Epitope-specific T-cell Research Articles

Adoptive transfer of Porphyromonas gingivalis heat shock protein epitope-specific T-cell lines into SCID mice in experimental atherosclerosis

Adoptive transfer of Porphyromonas gingivalis heat shock protein epitope-specific T-cell lines into SCID mice in experimental atherosclerosis

Epitope down-modulation as a mechanism for the coexistence of competing T-cells

Efficient immune responses against pathogens are frequently characterized by the simultaneous targeting of multiple epitopes. However, it remains unclear how the targeting of multiple epitopes is maintained in the face of competition for antigenic stimulation. Here, we investigate this question by using mathematical models of the population dynamics of a viral pathogen, antigen presentation sites and T-cells. We first show that direct competition for access to antigen presenting sites and indirect competition through killing of the pathogen select for dominance of the T-cell response with the highest affinity for its epitope. We then incorporate in our model that epitopes can become down-modulated following interaction with epitope specific T-cells. We demonstrate that epitope down-modulation leads to differentiation of epitope presentation on antigen presenting sites. This differentiation promotes the coexistence of multiple epitope specific responses. Hence, we propose that the functional relevance of epitope down-modulation may be to enable the persistence of a broad immune response despite competition for antigenic stimulation.

Simultaneous Reconstitution of Multiple CMV-Specific CD8+ T-Cell Populations with Divergent Functionality in Hematopoietic Stem Cell Transplant Recipients.

Recent studies using direct stimulation of PBMC from CMV-positive individuals with optimal CTL epitope peptides have identified new antigens that are targets of the host immune response. The growing number of targets of the cellular immune response has prompted an evaluation of which antigens are potentially associated with protective immunity. A panel of seven human cytomegalovirus (CMV) epitope peptides and the corresponding MHC-I tetramers was used to evaluate cellular immunity in six healthy seropositive donors and in six hematopoietic stem cell transplant recipients (HSCT). Broad CMV-specific responses were found to epitopes within several CMV polypeptides that were restricted by multiple HLA alleles in several individuals. The results document the simultaneous expansion of several of these CD8+ T-lymphocyte populations following allogeneic HSCT. The combined levels of CMV epitope-specific T-cells exceeded 20% of CD8+ T-lymphocytes in some individuals. The cytotoxic functionality of 26 different populations of CMV-specific T-lymphocytes detected within this group of 12 individuals was addressed by utilizing a recently described assay that measures transient surface levels of the lysosomal membrane proteins LAMP-1 (CD107a) and LAMP 2 (CD107b) after peptide stimulation. This degranulation/mobilization assay can be combined with tetramer staining of antigen-specific CD8+ T-lymphocytes, and has potential as a surrogate marker for cytotoxic function. We found that a significant proportion of CD8+ T-lymphocytes specific for epitopes within the CMV pp65 and pp50 gene products had functional potential as measured by this assay (median percentage of cells within 14 T-cell populations staining with pp65 or pp50 tetramers that degranulated on stimulation with cognate peptide = 26.0% and 19.8%). By contrast, CD8+ T-lymphocytes specific for epitopes within the CMV IE-1 gene product had markedly reduced functionality (median percentage of cells within 12 T-cell populations staining with IE-1 tetramers that degranulated on stimulation with cognate peptide = 5.6%). This difference was significant (p= 0.003 by an F-test after adjusting for HLA and using interaction of subjects and epitopes as the error). This reduced degranulation efficiency of IE-1-specific T cells is consistent with their inefficient cytotoxic recognition of CMV-infected autologous fibroblasts. Further characterization of this functional dichotomy includes comparison of cell surface marker phenotype and cytokine release in response to antigenic presentation. These functional differences between T-lymphocyte populations within the same individual have implications for choosing antigens that are both protective and necessary to include in a CMV vaccine for transplant patients at risk for infectious complications after viral reactivation.

