Transgene IL-21-Engineered T Cell-Based Vaccine Potently Converts CTL Exhaustion via the Activation of the mTORC 1 Pathway in Chronic Infection

CD8 cytotoxic T lymphocyte (CTL) exhaustion is one of the major obstacles for the effectiveness of virus control in chronic infectious diseases. We previously generated novel ovalbumin (OVA)-specific 41BBL-expressing OVA-TEXO and human immunodeficiency virus (HIV-1) Gag-specific Gag-TEXO vaccines, inducing therapeutic immunity in wild-type C57BL/6 (B6) mice, and converting CTL exhaustion in recombinant OVA-specific adenovirus AdVOVA-infected B6 (AdVOVA-B6) mice with chronic infection. IL-21 cytokine plays an important role in controlling chronic infections. Therefore, in this study, we constructed recombinant transgene IL-21-expressing AdVIL-21, and generated IL-21-expressing OVA-TEXO/IL-21 and Gag-TEXO/IL21 vaccines, or control vaccines (OVA-TEXO/Null and Gag-TEXO/Null) by infecting OVA-TEXO and Gag-TEXO cells with AdVIL-21 or the control AdVNull, lacking transgene, and assessed their effects in B6 or AdVOVA-B6 mice. We demonstrate that both OVA-TEXO/IL-21 and control OVA-TEXO/Null vaccines are capable of converting CTL exhaustion in chronic infection. However, the OVA-TEXO/IL-21 vaccine more efficiently rescues exhausted CTLs by increasing stronger CTL proliferation and effector cytokine IFN-γ expression than the control OVA-TEXO/Null vaccine in AdVOVA-B6 mice with chronic infection, though both vaccines stimulated comparable OVA-specific CTL responses and protective immunity *Xu and Zhang made the same contribution to the work. How to cite this paper: Xu, A.Z., Zhang, X.Y., Chibbar, R., Freywald, A., Tikoo, S., Zheng, C.Y. and Xiang, J. (2019) Transgene IL-21-Engineered T Cell-Based Vaccine Potently Converts CTL Exhaustion via the Activation of the mTORC1 Pathway in Chronic Infection. World Journal of Vaccines, 9, 1-21. https://doi.org/10.4236/wjv.2019.91001 Received: November 6, 2018 Accepted: December 22, 2018 Published: December 25, 2018 Copyright © 2019 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
During acute viral infections, both innate and adaptive immunity work together contribute to the clearance of the pathogens [1].Many acute infections stimulate massive CD8 + cytotoxic T lymphocyte (CTL) responses that play an important role in controlling invading viruses and these responses are divided into three phases: expansion, contraction and memory [1].In the expansion phase, the infectious pathogen triggers proliferation of effector CTLs cytolytic to virus-infected cells.This is followed by the contraction phase, where 90% -95% of effector CTLs die of apoptosis induced by cytolytic granzyme-B (GB)-mediated lethal hit, which is produced by regulatory T (Treg) cells [2].Typically, only 5% -10% of the expanded ensemble of CTLs survive and proceed in the final stage to constitute the long-term memory CTLs capable of turning on rapid responses, when re-encounting the same pathogen [1].
Severe CTL exhaustion often strongly correlates with high viral loads [12].It has been found that longer duration of the chronic infection or severe loss of CD4 + T cell help often leads to more serious CTL exhaustion [1] [13], and the final stage of CTL exhaustion often results in depletion of virus-specific CTLs [8]  Consistent with this, the CTL exhaustion is one of the major reasons for ineffective HIV control in infected patients.
