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Antigen-specific CTLs: to produce autologous cells product for adoptive cellular therapy

Open AccessPublished:March 22, 2017DOI:https://doi.org/10.1016/j.ijid.2017.03.015

      Highlights

      Major revision version
      • Cell cultivation medium used in this research was serum free to ensure safety for further application in vivo; the result showed this medium could perfectly support the expansion of cells.
      • Different cytokine environments were evaluated for their effect of supporting the expansion of cytotoxic lymphocyte expansion for both general and antigen-specific quantity.
      • The method is capable to manufacture qualified cell product for in vivo cellular therapy, some profiles of the final product for each participant of the cellular therapy clinical trail is provided.
      Minor revision version:
      • Implement of reference is done to prove the peptide specific CTLs recognize naturally processed and represented antigen.
      • Table 1 is divided into 2 tables to make it more convenient and streamlined.

      Abstract

      As antiretroviral therapy provides long term viral suppression but no cure, alternative therapies such as adoptive cellular therapy have thus been investigated in the anti-AIDS field.

      Objective

      This study sought to establish a HLA-A02 specific CTL cell culture method with comparison of the effects of different cytokines used in CTL cultivation to decide the best cultivation environment. In order to produce CTLs with targeted HLA-A02 restricted antigen specificity for adoptive cellular therapy, we evaluated autologous PBMC cultivation in different cytokine environment to select a better expansion condition to produce qualified CTL production.

      Methods

      We co-cultivated PBMC and peptides of these patients with HLA-A02 allele with different cytokines. After cultivation, multiple parameters were tested.

      Results

      1) Cytokines IL-2 alone can effectively amplify HLA-A02 specific CTL cells, and the count of CTLs was > 85% all through the process. 2) The HLA-A02 specific cells at the end of the cultivation were mainly CD3 + CD8+ cells. 3) The interferon stimulation test had shown that the expanded CTLs secreted more IFN-γ than before cultivation (0.9% -11.70%).

      Conclusion

      This model of CTL cultivation is successful in redirecting the specificity of antigen recognition and safely for HLA-A02+ patients cell adoptive therapy.

      Keywords

      Introduction

      HIV/AIDS is now one of the most important global public health issues. Although cART (combined anti-retrovirus therapy) has decreased the morbidity and mortality of HIV/AIDS, it cannot achieve a sterile cure due to the existence of HIV reservoirs. Within a decade, cellular therapy has been highlighted with the progression of medical science and technology; with utilization of antigen specific cytotoxic T lymphocytes(CTLs), cellular therapy has succeed in curing EBV and CMV infections (
      • Fujita Y.
      • Rooney C.M.
      • Heslop H.E.
      Adoptive cellular immunotherapy for viral diseases.
      ,
      • Mackinnon S.
      • Thomson K.
      • Verfuerth S.
      • Peggs K.
      • Lowdell M.
      Adoptive cellular therapy for cytomegalovirus infection following allogeneic stem cell transplantation using virus-specific T cells.
      ). Previous study had shown that CTLs correlated to HIV viral load decrease, especially in the viral decline phase of acute infection (
      • Walker B.D.
      • Chakrabarti S.
      • Moss B.
      • Paradis T.J.
      • Flynn T.
      • Durno A.G.
      • et al.
      HIV-specific cytotoxic T lymphocytes in seropositive individuals.
      ). Moreover, the study from our team had demonstrated not only quantity but also quality of CTLs matters in HIV control of natural immunity (
      • Dong T.
      • Zhang Y.
      • Xu K.Y.
      • Yan H.
      • James I.
      • Peng Y.
      • et al.
      Extensive HLA-driven viral diversity following a narrow-source HIV-1 outbreak in rural China.
      ). Thus it has drawn attention for further investigation of adoptive cellular therapy for HIV and its potential in the cure of HIV (
      • Patel S.
      • Jones R.B.
      • Nixon D.F.
      • Bollard C.M.
      T-cell therapies for HIV: Preclinical successes and current clinical strategies.
      ).
      Human leucocyte antigen (HLA) is the most important genetic product for immunity; it has a very close relationship to HIV infection. Located on the short arm of the 6 chromosome (6p21.31), it can be categorized into HLA-I, HLA-II, and HLA-III molecules based on its function and transcriptional production. With the three sites (HLA-I A site, HLA-I B site, HLA-I C site) polymorphism of HLA-I molecule, it is able to encode up to 4269 proteins (
      • Alter G.
      • Heckerman D.
      • Schneidewind A.
      • Fadda L.
      • Kadie C.M.
      • Carlson J.M.
      • et al.
      HIV-1 adaptation to NK-cell-mediated immune pressure.
      ). In the field of infectious diseases, HIV infection is one of the very few diseases that is correlated with the HLA molecule (
      • Rajapaksa U.S.
      • Li D.
      • Peng Y.C.
      • McMichael A.J.
      • Dong T.
      • Xu X.N.
      HLA-B may be more protective against HIV-1 than HLA-A because it resists negative regulatory factor (Nef) mediated down-regulation.
      ). In the Han Chinese population, HLA-A02 is the dominant subtype of HLA-A allele, and occupies up to 50% of the population (
      • Dong T.
      • Zhang Y.
      • Xu K.Y.
      • Yan H.
      • James I.
      • Peng Y.
      • et al.
      Extensive HLA-driven viral diversity following a narrow-source HIV-1 outbreak in rural China.
      ,
      • Zhang Y.
      • Peng Y.
      • Yan H.
      • Xu K.
      • Saito M.
      • Wu H.
      • et al.
      Multilayered defense in HLA-B51-associated HIV viral control.
      ). With the prevalence of HLA-A02 haplotype, therapy based on it has the potential to be utilized on the largest proportion of people.
      In order to investigate the safety of the HIV antigen-specific adoptive cellular therapy that is currently being carried out (registration number: ChiCTR-ICR-15005775, China registration center of clinical trial), we established this method to cultivate HIV antigen specific CTLs for adoptive refusion to patients by co-cultivating CTLs with peptides of HLA-A02 restricted dominant epitopes of HIV.

