|Year : 2014 | Volume
| Issue : 4 | Page : 998-1003
Changes in lymphocytes' telomerase activity by 4-1BB costimulation
Hosseini Tashnizi Ahmad1, Jaberipour Mansooreh1, 2, Masoodi Fereshteh2, Habibagahi Mojtaba3
1 Gene therapy laboratory, Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences, Shiraz, Iran
2 Department of Immunology, Immunotherapy Laboratory, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
|Date of Web Publication||9-Jan-2015|
Dr. Habibagahi Mojtaba
Department of Immunology, Immunotherapy Laboratory, School of Medicine, Shiraz University of Medical Sciences, Zand Blvd. Shiraz
Source of Support: None, Conflict of Interest: None
Aim: Lymphocytes are exceptional among somatic cells as these cells can induce telomerase enzyme after antigen stimulation to compensate chromosomal loss during rapid cell division. Activation of telomerase in lymphocytes needs CD28 signal, which simultaneously costimulates T cells during activation. 4-1BB of tumor necrosis factor superfamily also has been shown to costimulate T cells. Herein, we investigated changes in telomerase activity of lymphocytes during longitudinal cultures when T cells costimulated by CD80 or 4-1BB ligand or both molecules in conjunction with anti-CD3 stimulation.
Materials and Methods: Artificial antigen presenting cells (aAPCs) were produced by transduction of CD80, 4-1BB ligand or green fluorescent protein genes into A549 carcinoma cells using recombinant adenoviral vectors. Peripheral blood mononuclear cells were stimulated with anti-CD3 and co-cultured with the aAPCs. Cellular growth, expression of telomerase and production of interferon gamma (IFN-γ) were assessed at different time points and followed up to day 35.
Results: 4-1BBL provided effective costimulation for lymphocytes' activation, cytokine production or long-lasting growth where its effects exceeded over CD80 after the first week. 4-1BBL also promoted the telomerase activity and more importantly was able to re-induce the enzyme in the cells that stopped to grow with CD80 costimulation. Although combination of CD80 and 4-1BBL has additional effect on cellular growth or initial telomerase activity, it could not support telomerase activity in later time points.
Conclusion: Our results underscored unique features of 4-1BB over CD28 for prolonged support of lymphocytes' costimulation, which can be recruited for in vitro or ex vivo propagation of T cells for cancer immunotherapy purposes.
Keywords: 4-1BB, CD28, costimulation, proliferation, T cell, telomerase
|How to cite this article:|
Ahmad HT, Mansooreh, J, Fereshteh M, Mojtaba H. Changes in lymphocytes' telomerase activity by 4-1BB costimulation. J Can Res Ther 2014;10:998
| > Introduction|| |
Most somatic cells do not express telomerase enzyme and perform cell division with the expense of losing 50-100 base pairs of the chromosomal telomere sequences in each division. This phenomenon ultimately takes cells to the state of replicative senescence.  Lymphocytes, however, are able to reactivate the expression of the enzyme in a precisely controlled manner just after activation. In case of B cells, engagement of CD40 in the presence of IL-4 pushes cells forward for cell division until cells stop to respond to further stimulation.  Full activation of naïve T cells needs extra costimulatory signals in conjunction with Ag exposure where CD28 engagement was found the most essential costimulation. In fact, interaction of CD80/CD86 with CD28 during T cell activation results in interleukin-2 (IL-2) production, stabilization of mRNA of several cytokines, increase in glucose metabolism as well as upregulation of telomerase expression. , Direct evidence has shown that blocking CD28 signaling could abrogate the activation-induced expression of telomerase in cultured T cells.  However, activated lymphocytes have a limited time to divide as telomerase cannot compensate for all the telomeric lost in rapidly dividing T cells for long time even by Ag stimulation and CD28 signal. At this time, cells stop dividing and become senescent. In that regard, in vitro kinetic assays for the telomerase activity in T cells have shown that CD8 and CD4 T cells progressively lose telomere repeats after third to fifth and seventh rounds of re-stimulation, respectively. , At this stage, lymphocytes significantly upregulate the expression of cell cycle inhibitors like, p21 and stop cell cycling.  