| In the past decade, some important progress in the field of allogeneichematopoietic stem cell transplantation has improved the survival rate of the patients,but the virus infection is still an important risk factor for transplant-related death. Thepatients is severely immunosuppressed,opportunistic viral infection such asherpesvirus,especially the Epstein–Barr virus (EBV) and Cytomegalovirus(CMV) arethe most common viral infection after allogeneic hematopoietic stem celltransplantation. Regardless primary infection of EBV or reactivation of CMV mayendanger the lives of patients.The conventional treatment of EBV infection after transplantation, includingreduction of immunosuppressive drugs, antiviral agents, CD20monoclonal antibodyand chemotherapy, as well as antigen-specific T-cell infusion, etc. Reduction ofimmunosuppression proves largely ineffective in HSCT recipients owing to thecomplete ablation of the immunesystem and carries the risk of inducing GVHD.There is no specific antiviral drugs, acyclovir or ganciclovir targets lytically infectedcells. The lack of viral thymidine kinase expression during viral latency, makesantiviral therapy ineffective.Numerous case reports demonstrated rituximab’seffectiveness in preventing and treating post-transplant lymphoproliferative diseases,but it can deplete B cells for more than6months in these already immunosuppressedpatients. Adoptive transfer of virous-specific T cells can restore immunity in allogeneic stem cell transplant recipients, providing protection against EBV and CMVdisease.Adoptive immunotherapy with antigen-specific cytotoxic T lymphocytes(CTL)was clinically efficient and safe in prophylaxis and therapy for EBV and CMVinfection after HSCTDespite a high efficacy of adoptive T cell immunotherapy, the procedure islaborious and time consuming and is often too late to administer to the patient. In thecase of EBV-CTL, the generation of the EBV-LCL used as APCs requires4to6weeks followed by an additional4to6weeks for CTL activation and expansion. Thisprecludes urgent treatment of seriously ill patients. Successful strategies to reduce theproduction time of EBV-specific CTLs include the use of peptide stimulated bloodcells combined with IFN-γ capture to select specific CTLs and the use of antigenspeptide laoded dendritic cells to rapidly generate peptide-specific CTL. Thesemethods are effective but expensive, labor-intensiveWe report a simple modified peptide stimulation method using EBV andCMV-specific peptides to stimulate PBMCs for generation of EBV and CMV-specificCTLs.We compared different concentrations of peptides and dirrerent cultureconditions on the impact of the CTL cell proliferation and function in vitro.Theexpanded cells were the base for its clinical use. This optimization decreases thecomplexity and cost of CTL manufacture, making adoptive cellular therapy moreaccessible.Object：The lack of an optimal method to produce antigen-specific CTLs has hinderedthe further development of adoptive cell therapy.To simplify the culture procedures ofvirus-specific cytotoxic T lymphocytes, we explored a novel method under goodmanufacturing practice (GMP), a rapid expansion protocol, that provided a newpossibility for the treatment of life-threatening EBV and CMV diseases after HSCT.Method：Peripheral blood mononuclear cells from96healthy donors were isolated by Ficoll gradient centrifugation of200ml heparinized blood.High-resolution HLAtyping for HLA-A was identified by PCR-SBT methods,the most frequent alleles wasenrolled in this study.We used the same factors but different concentrations ofpeptides or the same concentrations of peptides but different culture conditions toculture CTL to compare the impact of the CTL cell proliferation and function invitro.In the optimization of culture conditions, we explored other high frequencyallele type as well as the amplification effect of CMV-CTL, in order to establish areliable amplification procedure.Result：1. In62healthy donors of Zhengzhou Han group,18alleles were found in HLA-A.The most frequent alleles at A locus were