Mesenchymal Stem Cell-Derived Exosomes Attenuate Murine Cytomegalovirus-Infected Pneumonia via NF-κB/NLRP3 Signaling Pathway.

Reactivation and infection with cytomegalovirus (CMV) are frequently observed in recipients of solid organ transplants, bone marrow transplants, and individuals with HIV infection. This presents an increasing risk of allograft rejection, opportunistic infection, graft failure, and patient mortality. Among immunocompromised hosts, interstitial pneumonia is the most critical clinical manifestation of CMV infection. Recent studies have demonstrated the potential therapeutic benefits of exosomes derived from mesenchymal stem cells (MSC-exos) in preclinical models of acute lung injury, including pneumonia, ARDS, and sepsis. However, the role of MSC-exos in the pathogenesis of infectious viral diseases, such as CMV pneumonia, remains unclear. In a mouse model of murine CMV-induced pneumonia, we observed that intravenous administration of mouse MSC (mMSC)-exos reduced lung damage, decreased the hyperinflammatory response, and shifted macrophage polarization from the M1 to the M2 phenotype. Treatment with mMSC-exos also significantly reduced the infiltration of inflammatory cells and pulmonary fibrosis. Furthermore, in vitro studies revealed that mMSC-exos reversed the hyperinflammatory phenotype of bone marrow-derived macrophages infected with murine CMV. Mechanistically, mMSC-exos treatment decreased activation of the NF-κB/NLRP3 signaling pathway both in vivo and in vitro. In summary, our findings indicate that mMSC-exo treatment is effective in severe CMV pneumonia by reducing lung inflammation and fibrosis through the NF-κB/NLRP3 signaling pathway, thus providing promising therapeutic potential for clinical CMV infection.

Adoptive transfer of CMV-specific TCR-T cells for the treatment of CMV infection after haploidentical hematopoietic stem cell transplantation.

BACKGROUND: Cytomegalovirus (CMV) reactivation after unmanipulated haploidentical stem cell transplantation (SCT) frequently occurs, causing life-threatening morbidities and transplantation failure. Pre-emptive therapy upon the detection of CMV viremia using antiviral agents is currently the standard of care but it was associated with significant toxicity. The CMV antigen-specific cytotoxic T lymphocyte therapy was limited by the time-consuming manufacture process and relatively low success rate. More effective and safer approaches for the treatment of CMV reactivation after haploidentical SCT are in urgent need. METHODS: A single-arm, open-label, phase I clinical trial evaluating the safety and efficacy of CMV-targeting T cell receptor-engineered T (CMV-TCR-T) cell therapy as the first-line pre-emptive therapy for patients with CMV reactivation after haploidentical peripheral blood SCT (PBSCT) was conducted in the Chinese PLA General Hospital. Six patients with CMV reactivation after haploidentical SCT were adoptively transferred by one to three doses of SCT donors-derived CMV-TCR-T cells. This trial was a dose-escalation study with doses ranging from 1×103 CMV-TCR-T cells/kg body weight per dose to 5×105 CMV-TCR-T cells/kg per dose. RESULTS: Except for the grade 1 cytokine release syndrome observed in one patient and mild fever in two patients, no other adverse events were observed. Four patients had response within a month after CMV-TCR-T cell infusion without the administration of any antiviral agents. The other two patients who initially did not respond to CMV-TCR-T cell therapy had salvage ganciclovir and foscarnet administration and then had rapid CMV clearance. The CMV-TCR-T cells displayed overall robust expansion and persistence in the peripheral blood after infusion. The CMV-TCR-T cells were first detected in the peripheral blood of these patients 3-7 days after the first dose of CMV-TCR-T infusion, rapidly expanded and persisted for at least 1-4 months, providing long-term protection against CMV reactivation. In one patient, the CMV-TCR-T cells started to expand even when the anti-graft-versus-host disease reagents were still being used, further indicating the proliferation potential of CMV-TCR-T cells. CONCLUSIONS: Our study first showed CMV-TCR-T cell as a highly feasible, safe and effective first-line pre-emptive treatment for CMV reactivation after haploidentical PBSCT. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT05140187).

Adoptive therapy with cytomegalovirus-specific cytotoxic T lymphocytes for refractory cytomegalovirus DNAemia and disease after allogeneic haematopoietic stem cell transplantation.

Cytomegalovirus (CMV) DNAemia and disease are common complications in patients undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT). Few studies have compared the efficacy and safety of the HSCT donor and third-party CMV-specific cytotoxic T lymphocytes (CMV-CTLs) in the treatment of CMV DNAemia and disease. In this study, we retrospectively compared the efficacy and safety of HSCT donor and third-party CMV-CTLs in patients with refractory CMV DNAemia or disease after allo-HSCT at our centre from January 2017 to September 2021. Fifty-three patients who received CMV-CTL therapy were enrolled, including 40 in the donor group and 13 in the third-party group, and they were adults aged 18 years or older. Within 6 weeks of treatment, 26 (65.0%) and 9 (69.2%) patients achieved complete response in the donor and third-party groups (p = 1.000). The 2-year overall survival was 59.6% (95% CI 46.1%-77.1%) and 53.8% (32.6%-89.1%) in the donor and third-party groups (p = 0.860). Four (10.0%) patients in the donor group and two (15.4%) patients in the third-party group developed acute graft-versus-host disease within 3 months after CMV-CTL infusions. In conclusion, our data suggest that donor and third-party CMV-CTLs have comparable efficacy and safety for refractory CMV DNAemia and disease.