Mice with lymphocytic choriomeningitis virus (LCMV) infection are frequently used for investigating LCMV-specific CTL responses, since the nature of the virus makes it an excellent mouse infection model.For example, different LCMV strains can cause distinct viral infections both acute and chronic.The Armstrong strain induces an acute viral infection, whereas LCMV clone 13 infection results in viremia that can last up to 3 months with virus persisting in the brain and kidneys [10], leading to a chronic viral infection [14].Interestingly, these two strains only differ from each other in 2 amino acids, but preserve all epitopes for T-cell receptors, thus allowing us to easily compare CTL responses between dominant and subdominant LCMV viral epitopes [15] [16].In addition, the latter strain has been extensively applied to study the dynamics of CTL responses and CTL exhaustion, as well as to assess immune therapeutics for the conversion of CTL exhaustion in chronic infections [14] [17].We have recently established an adenovirus-induced chronic infection model by i.v.infection of C57BL/6 mice with a recombinant adenovirus (AdV OVA ) expressing ovalbumin (OVA).Similar to the situation in the LCMV clone 13-induced chronic infection, our mice with the AdV OVA -induced chronic infection demonstrated OVA-specific CD44 + PD-1 + LAG-3 + memory CTL (mCTL) inflation.These mCTLs were also functionally defective and exhausted [18].We also found that the PD-1 blockade efficiently converts CTL exhaustion in the OVA-specific chronic infection model [19].
IL-21 cytokine was originally found to be produced by CD4 + T cells [20] and to serve as a "third" signal, functioning in concert with T cell receptor (TCR) activation and T cell co-stimulation to trigger CD8 + T cell responses [21].Recently, it has been shown that IL-21 plays a significant role in controlling chronic LCMV infection [22] [23] [24].IL-21 enhances cytolytic and virus control abilities of HIV-specific CTLs in vitro [25] [26], and enhances the viral control in immunodeficiency virus [4]-infected rhesus macaques and HCV-and HIV-1-infected patients [7] [27] [28].
We previously developed a novel ovalbumin (OVA)-specific exosome (EXO)-targeted T cell-based (OVA-T EXO ) vaccine by using non-specific polyclonal T cells with the uptake of OVA-specific dendritic cell (DC)-released EXO via the CD54/LFA-1 interaction [29].We demonstrated that the OVA-Texo vaccine was able to directly stimulate potent OVA-specific CTL responses in the absence of CD4 + T cell help by counteracting CD4 + 25 + FoxP3 + regulatory T (Treg) cell suppression [29] [30].We also developed an HIV-1 Gag-specific T cell-based vaccine, Gag-T EXO , by using non-specific polyclonal T cells with the uptake of Gag-specific DC-released EXO and demonstrated that the Gag-T EXO vaccine triggered potent Gag-specific immunity against Gag-expressing tumors in transgenic HLA-A2 mice [31].To enhance its immunogenicity, we generated 4-1BBL-expressing OVA-T EXO and Gag-T EXO vaccines, and demonstrated that A. Z. Xu et al.
the former one triggered potent therapeutic immunity [32].It also induced an efficient conversion of CTL exhaustion via its CD40L-dependent signaling activation of the mTORC1 pathway in chronic infection models [18].
In this study, we constructed a recombinant adenovirus (AdV IL-21 ) expressing mouse IL-21 and generated new OVA-T EXO/IL-21 and Gag-T EXO/IL-21 vaccines engineered to express IL-21 by infection of the above OVA-T EXO and Gag-T EXO cells with AdV IL-21 as previously described [32].We assessed the effectiveness of the OVA-T EXO/IL-21 vaccine in the conversion of CTL exhaustion and examined the effectiveness of the Gag-T EXO/IL-21 vaccine in therapeutic immunity against Gag-expressing tumors in chronic infection model.We found that the OVA-T EXO/IL-21 vaccination stronger rescued CTL exhaustion than the OVA-T EXO vaccine in chronic infection, while Gag-T EXO/IL-21 vaccination triggered more potent therapeutic immunity against established Gag-expressing BL6-10 Gag tumor lung metastases, when compared to the Gag-T EXO vaccine in chronic infection.

