Functional profile of human influenza virus-specific cytotoxic T lymphocyte activity is influenced by interleukin-2 concentration and epitope specificity

The ability of influenza A virus-specific cytotoxic T lymphocytes (CTL) to degranulate and produce cytokines upon antigenic restimulation was studied in four HLA-A*0101 and HLA-A*0201 positive subjects. Peripheral blood mononuclear cells of these subjects were stimulated with influenza A virus in the presence of high or low interleukin (IL)-2 concentrations. CD8(+) T cell populations specific for the HLA-A*0101 restricted epitope NP(44-52) and the HLA-A*0201 restricted epitope M1(58-66) were identified by positive staining with tetramers of peptide major histocompatibility complexes (MHC) (NP-Tm and M1-Tm, respectively). Within these populations, the proportion of cells mobilizing CD107a, or expressing interferon (IFN)-gamma and tumour necrosis factor-(TNF)-alpha upon short-term peptide restimulation was determined by flow cytometry. Independent of IL-2 concentrations, large subject-dependent differences in the mobilization of CD107a and expression of IFN-gamma and TNF-alpha by both NP- and M1-specific T cells were observed. In two of the four subjects, the functional profile of NP-Tm(+) and M1-Tm(+) cells differed considerably. Overall, no difference in the proportion of NP-Tm(+) or M1-Tm(+) cells expressing CD107a was observed. The proportion of M1-Tm(+) cells that produced IFN-gamma (P < 0.05) was larger than for NP-Tm(+) cells, independent of IL-2 concentration. When cultured under IL-2(hi) concentrations higher TNF-alpha expression was also observed in M1-Tm(+) cells (P < 0.05). The IL-2 concentration during expansion of virus-specific cells had a profound effect on the functionality of both M1-Tm(+) and NP-Tm(+) cells.     

病毒逃避细胞毒性T细胞免疫研究进展

病毒感染期间,病毒与宿主免疫系统之间发生一系列复杂的互相作用,宿主的目的是清除感染及将病理影响减少至最低限度;而病毒则试图逃避清除,以便长期存在及播散至其他宿主.细胞介导的免疫反应特别是病毒特异性CD8细胞毒T细胞(cytotoxicTlymphocyte,CTL)反应,常常在清除病毒感染中起关键作用,故许多病毒的逃避策略即针对于此.在感染过程中,病毒蛋白上氨基酸改变可影响CTL对其表位的识别,而这种逃避机制在病毒感染过程中可引起发病/病情迁延.     

丙型肝炎病毒非结构区5个细胞毒性T细胞表位的鉴定

目的 采用T细胞表位预测软件结合体外实验鉴定丙型肝炎病毒(HCV)特异性细胞毒性T细胞(CTL)表位.方法 采用T表位预测软件Rankpep预测HCV特异性CTL表位,选择候选CTL表位加以合成;用候选CTL表位肽分别刺激HCV感染者以及健康志愿者的外周血单个核细胞(PBMC),采用酶联免疫斑点试验(ELISPOT)检测PBMC中肽特异性分泌IFN-γ的斑点形成细胞(spots forming cells,SFC)的水平,采用细胞内细胞因子染色(intracellular cytokine staining,ICS)检测PBMC中肽特异性IFN-γ+CD8+T细胞的水平.结果 用5条候选CTL表位肽[NS3 450(TVPQDAVSR)、NS3 594(GPTPLLYRL)、NS4b 78(SMMAFSAAL)、NS5a 416(SEENVSVVF)和NS5a 367(TVSSALAEL)]分别刺激10个HCV感染者和2个健康者的PBMC后,健康者的PBMC不产生IFN-γ而7个HCV感染者的PBMC产生IFN-γ;HCV感染者的PBMC中肽特异性分泌IFN-γ的细胞的频率为(5-36)SFC/105 PBMC,肽特异性IFN-γ+CD8+T细胞占总CD8+T细胞的百分比为0.02%～0.25%.结论 ELISPOT结果和ICS结果证实5条肽NS3 450、NS3 594、NS4b 78、NSSa 416和NS5a 367为全新的HCV特异性CTL表位.     

