Induction of CD8+ T cell-mediated protective immunity against Trypanosoma cruzi

Trypanosoma cruzi was transformed with the Plasmodium yoelii gene encoding the circum-sporozoite (CS) protein, which contains the well-characterized CD8+ T cell epitope, SYVPSAEQI. In vivo and in vitro assays indicated that cells infected with the transformed T. cruzi could process and present this malaria parasite-derived class I MHC-restricted epitope. Immunization of mice with recombinant influenza and vaccinia viruses expressing the SYVPSAEQI epitope induced a large number of specific CD8+ T cells that strongly suppressed parasitemia and conferred complete protection against the acute T. cruzi lethal infection. CD8+ T cells mediated this immunity as indicated by the unrelenting parasitemia and high mortality observed in immunized mice treated with anti-CD8 antibody. This study demonstrated, for the first time, that vaccination of mice with vectors designed to induce CD8+ T cells is effective against T. cruzi infection.     

DNA sequences encoding CD4+ and CD8+ T-cell epitopes are important for efficient protective immunity induced by DNA vaccination with a Trypanosoma cruzi gene

Immunization of BALB/c mice with a plasmid containing the gene for Trypanosoma cruzi trans-sialidase (TS) induced antibodies that inhibited TS enzymatic activity, CD4+ Th1 and CD8+ Tc1 cells, and protective immunity against infection. We used this model to obtain basic information on the requirement of CD4 or CD8 or B-cell epitopes for an effective DNA-induced immunity against T. cruzi infection. For that purpose, mice were immunized with plasmids containing DNA sequences encoding (i) the entire TS protein, (ii) the TS enzymatic domain, (iii) the TS CD4+ T-cell epitopes, (iv) the TS CD8+ T-cell epitope, or (v) TS CD4+ and CD8+ T-cell epitopes. Plasmids expressing the entire TS or its enzymatic domain elicited similar levels of TS-inhibitory antibodies, gamma interferon (IFN-gamma)-producing T cells, and protective immunity against infection. Although the plasmid expressing TS CD4 epitopes was immunogenic, its protective efficacy against experimental infection was limited. The plasmid expressing the CD8 epitope was poorly immunogenic and provided little protective immunity. The reason for the limited priming of CD8+ T cells was due to a requirement for CD4+ T cells. To circumvent this problem, a plasmid expressing both CD4+ and CD8+ T-cell epitopes was produced. This plasmid generated levels of IFN-gamma-producing T cells and protective immunity comparable to that of the plasmid expressing the entire catalytic domain of TS. Our observations suggest that plasmids expressing epitopes recognized by CD4+ and CD8+ T cells may have a better protective potential against infection with T. cruzi.     

Diversity and complexity of CD8+ T cell responses against a single epitope of adenovirus E1B

This study describes the characteristics of the immune responses against adenovirus in C57BL/6 mice. CTL responses could be induced against E1Bp of adenovirus type 5, when whole viruses were immunized. A panel of E1Bp-specific CTL clones showed a wide range of T cell avidity. Recognition of the E1Bp peptide and a panel of variant peptides containing a single alanine substitution by CTL clones revealed that the fine specificity of the CTL response was quite diverse, rather than being limited to a certain clonal preference. Moreover, the variant peptides with a substitution at the TCR contact residue had antagonistic properties to some of the CTL clones, while being agonistic to others, reflecting the extensive diversity of the T cells. These results imply that the functional diversity of T cells to even a single epitope should be considered in manipulating immunity to viruses and in developing adoptive immunotherapy for immunocompromised individuals.     