      Method

      Experiment materials

      Based on a previous study by our team, HLA-A02 recognized dominant epitope peptides were used for CTLs cultivation ex vivo. The peptides were A2-GAG-YL9 YVDRFYKTL; A2-POL-IV9 ILKEPVHGV; A2-GAG-SL9 SLYNTVATL; A2-POL-YI9 YTAFTIPSI; A2-POL-FK10 FLGKIWPSYK; A2-GAG-VV9 VLAEAMSQV; A2-POL-LI9 LVGPTPVNI; A2-POL-VL9 VIYQYMDDL; A2-POL-AM9 ALVEICTEM. All peptides were synthesized by GL Biochem Co, Shanghai, purity >95%. Cytokines/Chemokines used included IL-2,IL-7,IL-4.
      Fetal cow serum (FCS) is usually required as an additive to regular cell culture mediums such as PRMI-1640. However, it brings about concerns like contamination of pathogen from FCS, batch differences of FAC, risk of immune rejection, etc. The culture medium used in this study was serum free, including IMSF100 (Beijing immunotech applied science limited) and CCM (Tianjin H&J Medical Ltd).

      Participants

      Participants were all HIV infected people possessing HLA-A02 alleles. The HLA typing was performed by SinoGenoMax Co., Ltd; written approvals obtained from the ethics committee of Beijing You’An hospital; written consent forms were signed by all participants. Clinical and laboratory data are shown in Table 1A (participant for method development) and Table 1B (participants of clinical trial).
      Table 1AClinical and laboratory characteristics of participants of the method development.
      Pt. IDGenderAgeViral Load
      Unit for Viral loads: Copies/ml. Unit for CD8 and CD counts: cell/μl.
      cARTCD8 counts
      Unit for Viral loads: Copies/ml. Unit for CD8 and CD counts: cell/μl.
      CD4 counts
      Unit for Viral loads: Copies/ml. Unit for CD8 and CD counts: cell/μl.
      HLA-

      A1
      HLA-A2HLA-B1HLA-B2
      481M50<50Y48845702245148
      55M50<50Y119629102023551
      429F51<50Y69021602025158
      524F48<50Y83029902245154
      349F376764N1303434021151
      215F50<50Y86545902305151
      446M563217N39418702263851
      565M65<50Y65023302241551
      15F61<50Y128496602244051
      * Unit for Viral loads: Copies/ml. Unit for CD8 and CD counts: cell/μl.
      Table 1BClinical and laboratory characteristics of participants of the adoptive cellular therapy.
      Pt.