Preparation of Dendritic Cells and Dendritic Cell-Released Exosomes
Bone marrow-derived dendritic cells (DCs) were obtained by culturing bone marrow cells of wild-type (WT) B6 mice in culture medium containing granulocyte monocyte colony-stimulating factor (GM-CSF) (20 ng/ml) and IL-4 (20 ng/ml) for six days, as previously described [29].DCs were pulsed with OVA (0.5 mg/ml) for overnight and termed DC OVA .DC OVA stained with various Abs were analysed by flow cytometry.DC OVA -released exosomes (EXO OVA ) were then purified from DC OVA culture supernatants by differential ultracentrifugation [29].
In addition, DCs were also infected with AdV Gag (100 pfu/cell) for overnight in a humidified 37˚C, 5% CO 2 incubator, and termed DC Gag .DC Gag -released EXOs were purified from DC Gag culture supernatants by ultracentrifugation and termed EXO Gag .

Preparation of the OVA-TEXO/IL-21 and Gag-TEXO/IL-21 Vaccines and the Control OVA-TEXO/Null and Gag-TEXO/Null Vaccines
The OVA-T EXO and Gag-T EXO vaccines were generated by the incubation of CD8 + ConA-T cells with EXO OVA or EXO Gag at 3 × 10 6 cells/10 µg for 1 hour at 37˚C, followed by the transfection of T cells with the uptake of EXO OVA or EX-O Gag with AdV 41BBL at 100 pfu/cell for another 2 hours to form the vaccines, as previously described [32].

Establishment of AdVOVA-Induced Chronic Infection Animal Model
To develop a chronic infection model, B6 mice (4/group) were intravenously (i.v.) injected with anti-CD4 Ab (300 µg/mouse) to deplete CD4 + T cells, followed by i.v.infection of mice with AdV OVA (1 × 10 6 pfu/mouse) one day after the anti-CD4 Ab treatment performed for CD4 + T cell depletion.This experimental procedure was aimed to develop a more stringent mouse model of chronic infection, since AdV OVA infection of mice was performed under the condition of CD4 + T cell depletion such that the 'helpless' CD8 + T cells activated by AdV OVA infection became functionally exhausted with severe defects in IFN-γ production and cytotoxicity [36].These mice were chronically AdV OVA -infected B6 (AdV OVA -B6) mice and termed AdV OVA -B6 mice with chronic infection.

Flow Cytometric Analysis
Peripheral blood samples derived from AdV OVA -B6 and rLmOVA-B6 mice were

Western Blot Analysis
EXO (10 µg/well) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred onto the nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA).The membrane was blocked with 5% bovine serum albumin (BSA) in PBS, immunoblotted with rabbit anti-CD9 or anti-LAMP-1 Ab, followed by the incubation with anti-rabbit IRDyeR800CW Ab, and then, scanning with the ODYSSEY imager according to manufacturer's instruction (Li-COR Bioscience, Lincoln, NE).

Electron Microscopy
EXO were fixed in 4% paraformaldehyde.The pellets were loaded onto carbon-coated formvar grids.The exosome sample-loaded grids were stained with saturated aqueous uranyl and then, examined with a JEOL 1200EX electron microscope at 60 kV.

Animal Studies
To cells/mouse).The mice were sacrificed 3 weeks after tumor cell injection, and the lung metastatic B16 melanoma colonies were counted in a blind fashion.Metastatic B16 melanoma colonies on freshly isolated lungs appeared as black color foci that were easily distinguishable from normal lung tissues.Metastasis was also confirmed by histological examination.ferent groups in flow cytometric analysis [32].Unless stated otherwise, data are expressed as mean (with SD).A value of p < 0.05 was considered statistically significant. .OVA-specific exosomes (EXO OVA ) were purified from DC OVA culture supernatants by ultracentrifugation, and analyzed by electron microscopy and Western blotting analyses, respectively.We demonstrated that EXO OVA had exosomal "saucer" or round shape with 50 -90 nm in diameter (Figure 1(b)) and contained EXO-associated proteins such as CD9 and LAMP-1 (Figure 1(c)) [37].We infected the OVA-T EXO cells [32] with AdV IL-21 to generate a transgene IL-21-engineered T cell-based vaccine, OVA-T EXO/IL-21 .IL-21 secretion in OVA-T EXO/IL-21 culture supernatants was estimated to be ~200 pg/ml using IL-21 ELISA kit.In contrast, there was no IL-21 detected in OVA-T EXO culture supernatants.