Recognition of endogenously synthesized HLA-DR4 restricted HCV epitopes presented by autologous EBV transformed B-lymphoblastoid cell line

Hepatitis C virus (HCV) causes non-A, non-B hepatitis and infects an estimated 170 million people worldwide. The treatment for HCV infection is often unsuccessful with high costs and many side-effects. There is a great need for alternative therapies including preventive and therapeutic vaccination for HCV infection. The experiments in this study were carried out to elucidate whether endogenously expressed antigen can be presented to helper T-cells restricted by class II molecules and to determine whether responses to plasmid-derived antigen resemble those that we have reported for recombinant antigens or synthetic peptides. To address these issues, a multi-epitope minigene was expressed in 293T-cells and Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells (BLCL). The transfected BLCLs were employed as APCs to stimulate epitope-specific T-cell hybridomas (THC). The results demonstrated that the endogenously expressed minigene antigens could be processed and presented to T-cell hybridomas by HLA matched BLCL. Five out of seven incorporated epitopes were recognized. Blockade of HLA DR could abolish the release of IL-2, which demonstrated that the endogenously expressed minigene antigens were presented by MHC class II molecules. The presentation of endogenously expressed antigens was much more efficient than that of exogenous antigens, at least in the present study. The findings obtained here have important significance for the development of an HCV DNA vaccine.

HIV-1–specific cytotoxicity is preferentially mediated by a subset of CD8+ T cells producing both interferon-γ and tumor necrosis factor–α

AbstractCD8+ T cells play a crucial role in the control of viral infections by direct elimination of infected cells and secretion of a number of soluble factors. Recent data suggest that HIV-1-specific CD8+ T cell subsets may differ in their ability to exert these effector functions. Here, we directly compared the cytokine secretion patterns and cytotoxic capacity of HIV-1-specific CD8+ T cells, using a flow-cytometric cytotoxicity assay based on caspase-3 activation in dying target cells. These experiments revealed considerable intraindividual and interindividual differences among epitope-specific T-cell effector functions: while the frequency of HIV-1-specific CD8+ T cells secreting interferon-γ but no tumor necrosis factor-α (TNF-α) following antigenic stimulation was only weakly correlated to their cytotoxic activity (R = 0.05, P = .57), a subset of CD8+ T cells secreting both inter-feron-γ and TNF-α was substantially more strongly associated with cytotoxicity (R = 0.67, P < .001). This subset of CD8+ T cells also exhibited stronger intracellular perforin expression and more pronounced direct ex vivo HIV-1-specific cytoxicity than CD8+ T cells secreting solely interferon-γ following sorting of these subpopulations according to their cytokine profile. These results suggest that HIV-1-specific cytotoxicity of CD8+ T cells is preferentially mediated by a subset of CD8+ T cells secreting both interferon-γ and TNF-α. (Blood. 2004;104:487-494)

Genetic variability of hepatitis C virus non-structural protein 3 and virus-specific CD8+ response in patients with chronic hepatitis C.

Hepatitis C virus (HCV) variation in specific T-cell epitopes may represent a mechanism of viral persistence in chronic infection. We examined the HCV non-structural protein 3 (NS3), including the immunologically relevant epitopes HCV NS3-2 KLVALGINAV (human leukocyte antigen [HLA]-A2-restricted) and HCV NS3-1391 LIFCHSKKK (HLA-A3-restricted), in 22 HLA-A2+ patients with chronic infection. Significant amino acid variation was found in HCV NS3-2 epitope sequences when compared to the HCV-1 prototype virus. Six of the nine different HCV NS3-2 peptide variants were identified in patients with HCV NS3-2-specific CD8+ cells, detected with an HLA-A2 tetramer made with the HCV-1 prototype peptide. Phylogenetic analysis, including HCV reference sequences other than HCV-1, suggested however that most of the variations in the HCV NS3-2 epitope could be related to genetic heterogeneity between HCV reference subtypes. Variation was less common when comparing HCV NS3-2 epitope sequences from the clinical isolates to the most-closely related HCV reference subtype in each case. Some subtype-independent variations were found in epitopic residues probably important for T-cell receptor interaction. In contrast, no significant variation was found in HLA primary anchor sites, flanking regions, or in the contiguous HLA A3-restricted CD8+ T-cell epitope. Ongoing variation was not evident in two selected patients with follow-up. In conclusion, (i) the HCV NS3-2 epitope is not conserved between different HCV strains/subtypes, and (ii) an HLA-A2 tetramer loaded with the HCV-1 prototype NS3-2 peptide may still detect NS3-specific CD8+ cells in some patients with variant viruses. These data may be useful to improve T-cell assays using HCV NS3 peptides, taking into account the genetic diversity of this virus.