Prediction and identification-based prediction of Chinese hepatitis C viral-specific cytotoxic T lymphocyte epitopes

Cytotoxic T lymphocytes (CTLs) play a critical role in the host immune response to infection by the Hepatitis C Virus (HCV). In the current study, a number of HCV CTL epitopes that represent the HLA polymorphisms found in the majority of Chinese people were predicted based on genomic and bioinformatic approaches. The predicted epitopes were evaluated for validity by examining the peptide-binding affinity for MHC class I molecules, the stability of peptide-MHC complexes, and frequencies of IFN γ-positive T cells. Among the predicted epitope peptides, HLA-A2 restricted epitopes [NS4B (1793-1801) SMMAFSAAL] and HLA-B7 restricted epitopes [P7 (774-782) AAWYIKGRL] were able to induce high frequencies of IFN γ-producing T cells, and the specific CTLs for other epitopes were not detected in peripheral blood lymphocytes from patients with HCV. Moreover, NS4B (1793-1801) exhibited high binding affinity for HLA-A2 molecules, and its stability of peptide-MHC class I complexes was sufficient, indicating that the high binding affinity for MHC class I molecules is an important factor for immunogenicity. Primary analyses of the immunogenicity of predicted epitopes, such as in the current study, will contribute to the future design of an efficient vaccine that will be able to induce vigorous, sustainable, and broad HCV-specific CTL responses for the Chinese population.     

Functional differences between influenza A-specific cytotoxic T lymphocyte clones expressing dominant and subdominant TCR.

We have shown that the dominance of CD8+ T cells expressing TCR Vbeta17 in the adult HLA-A*0201-restricted influenza A/M1(58-66)-specific response is acquired following first antigen exposure. Despite the acquired dominance of Vbeta17+ cells, subdominant M1(58-66)-specific clones expressing non-Vbeta17+ TCR persist in all individuals. To determine whether the affinity of the expressed TCR for the HLA-A*0201/M1(58-66) complex could influence functional properties, M1(58-66)-specific clones expressing subdominant (non-Vbeta17+) TCR were compared to cytotoxic T lymphocyte (CTL) clones expressing dominant (Vbeta17+) TCR. The Vbeta17+ CTL required up to 10,000-fold lower amounts of M1 peptide to mediate lysis compared to CTL clones expressing other Vbeta gene segments. All Vbeta17+ CTL clones tested bound HLA-A*0201/M1(58-66) tetramer, but two of three CTL clones expressing other TCR did not bind tetramer. The inability of non-Vbeta17+ CTL to bind tetramer did not correlate with phenotype, CD8 dependence or with cytokine production profiles. This suggests a limitation for the use of tetramers in examining subdominant T cell responses. Together these findings suggest that Vbeta17+ CTL which dominate the HLA-A*0201-restricted CTL response against influenza A are not functionally distinct from subdominant non-Vbeta17+ CTL. The dominance of Vbeta17+ CTL is likely to result from a competitive advantage due to superior CTL avidity for the HLA-A*0201/M1(58-66) complex.     

Absence of Epstein-Barr virus-specific, HLA class II-restricted CD4+ cytotoxic T lymphocytes in infectious mononucleosis.

Cytotoxic T lymphocytes (CTL) with the CD4+ phenotype that recognize major histocompatibility complex (MHC) class II antigens are detectable very frequently in cultures of human alloreactive or virus-specific T cells. The significance of these CD4+ CTL for an immune reaction in vivo is not clear. Since Epstein-Barr virus (EBV) transformed B cells express HLA-class I and class II antigens equally well both CD8+ and CD4+ CTL should be stimulated during an acute EBV infection. We analysed the MHC specificity and the phenotype of EBV-specific CTL from patients with infectious mononucleosis (IM). When tested directly without any previous culture, T cells from patients in the acute phase of IM showed specific MHC-restricted cytotoxicity against the autologous B cell line. Addition of a HLA class I specific monoclonal antibody (MoAb) but not of a HLA class II specific MoAb resulted in a complete blocking of the lytic activity. Cell sorting revealed that the entire cytotoxic activity was present in the CD8+ fraction whereas no specific CTL were detectable in the CD4+ fraction. The absence of cytotoxicity in CD4+ cells was not due to a lack of activation of these cells since both CD8+ and CD4+ cells were activated in situ, showing spontaneous growth in interleukin-2 (IL-2) and expressing the activation marker TP103. Frequency estimation revealed that 1/300-1/600 CD8+ but only 1/2000-1/4000 CD4+ T cells gave rise to a specific CTL colony after 10 days. If CD4+ colonies were tested repeatedly for cytotoxicity we found that CD4+ CTL acquired their cytotoxicity during in vitro culture. In addition, we isolated EBV-specific CD4+ T cell clones able to lyse their stimulator cells in the presence but not in the absence of lectin, even after a long period of culture. Taken together our results show that cytotoxicity mediated by CD4+ T cells does not play a role in an anti-viral immune response.     