OVA-TEXO/IL-21 Vaccine Stimulates OVA-Specific CTL Responses in Wild-Type C57BL/6 Mice
To examine the immunogenicity, we i.v.immunized B6 mice with OVA-T EXO/IL-21 or the control OVA-T EXO/Null vaccine.We demonstrated that both OVA-T EXO/IL-21

OVA-Expressing Adenovirus Induces an OVA-Specific Chronic Infection in Mice with CTL Exhaustion
We previously found that infection of B6 mice with AdV OVA, led to establishment of an OVA-specific chronic infection mouse model with OVA-specific CTL inflation and exhaustion [18].To develop acute and chronic infection models, B6 mice were i.v.infected with OVA-expressing recombinant rLmOVA bacteria and AdV OVA , respectively, followed by the kinetic analysis of OVA-specific CTL responses [18].We demonstrated that AdV OVA infection resulted in OVA-specific memory CD8 + T cell inflation, when compared to CTLs developed in rLmO-VA-immunized mice (Figure 3(a)), suggesting that rLmOVA induces an acute infection, while AdV OVA induces a chronic infection with CD8 + CTL inflation

The OVA-TEXO/IL-21 Vaccine Converts CTL Exhaustion in Chronic Infection
We examined in our next set of experiments, whether the OVA-T EXO/IL-21 vaccine converts CTL exhaustion in chronic infection.The AdV OVA -B6 mice were boosted with OVA-T EXO/IL-21 and the control OVA-T EXO/Null vaccine.Four days after the boost, cell proliferation and intracellular IFN-γ expression of OVA-specific CTLs were assessed by flow cytometry.We demonstrated that there were ~6-fold (5.20% vs 0.81%) of CTL increase in OVA-T EXO/IL-21 -boosted mouse peripheral blood, which is more than ~3-fold of CTL increase in the control OVA-T EXO/Null -boosted mouse peripheral blood at day 4 after the boost (Figure 4(a)), indicating that OVA-T EXO/IL-21 vaccine can more potently convert CTL exhaustion by significantly stimulating the proliferation of previously exhausted CTLs.We next assessed expression of an effector T cell cytokine, IFN-γ, in exhausted CTLs on a "per-cell" basis by intracellular staining of T cell IFN-γ.We demonstrated that only ~20% of OVA-specific CTLs were IFN-γ positive in AdV OVA -B6 mice (Figure 4(b)).In OVA-T EXO/Null -and OVA-T EXO/IL-21 -boosted AdV OVA -B6 mice, however, we found ~60% and ~75% of IFN-γ-producing CTLs in the total OVA-specific CTLs, respectively (Figure 4(b)), confirming that OVA-T EXO/IL-21 vaccine more potently converts CTL exhaustion in chronic infection by not only increasing the number of CTLs, but also restoring CTL functional effect.

OVA-Texo Converts CTL Exhaustion through the Activation of the mTORC1 Pathway
We previously showed that the OVA-T EXO vaccine rescued exhausted CTLs in chronic infection via its CD40L signaling, inducing the activation of the mTORC1 pathway [18].Four days after the boost, cell phenotypes of the OVA-specific CTLs were assessed by flow cytometry to examine, whether the OVA-T EXO/IL-21 vaccine activates the mTORC1 pathway.We analyzed CTLs for the phosphorylation status of mTORC1-regulated EIF4E (pEIF4E) and for the intracellular expression of the transcription factor T-bet by flow cytometry.We determined that OVA-specific CTLs up-regulated levels of phospho-EIF4E and T-bet in OVA-T EXO/Null -boosted AdV OVA -B6 mice (Figure 4(a)), which was consistent with our previous report [18].Interestingly, the intracellular expression of pEIF4E and T-bet in OVA-specific CTLs was significantly higher in the OVA-T EXO/IL-21 -boosted AdV OVA -B6 mice than that in the OVA-T EXO/Null -boosted AdV OVA -B6 mice (pEIF4E, p = 0.02; T-bet, p = 0.001) (Figure 4  that OVA-T EXO/IL-21 vaccine converts CTL exhaustion mostly through the activation of the mTORC1 pathway by both CD40L and IL-21 signaling.