TGF-beta regulates pathology but not tissue CD8+ T cell dysfunction during experimental Trypanosoma cruzi infection

Infection with the protozoan parasite Trypanosoma cruzi leads to chronic infection, with parasite persistence primarily in muscle tissue. CD8(+) T cells isolated from muscle tissue of T. cruzi-infected mice display decreased production of IFN-gamma in response to T cell receptor engagement. The expression of TGF-beta at the site of CD8(+) T cell dysfunction and parasite persistence suggested that this immunoregulatory cytokine might play a role in these processes. Mice expressing a T cell-specific dominant negative TGF-beta receptor type II (DNRII) were therefore infected with T. cruzi. Infection of DNRII mice resulted in massive CD8(+) T cell proliferation, leading to increased numbers but decreased frequencies of antigen-specific CD8(+) T cells in the spleen compared to wild-type mice. However, TGF-beta unresponsiveness failed to restore effector functions of CD8(+) T cells isolated from muscle tissue. Histological examination of skeletal muscle from T. cruzi-infected DNRII mice revealed an extensive cellular infiltrate, and DNRII mice displayed higher susceptibility to infection. Overall, while TGF-beta does not appear to be responsible for CD8(+) T cell unresponsiveness in peripheral tissue in T. cruzi-infected mice, these data suggest a role for TGF-beta in control of immunopathology in response to T. cruzi infection.     

CD8+-T-cell responses of Mycobacterium-infected mice to a newly identified major histocompatibility complex class I-restricted epitope shared by proteins of the ESAT-6 family

Here we describe the identification of a new CD8(+)-T-cell epitope, the GYAGTLQSL nonamer, shared by the TB10.3 and TB10.4 proteins of the Mycobacterium tuberculosis ESAT-6 family. Cytotoxic T cells from mycobacterium-infected mice efficiently recognized this epitope. GYAGTLQSL-specific T-cell hybridomas, which were able to recognize Mycobacterium bovis BCG-infected macrophages, were generated and now allow investigation of mycobacterial-antigen processing through the major histocompatibility complex class I pathway.     

CD8+ T lymphocyte responses are induced during acute hepatitis C virus infection but are not sustained

Cellular immune responses are likely to play a key role in determining the clinical outcome in acute infection with hepatitis C virus (HCV), but the dynamics of such responses and their relationship to viral clearance are poorly understood. In a previous study we have shown highly activated, multispecific cytotoxic T lymphocyte responses arising early and persisting in an individual who subsequently cleared the virus. In this study the HCV-specific CD8+ lymphocytes response has been similarly analyzed, using peptide-HLA class I tetramers, in a further nine individuals with documented acute HCV infection, six of whom failed to clear the virus. Significant populations of virus-specific CD8+ lymphocytes were detected at the peak of acute hepatic illness (maximally 3.5% of CD8+ lymphocytes). Frequencies were commonly lower than those seen previously and were generally not sustained. Early HCV-specific CD8+ lymphocytes showed an activated phenotype in all patients (CD38+ and HLA class II+), but this activation was short-lived. Failure to sustain sufficient numbers of activated virus-specific CD8+ lymphocytes may contribute to persistence of HCV.     

CD8+-T-cell-dependent control of Trypanosoma cruzi infection in a highly susceptible mouse strain after immunization with recombinant proteins based on amastigote surface protein 2

We previously described that DNA vaccination with the gene encoding amastigote surface protein 2 (ASP-2) protects approximately 65% of highly susceptible A/Sn mice against the lethal Trypanosoma cruzi infection. Here, we explored the possibility that bacterial recombinant proteins of ASP-2 could be used to improve the efficacy of vaccinations. Initially, we compared the protective efficacy of vaccination regimens using either a plasmid DNA, a recombinant protein, or both sequentially (DNA priming and protein boosting). Survival after the challenge was not statistically different among the three mouse groups and ranged from 53.5 to 75%. The fact that immunization with a recombinant protein alone induced protective immunity revealed the possibility that this strategy could be pursued for vaccination. We investigated this possibility by using six different recombinant proteins representing distinct portions of ASP-2. The vaccination of mice with glutathione S-transferase fusion proteins representing amino acids 261 to 500 or 261 to 380 of ASP-2 in the presence of the adjuvants alum and CpG oligodeoxynucleotide 1826 provided remarkable immunity, consistently protecting 100% of the A/Sn mice. Immunity was completely reversed by the in vivo depletion of CD8(+) T cells, but not CD4(+) T cells, and was associated with the presence of CD8(+) T cells specific for an epitope located between amino acids 320 and 327 of ASP-2. We concluded that a relatively simple formulation consisting of a recombinant protein with a selected portion of ASP-2, alum, and CpG oligodeoxynucleotide 1826 might be used to cross-prime strong CD8(+)-T-cell-dependent protective immunity against T. cruzi infection.     

