Virus-Specific T Cells: Promising Adoptive T Cell Therapy Against Infectious Diseases Following Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HSCT) is a life-saving therapy for various hematologic disorders. Due to the bone marrow suppression and its long recovery period, secondary infections, like cytomegalovirus (CMV), Epstein-Bar virus (EBV), and adenovirus (AdV), are the leading causes of morbidity and mortality in HSCT cases. Drug resistance to the antiviral pharmacotherapies makes researchers develop adoptive T cell therapies like virus-specific T cell therapy. These studies have faced major challenges such as finding the most effective T cell expansion methods, isolating the expected subtype, defining the functionality of the end-cell population, product quality control, and clinical complications after the injection. This review discusses the viral infections after HSCT, T cells characteristics during chronic viral infection, application of virus-specific T cells (VSTs) for refractory infections, standard methods for producing VSTs and their limitation, clinical experiences on VSTs, focusing on outcomes and side effects that can be helpful in decision-making for patients and further researches.


T cells in chronic viral infection
Several factors can result in T cell exhaustion, like permanent antigen exposure or reduced crosspresentation to CD4 + T cells. 15Continuous exposure to antigens affects cell function, and antigen over-exposure or increased viral load causes more severe T cell exhaustion.Antigen exposure must last at least two weeks to one month to cause T cell exhaustion.Some other factors can also stimulate the cell exhaustion, including inhibitory receptors such as programmed cell death protein 1 (PD1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T cell immunoglobulin and mucin domain 3 (TIM3), lymphocyte activation gene 3 protein (LAG3), 16 and soluble molecules such as interleukin (IL)-10 (released by tumor cells) and transforming growth factor ꞵ (TGF-ꞵ). 17egulatory T cells, an important source for IL-10 and TGF-ꞵ, also serve a key role in T cell exhaustion. 18herefore, T cell exhaustion can be eliminated by reducing regulatory T-cells and blocking inhibitory receptors.T-cell exhaustion can also be caused by infections, especially sepsis, resulting in cytokine storms. 19lthough cell exhaustion in cancer cases can be relieved by blocking the inhibitory receptors (such as PD-1 and CTLA4) and chemotherapy, this method cannot be considered effective for patients with recurrent CMV infections; since the cause of infection cannot be eliminated, and it is not possible to use blocking agents for a lifetime.Thus, it can be concluded that adoptive T cell therapy can be considered a promising approach to control recurrent viral infections in patients who underwent allo-HSCT (Figure 1).

Common methods for producing VSTs
Over the past 20 years, using VSTs has been significantly increased to reduce the uncontrolled proliferation of viruses in HSCT patients or other immune-suppressed patients.Numerous in vitro studies have been performed to reach an optimal condition and method for inducing T cell proliferation and selecting VSTs for clinical use. 20n the first immunotherapy studies using VSTs, T cells were cultured with CMV lysates and CMV-infected fibroblasts, requiring cleanroom, quality control, and quality assurance to provide good manufacturing practice (GMP) grade production.Nowadays, one of the standard methods applied for VSTs production is using tetramers to select and isolate specific T cells from the patient's entire T population. 21The main advantage of this method is the simplicity of the cell selection process.Besides, it does not need antigen-presenting cells (APCs), exogenous cytokines, or ex vivo manipulations.Additionally, since this process is performed using closed-system devices, there is no need for cleanroom and GMP equipment.However, this method only selects the T cells possessing a specific epitope for one type of HLA.Thus, it is only applicable to HLA-matched donors.Sometimes focusing on viral reactions with a particular epitope can lead to antigen escape, as observed in EBV cases. 22nother method for VST isolation is based on immunomagnetic cell sorting, which is specified for isolating interferon-gamma (IFN-γ) secreting T cells created via culturing T cells with viral peptides. 23In addition to the fast proliferation, which is the most significant advantage, this method does not require much manipulation.This method is superior to previous ones because it can cover all viruses and antigens based on the stimulation. 20timulating peripheral blood mononuclear cells (PBMCs) by APCs is another technique to produce GMP-grade VSTs.In 1990, this method was developed to produce EBV-specific T cells.It is initiated via stimulating CD8 + T cells with EBV 24 and followed by coculture with dendritic cells (DCs) transduced by AdV vectors specific for CMV and EBV. 25 Cytotoxic T cells (CTLs) generated by this method can enable T cells to detect all three viruses, including CMV (engineered from adenovirus vectors), EBV (from lymphoblastoid cell lines (LCLs)), and adenovirus (from adenovirus vectors).All the processes can be performed in a culture medium with a low blood amount (50-60 mL).This method is very time-consuming and takes long, even three months.Besides, it requires very expensive clinical viral vectors.In order to replace the viral vectors, nucleofected DCs with DNA plasmids of different viruses were used to create multi-specific VSTs.Thus, the CTLs were ready to be used after a single stimulation. 26espite the mentioned improvements, none of these methods can generate VSTs from seropositive donors, which is considered a major limitation.Because one of the greatest risks in viral infections is the absence of memory T cells in transplanted cells (like cells derived from the umbilical cord blood (UCB) sources or seropositive donors), leading to recipient infection infected with the pathogens. 27In this regard, numerous studies have tried to isolate and proliferate naive T cells from the UCB to solve this problem. 28Proliferating UCB-derived T cells using the G-Rex gas permeable device 29 to sufficient amounts for clinical applications indicates the possibility of using a system that has not experienced viral conditions to produce VSTs. 30Clinical use of T cells to treat or prevent post-HSCT viral infections is limited by factors such as the time-consuming and complicated nature of the cell production process.Therefore, few immunotherapy centers can provide such services.Many different groups have conducted studies to overcome these limitations.
Tetramer-based isolation is an up-and-coming method.However, it is costly for routine clinical applications 31 (Figure 2).