The Gag-TEXO/IL-21 Vaccine Induces Gag-Specific Therapeutic Immunity in Chronic Infection Model
To assess a potential therapeutic immunity of the Gag-T EXO/IL-21 vaccine in chronic infection, AdV OVA -B6 mice with chronic infection of AdV OVA were first i.v.injected with Gag-expressing BL6-10 Gag melanoma cells, and four days post

Discussion
The inhibitory PD-1 molecule was originally found on active T cells following cell functions through inhibiting TCR signaling by recruiting phosphatases [40] [41] and modulating the mTORC1 pathway responsible for T cell proliferation and effector function [38] [39].Later, expression of other inhibitory molecules such as LAG-3 and TIM-3 has also been found on exhausted CTLs [42].It has been demonstrated that PD-1 blockade rescued exhausted CTLs in chronic viral infection [17].An enhanced effect on conversion of CTL exhaustion was observed upon combination treatments with antagonists of various inhibitory molecules, such as PD-1, LAG-3 and Tim-3 [42] [43].In addition to blockade therapies, costimulatory signaling, such as 41BB-, OX40-and CD27-mediated costimulation also synergized with PD-L1 blockade by forcing exhausted CTLs to exit quiescence [4] [44] [45].Moreover, costimulating CD40L signaling has been shown to assure T cell activation by recruiting tumor necrosis factor (TNF) receptor-associated factor (TRAF), which leads to the activation of the mTORC1 pathway [46].Consistent with this, it has recently been demonstrated that CD40 agonist enhances PD-1 blockade's effect in rescuing exhausted CTLs in chronic infection [19].
IL-21 belongs to the common γ-chain cytokine family and is closely related to another family member, IL-2, which is encoded upstream of IL-21 on chromosome 3 [47].IL-21 binds to a heterodimeric receptor CD123 encoded on chromosome 7 [48], which is widely expressed by B cells, natural killer cells, DCs, macrophages and T cells [47] [48].The IL-21 cytokine was originally found to be produced by CD4 + T cells [20] and to serve as a "third" signal, functioning in We previously developed a novel OVA-specific EXO-targeted T cell-based OVA-T EXO vaccine by using non-specific polyclonal T cells with the uptake of OVA-specific DC-released EXO [29].We found that the OVA-Texo vaccine was able to directly stimulate potent OVA-specific CTL responses by counteracting CD4 + 25 + FoxP3 + Treg-induced CTL suppression [29] [30].We also demonstrated that the 4-1BBL-expressing OVA-T EXO vaccine triggered an enhanced therapeutic immunity in WT B6 mice [32] and induced an efficient conversion of CTL exhaustion in chronic infection model via the CD40L-initiated signaling through the mTORC1 pathway [18].In this study, we generated new OVA-T  signals.Therefore, this study is likely to have a strong impact on developing new therapeutic vaccines that might be used as monotherapies or in combination with other HIV-1 blockades for treating immune deficiency syndrome patients.
To prepare the OVA-T EXO/IL-21 or Gag-T EXO/IL-21 and the control OVA-T EXO/Null or Gag-T EXO/Null vaccines, the above OVA-T EXO or Gag-T EXO cells were further transfected with AdV IL-21 or AdV Null [100 pfu/cell] for 2 hours to form OVA-T EXO/IL-21 or Gag-T EXO/IL-21 and the control OVA-T EXO/Null or Gag-T EXO/Null vaccine respectively, as we previously described [32].DOI: 10.4236/wjv.2019.91001 5 World Journal of Vaccines A. Z. Xu et al.