A novel peptide vaccination augments cytotoxic CD8+ T-cell responses against Mycobacterium tuberculosis HspX antigen

Cellular immunity is a critical factor determining the safety and efficacy of newly developed vaccines against Mycobacterium tuberculosis infection. Crosstalk between CD4⁺ and CD8⁺ T-lymphocytes plays central roles in perpetuating the cytotoxic killing to the infected cells for host clearance. Our study proposed a novel alternating MHC-class II restricted peptide vaccination strategy to enhance the antigen-specific CD8⁺ T-cell activity against alpha-crystalline heat-shock protein (HspX) in C57BL/6 mice. Alternating peptide vaccination significantly stimulated a prominent HspX-specific CD8⁺ T-cell response with elevated Th1 and Th17 responses, without interference from Tregs suppression. Heightened central and effector CD8 memory were apparent in mice receiving alternating peptide vaccine, indicating a persisting recall immunity against HspX antigen. It was unlikely for alternating peptide vaccine to cause dysregulation in CD8⁺ T-cells as shown by minimal expression of KLRG1, PD1, LAG3, and CTLA-4 markers. Strong cytotoxic T-lymphocyte (CTL) responses were demonstrated in mice administrated with alternating peptide vaccines, suggesting its capacity in executing killing effector function against targeted cells. In conclusion, our novel vaccination strategy delineated potential benefits of alternating MHC-II peptides to invigorate efficient cytotoxic CD8⁺ T-cell responses against HspX antigen. Such approach might be applicable to serve as alternative immunotherapy for latent tuberculosis infection in future.     

Distinct kinetics of effector CD8+ cytotoxic T cells after infection with Trypanosoma cruzi in naive or vaccinated mice

The kinetics of effector CD8+-T-cell responses to specific Trypanosoma cruzi epitopes was investigated after challenge. Our results suggest that the delayed kinetics differs from that observed in other microbial infections and facilitates the establishment of the disease in na?ve mice. In contrast, in vaccinated mice, the swift CD8+-T-cell response helps host survival after challenge.     

Clearance of Theiler's virus infection depends on the ability to generate a CD8+ T cell response against a single immunodominant viral peptide

Theiler's murine encephalomyelitis virus (TMEV) induces a chronic demyelinating disease in the central nervous system of susceptible mice. Resistance to persistent TMEV infection maps to he D locus of the major histocompatibility complex suggesting a prominent role of antiviral CTL in the protective immune response. Introduction of the D(b) gene into the FVB strain confers resistance to this otherwise susceptible mouse line. Infection of the FVB/D(b) mouse with TMEV provides a model where antiviral resistance is determined by a response elicited by a single class I molecule. Resistant mice of the H-2(b) haplotype mount a vigorous H-2D(b)-restricted immunodominant response to the VP2 capsid protein. To investigate the extent of the contribution of the immunodominant T cell population in resistance to TMEV, FVB/D(b) mice were depleted of VP2-specific CD8(+) T cells by peptide treatment prior to virus infection. Peptide-treated mice were not able to clear the virus and developed extensive demyelination. These findings demonstrate that the D(b)-restricted CD8(+) T cells specific for a single viral peptide can confer resistance to TMEV infection. Our ability to manipulate this cellular response provides a model for investigating the mechanisms mediating protection against virus infection by CD8(+) T cells.     