Different stimulation ways for T cell expansion
The main strategy to proliferate T cells performs using magnetic beads with immobilized monoclonal antibodies to CD3 32 or CD3 and CD28, 33 along with cytokines.T cells in different maturation states and memory T cells show different proliferation and activation abilities; for instance, memory T cells have more proliferation potential than naive T cells.In addition, the proliferative potential of memory T cell subsets differs from central memory T cells, which show the highest proliferative rate.While T cell stimulation with the CD3 receptor (T-cell receptor) serves an important part in the differentiation fate of T cells, 34 hemostatic cytokines are involved in proliferation, differentiation, and viability of T cells in vivo 35 and in vitro. 36L-2 is an essential growth factor in T cell proliferation, used at concentrations of 20-1800 IU/mL. 37IL-7 plays a crucial part in the T cell viability and antigen-dependent proliferation of Naïve T cells. 38IL-15 is involved in the proliferation rate of CD8 + 35 and CD4 + memory cells in the absence of IL-7. 39IL-15 shares many biological features with IL-2, 40 like stimulating the differentiation and proliferation of central memory T cells to effector memory T cells. 34A variety of studies that used IL-2 as a proliferation agent showed favorable results.Thus, IL-2 is considered the gold standard for stimulating T cell proliferation in most clinical trials (Table 1).

Using VSTs in post-HSCT recurrent viral infections
HSCT can be considered one of the best treatments for malignant blood disorders.In selecting an appropriate donor, reducing the number of cytotoxic T cells should be considered, as it can reduce the risk of graft versus host disease (GVHD).GVHD prevention strategy introduces other hematopoietic stem cell sources, like haploidentical donors or UCBCs, which still result in infection, one of the most important reasons for transplant-related mortality. 70CMV, EBV, and AdV are the most prevalent virus pathogens in HSCT patients. 71espite the routine use of antiviral drugs, their prescription is accompanied by limitations.First, antiviral medications may suppress the immune system 72 and cause complications in chemotherapy and radiotherapy patients.Second, although clinical trials revealed the ability of the anti-CMV and anti-EBV drugs, the efficacy of anti-AdV drugs has not been reported in trials. 73ntiviral drugs, especially those applied for CMV, can cause late-onset CMV infection.As soon as antiviral therapies cease, late-onset CMV may initiate, more severe than normal, with delayed immune recovery. 74As a result, HSC transplanted patients with viral infections may need several courses of antiviral treatments that are very costly and may lead to drug resistance.Approximately 94% of CMV species resist ganciclovir due to their mutations in the UL97 gene. 75In addition, Nichols et al. reported that about one-third of allo-HSCT recipients experienced an increased viral load after antiviral therapy. 76Many patients show drug resistance to antiviral drugs due to their continuous use, leading to severe complications like liver encephalopathy. 77Therefore, adoptive immunotherapy with VSTs can be applied as one of the most attractive and creative substitutes for pharmacological antivirals.

CMV-specific T cells
The first clinical trials were implemented in the early     1990s, in which CMV-specific T cells were obtained from donors, cultured, and injected into the recipients.T cell clones derived from donors were injected into fourteen HSCT patients; no recipient showed CMV infection. 42arallel to efforts to produce CMV-specific T cells, many attempts were made to reduce the ex vivo expansion time.
Peggs et al generated VSTs by targeting IFNγ-secreting cells and managed 18 patients by pre-emptive therapy.
Their findings showed that this treatment significantly benefited patients on prophylaxis regimens, as six out of the seven patients did not experience reactive CMV.However, this method appears very effective in primary infections because nine of the eleven patients who received antiviral prophylaxis needed additional antiviral drugs later.In addition, many patients showed GVHD symptoms due to the active T cells. 20sing T-cells isolated by tetramers was first performed by Cobbold and colleagues.They treated nine transplant patients with CMV reactivity.Following CMV-specific T cells prescription, eight patients were treated, and two developed GVHD. 21These findings were supported by other clinical trials and confirmed that VST is a safe therapeutic strategy and can solve many limitations of antiviral drugs. 78Qasim et al treated adenoviremia in HSCT pediatric patients by isolating IFN-γ-secreting T cells.However, a third-party donor was required for two out of five patients. 51V-specific T cells Feuchtinger et al first reported using AdV-specific T cells for HSCT patients with resistant infection.AdV-specific T cells were generated based on isolating IFN-γ -secreting T cells and proliferating by IL-2 and feeder cell stimulation.According to their results, AdV disappeared in five cases, and GVHD was seen in one case.79

