Study On The Infection Of T Lymphocytes By Chimeric Adenovirus Ad5F35L

T lymphocytes are the core of the cellular immune and play a important role in the regulation and elimination of tumors.Adoptive transfer of tumor antigen-specific T cells is a promising approach for successful tumor therapy.Currently,tumor-infiltrating lymphocytes(TIL)isolated from patients and peripheral blood lymphocytes(PBLs)stimulated with tumor antigens in vitro are the main source of tumor specific T cells.However,it is diffrcult to isolate high-affinity tumor-reactive T cells from tumor-bearing patients and effectively expand in vitro to obtain adequate cells,which becomes an obstacle to the clinical application.TCR molecule comprised of a and p chains on the surface of T cell recognizes peptide antigen bound to MHC molecules on the surface of target cells and T cell specificity is solely determined by the TCRap gene.Consequently,the introduction of TCR gene that are derived from one tumor-specific T-cell clone into T lymphocytes can confer tumor specificity to the transduced T cells.Although there is now clear evidence for the clinical feasibility of TCR gene therapy,clinical effectiveness of TCR gene transfer is clearly low and further improvements are needed.One of the problems to be solved is the optimization of TCR expression,because the low efficacy may be related to the low level of the introduced TCR expression on gene-modified T cells.Many efforts have been focused on high-level TCR expression including the optimal arrangement of the transgene cassette and codon optimization of the TCR genes.However,a transfer system that yields a high infection efficiency is a prerequisite for the high-level TCR expression.Many studies have employed retroviral vectors(RV)and lentiviral vectors(LV)to transfer TCR gene.However,risk of cellular trangformation in peripheral T cells due to the retroviral genome integration is the greatest obstacle in clinical application.Replication-defective adenoviruses(Ad)are nonintegrating vectors and transduce both proliferating and quiescent cell types with high efficiency.Because of these advantages,they are more advantageous compared to retroviral 'vectors in TCR-gene modification.Currently,Ad vector based on well-characterized,nontumorigenic serotype5(Ad5)is used widely in gene therapy.Unfortunately,Ad5 transduces T lymphocytes poorly because of the lack of sufficient expression of coxsackie/adenovirus receptors(CAR)and aV integrins on the surface of T cells,which results in failure of adenoviral attachment and cell entry.To overcome the deficiency,many alterations have been made to improve the adenovirus-mediated gene expression in T cells.The adenovirus fiber is comprised of tail,shaft and knob.The shaft connects the N-terminal tail with the globular knob structure,which is responsible for interaction with the target cellular receptor.One of these attempts was replacement of fibers on serotype 5 with that from 35 to target CD46 ubiquitiously expressed complement regulatory protein,which obtained a chimeric vector(Ad5F35S)containing the Ad35 natural short-shaft and knob and consequently increased the infection efficiency.Here,we developed a chimeric vector(Ad5F35L),which contained the natural long-shaft of Ad5 and Ad35 knob.We investigated the ability of Ad5F35L to transduce human lymphocytes in comparison to Ad5 and Ad5F35S vectors.The chimeric adenoviral vector Ad5F35L and Ad5F35S was generated by using homologous recombination.To compare the infection efficiency of Ad5,Ad5F35S and Ad5F35L in human T cells,Jurkat cell line,normal CD3+ T cells and prestimulated CD3+ T cells were used in our study.K562(low CAR,as a control)and Jurkat were transduced at multiplicities of infection(MOI)ranging from 5 to 400.Compared with Ad5,obviously higher infection rates were observed when using Ad5F35 in both Jurkat and K562.Compared with Ad5F35S,statistically higher infection rates was found when using Ad5F35L in K562 and Jurkat at MOI of 100,200 and 400.It is critical to compare the infection rate in primary T cells.Primary human T lymphocytes isolated from two healthy donors were transduced with adenovectors for 12 hours at various MOIs ranging from 20 to 400.For all MOI tested with Ad5,the percentage of GFP-expressing cells in CD4+and CD8+T lymphocytes were found to be less than 10%.By contrast,the proportion of GFP-expressing cells in CD8+ T lymphocytes reached 30%after infection with Ad5F35L using MOI 100.The proportion of GFP positive T lymphocytes increased to 50%and 69%respectively when the MOI of the Ad5F35L virus was increased from 200 to 400.Similar results were observed in CD4+T lymphocytes,the proportion of GFP positive T lymphocytes increased to 50%and 65%respectively when the MOI of the Ad5F35L virus was increased from 200 to 400.when cells were transduced at MOI of 200 and 400,Ad5F35L showed statistically higher infection rates than Ad5F35S in both CD4+and CD8+T lymphocytes.To investigate the influence on infection efficiency following activation of T lymphocytes,enhanced Ad5F35L infection efficiency was found in activated CD4+T lymphocytes.An average infection rate of 73.5%(SD 3%)was observed in activated CD4+T cells when using Ad5F35L at an MOI of 400.By comparison,about 63%(SD 2.3%)of unactivated CD4+T cells were transduced with Ad5F35L.Transgene expression in CD8+ T and CD4+ T cells was followed over time after adenoviral infection with Ad5F35L at an MOI of 400.The proportion of T cells positive for GFP expression peaked at 2 to 3 days and thereafter declined by approximately one-half within 6 to 7 days.After 11 to 13 days the GFP expression levels were indistinguishable from background fluorescent signals.To validate the functional utility of the chimeric fiber,we tried to show that entry of Ad5F35L occurred via the pathway dicated by the konb domain of the fiber.Ad5 and Ad35 knobs were expressed in E.coli.Our results demonstrated that competition with the recombinant type 5 knob inhibited specifically the infectivity of Ad5 in a dose-dependent manner.When used at a concentration of 50(Pg/ml,the type 5 knob inhibited almost all of Ad5-mediated GFP gene expression and had no effect on Ad5F35L-mediated gene transfer.When the type 35 knob was used,it was observed that the Ad5F35L-mediated gene transfer could be blocked in a dose-dependent manner.At a concentration of 50 ?g/ml,the type 35 knob inhibited 50%of-Ad5F35L-mediated GFP expression and was not capable of blocking the gene transfer by Ad5 in competition experiments.These findings thus con:firm that Ad5F35L,which contains a chimeric fiber protein with the knob domain derived from Ad35,achieved cellular entry via the Ad35 pathway.To investigate the influence of cell proliferation on infection efficiency.Jurkat cells were cultured in RPMI-1640 medium supplemented with 10%or 1%fetal calf serum for 3 days,respectively.The cell cycle phase was determined using propidium iodide staining and the apoptosis in Jurkat cells was detected by annexin V/7-AAD staining.Trypan blue staining assay was used to determine survival cell numbers.Low serum culture could result in decreasement of cell proliferation.Jurkat cells cultured in different serum concentration were transduced with Ad5F35L-GFP at an multiplicities of infection(MOI)of 100.Low serum culture could inhibit Ad5F35 infection efficiency in Jurkat cells.Virus internalization and endosome escape was significantly inhibited.Inhibition of virus internalization and endosome escape are possible reasons for decreasement of Ad5F35 infection efficiency in Jurkat cells cultured with low serum concentration.In the present study,we have analyzed procedures from 3 aspects including binding to receptors in the cell surface,endocytosis and lysis of the endosomal membrane.The study on the influence of fiber shaft in infection efficiency and its mechanism will provide a foundation for the development of clinical adenoviral vector.