytes by immunostimulatory DNA-based vaccines

Immunostimulatory sequence (ISS) DNA containing unmethylated CpG dinucleotides stimulate NK and APC to secrete proinflammatory cytokines, including IFN-alphabeta and -gamma, TNF-alpha, and IL-6 and -12, and to express costimulatory surface molecules such as CD40, B7-1, and B7-2. Although ISS DNA has little direct effect on T cells by these criteria, immunization of wild-type mice with ISS DNA and OVA results in Ag-specific CTL and Th1-type T helper activity. This investigation examines the mechanisms by which ISS DNA primes CD8(+) and CD4(+) lymphocyte activities. In this report we demonstrate that ISS DNA regulates the expression of costimulatory molecules and TAP via a novel autocrine or paracrine IFN-alphabeta pathway. Coordinated regulation of B7 costimulation and TAP-dependent cross-presentation results in priming of Ag-specific CD8(+) CTL, whereas CD40, B7, and IL-12 costimulation is required for priming of CD4(+) Th cells by ISS-based vaccines.     

Synthesis of protein-loaded hydrogel particles in an aqueous two-phase system for coincident antigen and CpG oligonucleotide delivery to antigen-presenting cells

Materials that effectively deliver protein antigens together with activating ligands to antigen-presenting cells are sought for improved nonviral vaccines. To this end, we synthesized protein-loaded poly(ethylene glycol) (PEG)-based hydrogel particles by cross-linking PEG within the polymer-rich phase of an emulsion formed by a poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) triblock copolymer in saturated aqueous salt solution. These particles (500-nm diameter) contained high levels of encapsulated protein (approximately 75% of dry mass), which was selectively released by proteolytic enzymes normally present in the phagosomal/endosomal compartments of dendritic cells (DCs). For co-delivery of cellular activation signals, gel particles were surface-modified by sequential adsorption of poly(l-arginine) and CpG oligonucleotides. DCs pulsed with protein-loaded particles activated na?ve T cells in vitro approximately 10-fold more efficiently than DCs incubated with soluble protein. This organic solvent-free strategy for protein encapsulation within submicron-sized hydrophilic particles is attractive for macromolecule delivery to a variety of phagocytic and nonphagocytic cells.     

DNA vaccines encoding interleukin-8 and RANTES enhance antigen-specific Th1-type CD4(+) T-cell-mediated protective immunity against herpes simplex virus type 2 in vivo

Chemokines are inflammatory molecules that act primarily as chemoattractants and as activators of leukocytes. Their role in antigen-specific immune responses is of importance, but their role in disease protection is unknown. Recently it has been suggested that chemokines modulate immunity along more classical Th1 and Th2 phenotypes. However, no data currently exist in an infectious challenge model system. We analyzed the modulatory effects of selected chemokines (interleukin-8 [IL-8], gamma interferon-inducible protein 10 [IP-10], RANTES, monocyte chemotactic protein 1 [MCP-1], and macrophage inflammatory protein 1 alpha [MIP-1 alpha]) on immune phenotype and protection against lethal challenge with herpes simplex virus type 2 (HSV-2). We observed that coinjection with IL-8 and RANTES plasmid DNAs dramatically enhanced antigen-specific Th1 type cellular immune responses and protection from lethal HSV-2 challenge. This enhanced protection appears to be mediated by CD4(+) T cells, as determined by in vitro and in vivo T-cell subset deletion. Thus, IL-8 and RANTES cDNAs used as DNA vaccine adjuvants drive antigen-specific Th1 type CD4(+) T-cell responses, which result in reduced HSV-2-derived morbidity, as well as reduced mortality. However, coinjection with DNAs expressing MCP-1, IP-10, and MIP-1 alpha increased mortality in the challenged mice. Chemokine DNA coinjection also modulated its own production as well as the production of cytokines. These studies demonstrate that chemokines can dominate and drive immune responses with defined phenotypes, playing an important role in the generation of protective antigen-specific immunity.     

The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools

The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25?% of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50?% of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.     

ISCOMATRIX vaccines mediate CD8+ T-cell cross-priming by a MyD88-dependent signaling pathway

Generating a cytotoxic CD8(+) T-cell response that can eradicate malignant cells is the primary objective of cancer vaccine strategies. In this study we have characterized the innate and adaptive immune response to the ISCOMATRIX adjuvant, and the ability of vaccine antigens formulated with this adjuvant to promote antitumor immunity. ISCOMATRIX adjuvant led to a rapid innate immune cell response at the injection site, followed by the activation of natural killer and dendritic cells (DC) in regional draining lymph nodes. Strikingly, major histocompatibility complex (MHC) class I cross-presentation by CD8α(+) and CD8α(-) DCs was enhanced by up to 100-fold when antigen was formulated with ISCOMATRIX adjuvant. These coordinated features enabled efficient CD8(+) T-cell cross-priming, which exhibited prophylactic and therapeutic tumoricidal activity. The therapeutic efficacy of an ISCOMATRIX vaccine was further improved when co-administered with an anti-CD40 agonist antibody, suggesting that ISCOMATRIX-based vaccines may combine favorably with other immune modifiers in clinical development to treat cancer. Finally, we identified a requirement for the myeloid differentiation primary response gene 88 (MyD88) adapter protein for both innate and adaptive immune responses to ISCOMATRIX vaccines in vivo. Taken together, our findings support the utility of the ISCOMATRIX adjuvant for use in the development of novel vaccines, particularly those requiring strong CD8(+) T-cell immune responses, such as therapeutic cancer vaccines.     