double stained with FITC-CD8 Ab and PE-tetramer and analyzed by flow cytometry to assess CD8 + T cell responses at different days after infection.To increase the amount of OVA-specific CD8 + memory T (Tm) cells formed 30 days or over 30 days post infection, such that phenotypes of OVA-specific CD8 + Tm cells can be more accurately analyzed, B6 mice were infected with an increased amount of AdV OVA (1.2 -1.4 × 10 6 pfu/mouse) for the development of chronic infection AdV OVA -B6 mice, while B6 mice with prior i.v.injection of naïve CD8 + T cells (1 × 10 3 /mouse) purified from OTI mouse spelnocytes were infected with rLmOVA bacteria for the development of acute infection rLmOVA-B6 mice[18].To assess the phenotype of OVA-specific CD8 + Tm cells, peripheral blood samples derived from AdV OVA -B6 and rLmOVA-B6 mice 60 days post the primary infection were triply stained with FITC-CD8 Ab, PE-tetramer and PE-Cy5-Abs for various immune molecules, and then analyzed by flow cytometry.To assess the conversion of CTL exhaustion, AdV OVA -B6 mice with chronic infection were i.v.immunized with OVA-T EXO/IL-21 or the control OVA-T EXO/Null vaccine (1 × 10 6 cells/mouse), followed by the analysis of OVA-specific CD8 + T cell proliferation and phenotypes 4 days post immunization.To stain the intracellular molecules, mouse splenocytes derived from AdV OVA -B6 and rLmO-VA-B6 mice were first incubated with FcR blocking anti-16/32 Ab (eBioscience) for 30 min on ice for eliminating any nonspecific staining.The cells were then stained with FITC-CD8 Ab and PE-tetramer, followed by the fixation and permeabilization with Cytofix/Cytoperm (BD Biosciences) according to the manufacturer's instruction.The cells were further stained with PE-Cy5-antibodies DOI: 10.4236/wjv.2019.910016 World Journal of Vaccines A. Z. Xu et al. specific for various molecules such as EIF4E and T-bet, and the expression of these molecules was assessed by flow cytometry.For intracellular staining of IFN-γ, mouse splenocytes were first incubated in culture medium containing OVAI peptide (2 µg/ml) and Golgi-stop (0.7 µg/ml) (BD Biosciences) at 37˚C for 5 hrs, followed by the incubation with FcR blocking anti-16/32 Ab for 30 min on ice.The cells were then stained with FITC-CD8 Ab and PE-tetramer, followed by fixation and permeabilization with Cytofix/Cytoperm (BD Biosciences).Intracellular staining of IFN-γ was conducted using PE-Cy5-anti-IFN-γ Ab, and the expression of IFN-γ was assessed by flow cytometry.Data were acquired by CytoFlex (Beckman Coulter) and analyzed with FlowJo software (TreeStar, San Diego, CA).

Transgene IL- 21 -
Engineered T Cell-Based OVA-TEXO/IL-21 VaccineWe constructed a replication-deficient transgene IL-21-expressing recombinant adenovirus AdV IL-21 by recombinant DNA technology as we described in MATERIALS & METHODS (Figure1(a))[18] [32].B6 mouse dendritic cells were generated by culturing B6 mouse bone marrow cells in the culture medium containing GM-CSF and IL-4 for six days, pulsed with OVA for overnight and termed DC OVA[18] [32]

