Establishment of anti-Epstein–Barr virus (EBV) cellular immunity by adoptive transfer of virus-specific cytotoxic T lymphocytes from an HLA-matched sibling to a patient with severe chronic active EBV infection

We describe an experience of a specific immune transfer treatment in a patient with chronic active EBV infection. The patient had low anti-EBV T cell-mediated cytotoxic activity in his peripheral blood mononuclear cells (PBMC), which may have been the primary cause of the disease. An EBV-specific cytotoxic T lymphocyte (CTL) line was established from PBMC obtained from the patient’s sister whose human leucocyte antigens (HLA) are identical to patient's. The patient received three courses of intravenously administered CTL at 3-week intervals. The number of the cells was increased with each course of treatment. After infusion of the T cell line, anti-EBV CTL activity was detected in the patient's PBMC. CTL activity increased markedly after the second course of immune transfer therapy. The amount of EBV DNA in the patient's plasma showed transient but repeated decreases. Serum levels of tumour necrosis factor-alpha (TNF-α), which had elevated before treatment, began to decrease after initiation of treatment. No adverse effects were directly associated with CTL infusions. Despite having previously received a pneumococcal vaccine and prophylactic antibodies, the patient died of infection caused by Streptococcus pneumoniae bacteraemia 27 days after the third infusion. Although the long-term efficacy and safety of this therapy remains to be established, our findings suggest that adoptive transfer of CTL specific for EBV obtained from an HLA-matched donor might be a promising treatment for patients with chronic active EBV infection.     

Effective anti-tumor adoptive immunotherapy: utilization of exogenous IL-2-independent cytotoxic T lymphocyte clones

To attain one of the final goals for cancer immunotherapy, cytotoxic T lymphocyte (CTL) clones were selected on the basis of exogenous IL-2 independence after limiting dilution culture from mixed lymphocyte tumor cell culture cells of FBL-3 tumor-immune spleens. About 10% of the clones could be propagated up to >5 times by weekly passages in the presence of splenic feeder and stimulating tumor cells. Two of the representative FBL-3-specific CTL clones that were able to undergo the fifth passage were expanded in large numbers for adoptive transfer by two rounds of a weekly passage with medium containing IL-2. FBL-3-specific CTL clones thus obtained showed a strong ability to eliminate the established tumors when transferred into tumor-bearing nude mice. In addition, the cells were recovered from the mouse spleen even 8 months after the transfer. The most striking differences between the CTL clones used in this experiment and those maintained conventionally in the presence of IL-2 were the abilities to produce IL-2 by themselves and the high expression level of the integrin molecule, VLA-4, that disappeared when cultured completely in the continuous presence of IL-2 in vitro during 12 weeks. In addition, concomitant with the disappearance of exogenous IL-2 independence and VLA-4 expression, the CTL clones lost their capacity to eradicate the tumor in vivo. Thus, the higher capacity of CTL clones to produce IL-2 on their own seemed to be correlated with the in vivo efficacy for tumor eradication and the long-term maintenance of their physiological profiles typical of memory T cells.     

MUC1基因疫苗诱导小鼠特异性CTL和体液免疫应答

目的 :观察MUC1基因疫苗诱导小鼠特异性杀伤性T细胞及体液免疫应答的作用。方法 :采用股四头肌肌肉注射 ,将构建的MUC1基因疫苗pcDNA3.1 MUC1免疫雌性BALB/c小鼠 ,每次间隔 3wk ,共 3次。最后 1次免疫后第 3周 ,接种表达MUC1的EMT6乳腺癌细胞进行免疫保护实验。用 4h51Cr释放法检测小鼠脾细胞特异性CTL杀伤活性 ;免疫组化染色法检测小鼠血清特异性抗体的水平。结果 :在效靶比为 10 0∶1、5 0∶1、2 5∶1、12 .5∶1时 ,MUC1基因疫苗免疫组特异性CTL对EMT6靶细胞杀伤活性分别为 5 4 .1%、39.8%、2 6 .4 %和2 0 .1% ,对照组分别为 13.2 %、10 .0 %、8.2 %、7.2 %和 11.7%、9.8%、7.7%、7.0 % ,前者与后二者差异显著 (P <0 .0 1)。免疫组化染色检测显示 ,人乳腺癌组织MUC1呈染色阳性 ;MUC1基因疫苗免疫组仅见 4 0 % (4/ 10 )的小鼠有肿瘤形成 ,而 pcDNA3.1对照组和生理盐水阴性对照组 10 0 %可见肿瘤形成、生长 ,表明MUC1基因疫苗免疫组小鼠具有一定的免疫保护作用。结论 :MUC1基因疫苗可诱导小鼠产生特异性CTL及体液免疫应答 ,对小鼠体内荷瘤可能具有一定的预防作用     

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.     