Cytotoxic T-Lymphocyte-, and Helper T-Lymphocyte-Oriented DNA Vaccination

DNA vaccines have advantages over other types of vaccines in that they can induce strong cellular immune responses, namely cytotoxic T lymphocytes (CTL) and helper T lymphocytes (Th). DNA vaccines are therefore considered a promising alternative to attenuated live vaccines in the field of infectious diseases. So far, various DNA vaccines have been generated and tried to induce a particular cellular immune response by virtue of recombinant DNA technology. DNA vaccines have been designed for efficient transcription and translation of target genes by a variety of strategies. Also, various DNA vaccine strategies for induction of specific CTL and Th have been reported by taking into consideration antigen presentation pathways and the strategies have been shown to be effective to elicit particular T-cell responses. In this paper, we have reviewed these strategies, including our study on epitope-specific T-cell induction by DNA vaccination against Listeria monocytogenes infection. From this review, it has been surmised that, to induce strong immune responses by DNA vaccines, the immunization route and the immunization regimen, such as heterologous "prime-boost" regimen, should also be considered.

PPE Protein (Rv3873) from DNA Segment RD1 ofMycobacterium tuberculosis: Strong Recognition of Both Specific T-Cell Epitopes and Epitopes Conserved within the PPE Family

Proteins encoded by DNA segment RD1 of Mycobacterium tuberculosis have recently been demonstrated to play important roles in bacterial virulence, vaccine development, and diagnostic reagent design. Previously, we characterized two immunodominant T-cell antigens, the early secreted antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP10), which are encoded by the esx-lhp operon in this region. In the present study we characterized a third putative open reading frame in this region, rv3873, which encodes a PPE protein. We found that the rv3873 gene is expressed in M. tuberculosis H37Rv and that the native protein, Rv3873, is predominantly associated with the mycobacterial cell or wall. When tested as a His-tagged recombinant protein, Rv3873 stimulated high levels of gamma interferon secretion in peripheral blood mononuclear cells isolated from tuberculosis (TB) patients, as well as from healthy tuberculin purified protein derivative-positive donors. In contrast to other RD1-encoded antigens, Rv3873 was also found to be recognized by a significant proportion of Mycobacterium bovis BCG-vaccinated donors. Epitope mapping performed with overlapping peptides revealed a broad pattern of T-cell recognition comprising both TB-specific epitopes and epitopes also recognized by BCG-vaccinated donors. The immunodominant epitope (residues 118 to 135) for both TB patients and BCG-vaccinated individuals was found to be highly conserved among a large number of PPE family members.

How well can a T-cell epitope replace its parent carrier protein? A dose-response study.

This work examines the effectiveness of synthetic peptide immunogens derived from immunodominant T-cell epitopes as replacements for their intact parent protein in vaccines. Fluorescein was conjugated to hen egg lysozyme (FL-HEL, positive control) and three synthetic peptide immunogens: (a) murine B10.A (H-2a) immunodominant T-cell epitope of HEL [FL-(T-cell epitope)]; (b) multiple antigenic peptide (MAP) multimer of this epitope ([FL-(T epitope)]n-MAP, n = 2-4); and (c) negative control MAP with T-cell epitope residues replaced with glycine [(FL-Gly18)4-MAP]. The dose response of each immunogen was examined over a 300-fold range in B10.A mice. The immune response was monitored using antifluorescein ELISA assays. FL-(T epitope)'s immune response correlated positively with dose, with maximum response comparable to that of [FL-(T epitope)]n-MAP, or FL-HEL. This trend was consistent across 1 degrees, 2 degrees, and 3 degrees responses, although interanimal variability was higher in the latter two because of an all-or-none response in mice immunized with this peptide. [FL-(T epitope)]n-MAP's immune response was consistently high and nearly dose independent, a trend observed across 1 degrees, 2 degrees, and 3 degrees responses. FL-HEL's immune response correlated negatively to dose in the 1 degrees response but was nearly dose independent in the 2 degrees and 3 degrees responses. The magnitude of these latter responses was comparable to that observed for [FL-(T epitope)]n-MAP. (FL-Gly18)4-MAP did not elicit an immune response except at the highest dose. This trend was consistent across 1 degrees, 2 degrees, and 3 degrees responses. The monomeric epitope was 300-fold less potent than its parent carrier protein, but increasing immunogen valency using MAP technology compensated totally for reduced potency. (FL-Gly18)4-MAP's lack of response at all but the highest dose strongly suggests that a specific immunodominant T-cell epitope sequence for HEL is necessary for successful peptide mimicry of HEL. This work also demonstrates the importance of quality assessment of commercial MAP core resins.