      ID
      Sample IDGenderAgeViral Load
      Unit for Viral loads: Copies/ml. Unit for CD8 and CD counts: cell/μl.
      cARTCD8 counts
      Unit for Viral loads: Copies/ml. Unit for CD8 and CD counts: cell/μl.
      CD4 counts
      Unit for Viral loads: Copies/ml. Unit for CD8 and CD counts: cell/μl.
      HLA-

      A1
      HLA-A2HLA-B1HLA-B2
      HBXHIVA2-00101-20150520M41<50Y98651202:0602:0615:1835:01
      HIVA2-00102-20150602M41<50Y998350
      MJHIVA2-00201-20150707M28<50Y105229802:0111:0135:0138:01
      HIVA2-00202-20150727M28<50Y498245
      WHCHIVA2-00501-20150717M54<50Y50676502:0731:0146:0151:01
      HIVA2-00502-20150730M54<50Y590899
      ZZYHIVA2-00601-20150724M25<50Y41430302:0711:0115:0246:01
      HIVA2-00602-20150807M25<50Y789464
      WYPHIVA2-00701-20150724M31<50Y67143702:0111:0115:1139:01
      HIVA2-00702-20150807M31<50Y630397
      ZZQHIVA2-00803-20150910M29<50Y69591502:0111:0127:0440:01
      HIVA2-00804-20150924M29<50Y612879
      MQQHIVA2-00901-20150730M41<50Y64649002:0111:0115:1851:01
      HIVA2-00902-20150813M41<50Y880648
      YGJHIVA2-01101-20150812M45<50Y55549102:0102:0740:0146:01
      HIVA2-01103-20150910M45<50Y383387
      PYHHIVA2-01501-20150907M51<50Y52473702:0726:0108:0146:01
      HIVA2-01502-20150923M51<50Y560774
      XWJHIVA2-01601-20150907M42<50Y113254002:0102:0715:1835:01
      HIVA2-01602-20150923M42<50Y938494
      HJJHIVA2-01701-20150923M58<50Y38423302:0602:0740:0646:01
      HIVA2-01702-20151010M58<50Y389247
      LJZHIVA2-01801-20150925M26<50Y114074002:0733:0307:0240:01
      HIVA2-01802-20151010M26<50Y970589
      DWHIVA2-01901-20151013M31<50Y219551901:0102:0137:0146:01
      HIVA2-01902-20151028M31<50Y2120522
      ZLHIVA2-02001-20151013M34<50Y68860602:0126:0135:0155:02
      HIVA2-02002-20151028M34<50Y786629
      * Unit for Viral loads: Copies/ml. Unit for CD8 and CD counts: cell/μl.

      Separation of peripheral blood mononuclear cells (PBMCs)

      Peripheral blood samples of each participant were mixed with natural saline (NS) of equal volume, then added slowly into 15 ml Ficoll centrifuge tubes (GE Co., USA). After centrifuge, the interface layer of cell was drained out and mixed with NS of equal volume then centrifuged again. After that, supernatant was discarded and the cells were resuspended with 50 ml of NS and counted for the amount of cells, then centrifuged for the last time.

      Cultivation of HLA-A02 restricted antigen specific CTLs

      The PBMCs were suspended with serum free medium CCM then transferred into 6-well plates (Corning Co., USA), then HLA-A02 restricted epitopes peptides were added. After 2 days of incubation in 37 °C, 5% CO2 environment, serum free medium CCM with cytokines were added again. At day 5, and day 9 serum free medium IMSF100 with cytokines was added. At day 9 and day 12, sterile test was performed for each sample. At day 14, cultivation liquid was collected together for each participant, supernatant was discarded, and sediments were blown. 10 ml NS was used to suspend the cells for centrifuge for 2 times, then 50 ml NS with 1% of human serum albumin was used to resuspend the cells. The final product would then be transferred into disposable sterile plastic cell transfusion bag.