B cells modulate T cells so as to favour T helper type 1 and CD8+ T-cell responses in the acute phase of Trypanosoma cruzi infection

In this study, we have evaluated the production of pro- and anti-inflammatory cytokines and the formation of central and effector memory T cells in mice lacking mature B cells (mu MT KO). The results show that Trypanosoma cruzi infection in C57Bl/6m mu MT KO mice is intensified in relation to control mice and this exacerbation is related to low levels of inflammatory cytokines produced during the acute infection and the lower numbers of central and effector memory CD4(+) and CD8(+) T cells generated during the acute phase of the infection. In addition, a marked reduction in the CD8(+) T-cell subpopulation was observed in mu MT KO infected mice. In agreement to this, the degree of tissue parasitism was increased in mu MT mice and the tissue inflammatory response was much less intense in the acute phase of the infection, consistent with a deficit in the generation of effector T cells. Flow cytometry analysis of the skeletal muscle inflammatory infiltrate showed a predominance of CD8(+) CD45Rb low in B-cell-sufficient C57Bl/6 mice, whereas the preponderant cell type in mu MT KO skeletal muscle inflammatory infiltrate was CD4(+) T cells. In addition, CD8(+) T cells found in skeletal muscle from mu MT KO infected mice were less activated than in control B-cell sufficient infected mice. These results suggest that B cells may participate in the generation of effector/memory T cells. In addition and more importantly, B cells were crucial in the maintenance of central and effector memory CD8(+) T cell, as well as the determination of the T cell cytokine functional pattern, and they may therefore account for critical aspects of the resistance to intracellular pathogens, such as T. cruzi.     

Contribution of HLA class I allele expression to CD8+ T-cell responses against Epstein-Barr virus

Most immune responses to viral infections involve CD8+ T cells recognizing viral peptides of typically 9-10 amino acids in the groove of major histocompatibility complex (MHC) class I. Importantly, CD8+ T-cell responses appear to focus on few viral epitopes, a phenomenon termed immunodominance. While the understanding of this phenomenon has been based largely on experimental mice models, it is imperative to evaluate its contribution in humans, as the design of peptide-based vaccines may be influenced by immunodomination. Here, we present evidence that immunodominance can be detected among Epstein-Barr virus (EBV) epitopes associated with two of the most frequent class I alleles in Western Europe, human leucocyte antigen (HLA)-A2 and HLA-B7. CD8+ T-cell responses to HLA-A2-associated EBV epitopes were significantly reduced in individuals coexpressing HLA-B7. The impairment of HLA-A2-associated responses correlated with a dominant response to an HLA-B7 epitope. The data demonstrate a hierarchy in the human cellular immune response to immunodominant EBV epitopes presented by separate HLA class I alleles. This may have implications for EBV vaccine development as well as for the interpretation of isolated analysis of immunodominant responses to EBV.     

CD8+ T-cell recognition of a synthetic epitope formed by t-butyl modification

We set out to clone Bax-specific CD8⁺ T cells from peripheral blood samples of patients with primary chronic lymphocytic leukaemia. A number of clones were generated using a Bax peptide pool and their T-cell epitope was mapped to two peptides sharing a common 9-amino-acid sequence (LLSYFGTPT), restricted by HLA-A*0201. However, when these T-cell clones were tested against highly purified syntheses (> 95%) of the same peptide sequence, there was no functional response. Subsequent mass spectrometric analysis and HPLC fractionation suggested that the active component in the original crude peptide preparations (77% pure) was a peptide with a tert-butyl (tBu) modification of the tyrosine residue. This was confirmed by modification of the inactive wild-type sequence to generate functionally active peptides. Computer modelling of peptide:HLA-A*0201 structures predicted that the tBu modification would not affect interactions between peptide residues and the HLA binding site. However, these models did predict that the tBu modification of tyrosine would result in an extension of the side chain out of the peptide-binding groove up towards the T-cell receptor. This modified product formed < 1% of the original P603 crude peptide preparation and < 0·05% of the original 23-peptide mixture used for T-cell stimulation. The data presented here, illustrate the potential for chemical modifications to change the immunogenicity of synthetic peptides, and highlight the exquisite capacity of T-cell receptors to discriminate between structurally similar peptide sequences. Furthermore, this study highlights potential pitfalls associated with the use of synthetic peptides for the monitoring and modulating of human immune responses.     