Administration of different antigenic forms of altered peptide ligands derived from HIV-1 RTase influences their effects on T helper cell activation

Genetic hypervariability of viruses such as HIV-1 facilitates appearance of escape mutants for immune response. HIV-1 isolates display variant epitopes, which may fail to stimulate T-lymphocyte responses or act as natural T-cell receptor antagonists, contributing to viral persistence. We evaluated the effect on epitope specific T-cell reactions of different amino acid substitutions in a residue of the 248–262 sequence of HIV-1 reverse transcriptase (peptide 23), showing variability in different viral isolates. Responses against such a determinant have been detected in long-term nonprogressive patients. The modified antigenic determinant was administered either as synthetic peptide or as recombinant protein. Our results show that certain amino acid substitutions abolished peptide binding to major histocompatibility complex (MHC); other modifications, although not affecting the formation of the MHC/peptide complex, either abrogated T-cell proliferation or exhibited an antagonistic effect. The results suggest that residue 11 of peptide 23 exhibits a double function; its alteration affects both the peptide affinity for the MHC and the MHC/peptide complex affinity for the T-cell receptor. Furthermore, we demonstrated that synthetic ligands and recombinant proteins may produce distinct functional effects, providing evidence that synthetic peptides, compared with corresponding epitopes generated by intracellular processing of recombinant proteins, may bind to the MHC groove in a different conformation.

Administration of different antigenic forms of altered peptide ligands derived from HIV-1 RTase influences their effects on T helper cell activation

Genetic hypervariability of viruses such as HIV-1 facilitates appearance of escape mutants for immune response. HIV-1 isolates display variant epitopes, which may fail to stimulate T-lymphocyte responses or act as natural T-cell receptor antagonists, contributing to viral persistence. We evaluated the effect on epitope specific T-cell reactions of different amino acid substitutions in a residue of the 248–262 sequence of HIV-1 reverse transcriptase (peptide 23), showing variability in different viral isolates. Responses against such a determinant have been detected in long-term nonprogressive patients. The modified antigenic determinant was administered either as synthetic peptide or as recombinant protein. Our results show that certain amino acid substitutions abolished peptide binding to major histocompatibility complex (MHC); other modifications, although not affecting the formation of the MHC/peptide complex, either abrogated T-cell proliferation or exhibited an antagonistic effect. The results suggest that residue 11 of peptide 23 exhibits a double function; its alteration affects both the peptide affinity for the MHC and the MHC/peptide complex affinity for the T-cell receptor. Furthermore, we demonstrated that synthetic ligands and recombinant proteins may produce distinct functional effects, providing evidence that synthetic peptides, compared with corresponding epitopes generated by intracellular processing of recombinant proteins, may bind to the MHC groove in a different conformation.

Identification of immunodominant hepatitis C virus (HCV)-specific cytotoxic T-cell epitopes by stimulation with endogenously synthesized HCV antigens

