Cytolytic mechanisms and T-cell receptor Vβ usage by ex vivo generated Epstein–Barr virus-specific cytotoxic T lymphocytes

Ex-vivo-generated Epstein–Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) have been used for cellular adoptive immunotherapy of EBV-associated lymphomas. Here we investigated the phenotypes, cytolytic mechanisms, polyfunctionality and T-cell receptor (TCR) usage in growing and established CTL, generated by weekly stimulation with an EBV-transformed autologous lymphoblastoid cell line (LCL). Our results showed that phenotypically mature CTL developed within the first 4 weeks of culture, with an increase in CD45RO and CD69, and a decrease in CD45RA, CD62L, CD27 and CD28 expression. Spectratyping analysis of the variable β-chain of the TCR revealed that TCR repertoire remained diverse during the course of culture. Cytotoxicity of CTL was significantly inhibited by concanamycin A (P < 0·0001) and ethylene glycol-bis tetraacetic acid (P < 0·0001), indicating that a calcium and perforin-mediated exocytosis pathway with the release of granzyme B was the principal cytotoxic mechanism. The CTL mainly produced interferon-γ (IFN-γ) or tumour necrosis factor-α (TNF-α) upon restimulation with autologous LCL, although there were some polyfunctional cells producing IFN-γ and TNF-α. Granzyme B, perforin and Fas ligand were detected in CD8⁺ and CD4⁺ cells in all CTL; however, a greater proportion of CD8⁺ than CD4⁺ T cells expressed granzyme B (P < 0·0001) and more granzyme B was detected in CD8⁺ T cells than in CD4⁺ T cells (P = 0·001). This difference was not observed with Fas ligand or perforin expression. Our results provide insight into the basic characteristics of ex-vivo-generated CTL.     

Virus-specific cytotoxic T cell responses are associated with immunity of the cottontop tamarin to Epstein–Barr virus (EBV)

The cytotoxic responses of peripheral blood lymphocytes from cottontop tamarins to in vitro re-stimulation with autologous lymphoblastoid cell lines (LCL) were assayed. Lymphocytes from immune tamarins that had recovered from EBV challenge developed potent cytotoxicity for natural killer (NK) cell targets and for autologous LCL. The cytotoxicity for LCL targets was EBV-specific, as B cell blasts uninfected with EBV were not killed. The cell lines could be maintained by repeated stimulation with LCL and the addition of IL-2. Flow cytometry showed that they were T cell lines expressing CD2, CD3, CD4, CD8 and CD25. Dual-colour flow cytometry revealed two subpopulations, one CD4⁺ CD8⁺ population and the other CD4⁻ CD8⁺. After separation by magnetic cell sorting both subpopulations were shown to be cytotoxic and the CD4⁺ CD8⁺ fraction was also shown to be MHC class II-restricted; the MHC restriction of the CD8⁺ subpopulation could not be determined. The unseparated T cells and both the subpopulations were able to inhibit LCL outgrowth in vitro. In contrast, PBL from naive tamarins stimulated by autologous LCL developed less NK cell cytotoxicity and little cytotoxicity for LCL. The cytotoxic response was enhanced at higher levels of LCL stimulation, but the cells were unable to inhibit LCL outgrowth in vitro. We conclude that cytotoxic responses capable of inhibiting LCL growth in vitro correlate with in vivo immunity in the tamarin model and provide a basis for understanding the mechanism of vaccine-induced immune protection.     

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV‐specific T cell therapy

Adoptive immunotherapy using Epstein–Barr Virus (EBV)‐specific T cells is a potentially curative treatment for patients with EBV‐related malignancies where other clinical options have proved ineffective. We describe improved good manufacturing practice (GMP)‐compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)‐driven EBV‐specific T cell manufacture, and describe an improved phenotyping approach for analysing T cell products. We optimized the current LCL‐mediated clinical manufacture of EBV‐specific T cells to establish an improved process using xenoprotein‐free GMP‐compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL:T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analysed using a t‐stochastic neighbour embedding (t‐SNE) algorithm. The optimized GMP‐compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi‐parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using interleukin (IL)‐2, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP‐compliant closed‐process culture of LCL‐mediated EBV‐specific T cells is feasible, and profiling of T cells through cytokine expression gives improved characterization of start material, in‐process culture conditions and final product. Visualization of the complex multi‐parameter flow cytometric data can be simplified using t‐SNE analysis.     