Induction in vitro of a primary human antiviral cytotoxic T cell response

We report herein the successful priming of human anti-viral cytotoxic T cells (CTL) in vitro using two induction strategies based on the stimulation of peripheral blood mononuclear cells isolated from uninfected donors with synthetic viral peptides. The peptides used contain HLA-A2 binding motifs and have been identified as HLA-A2-restricted CTL epitopes in patients infected by the hepatitis B and C viruses. One approach uses repetitive long-term stimulation and the other uses bulk cultures containing large numbers of naive peripheral blood mononuclear cells. Both approaches successfully induce HLA-A2-restricted CTL specific for several viral epitopes. Some CTL recognize endogenously synthesized antigen on target cells infected with recombinant vaccinia virus expressing the corresponding viral proteins. This simple technique permits easy analysis of the primary human CTL repertoire, and may be exploitable for production of specific CTL effector cells for adoptive immunotherapy and dissection of the cellular and molecular requirements for priming of naive human CTL.     

Collagen-specific cytotoxic T lymphocyte responses in patients with ankylosing spondylitis and reactive arthritis

Both ankylosing spondylitis (AS) and reactive arthritis (ReA) are strongly associated with HLA-B27 although the mechanism for this association is still unknown. Here we examine the hypothesis that B27-restricted, joint antigen-specific cytotoxic T lymphocytes (CTL) may be the driving force of AS and ReA. Type II and type XI procollagens (CII and CXI, respectively), expressed almost exclusively in the articular cartilage of the joints, were chosen as the possible targets of autoimmune CTL. Type I procollagen (CI), expressed in many different tissues, was also included as control. Nineteen nonamer peptides bearing appropriate HLA-B27 binding motifs from human CI, CII and CXI were identified and synthesized. When analyzed for binding affinity to HLA-B27 in assembly assays, four (two from CII, two from CXI) were found capable of binding to HLA-B27 with high affinity. These B27-binding collagen peptides were then used to stimulate peripheral blood lymphocytes from eight B27-positive AS and three ReA patients for identification of possible B27-restricted autoimmune CTL. HLA-B27-restricted CTL specific for one of the CII peptides, P109 were found in one of the ReA patients, but in none of the others.     

Dengue virus-induced human cytotoxic factor: production by peripheral blood leucocytes in vitro.

During dengue type 2 virus (DV) infection of mice a unique cytokine, the cytotoxic factor (CF), is produced which reproduces the pathological lesions seen in patients of dengue haemorrhagic fever (DHF). Recently we have observed a CF-like protein in the sera of DHF cases. The present study was undertaken to investigate whether DV can stimulate human peripheral blood mononuclear cells (PBMC) in vitro to produce human CF (hCF). Cultures prepared from PBMC or its enriched subpopulations were inoculated with 1000 LD50 of DV and controls with normal mouse brain suspension (NMB). Aliquots of cultures were harvested daily from 24 h to 96 h and their supernatant (CS) and cells were separated. CS were screened for viral titres and for the presence of hCF by cytotoxicity assay, inhibition ELISA, dot blot and Western blot tests using anti-mouse-CF antibodies. The RNA from the cells was screened in Northern blot and dot blot tests for the presence of mRNA for CF. It was observed that hCF appeared in the CS of DV-infected culture of PBMC and T-enriched cells at 48 h and was present until 96 h; no CF was detected in CS of B cells or monocyte cultures. The production of hCF was abrogated by pretreatment of the T cells with anti-CD4 antibodies but not with anti-CD8 antibodies, indicating that hCF was produced by CD4+ T cells. The Northern blot analysis using oligonucleotide probes prepared on the basis of amino-terminal sequence of mouse CF, showed presence of mRNA for hCF in PBMC and T cell cultures. DV replicated in PBMC cultures with peak titres at 48 h. The findings of the present study demonstrate that DV-induced hCF is produced by human T cells.     

Analysis of the costimulatory requirements for generating human virus-specific in vitro T helper and effector responses

The present study analyzes the role of CD28-B7-mediated costimulation during in vitro human peripheral blood memory T cell activation by influenza A virus. Inhibition studies using the B7-binding fusion protein CTLA4Ig and antibodies against CD80 and CD86 demonstrate that CTLA4Ig and anti-CD86 inhibited influenza-specific T cell proliferation, interleukin (IL)-2 and interferon (IFN)- production, and generation of influenza-specific CD8+ CTL. The production of IL-10 and IL-18, which are known to modulate T cell immune responses, were not affected by blocking the CD28-B7 costimulatory pathway. Inhibition of diverse influenza-specific T cell functions could be reversed by the addition of exogenous IL-2 or IL-12 but not by the addition of IFN- or IL-18. Although IL-2 is known to overcome CD28-B7 costimulatory requirements, this is the first report showing that exogenous IL-12 is able to bypass CD28-B7 costimulatory blockade induced by CTLA4Ig in vitro. The induction of IFN- production with the recently described IFN- inducing cytokine IL-18 was not detected. In conclusion, these results demonstrate that CD86 represents a major costimulatory signal for the activation of resting peripheral blood memory T cells with recall antigens. These observations may have important implications for the development of immunotherapeutic strategies in diverse immunodeficiency diseases as well as in tumor immunotherapy.     