Hepatitis C virus (HCV)-specific CD8+ cytotoxic T lymphocytes (CTL) are believed to play an important role in the pathogenesis of liver cell injury and viral clearance in HCV infection. Because HCV does not efficiently infect human cells in vitro and primary infected hepatocytes cannot be used as stimulator/target cells for CTL analysis, development of efficient systems to activate and expand CTL in vitro, reproducing antigen presentation to CTL occurring during natural infection, is mandatory to study CTL activity and to define the hierarchy of immunodominance of CTL epitopes. To achieve this goal, 5 different defective adenoviruses carrying structural and nonstructural HCV genes (core, core-E1-E2, E2, NS3-NS4A, NS3-NS5A) were used to induce the endogenous synthesis of HCV proteins in human adherent mononuclear cells in vitro and to allow their entry into the HLA class I cytosolic pathway of antigen processing. The cytolytic activity of peripheral blood lympho-mononuclear cells (PBMC) from HLA-A2+ HCV-infected patients stimulated with recombinant adenovirus-infected cells was tested against target cells either pulsed with a panel of synthetic peptides containing the HLA-A2 binding motif or infected with recombinant vaccinia viruses carrying HCV genes. Our study defines a reproducible system to stimulate and expand HCV-specific CTL in vitro that mimics the conditions of antigen encounter in vivo. By this approach, we have identified several HLA-A2–restricted epitopes that should correspond to immunodominant HCV sequences recognized by CTL during natural infection. Therefore, these amino acid sequences represent ideal candidates for the design of therapeutic vaccines for chronic HCV infection. (HEPATOLOGY 2001;33:1533-1543.)

Regulatory functions of self-restricted MHC class II allopeptide-specific Th2 clones in vivo.

We studied T-cell clones generated from grafts of rejecting and tolerant animals and investigated the regulatory function of Th2 clones in vitro and in vivo. To prevent allograft rejection, we treated LEW strain recipient rats of WF strain kidney grafts with CTLA4Ig to block CD28-B7 costimulation. We then isolated epitope-specific T-cell clones from the engrafted tissue, using a donor-derived immunodominant class II MHC allopeptide presented by recipient antigen-presenting cells. Acutely rejected tissue from untreated animals yielded self-restricted, allopeptide-specific T-cell clones that produced IFN-gamma, whereas clones from tolerant animals produced IL-4 and IL-10. Adoptive transfer into naive recipients of Th1 clones, but not Th2 clones, induced alloantigen-specific delayed-type hypersensitivity (DTH) responses. In addition, Th2 clones suppressed DTH responses mediated by Th1 clones in vivo and blocked Th1 cell proliferation and IFN-gamma production in vitro. A pilot human study showed that HLA-DR allopeptide-specific T-cell clones generated from patients with chronic rejection secrete Th1 cytokines, whereas those from patients with stable graft function produce Th2 cytokines in response to donor-specific HLA-DR allopeptides. We suggest that self-restricted alloantigen-specific Th2 clones may regulate the alloimmune responses and promote long-term allograft survival and tolerance.

Detection of simian immunodeficiency virus Gag-specific CD8(+) T lymphocytes in semen of chronically infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex.

Evaluation of human immunodeficiency virus type 1-specific mucosal cytotoxic T lymphocytes can be hampered by limited cell yields from mucosal sites. We sought to characterize virus-specific CD8(+) T lymphocytes with cytotoxic activity in the male genital tracts of SIVmac-infected rhesus monkeys by using a peptide epitope-specific functional T-cell assay and a tetrameric major histocompatibility complex class I-peptide complex. This tetrameric complex was constructed with the rhesus monkey HLA-A homolog molecule Mamu-A*01 and a dominant-epitope 9-amino-acid fragment of SIVmac Gag (p11C, C-M). The proportion of tetramer-positive CD8(+) T cells in semen of SIVmac-infected monkeys ranged from 5.9 to 22.0%. By the use of a standard 51Cr release assay, these cells were found to have peptide epitope-specific cytolytic activity after in vitro expansion. Four-color flow-cytometric analysis of these seminal tetramer-positive CD8(+) T cells demonstrated that they express memory-associated (CD62L- CD45RA-) and activation-associated (CD11a+ Fas+ HLA-DR+) molecules. The present experiments illustrate the power of tetramer technology for evaluating antigen-specific CD8(+) T lymphocytes in a mucosal tissue compartment.

Characterization of and functional antigen presentation by central nervous system mononuclear cells from mice infected with Theiler's murine encephalomyelitis virus.