Stimulations, subsequent, to the primary activation can only induce modest amounts of the human telomerase reverse transcriptase (hTERT), and finally become undetectable. This observation is parallel to the loss of CD28 in T cells and concomitant with divisional senescence. , Although senescent T lymphocytes generally are considered unresponsive to divide by further Ag stimulation, are metabolically active and may exhibit other signs of responses. It has been shown that Ag stimulation in senescent T lymphocytes could keep the IL-2 receptor expression and more importantly can resume the telomerase expression in certain conditions. In fact, induction of telomerase could restart cell division in senescent CD28-negative T cells. , There are other molecular interactions rather than CD28 to serve as costimulation for T cell activation. Members of the tumor necrosis factor receptor (TNFR) family are well known to provide necessary costimulation signals for T cells concurrent or after CD28 interaction. Among them it has been shown that CD137 (4-1BB) could enhance TCR-induced T cell proliferation and increased the effector functions of helper and cytotoxic T lymphocytes. ,, There have been speculations if CD137 (4-1BB) and other members of the family could affect telomerase expression and activity.  We also previously reported the restoration of cell proliferation and re-expression of CD28 on T cells after switching the costimulation to 4-1BBL when cells were unresponsive to further Ag stimulation in conjunction with CD80 costimulation.  Therefore, in this study in addition to the growth capacity and IFN-γ production (as the major sign of T cell activation), we analyzed the expression of telomerase in the growing lymphocytes when they were in interaction with artificial antigen presenting cells (aAPCs) expressing CD80 and/or CD137 ligand (CD137L) in cultures and showed different time pattern of CD137 in terms of the activation of telomerase.
| > Materials and methods|| |
Preparation of aAPCs for T cell activation
Non-replicative adenoviral vectors (Ad5) were used to transfer and express human CD80, CD137L and GFP in A549 lung carcinoma cells to adapt them as artificial antigen presenting cell (aAPC). The production of the recombinant adenovirus vectors expressing the transgenes (Ad-CD80, Ad-4-1BBL, Ad-GFP) and the ability of the ligands to costimulate T cell activation were reported elsewhere. , A549 cells were infected with 300-600 particles per cell of Ad-CD80 and/or Ad-4-1BBL viruses to express one or both costimulatory ligands, or Ad-GFP, to express enhanced GFP, as control. The expression of the transgenes by the infected cells was analyzed with commercial antibodies against CD80 and CD137L (BD Pharmingen, US) and flow cytometry using four colors Beckton Dickinson FACS Calibur machines with CellQuest pro software for data acquisition.
T cell proliferation assays in coculture with aAPCs
Peripheral blood samples were collected from healthy donors with full informed consent approved by the ethics committee of Shiraz University of Medical Sciences. Neither of the participants had apparent infectious or autoimmune diseases nor history of major chronic diseases. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient then depleted from plastic-adherent cells. A549 cells were infected with the viral vectors to express the corresponding transgene (s) or the GFP control 48 h ahead of coculture with purified PBMCs in 24-well plates. Co-cultures of the aAPCs and 1 × 10 6 purified PBMCs in complete RPMI 1640 medium supplemented with 7% fetal-calf serum (Gibco, UK), 3% human pooled AB serum (Sigma, UK), 2 mM L-glutamine (Gibco, UK), 100 IU/mL penicillin and 100 μg/mL streptomycin (Gibco, UK) were stimulated with suboptimal concentration of 100 ng/ml soluble OKT3 anti-CD3 monoclonal antibody (Abcam, UK). Replicates of cultures were incubated at standard culture condition and half of the volumes of the culture media were replaced every three days. For the extended cultures, PBMCs were transferred to fresh wells and re-stimulated with anti-CD3 and aAPCs in weekly basis. At different time points, cells were harvested for cell enumeration by dye exclusion, assessing telomerase activity and flow cytometry studies. The effect of switching the costimulation (from B7-1 to 4-1BBL) on growth and telomerase activity of PBMCs was assessed on day 21 post-coculture. To this end, PBMCs harvested from replicates of B7-1-costimulated cultures were transferred to the plates with 4-1BBL or 4-1BBL + B7-1 or original B7-1 costimulation and analyzed in parallel with other replicates. For the purpose of intracellular gamma interferon staining, cells were re-stimulated with anti-CD3 for overnight and then harvested and processed for intracellular staining.