The onset of CD8+-T-cell contraction is influenced by the peak of Listeria monocytogenes infection and antigen display

The CD8+-T-cell response to infection with Listeria monocytogenes consists of expansion, contraction, and memory phases. The transition between expansion and contraction is reported to occur on different days postinfection with virulent (8 to 9 days) and attenuated (DeltaactA) (7 days) L. monocytogenes strains. We hypothesized that differences in the infectious courses, and therefore antigen (Ag) display, determine the precise time of the expansion/contraction transition in response to these infections. To test this, we infected BALB/c mice with 0.1 50% lethal dose of DeltaactA or virulent L. monocytogenes and measured bacterial numbers, Ag display, and Ag-specific CD8+-T-cell responses on various days after infection. We found that bacterial numbers and Ag display peaked between 12 and 36 h and between 36 and 60 h after infection with DeltaactA and virulent L. monocytogenes strains, respectively. Infection with DeltaactA L. monocytogenes resulted in a sharp peak in the Ag-specific CD8+-T-cell response on day 7, while infection with virulent L. monocytogenes yielded a prolonged peak with equivalent numbers of Ag-specific CD8+ T cells on days 6, 7, and 8 after infection. Truncating virulent infection with antibiotics on day 1 or 2 after infection resulted in a shift in the expansion/contraction transition from day 8 to day 7 after infection. However, antibiotic treatment beginning on day 3, after the peak of virulent L. monocytogenes infection and Ag display, had no effect upon the magnitude or timing of the CD8+-T-cell response. These results demonstrate a direct relationship between the course of infection and Ag display and that the timing of these events is important in shaping the T-cell response to infection.     

Generation, specificity, and function of CD8+ T cells in Trypanosoma cruzi infection

Summary: CD8⁺ T cells are crucial to the control of Trypanosoma cruzi infection and probably act via multiple mechanisms, the most important being the production of interferon‐γ (IFN‐γ). In the absence of CD8⁺ T cells, mice quickly succumb to the infection or develop a more severe chronic disease. Reduced production of IFN‐γ by CD8⁺ T cells is also associated with increased severity of chagasic disease in humans. CD8⁺ T cells in chronic T. cruzi infection are maintained as effector memory cells, undergo rapid expansion, and demonstrate effector functions following re‐exposure to antigen. However, the initial generation of T. cruzi‐specific CD8⁺ T‐cell responses appears to be relatively slow to develop. In addition, the expression of the effector function of the CD8⁺ T cells is compromised in some tissues, particularly in muscle. The targets of effective CD8⁺ T‐cell responses in T. cruzi infection are multiple and varied, and they represent some of the best vaccine candidates described to date. Further analysis of CD8⁺ T cells will provide insight into the disease process in T. cruzi infection and should identify methods to assess and enhance immunity to T. cruzi infection and protection from the symptoms of Chagas' disease.     

GM-CSF基因修饰同种异体肺癌细胞疫苗诱导CD8+T细胞的免疫反应

目的 探讨GM-CSF基因修饰同种异体肿瘤疫苗是否可诱导CUB T细胞的特异性免疫反应.方法 以接种Lewis肺癌(Lewsi Lung Cancer,LLC)细胞株的C57BL小鼠作为动物模型.利用含小鼠粒-巨细胞集落刺激因子(macrophage-colony-stmulating factor,GM-CSF)基因的重组质粒GM-CSF-plRKS2.EGFP,转染小鼠肺癌细胞株LA795,制备同种异体肿瘤疫苗.C57BL小鼠预防接种该疫苗3次后,皮下接种LLC细胞株,分别于免疫前、每次疫苗注射后采集小鼠脾淋巴细胞,采用ELLSPOT法检测淋巴细胞经LLC抗原刺激后的活化;脾细胞及其中的CD8 T细胞对LLC细胞株的杀伤活性;同时采用免疫组化方法检测注射部位淋巴细胞浸润情况.结果 疫苗注射部位可见明显的CD8 T细胞浸润;经ELISPOT方法检测,GM-CSF基因修饰LA795疫苗接种后,小鼠脾淋巴细胞经LLC抗原刺激后活化细胞比例均显著升高(P＜0.01);同时,脾细胞及其中的CD8 T细胞对LLC细胞的杀伤活性明显升高(P＜0.05).结论 GM-CSF分泌性同种异体瘤苗可诱导CD8 T细胞的活化,以及对自体肿瘤细胞的杀伤,即可诱导特异性抗肿瘤免疫反应.