Comparison of the frequency of peptide-specific cytotoxic T lymphocytes restricted by self- and allo-MHC following in vitro T cell priming

T cell recognition of antigenic peptides is thought to occur preferentially in the context of self-MHC. Here, we have tested the ability of four different K(b)-peptide combinations to stimulate self- and allo-restricted CTL responses in three different mouse stains. Responder T cells were primed in vitro with peptide-loaded stimulator cells, followed by limiting dilution assays to measure the number of peptide-specific cytotoxic T lymphocytes (CTL). For three peptides the number of CTL restricted by self-MHC was higher than for allo-MHC-restricted responses, although the difference was surprisingly small (3- to 5-fold). For the fourth peptide there was no detectable difference in the number of self- and allo-restricted CTL. Peptide titration experiments revealed that high avidity CTL were present in both the self- and allo-restricted setting. These data showed that the bias for preferred peptide recognition in the context of self-MHC imposed by positive thymic selection seems marginal. This raised the possibility that the TCR repertoire is inherently biased towards MHC restriction, independent of MHC-guided thymic selection. This was supported by the analysis of mature T cells generated from the thymus of MHC-deficient mice by lectin stimulation. K(b)-restricted CTL were found amongst these T cells at numbers similar to those of allo-restricted CTL. In summary, the data suggest that MHC-restricted peptide recognition is an inherent feature of the TCR repertoire and does not require thymic selection by MHC molecules.     

Recognition of ADP-ribosylation factor 4-like by HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes from patients with brain tumors

Although specific immunotherapy is one candidate treatment of brain tumor, the molecular basis of T-cell-mediated recognition of brain tumors has not yet been elucidated. In this study, we tried to identify brain tumor antigens using HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes (CTLs). As an HLA-A2-restricted OK-CTL line contained CTLs capable of responding to HLA-A2+ malignant glioma cells, this cell line was used for identification of brain tumor antigens. After screening a cDNA library from brain tumor cells, this CTL line was found to produce interferon (IFN)-gamma when cultured with COS-7 cells, which were cotransfected with both a cDNA clone (clone 1) and HLA-A0207 cDNA. Data base searches indicated that the clone 1 was 98% identical to that of the human ADP-ribosylation factor 4-like (ARF4L). Two peptides, ARF4L 15-24 and ARF4L 69-77, possessed the ability to induce HLA-A2-restricted and tumor-reactive CTLs from peripheral blood mononuclear cells of patients with brain tumors. Although ARF4L seemed to be ubiquitously expressed at the mRNA level, ARF4L-reactive CTLs failed to exhibit cytotoxicity against normal lymphoid blasts. These results indicate that these two ARF4L peptides could be targets for immunotherapy of HLA-A2+ patients with brain tumors.     

Allo- and self-restricted cytotoxic T lymphocytes against a peptide library: evidence for a functionally diverse allorestricted T cell repertoire

BALB/c-derived spleen cells were depleted of cytotoxic T lymphocytes (CTL) recognizing allogeneic (H2^b) and TAP-negative cells followed by stimulation with the same cells loaded with a synthetic library binding to H2-K^b. The resulting CTL lines were found to differ widely in peptide specificity and to exhibit an avidity towards the library as that demonstrated for syngeneic CTL. These results demonstrate that positive selection in the context of a certain MHC molecule does not seem to be required for generating high-avidity TCR that are restricted by the same molecule. However, positive selection increases the frequency of such CTL. By raising T cell lines from a repertoire which did not undergo negative selection by the restriction element in question, it becomes possible to produce effective self-peptide/MHC as well as nonself-peptide/MHC-specific CTL as tools for adoptive tumor immunotherapy.     

Limiting dilution analysis of the allo-MHC anti-paternal cytotoxic T cell response II: recurrent spontaneous abortion and the effect of immunotherapy

Using limiting dilution analysis (LDA) we determined anti-paternal cytotoxic T lymphocyte precursor (CTLp) frequencies in the peripheral blood of 10 women with unexplained recurrent spontaneous abortion (RSA) before and after immunization with paternal lymphocytes. The women and their partners were HLA tissue-typed and none of the women had anti-paternal cytotoxic antibodies (APCA) before immunization. All other known causes of RSA were excluded. All 10 women were found to have high frequencies of specific anti-paternal cytotoxic T cells before immunization (range I 1/1030 to 1/9574). Splitwell analysis showed that these cytotoxic cells were specific to paternal MHC antigens. These frequencies rose significantly following immunization (range 1/683 to 1/4652). The cytotoxic T lymphocyte frequencies against an HLA-mismatched third party varied from woman to woman, but were not affected by the immunization. The LDA data conformed lo single-hit kinetics, indicating that only cytotoxic T ceils were limiting in the assay. Our data are in sharp contrast to the previously held view that women with RSA may be hyporesponsive to paternal MHC antigens. Immunizing such women with paternal leucocytes further sensitizes them. These findings cannot be reconciled with a favourable outcome in the treatment of RSA with immunotherapy. We would argue that this treatment is al best of unproven value, and may even be harmful. Thai these women may sometimes have successful pregnancies following immunotherapy testifies to the effectiveness of the classical MHC antigen-deficient trophoblast as an immunological barrier between mother and fetus.     