检测Epstein-Barr病毒特异性细胞毒性T淋巴细胞方法的建立及其初步研究

目的建立一种非放射性、简便易行的可检测特异性细胞毒性Ｔ淋巴细胞的方法，并且初步应用于Ｅｐｓｔｅｉｎ－Ｂａｒ病毒的细胞免疫应答。方法用重组的ＥＢＶ－ＬＭＰ１痘苗病毒、ＴＫ＋痘苗病毒和杆状病毒系统表达的ＥＢＶ－ＬＭＰ１蛋白分别免疫Ｂａｌｂ／Ｃ小鼠，用Ｐ８１５细胞和乳酸脱氢酶法检测ＥＢ病毒特异性细胞毒性Ｔ细胞的杀伤效应。结果重组ＥＢＶ－ＬＭＰＩ痘苗病毒免疫组原发ＣＴＬ水平和体外诱生的二次ＣＴＬ水平均高于ＴＫ＋痘苗病毒免疫组和正常组；杆状病毒系统表达的ＥＢＶ－ＬＭＰ１蛋白免疫组的ＣＴＬ水平也明显高于正常鼠。结论本法可以较好的反映ＥＢ病毒特异性细胞毒性Ｔ细胞的水平，而且再一次说明ＬＭＰ１基因能够诱发特异性的细胞免疫。     

Activation of epitope-specific memory cytotoxic T lymphocyte responses by synthetic peptides

Cytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by their ability to bind to MHC class I molecules. Polyclonal Epstein–Barr virus (EBV)-specific memory CTL responses, reactivated from blood lymphocytes of HLA-A11-positive individuals by stimulation with the autologous EBV-transformed lymphoblastoid cell line (LCL), are often dominated by reactivities directed to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416–424 of EBV nuclear antigen-4 (EBNA4). We now report the selective activation of IVT-specific CTL by stimulation of lymphocytes with the corresponding synthetic peptide. A more than 10-fold increase in frequency of CTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented by HLA-A11-transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstrated that clones activated under these conditions are as efficient as clones activated by conventional LCL stimulations. Induction of memory CTL responses required low surface density of MHC : peptide complexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentrations of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealed the presence of IVT-specific CTL precursors in a donor that failed to mount an IVT-specific response upon stimulation with the autologous B95·8 virus-transformed LCL. The results suggest that stimulation with synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and may be particularly helpful for the selective expansion of subdominant CTL specificities.     

Differential dynamics of the peripheral and intrahepatic cytotoxic T lymphocyte response to hepatitis B surface antigen

The distribution and dynamics of the cytotoxic T lymphocyte (CTL) response to hepatitis B surface antigen (HBsAg) were studied in mice after intramuscular DNA immunization and after hepatic infection by a recombinant adenovirus that expresses the hepatitis B virus genome (Ad-HBV). CTLs specific for HBsAg accumulate preferentially in the spleen after DNA immunization but are primarily intrahepatic after Ad-HBV infection. The secondary CTL response to Ad-HBV in DNA-primed mice is characterized by rapid depletion of effector CTLs from the spleen, and their expansion in the liver where they cause hepatitis, secrete interferon gamma (IFNγ), and inhibit HBV gene expression. Suppression of HBsAg synthesis is accompanied by disappearance of intrahepatic IFNγ-producing CTLs and their reaccumulation in the spleen. The data suggest a possible explanation for the paucity and functional deficiency of HBV-specific CTLs in the periphery during chronic HBV infection, and that the severity of infection can be worsened by a preexisting CTL response if neutralizing antibody is not also present.     