Measurement of gamma interferon production by cultured lymphocytes
Production of IFN-γ was measured at single cell level and in secreted form during culture of PBMCs. At different time points, cultures were re-stimulated with anti-CD3 whilst the secretion of the cytokine through the golgi apparatus was inhibited by brefeldin A (5 μg/mL, Sigma, UK) in the last five hours. Cultured lymphocytes were counter-stained with anti-CD4 (PerCP-Cy5.5) or anti-CD8 (PE) antibodies (BD pharmingen, UK), and intracellular IFN-γ was stained with (fluorescein isothiocyanate) FITC conjugated anti-h IFN-γ antibody after cell fixation and permeabilization (BD Pharmingen, UK). Staphylococcal enterotoxin B (SEB, Sigma, UK) at 50 ng/mL was used as a positive control in the experiments. To rule out non-specific staining, isotype matched control antibodies were used (BD Pharmingen, UK). Commercial ELISA kit (Bendermed Systems, Austria) was used to measure the concentration of IFN-γ in the culture supernatant of the growing cells at two time points of days 3 and 7 of cultures.
PCR ELISA Telomerase activity assay
The telomerase activity in the cultured cells was assessed by Telo TAGGG Telomerase polymerase reaction chain (PCR) ELISA kit (ROCHE, Germany) according to the manufacturer's instruction. Briefly, the harvested cells (2 × 10 5 ) at different time points were lyzed and the PCR amplification was performed in a reaction mixture contained telomerase substrate, primer mixes, dNTPs, and Taq DNA polymerase enzyme. The reaction mixture was incubated at 25°C for 20 min allowing the telomerase mediated extension of the biotin-labeled primer. The amplification cycle of 94˚C/30S, 50˚C/30S, and 72˚C/90S for 30 rounds was repeated afterward. The PCR products were denatured and hybridized with a digoxigenin-labeled telomeric repeat-specific probe. The PCR products were visualized using peroxidase enzyme conjugated antibodies against digoxigenin and the optical density was measured at 450 nm after processing the plates. Preparation of negative controls was done by treating the cells with high temperature (85°C, 10 min) or by RNase before processing the samples. The calculation of the relative telomerase activity was performed by dividing the optical density of the tests to the control samples.
Two-tail student t test and two-way analysis of variance (ANOVA) test were used to determine the significance of differences between the experimental conditions. A P value of 0.05 was considered statistically significant.
| > Results|| |
Long-term T cell expansion in response to aAPCs
To activate lymphocytes, PBMCs were grown in coculture with A549 surrogate Ag presenting cells expressing either CD80 or 4-1BBL or the combination of both molecules as the dual costimulation. For the long-term cultures, cells were re-stimulated every week. Viable cells were counted at different time points by trypan blue dye exclusion or harvested for different downstream assays. [Figure 1]a shows normalized numbers of PBMCs in different culture conditions in logarithmic scale from 4 independent experiments. As it was reported earlier,  and herein, we showed again that the dual costimulation regimens had greater effect to support the growth of PBMCs in response to anti-CD3 antibody even in the absence of exogenous IL-2 in the cultures. PBMCs were not responding to anti-CD3 stimulation with CD80 costimulations after the second passage as cell numbers declined. However, presence of 4-1BBL was advantageous for greater cell growth compared with CD80 costimulation. Continuation of the cultures up to 35 days, with weekly re-stimulation, resulted in the expansion of the cells up to 7 and 41 fold of the starting number with 4-1BBL and CD80 + 4-1BBL costimulation, respectively. The magnitudes of the growths at that time were 14 and 82 fold greater than CD80 costimulated cultures (P < 0.02 and P < 0.008, respectively). Transfer of growth arrested B7 costimulated cells to the wells contained fresh aAPCs with 4-1BBL or CD80 + 4-1BBL resulted in revival of their growth after re-stimulation with anti-CD3. Cell counts of the cultures showed significant increase mainly after the second round of the re-stimulation in switched condition (day 14 post-transfer, P < 0.05). In this way, cell counts were 41 and 53-fold higher than the number of cells in CD80 costimulated cells in 4-1BBL and the dual-costimulated cultures, respectively [Figure 1]b.
