人外周血中SLA-P1抗原特异性T细胞前体频率及其细胞因子分泌格局分析

用基因工程表达并纯化的版纳猪SLAI类P1为抗原 ,经两轮刺激PBMC以3H TdR法检测 1 0名健康人外周血中的特异性T细胞前体频率在 6 67× 1 0 7～ 1 0 8× 1 0 6 之间。用ELISA与RT PCR法进一步分析了抗原特异性细胞的细胞因子分泌特点 ,发现所有抗原阳性孔都呈现IFN γ (+) /IL 4( )的典型Th1型格局。以上结果部分明确了外周血SLA P1特异性T细胞的特征 ,为揭示SLA P1特异性T细胞在猪 人急性细胞性排斥中的作用提供了实验依据     

TLSFJM对同种异体抗原诱导的小鼠杀伤性T细胞分化及？…

本文以小鼠混合淋巴细胞反应为模型,研究了ＪＭ急性Ｔ淋巴细胞白血病细胞分泌的抑制因子（ＴＬＳＦＪＭ）在体外对同种异体抗原诱导小鼠Ｔ细胞增殖和杀伤性Ｔ细胞分化的影响。结果表明：（１）ＴＬＳＦＪＭ可明显抑制同种异体抗原刺激的Ｔ细胞增殖,且具有剂量依赖性;（２）ＴＬＳＦＪＭ能有效地抑制同种异体抗原诱导的小鼠ＣＴＬ分化;（３）ＴＬＳＦＪＭ对杀伤相ＣＴＬ的杀伤活性没有抑制作用。     

T细胞识别同种异型移植抗原的研究进展

移植是末期组织、器官功能衰竭最彻底的治疗措施。同种异体移植(allogeneic transplantation)是临床最常见的移植类型。在本质上,人类的同种异体移植排斥反应是由受者的T细胞介导的、针对移植抗原的免疫应答。这一免疫应答是通过受者T细胞表面的T细胞受体(T cell receptor,TCR)识别移植物     

同种特异T细胞疫苗诱导移植物存活期延长的研究：Ⅰ.同种特异T…

在体外实验中观察了同种特异Ｔ细胞疫苗免疫鼠Ｔ淋巴细胞对同种抗原和丝分裂原的反应能力，Ｂ６同种抗原特异ＴＣＶ免疫的ＢＡＬＢ／ｃ小鼠对Ｂ６同种抗原的反应能力明显受抑制。对无关第三者同种抗原ＡＫＢ的反应能力也显著下降，表明存在抗原非特异性的抑制作用。     

人食管癌TE-1细胞MHC-Ⅰ相关抗原肽的初步研究

目的寻找TE-1细胞MHC-Ⅰ类分子提呈的抗原肽。方法用弱酸洗涤法获得TE-1细胞表面与MHC-Ⅰ类分子结合的抗原肽，经SepPak-C18柱、Centrion-3超滤器和RP-HPLE去盐纯化后，用负载该抗原肽的树突状细胞刺激生成肿瘤特异性杀伤细胞，以Cr^51释放法测定CTL的杀伤活性。结果高效液相色谱图显示多个峰；负载抗原肽DC刺激的CTL比未负载抗原肽DC刺激的CTL对TE-1细胞杀伤率高，两者差异有统计学意义；负载抗原肽DC刺激的CTL对与抗原肽孵育的T2细胞比对未经与抗原肽孵育的T2细胞杀伤率高，两者差异有统计学意义。结论弱酸洗涤法可从TE-1细胞上获得有效的抗原肽；混合抗原肽中有可与HLA-A2分子结合并能激发CTL的抗原肽。     