Unconventional cytotoxic T lymphocyte recognition of synthetic peptides corresponding to residues 1—23 of Ras protein

A peptide corresponding to amino acids 1 through 23 of Ras protein containing a mutation at position 12 was used to induce cytotoxic T lymphocytes (CTL) in mice. Although the CTL were CD8⁺ and expressed α, β T cell antigen receptors (TCR), their major histocompatibility complex (MHC)-restriction was unconventional. They recognized peptide-treated murine cells of different H-2 haplotypes, but not MHC class I-negative cells. Human HLA class I molecules did not present Ras peptides and hybrid human/mouse MHC molecules revealed that all three extracellular domains α1, α2 and α3 were required for recognition by peptide-specific CTL. Shortening the 23-mer peptide by 5 residues at either the amino or carboxy terminus resulted in loss of CTL recognition. This demonstrates an unusual form of antigen recognition by mouse CTL in which peptide presentation requires murine H-2 class I molecules but is not class I allele restricted, and the peptides recognized are much larger than peptides in conventional class I-restricted responses.     

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.     

Proteasome inhibitors induce the presentation of an Epstein-Barr virus nuclear antigen 1-derived cytotoxic T lymphocyte epitope in Burkitt's lymphoma cells

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is generally expressed in all EBV-associated tumours and is therefore an interesting target for immunotherapy. However, evidence for the recognition and elimination of EBV-transformed and Burkitt's lymphoma (BL) cells by cytotoxic T lymphocytes (CTLs) specific for endogenously presented EBNA1-derived epitopes remains elusive. We confirm here that CTLs specific for the HLA-B35/B53-presented EBNA1-derived HPVGEADYFEY (HPV) epitope are detectable in the majority of HLA-B35 individuals, and recognize EBV-transformed B lymphocytes, thereby demonstrating that the GAr domain does not fully inhibit the class I presentation of the HPV epitope. In contrast, BL cells are not recognized by HPV-specific CTLs, suggesting that other mechanisms contribute to providing a full protection from EBNA1-specific CTL-mediated lysis. One of the major differences between BL cells and lymphoplastoid cell lines (LCLs) is the proteasome; indeed, proteasomes from BL cells demonstrate far lower chymotryptic and tryptic-like activities compared with proteasomes from LCLs. Hence, inefficient proteasomal processing is likely to be the main reason for the poor presentation of this epitope in BL cells. Interestingly, we show that treatments with proteasome inhibitors partially restore the capacity of BL cells to present the HPV epitope. This indicates that proteasomes from BL cells, although less efficient in degrading reference substrates than proteasomes from LCLs, are able to destroy the HPV epitope, which can, however, be generated and presented after partial inhibition of the proteasome. These findings suggest the use of proteasome inhibitors, alone or in combination with other drugs, as a strategy for the treatment of EBNA1-carrying tumours.     

Synthetic peptides based on Chlamydia trachomatis antigens identify cytotoxic T lymphocyte responses in subjects from a trachoma-endemic population

CD8⁺ cytotoxic T lymphocytes (CTL) recognize peptide antigens in the context of class I MHC antigen molecules. To identify peptides capable of eliciting anti-Chlamydia trachomatis CTL responses, 13 synthetic peptides conforming to human leucocyte antigen (HLA)-B8- or -B35-predicted binding motifs were synthesized using sequences based on C. trachomatis major outer membrane protein (MOMP) and heat shock protein 60 (hsp60). Two of 11 HLA-B35-predicted binding peptides were able to stabilize HLA-B35 in an in vitro binding assay. All peptides were tested in CTL assays using peripheral blood mononuclear cells (PBMC) isolated from 26 HLA-B8 or -B35 individuals resident in a trachoma-endemic community. Responses to MOMP and hsp60 peptides were identified in a minority of both HLA-B8 and -B35 individuals. Two of 12 HLA-B8 subjects responded to MOMP and 1/13 to hsp60 peptides. Responses in HLA-B35 subjects were similar, 1/13 subjects responding to MOMP and 2/13 to hsp60 peptides. CTL responses were observed only in children resolving current infection and in adults without scarring of the conjunctiva. These results suggest that anti-chlamydial CTL occur at low levels in peripheral blood, but may be important in the resolution of naturally acquired human ocular chlamydial infection.     

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.     