HLA-A*2402(17.74%), HLA-A*1101(16.13%), HLA-A*0201(15.32%).2. We compared different concentrations of peptides to culture CTL in vitro.Threedays before, the cells in sizes no significant changed under morphology. A weeklater, part of cells formed colonies. The cells of0μmol/L and1μmol/L group weregrowing well, with5μmol/L group significantly reduced the number of colonyformation. On day7, three groups showed no singnificant different in the totalnumber of cells, but on day14and21, the total number of cells in other groupcompared to5μmol/L were different(P<0.05).Further more, we detectedfrequency of the peptide-specific CTLs, and found the percentage of IFN-γ+CD8+T in1μmol/L group was gradually increased with culture. On day21it increased(11.72±5.03)%. These culture cells were selected to evaluate the killing abilitiesto auto-BLCL and K562. The effector cells that were expanded demonstratedHLA-restricted lysis against auto-BLCL, and the proportion of non-specifickilling was less than15%.3. Expansion of effector cells was compared using culture bags and flasks.After7,14,21days of ex vivo expansion, the two cell populations were analyzed. Phenotypicanalyses showed no differences between the expanded cells in the bags and flasks.In culture bags, there was a predominance of CD3+T cells（90.03±10.28）%,the majority of the CD3+T cells were CD3+CD8+T cells（52.27±25.38）%, there wasno difference in both culture conditions (P>0.05). The effector cells that wereexpanded in the culture bag demonstrated improved cell lysis against theauto-BLCL compared with cells expanded in flasks. There was no difference incell lysis when analyzing the expanded cells obtained from the bags or the flasksusing K562as the target. After21days of expansion, when testing for lysis ofauto-BLCL, there was no difference in the percentage lysis between cellsexpanded in culture bags and in flasks at the effector cells to target ratio:20: l(P>0.05),10:1(P>0.05) and5:1(P>0.05).4. After2weeks in culture, the average total cell expansion was5.01-fold, and thefrequency of EBV-specific CTL in HLA-A*2402,A*0201,A*1101was (13.27±2.35)%:(9.74±3.32)%:(10.49±2.56)%,respectively. On day0, the percentage ofCD3+T cell was (43.34±20.08)%, CD3+CD8+T cells was (18.25±16.23)%. After14days culture, CD3+Tcells increased to (88.28±12.23)%, CD3+CD8+T cellincreased to (45.83±21.59)%, B cells and NK cells were decreased.The effectorcells and target cells in the effector to target ratio of10:1for24hours, there was amarked increase in the number of IFN-gamma when comparing experimentalgroupd(4328±426) and the control(526±73), P <0.01.On day14, the effector totarget ratio of20: l, killing against autologous LCL was (54.22±21.14)%and(41.53±12.28)%at10: l respectively, killing autologous PHA-stimulatedlymphoblastoid cells, K562cells, and does not match the LCL was less than15%.5. After2weeks in culture, the median frequency of CMV-specific CTL after culturein3seropositive healthy donors was22.21（17.53-26.85）%,the average total cellexpansion for seropositive samples was7.18fold and the total CMV-specific CTLexpansion was1120fold(657to2598). CMV-seronegative cultures did not showany CMV-specific CTL expansion（P<0.00）. The effector cells generated fromCMV-seropositive in dividuals exhibited a strong NLV-specific response againstNLV-pulsed T2cells(78.34±12.06)%, but failed to kill T2cells that had beenpulsed with TLN peptide(7.00±1.03)%. Conclusion：In this study, we select several CD8+specific T cells recognizing the peptideepitopes, derived from different antigens, to expand CTLs ex vivo with recombinanthuman interleukin-2. Only30to50ml of peripheral blood after two weeks culture,canmeet therapeutic requirements for most recipients. The high frequency ofCD8+peptide-specific T cells recognizing the epitope occurred in EBV or CMVseropositive individuals. The high-concentration peptide-specific CTLs provide abasis for adoptive antigen-specific CTL immunotherapy. Furthermore, the resultsdemonstrate a reproducible and reliable procedure not only for EBV but also CMV. |
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