同种异体反应性细胞与调节性T细胞在新生期诱导的小鼠移植耐受中的作用探讨

目的:初步探讨同种异体反应性T细胞克隆清除与调节性T细胞在小鼠移植耐受中的作用。方法:雌性BALB/c小鼠与雄性C57BL/6小鼠杂交一代获得F1小鼠,不同剂量的F1小鼠脾脏细胞经眶静脉输注给新生24 h C57BL/6小鼠体内诱导耐受,成年后移植F1小鼠来源的皮肤,建立不同耐受程度的小鼠移植耐受模型;耐受小鼠脾脏细胞经CFSE标记后注射到F1小鼠体内,分析耐受小鼠来源的T细胞在体内对F1抗原的增殖能力;流式细胞术、过继转移实验分析CD4+Foxp3+调节性T细胞在移植耐受和移植排斥过程中的表达。结果:C57BL/6小鼠在新生期输注F1小鼠脾脏细胞可诱导移植耐受,耐受程度与输注的脾脏细胞剂量有关,3×107个F1小鼠脾脏细胞可诱导C57BL/76小鼠长期皮肤移植耐受,1×107个细胞诱导可使移植皮肤生存时间显著延长,但在50 d内完全排斥;体内混合淋巴细胞反应实验证明,长期耐受小鼠体内的同种异体反应性T细胞被完全克隆清除,但低剂量组小鼠体内仍存在一定数量的反应性T细胞;流式细胞分析发现,高剂量和低剂量组小鼠体内的CD4+Foxp3+调节性T细胞表达与初始小鼠相比没有显著差异;同种异体反应性T细胞过继转移给耐受小鼠,移植耐受的皮肤发生排斥反应,小鼠体内的调节性T细胞表达升高。结论:小鼠的移植耐受程度与小鼠体内的同种异体反应性T细胞的克隆清除程度有关,与CD4+Foxp3+调节性T细胞的表达没有直接关系;调节细胞在移植排斥过程中表达升高,可能作为一种反馈机制参与耐受的形成。     

超抗原与T细胞的作用

超抗原不需要抗原提呈细胞的加工处理,以MHCII类分子依赖或非依赖的方式递呈,结合于TCR的Vβ而激活T细胞,使其发生增殖、凋亡和免疫无反应性。研究超抗原与T细胞的作用,将有助于探讨淋巴细胞对自身抗原的耐受机制,HIV致病的机理,以及超抗原在肿瘤免疫治疗中的安全应用等。     

The precursor frequency of alloreactive CTLs is proportional to the number of T cell epitope specificities

The aim of this study is to find the experimental evidence that the precursor frequency of alloreactive CTLs is proportional to the number of the T-cell epitope specificities. The number of T-cell epitope specificities was manipulated by pulsing different number of HLA-A2 restricted peptide(s) onto the T2 cells, which acted as stimulating cells to elicit allo-reaction by co-culturing with peripheral blood lymphocytes (PBLs) of HLA-A2 negative individual. Ten HLA-A2 restricted peptides (all were normal cell components) were synthesized, and cell peptide extract was prepared by frozen and thawed.T2 cells loaded with different number of peptide(s) were co-cultured with PBLs of an HLA-A2 negative individual; the latter were stained with PKH67 in advance. Then the proliferation was monitored with flow cytometry, and the precursor frequency of the effector cells was analyzed by the ModFit Software. After 6 d of culture, no proliferation was observed in the bulk culture of PBL alone, and obvious proliferation took place when PBLs of the HLA-A2 negative were co-cultured with T2 cells loaded with or without loading peptide(s). The precursor frequency of the alloreactive CTLs was 0.052 819 for co-culture with T2 cells loaded without peptide; however it was 0.030 429 for T2 cells with EBV/ LMP2A and 0. 030 528 for T2 cells loaded with a single autogeneic peptide, and increased up to 0.144 942 for T2 cells loaded with 10 autogeneic peptides; the precursor frequency was 0.203 649 when co-cultured with T2 cells loaded with miscellaneous peptides extracted from the cytoplasm of T2 cells. This study reveals that the precursor frequency of alloreactive CTLs is proportional to the number of T-cell epitope specificities, and independent of the density of the allogeneic HLA ClassⅠmolecule. Our findings support the hypothesis that the alloreactive T cell populations comprise miscellaneous T cell clones; each is specific to corresponding pMHC. The novel constellation of peptides presented by allogeneic MHC molecules makes thousands of different epitopes, which account for the exceptional high precursor frequency of alloreactive T cells.     

Limited plasticity in the recognition of peptide epitope variants by an alloreactive CTL clone correlates directly with conservation of critical residues and inversely with peptide length