EBV-specific T cells
Due to APCs and LCLs delivering viral antigens to T cells, the conditions in which VSTs are obtained by LCLs and APCs can be critical.EBV-specific T cells were firstly established in 1996 and 1998. 48In a multi-institutional study in 2009, 114 HSCT patients were candidates for adoptive EBV-specific T cell therapy.A total of 101 patients received EBV-specific CTLs, and none of them (either prophylaxis or preemptive therapy) showed recurrent PTLD or de novo GVHD symptoms. 80MSKCC group treated 47 HSCT patients with EBV-specific CTLs derived from an HSCT donor or a third-party donor.They reported an overall response of 68% and no GVHD symptoms. 56Other studies with fewer patients were performed and admitted the potency and safety of the EBV-specific T cells obtained by LCLs. 81,82lti-virus-specific T cells MVSTs target the most common post-HSCT viruses.In order to enhance the specificity of CTLs against viruses and target more infections, different studies were conducted to target specific viruses, like targeting EBV and AdV, 80 CMV and AdV, 37 and CMV, AdV, and EBV. 25 HSCT recipients can also be infected with other viruses, including BK, human herpesvirus 6 (HHV6), influenza, parainfluenza, coronavirus, and respiratory syncytial virus, causing morbidity and mortality.In order to expand VSTs for other viruses, a group of scientists developed a way to create polyclonal T cells (CD4 + , CD8 + ) for different viruses, including Elizabethkingia, CMV, Adv, BK, HHV6, respiratory syncytial virus, and influenza, to face against other post-HSCT viral risk factors. 83VSTs were also used in the clinic.The most critical inclusion criterium in all clinical trials is donor seropositivity.Clinical experiments in T-cell immunotherapy with seropositive donors are minimal, but recently UCB-derived CTLs have been assessed in phase I clinical trials.Among the nine patients who used MVSTs against CMV, EBV, and Adv, only three represented active viral reactivation.One patient showed both active CMV and AdV infection, represented increased CMV and AdV-specific T cells and decreased CMV and AdV viruses following VSTs injections, and was successfully treated without antiviral pharmacotherapy.Two patients who experienced EBV reactivation or infection before or immediately after VSTs injection were treated without using antiviral drugs, and VSTs were detectable in their peripheral blood. 84

Third party donor's T cells
Although the frequency of refractory infections is relatively low in HSCT recipients, which makes it unreasonable to prepare VSTs for all at-risk patients, the aggressive character of the viral pathogens demands immediate availability to VSTs in antiviral therapy non-responder patients.Waiting 8-to 10-week to generate VSTs is too long for patients who developed an infection.This obstacle can be dominated by off-the-shelf third-party T cells.Production and storage of HLA-matched VSTs from third-party donors (bio-banking) could be a promising approach for adoptive VSTs therapy for patients with no HLA-matched donor.
Previous studies in this regard are summarised in Table 1.First, Haque et al. applied EBV-specific T cells to 33 HSCT or solid tumors surgery patients to treat their PTLD.They reported a total response of 64% and 52% in five weeks and six months, respectively.No evidence of GVHD or rejection was observed.Best results were obtained when patients received HLA-matched transplants. 45In 2011, Qasim et al. conducted a clinical trial on AdV-infected HSCT patients and applied VSTs obtained from thirdparty donors using isolating IFN-γ secretory cell method.They reported an increased risk of alloreactive T cells and GVHD. 85The Memorial Sloan-Kettering group evaluated the efficacy and safety of this method by comparing EBVspecific T cells and donor lymphocyte infusion (DLIs) in PTLD patients.Although the responsiveness was the same in both groups, GVHD incidence was higher in the DLI group recipients. 86inally, a multi-institutional study used previouslystored VSTs from a third-party donor and showed that the procedure was safe.According to their study, HLA incompatibility can lead to complications; in other words, the shared allele must identify the specific epitope of the virus.A professional team is required to isolate, store, and inject the virus-specific T cell products. 87

Conclusion
Trials using VSTs (mono-specific or multi-virus) demonstrate their ability to cure recurrent and pharmacotherapy refractory viral infections.No side effects such as GVHD were reported, even with alternative donors.Third-party donors offer a great opportunity to use the off-the-shelf product for many post-HSCT infections.It can be a promising therapeutic approach for many post-HSCT infections.Despite the limitations in generating VSTs, they have shown promising outcomes in clinical trials.

Figure 1 .
Figure 1.There are different factors causing cell exhaustion.This figure shows the effect of chronic viral exposure, inhibitory receptors, and their ligands, reduced cross-presentation with T CD4 + , and high levels of IL10 and TGFβ in T cells' environment.

Figure 2 .
Figure 2. Conventional methods for producing VSTs (classical expansion, multimer selection, gamma capture, and rapid CTL generation) are illustrated in this figure.