[ 18 ]Figure 2 .
Figure 2. AdV IL-21 does not enhance OVA-T EXO vaccine immunity in wild-type (WT) B6 mice.(a) WT B6 mice (n = 6) were injected with OVA-T EXO/Null or OVA-T EXO/IL-21.Six days after the injection, blood samples were collected, stained with FITC-CD8 Ab and PE-tetramer, and then analyzed by flow cytometry.The percentages of tetramer + CD8 + T cells in the total CD8 + T cell population are indicated.(b) WT B6 mice were immunized with OVA-T EXO/Null or OVA-T EXO/IL-21 , followed by injection with BL 6-10/OVA tumor cells 6 day later.Mice lungs were collected at 21 days after the tumor cell injection.Metastatic tumor colonies in lung tissues were counted.(c) The lung tissues of immunized mice were fixed in 10% neutral-buffered formalin and then embedded in paraffin.Tissue sections were stained with H&E and examined by microscopy.Magnification, ×100.One representative experiment of two is shown.

Figure 3 .
Figure 3. AdVova induces chronic infection in B6 mice.(a) OVA-specific CTL responses were analyzed at the indicated days post-AdVova or -rLmOVA infection by flow cytometry (n = 4).(b) Sixty days after the infection, flow cytometric analyses were performed.PE-tetramer and FITC-CD8 double positive cells were gated (rectangle) for further assessment of the expression of the indicated molecules (solid lines on the right).Mean fluorescence intensity (MFI) values are indicated in each panel.Dotted lines represent isotype controls.(c) Splenocytes were stained with PE-tetramer and FITC-CD8 or permeabilized for the assessment of intracellular IFN-γ by flow cytometry.The value in each panel represents the percentage of CD8 + T cells producing IFN-γ in the total CD8 + T-cell population.**p < 0.01.One representative experiment of two is shown.

Figure 4 .
Figure 4. AdV IL-21 enhance the capability of OVA-T EXO vaccine in converting CTL exhaustion.AdVova-infected B6 mice (n = 5) were immunized with OVA-T EXO/Null or OVA-T EXO/IL-21 at day 60 after the infection.(a) One day prior to (day 0) and 4 days post the immunization, periphery blood samples were analyzed for OVA-specific CTL responses by flow cytometry.The value in each panel represents a percentage of PE-tetramer-positive CD8 + T cells in the total peripheral CD8 + T cell population.Four days after immunization, mouse splenocytes were stained with PE-Tetramer, FITC-CD8, and PE-Cy5-labeled Abs.The tetramer + CD8 + T cells were gated and assessed for expression of pelF4E and T-bet (solid lines).MFI values are indicated in each panel.The fold increases (%Tetramer + T cells on day 4 post vaccine/%Tetramer + T cells on day 0) of tetramer + CD8 + T cells are indicated in the graph.(b) Splenocytes were permeabilized for the assessment of intracellular IFN-γ by flow cytometry at day 4 after immunization.*p < 0.05.One representative experiment of two is shown.
B16 melanoma cell challenge, mice were i.v.immunized with the Gag-T EXO/IL-21 or the control Gag-T EXO/Null vaccine (Figure5(a)).Seventeen days later, mouse lung tissues were collected for histopathological examination (Figure5(a)).Importantly, the Gag-T EXO/IL-21 vaccine demonstrated a complete eradication of established BL6-10 Gag lung metastases in 5/6 AdV OVA -B6 mice, thus stimulating more efficient therapeutic immunity against Gag-expressing BL6-10 Gag melanoma than the control Gag-T EXO/Null vaccination (Figure 5(b)).Our data indicate that Gag-T EXO/IL-21 vaccine is capable of inducing potent therapeutic immunity against established Gag-expressing tumors in the presence of chronic infection.