Although self-restricted T cells are peptide-specific and can distinguish among closely related ligands, they have some flexibility in the recognition of sequence variants of their natural peptide epitopes. Alloreactive cytotoxic T lymphocytes (CTL) can recognize specific peptides bound to the allo-major histocompatibility complex (MHC) molecule, but their plasticity in the recognition of related peptide variants has not been properly defined. The anti-B*2705 alloreactive CTL 27S69 specifically recognizes a natural octamer ligand of HLA-B*2705. In this study, we tested the recognition of a nested set of epitope variants by this CTL clone. Although none of these peptides was recognized equally as the natural epitope, two of the peptide variants were recognized with only slightly decreased efficiency. Peptide sensitization assays showed that CTL recognition of epitope variants correlated directly with conservation of two non-anchor residues that were critical for recognition of the natural epitope, and inversely with peptide length. Molecular modeling of the peptide variants complexed with B*2705 provided a rational explanation for their differential recognition. Location of the two critical peptide residues at the right three-dimensional space favored efficient recognition by CTL 27S69. The negative effect of increasing peptide length on recognition was due to the bigger bulging surface between the two critical residues, which precluded for optimal interaction with the specific T-cell receptors (TCR). Our results demonstrate that an alloreactive CTL has a degree of plasticity in the recognition of peptide epitope variants that is comparable to that of peptide-specific self-restricted CTL, and define the structural features determining crossreaction among related peptides.     

Precursor frequency can compensate for lower TCR expression in T cell competition during priming in vivo

The factors controlling clonal dominance of cytotoxic T lymphocyte (CTL) responses are currently not well understood. To study the functional impact of the strength of the interaction of a T cell with an antigen-presenting cell in this context, we established a new mouse model comprised of two T cell receptor (TCR)-transgenic strains expressing the identical TCR in differing amounts, hence providing two CTL clones with different avidities but identical specificity and affinity. Utilizing this new model, we show that upon antigen challenge higher-avidity CTL expand at the expense of moderate-avidity CTL in vivo if present in equal numbers. Beyond this, moderate-avidity T cells can also contribute to a CTL response when present in excess. These results suggest that in addition to a proposed affinity/avidity threshold, the precursor frequency is important in defining clonal dominance. A new model in which TCR density and precursor frequency define the outcome of a CTL response is discussed.     

Limiting dilution analysis of alloreactive T helper cells: precursor frequencies similar to that of alloreactive cytotoxic T cells

Precursor frequencies for alloreactive T helper cells involved in the generation of primary cytotoxic responses from thymocytes were determined in splenic T cells and selected Lyt-1 lymphocytes by limiting dilution analysis. T helper precursors at frequencies ranging from 1/5000 to 1/13,500 were found in individual experiments in unsensitized selected Lyt-1 populations reacting to H-2 alloantigens. After preactivation of Lyt-1 lymphocytes with antigen in limiting dilution, the frequencies of T helper cells were increased 2-3 fold when cultured in the absence and 10-50-fold when cultured in the presence of T cell growth factor. The frequencies for T helper precursors found in Lyt-1 cells were comparable to those of unselected T cells, indicating that a significant portion of T helper cells resides in the Lyt-123 population. Activation of T helper precursors with H-2 antigens or with H-2 and non-MHC (plus MLs) antigens resulted in similar frequencies, suggesting that the same T cell can respond to H-2 and non-MHC determinants. The data suggest that alloreactive T helper precursors exist at frequencies similar to that of CTL precursors. In addition, the results indicate that the induction of CTL by T helper cells is subject to regulation presumably by suppressive cells and that Lyt-1 inducer cells may be involved in the development of suppression for CTL responses.     

Establishment and epitope analysis of allo-specific cytotoxic T lymphocytes at a tumor site recognizing a spouse's HLA-A0206 molecule

PROBLEM: The molecular basis of allo-reactivity in reproductive immunity has not been fully clarified. METHOD OF STUDY: Cytotoxic T lymphocytes (CTLs) were established from tumor-infiltrating lymphocytes (TILs). The allo-reactivity of the CTLs against various tumor cell lines or human leukocyte antigen (HLA)-A allele-transfected COS-7 cells was measured by 51Cr-release or interferon-gamma production assay. RESULTS AND CONCLUSIONS: We have established CTLs reacting to an HLA-A0206 molecule that matched a spouse's HLA-A allele from the TILs of a 68-year-old multiparous patient with gastric cancer. The amino acids at positions 66 and 88 in the alpha1 domain of HLA-A0206, both of which were common in the other HLA-A2 subtypes, were involved in the recognition by the CTLs. Endogenous peptides in the groove were not involved in the recognition. These results suggest the presence of long-lasting memory CTLs raised by the reproduction process, and may facilitate a better understanding of the molecular basis of allo-recognition during reproduction.     