In vitro measurement of cytotoxic T cell activity does not predict clinical progression in paediatric HIV disease—two case studies

Cytotoxic T cells are believed to be an important immune response in HIV infection, both in the initial response to viraemia, and in controlling HIV replication and maintaining clinical stability. We report here the detailed findings in two vertically infected children, from the Edinburgh perinatal cohort. Both were clinically stable for the first 7 years of life. One had vigorous HIV-specific cytotoxic T lymphocyte (CTL) responses, and non-lytic suppression, measured in vitro, while the second had no CTL activity against HIV. Despite her HIV-specific immunity, the first child had a declining CD4 count, and a high and fluctuating viral load, whereas the second child maintained a stable CD4 count, a low viral load and had a virus which could not be cultured in peripheral blood mononuclear cells (PBMC) in vitro. The first child subsequently progressed to AIDS and has now died, while the second remains clinically well. More detailed investigations showed the clinically stable child to be heterozygous for the CCR5 receptor, and to be HLA-B49—both of which markers have been associated with slow HIV disease progression. These findings question the role of CTL in maintaining stable HIV disease, and stress the need for immunological investigations to be considered in the light of the genetic make-up of the patient. They may also reflect a different immunopathogenesis of HIV disease in children compared with adults.     

Influenza A antigen exposure selects dominant Vbeta17+ TCR in human CD8+ cytotoxic T cell responses.

During acute human viral infections, such as influenza A, specific cytotoxic T lymphocytes (CTL) are generated which aid virus clearance. We have observed that in HLA-A*0201+ subjects, CTL expressing Vbeta17+ TCR and recognizing a peptide from the influenza A matrix protein (M1(58-66)) dominate this response. In experimental models of infection such dominance can be due to inheritance of a restricted T cell repertoire or acquired consequent on expansion of CTL bearing an optimum TCR conformation against the MHC-peptide complex. To examine how influenza A infection might influence the development of TCR Vbeta17 expansion, we studied influenza A-specific CTL in a cross-sectional study of 82 HLA-A*0201+ individuals from birth (cord blood) to adulthood. Primary M1(58-66) -specific CTL were detected in cord blood, but their TCR were diverse and depletion of Vbeta17+ cells did not abrogate specific cytotoxicity. In contrast following natural influenza A infection, TCR Vbeta17+ CTL dominated to the extent that only one of nine adult CTL lines retained any functional activity after in vitro depletion of Vbeta17+ CTL. These results suggest that the dominance of Vbeta17+ TCR among adult M1(58-66)-specific CTL results from maturation and focussing of the response driven by exposure to influenza, and have implications for optimum immunization strategies.     

Successful in vitro generation of Epstein-Barr virus-specific cytotoxic T lymphocytes from severe chronic active EBV patients

Severe chronic active Epstein-Barr virus (EBV) infection (SCAEBV) is a rare but life-threatening disorder. Poor cytotoxic activity against the virus is widely believed to contribute to the development of this disease. We wished to determine whether it is possible to generate autologous EBV-specific cytotoxic T cells (CTLs) in vitro that can be infused back into the patient to treat his/her viremia. To do this, we first had to establish autologous EBV-transformed B cells (EBCL) as antigen-presenting cells, which is known to be difficult to do with B cells from SCAEBV patients. In one patient, the standard method of incubating B cells with EBV-containing B95-8 supernatant was sufficient. In a second patient, however, the B cells apoptosed too rapidly in culture to permit transformation. However, apoptosis could be blocked by the presence of CD40 ligand-transfectant cells, and EBV transformation was successful when performed with this transfectant. Indicating a native immune response to EBV, peripheral blood lymphocytes from both patients proliferated in response to autologous EBCL. Furthermore, patient T cells had higher frequencies of IFN-γ-producing CD8⁺ cells after stimulation with autologous EBCL than sero-positive healthy controls. EBV-specific CTLs could be generated from both patients after repeated stimulation with autologous EBCL. These CTL lines were predominantly composed of CD4⁺ cells, and autologous EBCL killing was largely inhibited by an antibody against HLA-DR. These findings support the possibility of adoptive immune therapy to treat SCAEBV patients. Received: 4 October 2000     

汉滩病毒核蛋白T细胞表位的鉴定及其特异性T细胞应答规律的初步探讨

目的:鉴定引起肾综合征出血热(HFRS)的汉滩病毒核蛋白(HTNV-NP)的T细胞表位,并探讨其特异性T细胞应答的规律。方法:合成覆盖HTNV-NP全长序列的全套部份重叠15肽,用ELISPOT方法筛选能刺激HFRS患者外周血单个核细胞(PBMC)产生IFN-γ的15肽,并测定其特异性T细胞的频率。结果:47例HFRS患者中,有18例可对HTNV-NP产生特异性T细胞应答,并发现在病程的早期就已开始产生T细胞应答。从11例患者PBMC中,鉴定出了17种HTNV-NP特异的T细胞表位,其中14种T细胞表位为首次报道,T细胞表位主要位于NP一级结构的中部。结论:HFRS病程早期即可产生特异性T细胞应答,T细胞表位主要位于NP序列的中部,T细胞应答可能在汉滩病毒感染过程中起重要的免疫保护作用。