We examined the phenotype and function of cells infiltrating the central nervous system (CNS) of mice persistently infected with Theiler's murine encephalomyelitis virus (TMEV) for evidence that viral antigens are presented to T cells within the CNS. Expression of major histocompatibility complex (MHC) class II in the spinal cords of mice infected with TMEV was found predominantly on macrophages in demyelinating lesions. The distribution of I-As staining overlapped that of the macrophage marker sialoadhesin in frozen sections and coincided with that of another macrophage/microglial cell marker, F4/80, by flow cytometry. In contrast, astrocytes, identified by staining with glial fibrillary acidic protein, rarely expressed detectable MHC class II, although fibrillary gliosis associated with the CNS damage was clearly seen. The costimulatory molecules B7-1 and B7-2 were expressed on the surface of most MHC class II-positive cells in the CNS, at levels exceeding those found in the spleens of the infected mice. Immunohistochemistry revealed that B7-1 and B7-2 colocalized on large F4/80(+) macrophages/microglia in the spinal cord lesions. In contrast, CD4(+) T cells in the lesions expressed mainly B7-2, which was found primarily on blastoid CD4(+) T cells located toward the periphery of the lesions. Most interestingly, plastic-adherent cells freshly isolated from the spinal cords of TMEV-infected mice were able to process and present TMEV and horse myoglobin to antigen-specific T-cell lines. Furthermore, these cells were able to activate a TMEV epitope-specific T-cell line in the absence of added antigen, providing conclusive evidence for the endogenous processing and presentation of virus epitopes within the CNS of persistently infected SJL/J mice.

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We evaluated relationships between critical Gag T-cell escape mutations and concomitant T-cell responses to determine whether HLA-restricted Gag mutations that confer protection, occur at similar rates in a population infected with mixed HIV-1 clades A1 and D viruses.Assessment of Gag selective pressure, and adaptive T-cell functions to KAFSPEVIPMF (KF11), ISPRTLNAW (ISW9) and TSTLQEQIGW (TW10) Gag epitopes were combined with host HLA to assess correlations with rates of critical epitope escape mutations in clades A1- (n = 23) and D- (n = 21) infected, untreated subjects. Infecting clades and selection pressure were determined from the gag sequences.Overall, Gag escape mutations A163X in KF11 were detected in 61% (14/23) A1- infected compared to 5% (1/21) in D-infected subjects (p = 0.00015). Gag mutations I147X in the ISW9 epitope were seen in 43%: (10/23) clade A compared to 5%: (1/21) clade D infected subjects, p = 0.007, Fisher's Exact test. Both mutations were more frequent in clade A1 infection. Frequencies of the measured epitope-specific T-cell responses were comparable across clades. Peptide binding affinities for the restricting HLA alleles did not differ across clades. Overall, selection pressure on the Gag protein was significantly greater in clade A than in clade D sequences.These findings imply that HIV-1 vaccine strategies designed to target structurally constrained T-cell epitopes may be further challenged by clade-driven outcomes in specific HLA-restricted Gag epitopes. Equally, the data are line with slower HIV-1 disease progression in clade A infection; and raise hope that increased selective pressure on Gag may be protective irrespective of host HLA alleles.

Production of thyroid-stimulating antibodies in mice by immunization with T-cell epitopes of human thyrotropin receptor.

We examined whether mice, immunized with TSH receptor (TSH-R) peptides, which are known to be T-cell epitopes in patients with Graves' disease, would show thyroid-stimulating antibody (TSAb). We immunized DBA/1J mice with TSH-R peptide amino acids 132-150, 145-163, 158-176, and 172-186 and with a pool of these four peptides. The antibodies produced in these mice were evaluated by measurement of TSAb activity using Chinese hamster ovary cells expressing human TSH-R. Seven of 20 mice showed TSAb activity that could be partially blocked with TSH-R peptides. To assess the role of T-cell epitope-specific T-cells in the production of TSAb, we transferred a T-cell line developed from a TSAb-positive mouse to other syngeneic DBA/1J mice. Two of 4 recipient mice showed TSAb activities. These findings suggest that specific T-cell epitopes of TSH-R play a crucial role in the production of TSAb.