|Figure 1: Enumeration of lymphocytes in longitudinal cultures with different costimulation (a) Lymphocytes co-cultured with aAPCs expressing single or dual costimulation (of CD80 and 4-1BBL) or GFP as control and stimulated with anti-CD3 antibody. Cells were counted at different time points while subcultured by weekly basis. Dual costimulation resulted in much higher cell expansion (more than 40 fold, P < 0.02); however, CD80 costimulated cells failed to proliferate after the second subculture. 4-1BBL costimulation resulted in delayed but long-lasting proliferation of lymphocytes. (b) When fractions of cells from CD80 costimulated cultures were transferred to fresh cultures with 4-1BBL or dual costimulation, the growth-arrested cells restart the growth and increase in number (P < 0.05)|
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IFN-γ production by costimulated cells
As a sign of effector function of the costimulated T cells, IFN-γ production was measured in the culture supernatants, and intracellularly by enzyme-linked immunosorbent assay (ELISA) and flow cytometry analysis, respectively. [Figure 2]a and b show the mean and individual concentrations of IFN-γ in the culture supernatant of PBMCs from seven lab donors in day 3 and day 7 of the cultures, respectively. Only small amounts of IFN-γ were accumulated in the GFP control cultures. In the cultures costimulated with CD80, IFN-γ secretion averaged 61 ng/mL and 72 ng/mL after three and seven days, respectively. In the cultures costimulated with 4-1BBL, however, the mean concentration of IFN-γ reached to more than 60 ng/mL after three days and 87 ng/mL after 7 days, which was significantly higher than CD80 costimulated cultures on day 7 (P < 0.04). Dual costimulation of the lymphocytes resulted in an enhanced production of IFN-γ in both time points compared with single costimulations (P < 0.03). In this way, there were 85 ng/mL and 118 ng/mL IFN-γ on day 3 and day 7, respectively.
|Figure 2: IFN-γ production by PBMCs in co-culture with aAPCs expressing different costimulation PBMCs were co-cultured with aAPCs and stimulated with anti-CD3 as described in 'Materials and Methods'. Culture supernatants were harvested on day 3 (a) and day 7 (b) and IFN-γ cytokine content was measured by ELISA. More IFN- γwas accumulated in the cultures with 4-1BBL costimulation (day 7, P < 0.04) or dual costimulation (day 3 and day 7, P < 0.03)|
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Results of intracellular staining for IFN-γ are shown in [Figure 3]. [Figure 3]a depicts typical flow cytometry plots out of seven independent experiments for CD4 + and CD8 + T cells with IFN-γ production after over-night re-stimulation. [Figure 3]b-e summarize data for two-time points of day 7 and day 14 of seven independent experiments. As shown, anti-CD3 re-stimulation of GFP control cultures led to the lowest numbers of IFN-γ positive cells in both cell populations and time points. On the seventh day of the culture [Figure 3]b and c, the mean frequencies of CD4 + IFN-γ+ and CD8 + IFN-γ+ T cells in the 4-1BBL costimulated cultures were about five-fold (6.4% vs 1.2%) (P < 0.0003) and two-fold (5.5% vs 3%) (P < 0.03), higher compared with the cultures of CD80 costimulation, respectively. The mean frequencies of IFN-γ producing CD4 and CD8 T lymphocytes in the cultures with dual costimulation increased up to 9%, which was about 7 and 3 times greater than CD80 costimulation alone, respectively (P < 0.002). On day 14, post-culture [Figure 3]d and e], the overall IFN-γ response of the CD4 and CD8 lymphocytes were weaker than responses on day 7 and the frequency of IFN-γ+ cells decreased in all cultures. However, cultures costimulated with 4-1BBL alone awarded the highest proportions of IFN-γ+ cells, which reached up to 2.5% and 2% among CD4 and CD8 cells of whole PBMC, respectively (P < 0.01). Surprisingly, in dual-costimulated cultures, which demonstrated the greatest cell proliferation, lower proportion of the cells revealed positive for intracellular IFN-γ production (P < 0.4) [Figure 3]d and e.