A role for IFN-gamma from antigen-specific CD8+ T cells in protective immunity to Listeria monocytogenes

Whether IFN-gamma contributes to the per-cell protective capacity of memory CD8(+) T cells against Listeria monocytogenes (LM) has not been formally tested. In this study, we generated LM Ag-specific memory CD8(+) T cells via immunization of wild-type (WT) and IFN-gamma-deficient (gamma knockout (GKO)) mice with LM peptide-coated dendritic cells and compared them phenotypically and functionally. Immunization of WT and GKO mice resulted in memory CD8(+) T cells that were similar in number, functional avidity, TCR repertoire use, and memory phenotype. The protective capacity of memory CD8(+) T cells from immunized WT and GKO mice was evaluated after adoptive transfer of equal numbers of WT or GKO cells into naive BALB/c mice followed by LM challenge. The adoptively transferred CD8(+) T cells from GKO donors exhibited a decreased ability to reduce bacterial numbers in the organs of recipient mice when compared with an equivalent number of Ag-matched WT CD8(+) T cells. This deficiency was most evident early (day 3) after infection if a relatively low infectious dose was used; however, transferring fewer memory CD8(+) T cells or increasing the LM challenge dose revealed a more pronounced defect in protective immunity mediated by the CD8(+) T cells from GKO mice. Our studies identified a decrease in Ag-specific target cell lysis in vivo by CD8(+) T cells from GKO mice as the mechanism for the decreased protective immunity after LM challenge. Further studies suggest that the lack of IFN-gamma production by the Ag-specific CD8 T cells themselves diminishes target cell sensitivity to cytolysis, thereby reducing the lytic potency of IFN-gamma-deficient LM-specific memory CD8(+) T cells.     

Plant viral genes in DNA idiotypic vaccines activate linked CD4+ T-cell mediated immunity against B-cell malignancies

DNA delivery of tumor antigens can activate specific immune attack on cancer cells. However, antigens may be weak, and immune capacity can be compromised. Fusion of genes encoding activating sequences to the tumor antigen sequence facilitates promotion and manipulation of effector pathways. Idiotypic determinants of B-cell tumors, encoded by the variable region genes, are clone-specific tumor antigens. When assembled as single-chain Fv (scFv) alone in a DNA vaccine, immunogenicity is low. Previously, we found that fusion of a sequence from tetanus toxin (fragment C; FrC) promoted anti-idiotypic protection against lymphoma and myeloma. We have now investigated an alternative fusion gene derived from a plant virus, potato virus X coat protein, a primary antigen in humans. When fused to scFv, the self-aggregating protein generates protection against lymphoma and myeloma. In contrast to scFv-FrC, protection against lymphoma is mediated by CD4+ T cells, as is protection against myeloma. Plant viral proteins offer new opportunities to activate immunity against linked T-cell epitopes to attack cancer.     

Vaccination with a Shigella DNA vaccine vector induces antigen-specific CD8(+) T cells and antiviral protective immunity

A prototype Shigella human immunodeficiency virus type 1 (HIV-1) gp120 DNA vaccine vector was constructed and evaluated for immunogenicity in a murine model. For comparative purposes, mice were also vaccinated with a vaccinia virus-env (vaccinia-env) vector or the gp120 DNA vaccine alone. Enumeration of the CD8(+)-T-cell responses to gp120 after vaccination using a gamma interferon enzyme-linked spot assay revealed that a single intranasal dose of the Shigella HIV-1 gp120 DNA vaccine vector elicited a CD8(+) T-cell response to gp120, the magnitude of which was comparable to the sizes of the analogous responses to gp120 that developed in mice vaccinated intraperitoneally with the vaccinia-env vector or intramuscularly with the gp120 DNA vaccine. In addition, a single dose of the Shigella gp120 DNA vaccine vector afforded significant protection against a vaccinia-env challenge. Moreover, the number of vaccinia-env PFU recovered in mice vaccinated intranasally with the Shigella vector was about fivefold less than the number recovered from mice vaccinated intramuscularly with the gp120 DNA vaccine. Since the Shigella vector did not express detectable levels of gp120, this report confirms that Shigella vectors are capable of delivering passenger DNA vaccines to host cells and inducing robust CD8(+) T-cell responses to antigens expressed by the DNA vaccines. Furthermore, to our knowledge, this is the first documentation of antiviral protective immunity following vaccination with a live Shigella DNA vaccine vector.     