Delivery of class I and class II MHC-restricted T-cell epitopes of listeriolysin of Listeria monocytogenes by attenuated Salmonella

Using a Salmonella vaccine-Listeria infection model of intracellular infection, we studied the capacity of an attenuated strain of Salmonella carrying T-cell epitopes of listeriolysin (LLO) of L. monocytogenes to elicit epitope-specific T-cell responses. Class II (LLO 215–226) or class I (LLO 91–99) MHC-restricted T-cell epitopes of LLO were inserted within a central, hypervariable domain of the flagellin protein of an attenuated ΔaroA Salmonella dublin strain. T cells from Listeria-immunized mice were activated by lysates or heat-killed preparations of Salmonella construct expressing the LLO 215–226 epitope, indicating that LLO 215–226 is processed and presented to T cells when offered to antigen-presenting cells as part of a flagellin-epitope fusion protein. The chimeric flagellin genes were integrated into the chromosome of the flagellin-negative S. dublin strain to obtain stable expression of the epitopes. Immunication with the living, chromosomally integrated Salmonella construct carrying LLO 215–226 epitope as part of the flagellin protein generated T cells reactive with the corresponding LLO peptide, indicating that this chimera can stimulate a class-specific immune response in vitro. The effect of flanking residues on the processing and presentation of MHC class I LLO 91–99 epitope was studied using Salmonella vaccine strains that express chimeric flagellins containing one of three LLO 91–99 inserts: 91–99 (normal flagellin amino acids as flanking residues); KK91–99KK (Lys-Lys flanking residues); and AAA91–99AAA (Ala-Ala-Ala flanking residues). Spleen cells from mice immunized with the KK91–99KK flagellin chimeric Salmonella strain showed the greater CTL response indicating that Lys-Lys flanking residues are associated with enhanced immunogenicity, possibly by facilitating the proteolytic excision of the epitope in the endosomal compartment of antigen-presenting cells. A statistically significant decrease in the number of hepatic and/or splenic bacteria was achieved after Listeria challenge in mice immunized with Salmonella constructs expressing either MHC class II LLO 215–226 or class I LLO KK91–99KK flagellin chimera, showing that the immunogenicity of these constructs is associated with an epitope-specific increased resistance to infection.

Immunogenicity of a mycobacterial T-cell epitope expressed in outer membrane protein PhoE of Escherichia coli

The outer membrane protein PhoE of Escherichia coli can be used for the expression of foreign antigenic determinants. Previously, a T-cell epitope of the 65 kDa heat shock protein (hsp65) of Mycobacterium tuberculosis, comprising amino acids 180 to 188, was expressed in PhoE. The hybrid protein induced proliferation of epitope-specific T-cell clones in vitro. In this report, the potential of the hybrid protein to induce an in vivo T-cell response against the 180—188 T-cell epitope was assessed. Popliteal lymph node cells, isolated from rats immunized with PhoE containing the hsp65 epitope, showed high proliferative responses to a synthetic peptide consisting of amino acids 180 to 188 of hsp65, indicating that the epitope is immunogenic in the PhoE-associated conformation.

Heat-killed Listeria monocytogenes and L. monocytogenes soluble antigen induce clonable CD4+ T lymphocytes with protective and chemotactic activities in vivo

In the present study we attempted to analyze the possibility to induce in mice a T-cell response using killed Listeria monocytogenes in adjuvant. Clearly, nonviable antigen is capable of inducing protective and granuloma-forming T cells in C57BL/6 mice when emulsified in complete Freund's adjuvant. These T cells were cloned in vitro by using antigen and irradiated splenocytes, as antigen-presenting cells, and the clones were characterized in vivo. Listeria-specific T-cell clones showed protective and chemotactic activity upon adoptive transfer, although some degree of functional heterogeneity among different clones was observed. The heterogeneous in vivo functions could not be correlated with the ability of the clones to produce gamma interferon or T-cell growth factor (interleukin-2 and/or interleukin-4). It was demonstrated that an in vivo relevant fraction of listeria-specific T lymphocytes can be induced by nonviable antigen in complete Freund's adjuvant. These results show that the low immunogenicity of heat-killed bacteria is not due to the expression of specific protective T-cell epitopes only by live bacteria.