|Figure 3: Intracellular staining for IFN-γ in PBMCs co-cultured with different costimulation PBMCs were co-cultured with aAPCs as described in 'Materials and Methods' and the presence of intracellular IFN-γ was assessed by flow cytometry. (a) Typical flow cytometry result of cells stained on day 7 from (a) SEB-activated control; (b) GFP control; (c) CD80 costimulated; (d) 4-1BBL costimulated; (e) dual costimulated cultures. (b and d) The frequency of CD4+IFN-γ+ cells on day 7 and 14, respectively; (c and e) The frequency of CD8+IFN-γ+ cells on day 7 and 14, respectively. Dual costimulation of CD80+4-1BBL supported more cells for cytokine production on day 7 (P < 0); however, more cells stained positive for IFN-γ on day 14 in cultures costimulated with 4-1BBL. Results from 7 independent experiments.|
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Effect of different costimulation on telomerase activity in PBMCs
Telomerase activity in the cultured lymphocytes with CD80 and/or 4-1BBL costimulations was assessed at different time points by PCR ELISA method. The mean telomerase enzyme activities in PBMCs from four independent experiments are presented in [Figure 4]. As it is shown, on day three post-culture, lymphocytes in GFP control cultures had least telomerase induction. At the same time, CD80 costimulation resulted in 6-fold increase in the telomerase activity, compared with the lymphocytes in the GFP control culture (P < 0.003), although there was a large variation among the samples. 4-1BBL costimulated lymphocytes at the same time point showed a moderate (up to 4-fold increase) telomerase activity (P < 0.05). Combination of both costimulations caused greater reactivity of telomerase in the lymphocytes with 20-fold increase (P < 0.001). On day 7, in spite of significant growth in the costimulated cells, the overall telomerase activity in all cultures declined significantly. At this time point, dual costimulated cells showed more variation for telomerase activity where the mean reactivity reached to 5 folds higher than the controls. The enzyme activity in the rest of the culture conditions on day 7 was not statistically different (P > 0.05). For the longitudinal study, equal numbers of the cultured cells subcultured on day 7 and then day 14 and re-stimulated with the corresponding treatments. The telomerase activity was assessed again on day 16 of the experiments. Compared with GFP controls, telomerase activity in 4-1BBL costimulated cells demonstrated up to 8-fold increase (P = 0.01) though single costimulation with CD80 did not cause considerable increase in the enzyme activity at this time point. Dual costimulation (of CD80 plus 4-1BBL) only increased the telomerase activity 3-fold higher than controls (P > 0.05). Surprisingly, at this point, cells treated with dual costimulation still were showing rapid proliferation.
|Figure 4: Telomerase activity in cultured lymphocytes with different costimulation (a) PBMCs were cultured and maintained as described in the 'Materials and Methods'. Telomerase activity was assessed by PCR ELISA methods at day 3, 7 and every two days after re-stimulation. Data were compared with the GFP-control cultures. Telomerase was induced in the cultures with CD80 and/or 4-1BBL costimulation, however, only 4-1BBL costimulation could sustain the induction of telomerase at late time points. (b) Telomerase could be re-induced in the growth- arrested cells from CD80 costimulated cultures by switching costimulation to 4-1BBL on day 21 or the third rounds of subculture.|
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To determine the effect of switching costimulation on telomerase activity, on day 21, some replicates of CD80 costimulated cells were split into three fresh culture conditions and costimulated with either 4-1BBL, CD80 + 4-1BBL or original CD80 while the rest of the cultures kept growing. Analyses of the telomerase activity of the cells on day 23 (or 4 days post transfer) showed similar patterns of greater enzyme activity in 4-1BBL costimulated cells even higher than dual costimulation (9.6 fold vs 5.7 fold, respectively, compared with GFP control). Switching to 4-1BBL or dual treatment resulted in reviving the enzyme activity (3 fold and 2.2 fold, respectively, higher than the CD80 costimulated cells). Although the change was marginally significant, keeping the cells with CD80 costimulation was not able to mobilize the enzyme activity at all (P < 0.07). Next round of the re-stimulation was done on day 28, and the telomerase activity was determined on day 35 (equivalent to day 14 post-transfer). Although telomerase activity declined in the cultured cells, 4-1BBL costimulation kept highest induction for the enzymatic activity and stayed above the dual and CD80 costimulations (4 fold and 2 fold, respectively, P < 0.05).