Figure 5 .
Figure 5. Gag-T EXO/IL-21 vaccine stimulates enhanced antitumor immunity in chronic infection mice.(a) Experimental set-up to examine the therapeutic antitumor immunity of T EXO vaccines.Chronic AdVova-B6 mice (n = 6) were i.v.injected with BL6-10 OVA cells.Four days after tumor challenge, mice were vaccinated with Gag-T EXO/Null , Gag-T EXO/IL-21 or control ConA-T.The mice were sacrificed 3 weeks after tumor cell challenge.(b) The average number of lung metastatic tumor colonies was counted.*p < 0.05 versus cohorts of ConA-T cells.One representative experiment of three is shown.
Gag-T EXO/IL-21 vaccines, expressing IL-21, by infecting the above 41BBL-expressing OVA-T EXO and Gag-T EXO cells with AdV IL-21 , as previously described[32].We assessed the effectiveness of the OVA-T EXO/IL-21 vaccine in the conversion of CTL exhaustion and examined the effectiveness of the Gag-T EXO/IL-21 vaccine in therapeutic immunity against Gag-expressing tumor in chronic infection.We discovered that in chronic infection, the OVA-T EXO/IL-21 vaccination more strongly rescued CTL exhaustion than the OVA-T EXO/Null vaccine.In addition, we also found that OVA-T EXO/IL-21 -boosted CTLs strongly up-regulated phosphorylation of mTORC1-controlled EIF4E.OVA-T EXO/IL-21 also very effectively enhanced mTORC1-controlled expression of T-bet that regulates T cell activation.These OVA-T EXO/IL-21 -induced responses were significantly stronger than OVA-T EXOboosted ones.It has been shown that IL-21 promotes CTL survival via activation of the PI3K signaling cascade [49].Our data showing that OVA-T EXO/IL-21 vaccination more strongly rescued CTL exhaustion than the OVA-T EXO one indicates that the IL-21 signaling of OVA-T EXO/IL-21 vaccine plays an important role in conversion of CTL exhaustion via the activation of the mTORC1 pathway in chronic infection.Our data thus provide the first evidence that our novel T cell-based vaccine is capable of converting CTL exhaustion in chronic infection via its CD40L and IL-21 signaling through the mTORC1 pathway.CD8 + CTLs are important effector T cells capable of directly destroying HIV-1-infected cells, and their activity correlates with acute viral control and long-term nonprogression [56] [57] [58] [59].CD8 + CTLs play a critical role in controlling HIV-1 proliferation and disease progression even in the absence of neutralizing antibodies [60] [61].Stimulation of HIV-1-specific CTLs has been also reported to facilitate elimination of latent viral reservoirs [62][63].We originally generated HIV-1 Gag-specific Gag-T EXO vaccine by using polyclonal ConA-T cells with the uptake of Gag-specific DC-released EXO, and demonstrated that Gag-T EXO stimulated Gag-specific CTL responses in transgenic HLA-A2 mice[31].We also generated a 4-1BBL-expressing Gag-T EXO vaccine capable of triggering more efficient CTL responses and therapeutic immunity against Gag-expressing tumor challenges than the original Gag-T EXO vaccine[32].In this study, we have generated a new Gag-T EXO/IL-21 vaccine engineered to express IL-21 by infection of the 41BBL-expressing Gag-T EXO cells with AdV IL-21 as[32], and demonstrated that the Gag-T EXO/IL-21 vaccine triggered more effective therapeutic immunity against established Gag-specific BL6-10 Gag melanoma lung metastases in chronic infection.We expect that combinations of similarly designed vaccines with blockades against various inhibitory molecules, such as PD-1, TIM-3 and LAG-3, may become new startegies for combined immunotherapies to covert CTL exhaustion in chronic infections such as HIV-1.Taken together, our data demonstrate that our novel transgene IL-21-engineered T cell-based vaccine OVA-T EXO/IL-21 is capable of strongly converting the exhaustion of CD40-expressing CTLs in chronic infection via the activation of the mTORC1 pathway caused by endogenous CD40L-and transgene IL-21-triggered DOI: 10.4236/wjv.2019.9100115 World Journal of Vaccines A. Z. Xu et al.
(GraphPad Software, San Diago, CA) to compare variables of different groups in animal studies or with the Student t test to compare variables of dif- Statistical analyses were performed with the Mann-Whitney U test using Prism DOI: 10.4236/wjv.2019.910017 World Journal of Vaccines A. Z. Xu et al. software