Activation of human alloreactive cytotoxic precursor T lymphocytes

The requirements for allogeneic T-cell activation have been studied in experiments with T and/or B cells as stimulator. Although target determinants (TDs, defined by CTL effectors in CML) are present on B and T cells used as target cells, this study indicates that TDs are functionally different when expressed on B and T cells used as stimulator cells, as only B cells can activate CTL precursors. Further, the study confirms that inducing TDs and strong lymphocyte-activating determinants (LADs, defined by proliferation in MLC) can be distinct structures found on two different stimulator B cells. The study suggests that binding of cytotoxic precursor T cells to TDs per se does not allow any detectable activation or start of proliferation and differentiation but requires another function of the stimulator cells in the non-T-cell compartment. The nature of this function is unknown, but it is the background for the first signal received by the TD-specific clones of CTL precursors, resulting in the expression of growth receptors for T-cell growth factor or interleukin 2 which is the second signal necessary for clonal expansion and differentiation.     

The use of endogenous T cells for adoptive transfer

Adoptive T-cell therapy involves the ex vivo enrichment and expansion of tumor-reactive T cells for infusion. As an immune-based approach, adoptive therapy has become an increasingly attractive modality for the treatment of patients with cancer due to its potential for high specificity, non-cross resistance with conventional therapies, and promise of long-term immunoprotection. In recent years, a resurgence in discoveries underlying T-cell recognition, tumor immune evasion, and T-cell memory and differentiation coupled with the development of several enabling technologies have facilitated a renewed focus in the field of adoptive therapy and its transition to the clinical arena as a treatment modality for patients with cancer. In this review, endogenous T cells derived from peripheral blood or tumor sites will be presented as a source of effector cells for adoptive therapy and strategies to isolate, manipulate, and enhance the function of antigen-specific T cells in vitro and to augment their in vivo efficacy and persistence by host immunomodulation are presented in the context of an ever-increasing inventory of preclinical and clinically available reagents. Optimizing the combination of adoptive cellular therapy and other immune-based and conventional approaches will herald a new generation of research and clinical opportunities for cancer immunotherapy.     

The renal cell carcinoma lysis by a specific cytotoxic T cell clone is independent of the Fas/Fas-L cytotoxic pathway

The expression of Fas antigen at the surface of renal cell carcinoma and the susceptibility to Fas-mediated lysis by a tumor specific CTL clone were investigated. Renal cell carcinoma cell lines expressed Fas antigen and were susceptible to apoptosis mediated by antibodies to Fas/APO1. Using RT-PCR, we further showed that these cell lines expressed mRNA for Fas deleted transmembrane region, corresponding to a soluble form of Fas/APO-1. To investigate the role of the Fas/FasL pathway in the cytotoxic response against RCC cells, we analyzed the induction of Fas-L on a tumor specific T cell clone (CTL 8C2), previously generated against one RCC cell line. Fas-L expression on CTL 8C2 was detected by RT-PCR after stimulation with autologous tumor cells. However, the cytotoxic activity of CTL 8C2 was completely abolished when EGTA was added, suggesting that the cytolysis was mainly mediated by a Ca++-dependent pathway, perforin/granzyme-based.     

T细胞受体基因转染过继性免疫治疗的研究进展

利用T细胞进行过继性免疫治疗是治疗病毒感染性疾病和肿瘤的理想方法,但是用于治疗的T细胞的特异性、亲和性和数量等限制了其应用,如何获得特异、高效、一定数量的T细胞是目前亟待解决的问题。采用TCR基因转染的方法,将特异性高亲和力TCR转移到受体的T细胞中,可以特异性杀伤受体体内的肿瘤细胞。     

Phenotype, cytokine production and cytolytic capacity of fresh (uncultured) tumour-infiltrating T lymphocytes in human renal cell carcinoma