Nasal Flt3 ligand cDNA elicits CD11c+CD8+ dendritic cells for enhanced mucosal immunity

The embryo expresses paternal Ags foreign to the mother and therefore has been viewed as an allograft. It has been shown that anergic T cells generated by blocking of the CD28/B7 costimulatory pathway with anti B7-1 and anti B7-2 mAbs can be transferred as suppresser cells to prevent allograft rejection. Little is known, however, about the in vivo function of anti-B7-treated T cells after their transfer into abortion-prone mice in the maintenance of materno-fetal tolerance. In the present study, abortion-prone CBA/J females mated with DBA/2 males were administered anti-B7-1 and anti-B7-2 mAbs on day 4 of gestation (murine implantation window). The anti-B7-treated T cells subsequently were adoptively transferred into abortion-prone CBA/J mice. We demonstrated that costimulation blockade with anti-B7 mAbs at the time of implantation resulted in altered allogeneic T cell response and overcame increased maternal rejection to the fetus in the CBA/JxDBA/2 system. The transferred anti-B7-treated T cells appeared to be regulatory, decreasing responsiveness and generating clonal deviation in maternal recipient T cells. The transferred CFSE-labeled T cells were found to reside in the spleen and uterine draining lymph nodes, and a few were localized to the materno-fetal interface of the maternal recipient. Our findings suggest that the anti-B7-treated T cells not only function as potent suppresser cells, but also exert an immunoregulatory effect on the maternal recipient T cells, which cosuppresses maternal rejection to the fetus. This procedure might be considered potentially useful for fetal survival when used as an immunotherapy for human recurrent spontaneous abortion.     

Rapid clearance of a recombinant Salmonella vaccine carrier prevents enhanced antigen-specific CD8 T-cell responses after oral boost immunizations

The type III secretion system of Salmonella enterica serovar Typhimurium can be used to target heterologous antigens directly into the cytosol of antigen-presenting cells. Our laboratory has previously reported that the single oral immunization of mice with a recombinant Salmonella strain expressing the translocated Yersinia outer protein E fused to the immunodominant antigen p60 from Listeria monocytogenes results in the efficient induction of p60-specific CD8 T cells and confers protection against a lethal wild-type Listeria challenge infection. In the present study, we investigated whether these antigen-specific cytotoxic T lymphocytes induced by the prime immunization contribute to a more rapid clearance of the vaccine carrier after subsequent boost immunizations and whether oral boost immunizations lead to an augmented p60-specific CD8 T-cell response. We found that the ability of recombinant Salmonella strains to colonize the intestine, mesenteric lymph nodes, and spleen was markedly impaired after boost immunizations but that this effect was independent of existing CD8 T cells reactive with p60(217-225). A significant elevation of antigen-specific CD8 T cells could not be detected by enzyme-linked immunospot assay after the second or the third oral immunization, possibly due to the rapid clearance of the bacterial vaccine carrier from lymphatic organs.     

Dendritic cells genetically modified to express CD40 ligand and pulsed with antigen can initiate antigen-specific humoral immunity independent of CD4+ T cells

We have investigated whether dendritic cells genetically modified to express CD40 ligand and pulsed with antigen can trigger B cells to produce antigen-specific antibodies without CD4+ T-cell help. Dendritic cells modified with a recombinant adenovirus vector to express CD40 ligand and pulsed with heat-killed Pseudomonas induced naive B cells to produce antibodies against Pseudomonas in the absence of CD4+ T cells in vitro, initiated Pseudomonas-specific humoral immune responses in vivo in wild-type and CD4-/- mice, and protected immunized wild-type and CD4-/-, but not B-cell -/- mice, from lethal intrapulmonary challenge with Pseudomonas. Thus, genetic modification of dendritic cells with CD40 ligand enables them to present a complex mixture of microbial antigens and establish CD4+ T cell-independent, B cell-mediated protective immunity against a specific microbe.     

Protective CD8 T cell immunity triggered by CpG-protein conjugates competes with the efficacy of live vaccines

In contrast to infectious (live) vaccines are those based on subunit Ag that are notoriously poor in eliciting protective CD8 T cell responses, presumably because subunit Ags become insufficiently cross-presented by dendritic cells (DCs) and because the latter need to be activated to acquire competence for cross-priming. In this study, we show that CpG-Ag complexes overcome these limitations. OVA covalently linked to CpG-DNA (CpG-OVA complex), once it is efficiently internalized by DCs via DNA receptor-mediated endocytosis, is translocated to lysosomal-associated membrane protein 1 (LAMP-1)-positive endosomal-lysosomal compartments recently shown to display competence for cross-presentation. In parallel, CpG-OVA complex loaded DCs become activated and acquire characteristics of professional APCs. In vivo, a single s.c. dose of CpG-OVA complex (10 mug of protein) induces primary and secondary clonal expansion/contraction of Ag-specific CD8 T cells similar in kinetics to live vaccines; examples including Listeria monocytogenes genetically engineered to produce OVA (LM-OVA) and two viral vector-based OVA vaccines analyzed. Interestingly, CpG-OVA complex induced almost equal percentages of Ag-specific memory CD8 T cells as did infection with LM-OVA. A single dose vaccination with CpG-OVA complex protected mice against lethal doses of LM-OVA. These data underscore that the synergy imparted by CpG-OVA complex-mediated combined triggering of innate and specific immunity might be key to initiate CD8 T cell-based immunoprotection by synthetic vaccines based on subunit Ag.