At low precursor frequencies,the T‐cell response to chronic self‐antigen results in anergy without deletion

The behavior of self‐reactive T cells in the peripheral immune system has often been studied by following the fate of adoptively transferred antigen‐specific T cells in antigen expressing mice. In most cases, after a period of expansion, such cells undergo a slow clonal deletion, accompanied by the onset of anergy and/or suppression in the remaining cells. Here, we demonstrate that at initial frequencies approaching those found in normal repertoires, it is possible to completely avoid deletion and still maintain peripheral tolerance. At starting numbers of <1000 T cells, stimulation by chronic self‐antigens resulted in a period of robust clonal expansion, followed by a steady plateau phase extending beyond 4 months. Despite their stable persistence, the self‐reactive T cells did not convert to a Foxp3+ fate. However, they displayed a considerable block in their ability to make IL‐2, consistent with the onset of anergy — in a precursor frequency or deletion independent fashion.     

Peptides of 23 residues or greater are required to stimulate a high affinity class II-restricted T cell response

Helper T cells recognize fragments of antigen bound to the class II molecules on the surface of antigen-presenting cells. Naturally processed antigenic fragments have been isolated from the class II molecules and shown to be heterogeneous in length, ranging from 13 to 25 residues, and to vary at both the N and C termini. A 15-residue peptide in an extended conformation is predicted to fit in an open peptide-binding cleft of the class II molecules. Thus, the longer peptides observed bound to class II presumably have regions which reside outside the cleft. It is not known if the additional length contributes significantly to T cell activation. We have carried out a systematic analysis of the antigenicity of peptides of increasing length beyond the minimally defined T cell antigenic peptide. Here we show that the full functional activities of peptides representing the major antigenic determinant of the protein antigen, cytochrome c, minimally require that the peptides be 23 amino acids long. The long peptides do not require processing and are presented by purified class II molecules incorporated into synthetic membranes, indicating that such peptides associate directly with class II and require no additional cellular machinery for presentation. We also show that a hybrid peptide, 51 residues in length, containing a 29-residue cytochrome c peptide and a “promiscuous” peptide of tetanus toxoid, is more antigenic than the 23-residue peptide alone and significantly, does not require processing. Thus, the additional peptide length, although not predicted to bind in the peptide-binding groove of the MHC class II molecule, has a significant impact on the ability of the peptides to stimulate T cell responses maximally.     

IL-12-Dependent enhancement of CTL response to weak class I-restricted peptide immunogens requires coimmunization with T helper cell immunogens

The effect of in vivo administration of rmIL-12 on the CTL response to immunization with a weakly immunogenic class I-restricted peptide emulsified in incomplete Freund's adjuvant was investigated. In the absence of IL-12, peptide-specific CTL responses were significantly greater following coimmunization with class I-restricted peptide and T helper cell antigens than following immunization with the class I-restricted peptide alone. IL-12-dependent enhancement of the CTL response to peptide immunization was demonstrated in the presence of, but not in the absence of, coimmunization with T helper cell antigen. These findings indicate that IL-12 enhancement of the CTL response to weak class I-restricted immunogens is T helper cell dependent. Treatment with rmIL-12 also enhanced the CTL response to immunization with cDNA encoding both CTL and T helper cell epitopes. These findings are relevant to the design of vaccines containing tumor-associated class I-restricted peptides currently being tested as an immunotherapy for cancer patients.     

Use of herpesvirus saimiri-immortalized macaque CD4(+) T cell clones as stimulators and targets for assessment of CTL responses in macaque/AIDS models

Herpesvirus saimiri (HVS), a nonhuman primate γ herpes virus, was used to immortalize pig-tailed macaque CD4⁺ T lymphocytes. The HVS-immortalized T cell lines were used to develop CD4⁺ T cell clones from two animals. Three CD4⁺ T cell clones were further characterized for the expression of cell surface markers. All expressed CD2, CD4, CD58, CD69 and CD80 and therefore resembled activated T cells. These clones required exogenous IL-2 for efficient growth and were found to be highly susceptible to infection by the challenge virus, Chimeric simian/human immunodeficiency virus (SHIV_KU-1). They could also be productively infected not only by the quasispecies of the challenge virus (SHIV_KU-1/PDJ and SHIV_KU-1/PNA, isolated from macaque PDj and PNa, respectively) but also by a different chimeric simian/human immunodeficiency virus (SHIV_89.6P) and simian immunodeficiency virus (SIV_MAC239). The virus-infected CD4⁺ T cell clones were also used as stimulators for generation of CTL effectors. These effectors exhibited excellent virus-specific lysis in chromium-release assays when syngenic SHIV_KU-1 infected autologous CD4⁺ T cell clones were used as targets. The target cell lysis was virus specific, as uninfected control cells showed no or minimal lysis.