The loss of immunodominant epitopes affects interferon-gamma production and lytic activity of the human influenza virus-specific cytotoxic T lymphocyte response in vitro

In the present study, we examined the effect of the loss of the human leucocyte antigen (HLA)-B*3501-restricted nucleoprotein (NP)(418-426) epitope on interferon (IFN)-gamma-production and lytic activity of the human cytotoxic T lymphocyte (CTL) response in vitro. Extensive amino acid variation at T cell receptor contact residues of the NP(418-426) epitope has led to repeated evasion from specific CTL. We generated recombinant influenza viruses with variants of the NP(418-426) epitope, which were used to stimulate peripheral blood mononuclear cells obtained from six HLA-B*3501-positive study subjects in order to expand virus-specific CTL. Loss of the NP(418-426) epitope resulted in a significant reduction of IFN-gamma-expressing CD8+ T cells, similar to that observed previously after the loss of the HLA-B*2705-restricted NP(383-391) epitope. In addition, the effect of the loss of the NP(418-426) epitope on the lytic activity of the virus-specific CTL response was assessed. Also this functional property of the virus-specific CTL response was affected significantly by the loss of this and the NP(383-391) epitope, as determined using the newly developed fluorescent antigen-transfected target cell (FATT)-CTL assay. These findings indicate that the loss of single immunodominant epitopes affects the functionality of the virus-specific CTL response significantly.     

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。     

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.     

慢性乙型肝炎患者HBV C、B基因型与特异性细胞毒性T淋巴细胞表面PD-1表达的关系

目的 探讨慢性乙型肝炎（CHB）患者HBV C、B基因型与HBV特异性细胞毒性T淋巴细胞（CTL）表面程序性死亡受体-1（PD-1）表达的关系和意义.方法 共研究CHB患者71例,人白细胞抗原（HLA）-A2阳性,HBV DNA ＞103拷贝/ml,其中C基因型34例（47.89％）,B基因型36例（50.70％）.比较C、B基因型感染者外周血HBV特异性CTL表面PD-1的表达水平、HBV特异性CTL水平、HBV DNA水平、ALT和TBil的水平.结果 CHB C基因型感染者的HBV特异性CTL表面PD-1的表达水平（37.30±3.05％）高于B基因型感染者（26.19±3.06％）,t=15.47,P＜0.001,HBV特异性CTL水平（0.25±0.03％）低于B基因型感染者（0.45±0.13％）,t=21.54,P＜0.001,HBV DNA水平（6.75±0.77lo910拷贝/ml）高于B基因型感染者（4.96±1.12 log10拷贝/ml）,t=7.93,P＜0.001,ALT水平（487.39±87.36IU/L）高于B基因型感染者（235.25±90.91IU/L）,t=12.32,P＜0.001,TBil水平（49.73±6.45）高于B基因型感染者（28.48±5.89％）,t=9.01,P＜0.001.结论 CHB C基因型感染者外周血HBV特异性CTL表面PD-1表达水平高于B基因型感染者,导致C基因型感染者HBV特异性CTL水平低于B基因型感染者,HBV DNA水平高于B基因型感染者,因此,肝功能损害比B基因型感染者重.     

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.     

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.     

IL-7 enhancement of antigen-driven activation/expansion of HIV-1-specific cytotoxic T lymphocyte precursors (CTLp).

CD8+ cytotoxic T lymphocytes are an important component in the immunologic control of human viral diseases. IL-7, a stromal cell-derived cytokine, has been demonstrated to enhance both anti-tumour and anti-viral CTL as well as lymphokine-activated killer (LAK) activity. We studied the ability of IL-7 to support the activation and the growth of in vitro antigen-specific CTL precursors (CTLp) present in peripheral blood mononuclear cells (PBMC) from HIV-infected patients. Results from these studies demonstrate that inclusion of IL-7 in a vaccinia/HIV-1 vector-based stimulation strategy greatly augmented overall CTL reactivities, whereas addition of IL-7 to unstimulated cultures failed to induce any significant anti-viral cytolytic activity. In four of six patients, HIV-specific lytic activities were significantly higher in cultures stimulated with antigen plus IL-7 compared with in vitro stimulation (IVS) with antigen alone. Cytotoxic activity was principally mediated by CD8+ effector cells, and CD3+/CD8+ cell expansion was increased by 2.7-fold in the presence of IL-7. In PBMC from seronegative donors, IL-7 enhanced anti-vaccinia CTL activities with less effect on cell proliferation. Furthermore, anti-gag CTL frequencies determined by limiting dilution analysis were increased by 2- and 10-fold in two asymptomatic patients following IVS plus IL-7 compared with antigen stimulation alone. Cytofluorimetric analysis revealed that IL-7 preferentially expanded CD8 memory cells (CD45RO+) and CD8+ lymphocytes expressing activation molecules. IL-7 was also able to support the growth of CD4+ lymphocytes, while having no effect on natural killer (NK)/K lymphocytes. Taken together, these data suggest that IL-7 acts cooperatively with the antigen supporting in vitro maturation of CTLp into functional cytotoxic effectors. Thus IL-7 may be an important biologic entity to consider as part of future immune-based therapies in which ex vivo expansion of antigen-driven CTL is an important determinant.     