      Flow cytometry of cell subtype

      0.5*106 cells were used for this panel, which included fluorescence antibodies (all BD Co.) of CD3-V500, CD8-PE, CD4-FITC, CD56-Percp, CD19-APC. After staining for 30 min at 4 °C, phosphate buffered saline (PBS) was used to suspend the stained cells for 2 times of centrifuging for Flow cytometry analysis.

      Flow cytometry for intracellular cytokine staining (ICS)

      Golgistop (BD Co.) were added into 0.5*106 cells and then co-stimulated with HLA-A02 restricted epitope peptides for 4 hours at 37 °C for the test tube; control tube was the same minored epitope peptides. Fluorescence antibody panel, which included (all BD Co.) CD3-V500, CD8-PE, CD4-FITC, CD56-Percp, CD19-APC, was added into both tubes. For the next step, Fixation/Permeabilization Solution (BD Co.) was added into each tube. Then PBS were used to wash the cells, then IFN-γ-APC fluorescence antibodies (BD Co.) were added to the tubes. For each step, both tubes were kept still at dark for 30 min at 4 °C. Finally, each sample was suspended with PBS and centrifuged two times.

      Flow cytometry for antigen specificity of CTLs.

      0.5*106 cells were used, with a fluorescence antibody stain panel that included CD3-V500, CD8-PE, CD4-FITC (BD Co.), the tetramer used for staining was tetramer –APC synthesized by Weatherall institute of molecular biology of Oxford University. Tubes then were kept still at dark for 30 min at 4 °C. Each sample was suspended with PBS and centrifuged 2 times. The control tube contained 0.5*106 cells stained by the same panel without tetramer.

      Results

      Cultivation outcomes under different cytokine environments

      In this section, we enrolled 2 HLA-A02 HIV infected patients (patient ID 55 and 418), 2*106 cells of each patient were assigned to two groups, named Group A and Group B. Group A contained only IL-2 (500U/ml). Group B contained IL-4 (400U/ml) and IL-7 (10 ng/ml).
      The results of cells cultivated in these two groups showed: 1) The count of CTLs was stable, maintained >85% all through the cultivation process. 2) At day 12, targeted antigen specific CTL emerged (determined by CD3 + CD8 + IFN+ % by IFN-γ secretion Flow cytometry test). At day 18, the CTLs was still expanding, but at day 20 antigen specific CTLs level started to decrease. Thus we recommend the timing for harvesting the targeted antigen specific CTLs is between day 12 to day 18. 3) Cytokine environment of Group A was better than Group B in stimulating CTLs to expand. Cell count was too low to harvest the required amount to do the IFN-γ secretion Flow cytometry test at day 20 for Group B. Details of cell cultivation for both group is shown in Table 2.
      Table 2Details of the subtypes of cells cultivated at different cytokine environment and time point.
      Pt. IDTime pointAmount

      (*106)
      Rate of survivalCD3 +%CD3 + CD8 +%CD3 + CD4 +%CD3 + CD8 + IFN-γ+%
      418-AD1210.8094.24%78.7365.116.731.50
      418-BD120.72100.00%93.1739.7840.220.20
      55-AD121.8093.75%96.3660.129.451.00
      55-BD120.3685.71%97.4872.8713.980.00
      418-AD1811.8095.16%81.8668.172.884.30
      418-BD180.8496.77%96.8350.2634.980.30
      55-AD183.0089.29%92.7043.8226.931.40
      55-BD181.18100.00%99.2174.3916.940.00
      418-AD2048.696.43%79.6164.062.550.2
      418-BD200.112100.00%nd*ndndnd
      55-AD201.896.77%93.3730.3845.830.6
      55-BD200.196100.00%ndndndnd
      A:IL-2(500U/ml);
      B: IL-4 (400U/ml) and IL-7 (10 ng/ml)
      nd: not done.