We investigated the phenotype and functional capacities of tumour-infiltrating lymphocytes (TIL), freshly isolated from primary renal cell carcinoma (RCC) specimens (n = 20). Three-colour flow cytometry immunophenotyping revealed that RCC TIL consist mainly of CD3⁺ T cells, with a clear predominance of CD4⁻CD8⁺ over CD4⁺ CD8⁻ T cells, and a marked population of CD4⁺ CD8⁺ T cells. Natural killer (NK) cells were also strongly represented (> 25% in 15 of 20 tumour samples), while B cells constituted a minor TIL subset (< 5% in 18 of 20 tumour samples). More importantly, the T and NK cells within the tumour displayed a significantly higher expression of the early activation marker CD69 than their counterparts in adjacent normal renal tissue and in peripheral blood. Expression of CD54 and of HLA-DR was also elevated on CD3⁺ TIL, and HLA-DR expression was further vigorously up-regulated following ex vivo stimulation with anti-CD3, all suggesting enhanced immune activity within the tumour microenvironment. CD3⁺ CD4⁺ TIL displayed a normal capacity to up-regulate CD25 expression and to secrete both Th1-type (IL-2, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ)) and Th2-type (IL-4, IL-5 and IL-10) cytokines upon triggering with anti-CD3. Furthermore, cytokine production was susceptible to modulation by CD28 costimulation. CD3⁺ CD8⁺ TIL, on the other hand, consistently demonstrated a poor up-regulation of CD25 upon triggering with anti-CD3, and displayed poor ex vivo cytolytic activity in an anti-CD3-redirected 4-h cytotoxicity assay against murine P815 cells. Collectively, our findings indicate that the CD3⁺ CD4⁺ TIL in RCC have normal functional capacities, whereas the proportionally major CD3⁺ CD8⁺ TIL are functionally impaired. The relevance of these findings to the in vivo local immune response in RCC is discussed.     

Subtypes of HLA-B27 detected by cytotoxic T lymphocytes and their role in self-recognition

In the present study cytotoxic T lymphocytes were generated in MLC of lymphocytes from two unrelated HLA-A, B, C-identical, B27-positive, but D/DR-different, individuals. These CTL were shown to detect subtypes of HLA-B27. CTL specific for influenza virus lysed infected target cells matched for HLA-B27 only when they shared the same subtype. This indicates that the two subtypes of HLA-B27 detected by CTL function also as distinct elements in a self-restricted CTL response. Both subtypes were found among patients with ankylosing spondylitis.     

Serine protease inhibitors and cytotoxic T lymphocytes

Summary: Serine proteases control a wide variety of physiological and pathological processes in multi-cellular organisms, including blood clotting, cancer, cell death, osmoregulation, tissue remodeling, and immunity to infection. Cytotoxic T lymphocytes (CTLs) are required for adaptive cell-mediated immunity to intracellular pathogens by killing infected cells and through the development of memory T cells. Serine proteases not only allow a CTL to kill but also impose homeostatic control on CTL number. Serine protease inhibitors (serpins) are the physiological regulators of serine proteases’ activity. In this review, I discuss the role of serpins in controlling the recognition of antigen, effector function, and homeostatic control of CTLs through the inhibition of physiological serine protease targets. An emerging view of serpins is that they are important promoters of cellular viability through their inhibition of executioner proteases. This view is discussed in the context of the T-lymphocyte survival during effector responses and the development and persistence of long-lived memory T cells. Given the important role serpins play in CTL immunity, I discuss the potential for developing new immunotherapeutic approaches based directly on serpins or knowledge gained from identifying their physiologically relevant protease targets.     

The flexibility of the TCR allows recognition of a large set of naturally occurring epitope variants by HIV-specific cytotoxic T lymphocytes.

Pathogens attempt to evade immune recognition by expressing mutated antigens. The present study shows that two mechanisms happen in vivo during the course of HIV infection to limit the escape of antigenic variants from cytotoxic T lymphocyte (CTL) recognition: recognition of several epitope variants by the same TCR and generation of several CTL populations specific for a single epitope but recognizing different variant sequences. We have studied two CTL populations directed towards the HIV-p24gag amino acids 176--184 QASQEVKNW epitope, presented by HLA-B5301. Both CTL populations were derived from a long-term asymptomatic HIV-infected child and they express different TCR. Each of the two CTL recognizes five of the 10 naturally occurring variants. These variants are distinct for both CTL and thus a total of eight variants are recognized. Thus, polyclonality of CTL specific for the same epitope but differing in variant sequences recognized may improve the control of variant viruses' replication in vivo. In addition to cross-recognition of several variant epitopes, promiscuous recognition of exogenous peptides complexed to allogeneic HLA-B molecules occurs, showing that the TCR can tolerate amino acid changes on both the peptide and the MHC molecule. This flexibility of the TCR is probably of great importance for control of viruses with high genetic variability, such as HIV.