Selective stimulation of murine cytotoxic T cell and antibody responses by particulate or monomeric hepatitis B virus surface (S) antigen

In the murine system, we tested in vivo the immunogenicity of different preparations of the yeast-derived surface antigen (S-antigen or S-protein) of hepatitis B virus (HBV). Native S-protein molecules self-assemble into stable 22-nm particles. BALB/c mice immunized with low doses of native S-particles without adjuvants efficiently generated an H-2 class I-restricted CD8⁺ cytotoxic T lymphocyte (CTL) response, and developed easily detectable serum antibody titers against conformational determinants of the native S-particle or linear epitopes of the denatured S-protein. Disruption of S-particles with sodium dodecyl sulfate and β-2-mercaptoethanol generated p24 S-monomers. Injection of an equal dose of S-monomers into mice efficiently primed CTL, but did not stimulate an antibody response against conformational or linear epitopes of the native or denatured S-protein. In vivo priming of CTL by S-particles or S-monomers required “endogenous” processing of the antigen because the injection of an equimolar (or higher) dose of an antigenic, S-derived 12-mer peptide into mice did not prime CTL. Native (particulate) or denatured (monomeric) S-antigen injected with mineral oil (incomplete Freund's adjuvant) or aluminum hydroxide failed to stimulate a CTL response. Hence, different preparations can be produced from a small protein antigen which specifically stimulate selected compartments of the immune system.     

In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild-type p53

The central role of the p53 tumor suppressor gene product in oncogenesis is gradually being clarified. Point mutations in the p53 tumor suppressor gene are common in most human cancers and are often associated with p53 protein overexpression. Overexpressed wild-type or mutant determinants of the p53 protein thus represent an attractive target for immunotherapy of cancer directed against a structure involved in malignant transformation. An important step towards this goal is identification of epitopes of p53 that can be recognized by human cytotoxic T lymphocytes. We identified peptides of (mutant) p53 capable of binding to HLA-A2.1 in an in vitro assay. These HLA-A2.1-binding peptides were utilized for in vitro induction of primary cytotoxic T lymphocyte responses using a human processing-defective cell line (174CEM.T2) as antigen-presenting cell. These cells display “empty” HLA class I surface molecules, that can efficiently be loaded with a single peptide. We obtained CD8⁺ cytotoxic T lymphocyte clones capable of specifically lysing target cells loaded with wild-type or tumor-specific mutant p53 peptides. This strategy allows the in vitro initiation of human cytotoxic T lymphocyte responses against target molecules of choice.     

点突变p53肽诱导肽特异性CTL的研究

目的探索点突变ｐ５３肽诱导细胞免疫应答的可能性,为其作为肽疫苗用于肿瘤免疫治疗提供实验依据。方法人工合成小鼠点突变ｐ５３肽Ｐ１３２Ｆ（ＬＮＫＬＦＦＱＬ,１３２Ｃｙｓ→Ｐｈｅ突变）,与不完全弗氏佐剂（ＩＦＡ）或ＲＩＢＩ佐剂（ＲＡＳ）混合,皮下免疫Ｃ５７ＢＬ／６小鼠,分离脾细胞体外用肽再刺激,诱导细胞毒Ｔ淋巴细胞（ＣＴＬ）,并以５１Ｃｒ释放法检测其杀伤活性。结果Ｐ１３２Ｆ肽加上ＩＦＡ或ＲＡＳ免疫小鼠,均能诱导肽特异性ＣＤ８＋ＣＴＬ;低剂量重组白细胞介素２（ｒＩＬ－２）能增强肽免疫效果。结论所用突变的ｐ５３蛋白具有免疫原性,提示来源于突变ｐ５３的抗原肽有可能作为肽疫苗用于临床肿瘤免疫治疗     

Epstein–Barr virus infection–Related hemophagocytic lymphohistiocytosis

We report a case of 27-year-old female diagnosed with hemophagocytic lymphohistiocytosis (HLH) following a recent Epstein–Barr virus (EBV) infection. A known case of relapsing remitting multiple sclerosis on corticosteroids for last 6 months presented to the critical care unit with fever, maculopapular rash and difficulty in breathing. A rapid and correct diagnosis with the precise treatment led to complete recovery of this patient. The HLH is a rare complication of primary EBV infection.