      Details of cells cultivated in Group A

      subtypes of cultivated cells

      The first panel of the flow cytometry test is to examine the proportion of each subtype of cell in the final product of the cultivation in order to determine if this method was able to produce an adequate amount of CTLs. The results of the test showed that the dominant subtype of the cultivated cells was CD3 + CD8+ T cells (67.2%), followed by CD3 + CD4 + T cells (17.8%), CD3-CD56+ NK cells (4.09%), CD3-CD19+ B cells (3.90%). Other subtypes of cells in total took about 7.01% of the whole product. Gating strategy of PBMCs and subtypes of cells are shown in Figure 1.
      Figure 1
      Figure 1subtypes of cells cultivated.
      The dominant subtype was CD3+CD8+T cells (67.2%), it were followed by CD3+CD4+ T cells(17.8%), CD3-CD56+ NK cells (4.09%), CD3-CD19+B cells(3.90%).

      Results of intracellular cytokine staining by flow cytometry test

      In order to determine the specificity of the cultivation product in an accurate manner, we performed intracellular cytokine staining (ICS) to cross match the antigen specificity both by active biological reaction and phenotype comparison. 1*106 cells were obtained from the cultivation, then centrifuged for 2 times to wash them from previous cultivation medium and cytokines. Then they were divided equally into test group and control group. After the steps mentioned in the Methods section for ICS, flow cytometry tests were performed to examine the proportion of the products with IFN-γ secretion. The array showed that in the control group, there were 1.5% of the IFN+ cells. In test group (peptide stimulation group), there were 7.86% of the IFN+ cells. With the 5 folds larger amount of cells showing IFN secretion between test and control group, the active immune response confirmed the success of expansion of CTLs with targeted antigen specificity. Results are showed graphically in Figure 2.
      Figure 2
      Figure 2result of intracellular cytokine staining by Flow cytometry test.
      Each tube was assigned 0.5*106 cells and Golgistop remedy was added in both tubes. The HLA-A02restricted epitope peptides were added only into test tube, then both tubes were incubated at 37 °C for 4 hours. After that each tube went through fluorescence antibodies staining.

      Flow cytometry for antigen specificity of CTLs

      Alongside the immune response test of ICS, we also performed tetramer staining to check the proportion of cell possessing T cell receptor (TCR) with the required antigen specificity to confer the ICS result and more adequately evaluate the antigen specificity of the cultivation product. Cell assignments were the same as in section I. After fluorescence antibody staining of lymphocyte marker and tetramer staining, flow cytometry was performed and the results showed that in the control group, there were 0.002% CD8 + tetramer+ cells, while in the test group the proportion is 0.86%. This result showed a 430-fold expansion of CTLs with targeted TCR, and cross-checked the capacity of antigen specificity induced by the cultivation method by phenotype of the cells. Results are shown graphically in Figure 3.
      Figure 3
      Figure 3Flow cytometry for antigen specificity of CTLs.
      Each tube was assigned 0.5*106 cells. Control tube: added CD3+CD4+CD8 fluorescence antibody but no tetramer; Test tube: added CD3+CD4+CD8+Tetramer fluorescence antibody. After fluorescence antibodies staining of lymphocyte marker and tetramer staining, flow cytometries were performed.

      Samples from different individuals cultivated under Group A environment

      9 cases of HLA-A02 alleles possessing HIV-1 infected individuals were recruited in this section; blood samples were processed as in the Methods section and under Group A environment (solely IL-2). At day 12, samples were acquired from the cultivated cells and tested byIFN-γ secretion. The result showed: 1) Rate of live cells was higher than 85% for all samples. 2) CTLs proportion of each cultivation ranged from 27.06% to 65.11%, median 52.44%. 3) Proportion of CD3 + CD8 + IFN-r+ cells in cultivations of different individuals ranged from 0.9% to 11.7%, median 1.5%. The detailed data are shown in Table 3.
      Table 3details of cell cultivation of different individuals.
      Pt. ID.Rate of survival

      at day 12
      CD3 +%CD3 + CD8 +%CD3 + CD4 +%CD3 + CD8 + IFN-γ+%
      42991.67%88.8742.8515.155.65
      52496.15%65.7935.0517.471.68
      34993.33%80.6763.528.805.28
      21590.91%90.4927.0647.960.90
      41894.24%78.7365.116.731.50
      5593.75%96.3660.129.451.00
      44695.83%85.5131.0127.741.40
      56595.00%68.1355.2115.181.10
      15100.00%74.8952.449.5011.70
      All participants were HLA-A02+, specimen were cultivated with IL-2(500U/ml) for 12 days and went through test.