| > Discussion|| |
In previous studies, we  and others , showed significant co-signal activities of CD137 of the TNFR family during Ag or mitogen stimulation of T cells. In addition to that we were able to demonstrate the capability of 4-1BB to awake T cells' proliferation after they became unresponsive to further anti-CD3 stimulation in conjunction with CD80 costimulation.  Here, in a similar model of study, we used recombinant adenoviruses and expressed natural ligands for 4-1BB and CD28, i.e. 4-1BBL and CD80, respectively, and investigated whether such T cell reactivation could be in association with changes in telomerase activity. In this model, aAPCs with overexpression of 4-1BBL boosted T cell proliferation, activation and increased IFN-γ cytokine production by the cultured cells even after two weeks long anti-CD3 stimulation and supported high volumes of cell proliferation. Moreover, it could induce telomerase reactivation even further than the induction by CD80/CD28 signal in long term. Literature shows that CD28 engagement is indispensable for full T cell activation during Ag stimulation (known as signal 2 and signal 1, respectively).  In addition to that CD28-CD80 interaction is the initial cellular contact to provide signal for telomerase re-expression in lymphocytes. , Similarly, in our study, all cultures including CD80 costimulated cells showed the upregulation of telomerase activity when assessed three days post anti-CD3 stimulation. The order of telomerase activity (dual costimulation > CD80 > 4-1BBL) was in line with literature and with the magnitude of cells proliferation. In spite of that the overall enzyme activity declined significantly when cells were tested again on the next time points. Literature shows that both CD4 and CD8 T cells lose the expression of CD28 after activation. , In fact, there is an accumulation of Ag activated CD28 - T cell population in the immune system throughout aging, chronic infections or during longitudinal in vitro T cell activation.  Direct experimental evidence shows that CD8 - CD28 - cells were not able to upregulate telomerase after anti-CD3 activation and CD28 ligand costimulation.  Parish et al., could temporarily enhance telomerase expression and delayed the process of cell senescence by sustained expression of CD28 via gene transduction into CD28 - cells.  However, beyond CD28, there is a considerable redundancy in costimulatory receptors for the activation of differentiated T cells, such as members of TNFR family. In our cultures, presence of the natural ligand to 4-1BB could re-induce the expression of telomerase even after 3 weeks culture when telomerase activity in the lymphocytes with either CD80 or CD80 + 4-1BBL costimulation was not considerable. Such cells were proliferative, as we showed their growth. In a study by Plunkett et al., signal transduction to T cells through chimeric receptors contained signaling domains of CD137 and some other receptors in tandem with CD3 zeta chain (not through interaction with natural ligands) were able to upregulate telomerase activity at least in some subsets of CD28 - T cells. 
We could not explain the poor reactivation of telomerase in the growing cells from dual costimulated cultures while they were rapidly growing. Whether the low level of telomerase activity detected in those cells demonstrates the overall downregulation of telomerase activity in every lymphocyte or different numbers of activated cells with high levels of enzyme activity were mixed with some non-dividing lymphocytes is not clear to us at this point. In that regard, it has been reported that the rapid cell proliferation in some conditions can force cells for premature cease of the telomerase activity and brings the ultimate fate of cellular senescence in the lymphocytes. 
We also showed that telomerase reactivation could happen in non-replicative cells from CD80 costimulated condition after transferred to the cultures with 4-1BBL costimulation on day 21 post-culture. Such activation could be the basis of the resume of cell proliferation in those cells as number of the cells grew after a week culture. These results showed that providing appropriate signal, like 4-1BB, can reactivate telomerase and other key controlling elements for its expression in cells that appear to be unreplicative or senescent. Literature shows that in a secondary immune response, where central memory T cells should differentiate into rapidly dividing effector cells, 4-1BB costimulation plays crucial roles. , Our findings here showed more unique features of 4-1BB costimulatory pathway for activation of T cells and highlighted its potentials to fill the gaps of CD28 costimulation for in/ex vivo expansion of T cells in immunotherapy approaches. In the conditions, where CD28 - tumor specific T cells should establish an effective immune response, further help from 4-1BBL may provide the required signals to revive the reaction. Nowadays, different methodologies are in use for ex vivo expansion of T cells and it seems that recapitulating the biological interactions during T cell activation by providing costimulatory contacts through multiple ligand receptors, not just CD28 interaction, could be more beneficial. Several molecules have been shown to costimulate immune system; however, selection of right combination needs more investigations to be done.
| > Acknowledgment|| |
This work was supported by grant from deputy of research of Shiraz University of Medical Sciences, Shiraz-Iran.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]