      The specific autologous CTL cells for adoptive cellular therapy

      Based on the above results, 14 cases of HLA-A02 alleles possessing HIV-1 infected individuals were recruited in our clinical trail (registration number: ChiCTR-ICR-15005775, China registration center of clinical trial). The clinical characteristics are shown in Table 1. Blood samples were processed as in Methods section under Group A environment (solely IL-2). Samples were acquired from the cultivated cells and tested for their IFN-γ secretion. The proportion of CD3 + CD8 + IFN-γ+ cells in cultivations of different individual ranged from 0.0% to 10.9%. The detailed data are shown in Table 4.
      Table 4details of cell cultivation of different individuals in clinical trials.
      Pt.IDdaysTotal cellRate of

      live cell
      CD3+CD3 + CD8 + IFN-γ+CTL count
      ×107%%%×105
      HBXHIVA2-00101-20150520133.5294.6076.310.401.072.93
      HIVA2-00102-20150602137.2291.7081.560.502.946.02
      MJHIVA2-00201-20150707204.0075.0088.350.000.002.00
      HIVA2-00202-20150727144.9590.0068.570.602.042.25
      WHCHIVA2-00501-20150717135.0090.9086.091.606.891.67
      HIVA2-00502-201507301631.5096.9272.352.2050.149.84
      ZZYHIVA2-00601-201507241410.5095.5082.640.907.814.77
      HIVA2-00602-20150807137.5093.7581.271.7010.363.13
      WYPHIVA2-00701-201507241411.1095.7059.182.7017.745.05
      HIVA2-00702-201508071310.5095.7059.063.1019.224.77
      ZZQHIVA2-00803-201509101415.4096.5068.100.202.106.42
      HIVA2-00804-201509241622.5090.0090.200.306.099.38
      MQQHIVA2-00901-201507301413.5096.4374.5910.90109.764.22
      HIVA2-00902-20150813138.0097.5679.629.1057.964.44
      YGJHIVA2-01101-201508121414.0093.3078.301.2013.156.36
      HIVA2-01103-201509101413.7589.3066.800.100.925.73
      PYHHIVA2-01501-201509071646.7595.5174.180.206.9412.99
      HIVA2-01502-201509231615.5091.2059.000.302.746.46
      XWJHIVA2-01601-201509071637.4095.7746.412.4041.6615.58
      HIVA2-01602-201509231619.0095.0064.500.506.137.92
      HJJHIVA2-01701-201509231717.5089.7092.100.101.617.29
      HIVA2-01702-20151010167.0087.5065.400.100.462.92
      LTSHIVA2-01801-20150925158.0084.2092.400.100.743.33
      HIVA2-01802-20151010134.0093.2079.000.100.321.67
      DWHIVA2-01901-20151013158.0094.1083.500.000.002.22
      HIVA2-01902-201510281420.0097.5067.501.0013.505.56
      ZLHIVA2-02001-201510131510.0090.9083.900.100.845.56
      HIVA2-02002-201510281413.0089.6569.900.201.827.22

      Discussion

      The Berlin patient had brought an outlook for sterile/functional cure for HIV infection (
      • Allers K.
      • Hütter G.
      • Hofmann J.
      • Loddenkemper C.
      • Rieger K.
      • Thiel E.
      • et al.
      Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation.
      ,
      • Hütter G.
      • Nowak D.
      • Mossner M.
      • Ganepola S.
      • Müssig A.
      • Allers K.
      • et al.
      Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation.
      ), which has drawn attention for cellular therapy for the disease. Given the important role of CTLs in natural immune reaction towards HIV (
      • Zhang Y.
      • Peng Y.
      • Yan H.
      • Xu K.
      • Saito M.
      • Wu H.
      • et al.
      Multilayered defense in HLA-B51-associated HIV viral control.
      ), CTL adoptive therapy has been highlighted.
      If the source of cultivation is the PBMCs, then T cells and antigen processing cells (APCs) exist simultaneously,and then only antigen peptides and multiple cytokine/chemokine are needed to form a whole cultivation system. Moreover, it has been proven that in vitro cultivated peptide specific CTLs are able to recognize naturally processed and presented epitopes (
      • Zhang Y.
      • Peng Y.
      • Yan H.
      • Xu K.
      • Saito M.
      • Wu H.
      • et al.
      Multilayered defense in HLA-B51-associated HIV viral control.
      ). Thus an in vitro expansion of antigen-specific CTL product would be valid for clinical usage. In this study, we used HLA-A02 restricted peptides, then compared the effects of IL-2 and the combination of IL-7 and IL-4 for cultivation support. According to previous studies, IL-2 could enhance CTLs’ expansion with high efficiency and specificity (
      • Kern D.E.
      • Gillis S.
      • Okada M.
      • Henney C.S.
      The role of interleukin-2 (IL-2) in the differentiatin of cytotoxic T cells: the effect of monoclonal anti-IL-2 antibody and absorption with IL-2 dependent T cell lines.
      ). IL-7 (
      • Ferrari G.
      • King K.
      • Rathbun K.
      • Place C.A.
      • Packard M.V.
      • Bartlett J.A.
      • et al.
      IL-7 enhancement of antigen-driven activation/expansion of HIV-1-specific cytotoxic T lymphocyte precursors (CTLp).
      ) and IL-4 (
      • Olver S.
      • Apte S.H.
      • Baz A.
      • Kelso A.
      • Kienzle N.
      Interleukin-4-induced loss of CD8 expression and cytolytic function in effector CD8 T cells persists long term in vivo.
      ) could affect lymphocytes expansion, such as maintaining memory cell survival and decreasing the death of cells after activation to achieve a better expansion and maintenance of cell activity. The results of our study indicated that IL-2 has a better effect than IL–7 + IL-4 in expanding CTLs. A triple or even bigger combination of cytokines such as IL–2 + IL–7 + IL-4 might have a greater effect in the cultivation, which is left for further investigation. Based on previous study, an addition of IL-15 may enhance the cultivation outcome (
      • Kanai T.
      • Thomas E.K.
      • Yasutomi Y.
      • Letvin N.L.
      IL-15 stimulates the expansion of AIDS virus-specific CTL.
      ).
      For the immune affect part, we used IFN-γ stimulation array and tetramer staining to test the antigen specificity both bio-actively and phenotypically. As it indicated, the IFN-γ secretion had a profound elevation after antigen peptide stimulation; the count of CD8 + tetramer+ cell was elevated at the end of cultivation compared with baseline. These results proved the capacity of this method of expanding CTLs with targeted antigen specificity. Moreover, through different time points we tested the level of HIV-p24, endotoxin, bacterial; all of them were negative, which proved this method could manufacture a product safe for medical use.
      In the laboratory, anti-CD3 antibody is usually used for large fold expansion (
      • Brennan C.A.
      • Ibarrondo F.J.
      • Sugar C.A.
      • Hausner M.A.
      • Shih R.
      • Ng H.L.
      • et al.
      Early HLA-B*57-restricted CD8+ T lymphocyte responses predict HIV-1 disease progression.
      ), but in concern for minimum additives and the most simplified cultivation environment to avoid adverse effects from unnecessary additives, we did not use it. However, due to this, the cells had expanded only 3 to 10 folds, which is left for further investigation.
      In summary, a method was established using non-serum cultivation medium, HLA-A02 restricted HIV antigen peptide and IL-2, to expand HLA-A02 antigen specific CTLs, which proved safe for medical use. We used this method to culture the autologous specific CTL for adoptive cellular therapy (registration number: ChiCTR-ICR-15005775, China registration center of clinical trial).

      Acknowledgments

      The work was supported by National S&T Major Project for Infectious Diseases Control (2012ZX10001006-001-008, 2013ZX10001004-001-002, 2015ZX10004801-001); Beijing Municipal Science & Technology Commission (D131100005313004, D131100005313005); Beijing Key Laboratory (No. BZ0373).

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