Circular mRNA-based TCR-T offers a safe and effective therapeutic strategy for treatment of cytomegalovirus infection.

Circular mRNA (cmRNA) is particular useful due to its high resistance to degradation by exonucleases, resulting in greater stability and protein expression compared to linear mRNA. T cell receptor (TCR)-engineered T cells (TCR-T) represent a promising means of treating viral infections and cancer. This study aimed to evaluate the feasibility and efficacy of cmRNA in antigen-specific-TCR discovery and TCR-T therapy. Using human cytomegalovirus (CMV) pp65 antigen as a model, we found that the expansion of pp65-responsive T cells was induced more effectively by monocyte-derived dendritic cells transfected with pp65-encoding cmRNA compared with linear mRNA. Subsequently, we developed cmRNA-transduced pp65-TCR-T (cm-pp65-TCR-T) that specifically targets the CMV-pp65 epitope. Our results showed that pp65-TCR could be expressed on primary T cells for more than 7 days. Moreover, both in vitro killing and in vivo CDX models demonstrated that cm-pp65-TCR-T cells specifically and persistently kill pp65-and HLA-expressing tumor cells, significantly prolonging the survival of mice. Collectively, our results demonstrated that cmRNA can be used as a more effective technical approach for antigen-specific TCR isolation and identification, and cm-pp65-TCR-T may provide a safe, non-viral, non-integrated therapeutic approach for controlling CMV infection, particularly in patients who have undergone allogeneic hematopoietic stem cell transplantation.

Development of a highly cytotoxic, clinical-grade virus-specific T cell product for adoptive T cell therapy.

At present, recipients of allogeneic hematopoietic stem-cells are still suffering from recurrent infections after transplantation. Infusion of virus-specific T cells (VST) post-transplant reportedly fights several viruses without increasing the risk of de novo graft-versus-host disease. This study targeted cytomegalovirus (CMV) for the development of an innovative approach for generating a very specific VST product following Good Manufacturing Practices (GMP) guidelines. We used a sterile disposable compartment named the Leukoreduction System Chamber (LRS-chamber) from the apheresis platelet donation kit as the starting material, which has demonstrated high levels of T cells. Using a combination of IL-2 and IL-7 we could improve expansion of CMV-specific T cells. Moreover, by developing and establishing a new product protocol, we were able to stimulate VST proliferation and favors T cell effector memory profile. The expanded VST were enriched in a closed automated system, creating a highly pure anti-CMV product, which was pre-clinically tested for specificity in vitro and for persistence, biodistribution, and toxicity in vivo using NOD scid mice. Our results demonstrated very specific VST, able to secrete high amounts of interferon only in the presence of cells infected by the human CMV strain (AD169), and innocuous to cells partially HLA compatible without viral infection.

Case Report: Fatal cytomegalovirus pneumonia after CAR-T cell therapy in the long-term follow-up.

Introduction: The rapidly developed CAR-T cell therapy has a unique profile of side effects, which perhaps has not been totally realized and understood, especially the late-phase toxicity. CMV is prevalent world-wide and establishes a life-long latency infection. It can lead to life-threatening complications in immunocompromised host, and little is known about CMV disease in patients after CAR-T cell therapy. Here, we report a patient who developed possible CMV-pneumonia three months after anti-CD19 and anti-CD22 CAR-T cell therapy for relapsed B-ALL, contributing to the understanding of severe side-effects mediated by virus infection or reactivation in patients receiving CAR-T cell infusion. Case presentation: A 21-year old male patient with relapsed B-ALL received anti-CD19/22 CAR-T cell therapy, and achieved complete remission 2 weeks after the infusion. However, three months later, the patient was hospitalized again with a 10-day history of fever and cough and a 3-day history of palpitations and chest tightness. He was diagnosed with possible CMV pneumonia. Under treatment with antiviral medicine (ganciclovir/penciclovir), intravenous gamma globulin and methylprednisolone and the use of BiPAP ventilator, his symptoms improved, but after removing penciclovir his symptoms went out of control, and the patient died of respiratory failure 22 days after admission. Conclusion: CMV infection/reactivation can occur in patients long after receiving anti-CD19/22 CAR-T cell therapy, and induce fatal pneumonia, which reminds us of the late side effects associated with immunosuppression after CAR-T cell infusion.

A systematic literature review of the economic and healthcare resource burden of cytomegalovirus.

OBJECTIVE: Cytomegalovirus (CMV) can infect individuals at any age, including infants, who may contract it from infected mothers (congenital CMV [cCMV]). Whereas CMV infection is typically asymptomatic or causes mild illness in healthy individuals, infection can result in severe outcomes in immunocompromised individuals and in infants with cCMV. This systematic review aims to characterize the economic impact of CMV and cCMV infections. METHODS: Medline, Embase, and LILACS databases were searched for publications reporting the economic impact of cCMV and CMV infections across all age groups. Manuscripts published between 2010 and 2020 from Australia, Latin America, Canada, Europe, Israel, Japan, the United States, and global (international, worldwide) studies were included; congress materials were excluded. Outcomes of interest included cCMV- and CMV-attributable direct costs/charges, resource utilization, and indirect/societal costs. RESULTS: Of 751 records identified, 518 were excluded based on duplication, population, outcome, study design, or country. Overall, 55 articles were eligible for full-text review; 25 were further excluded due to population, outcome, study design, or congress abstract. Two publications were additionally identified, resulting in economic impact data compiled from 32 publications. Of these, 24 publications reported cost studies of cCMV or CMV, including evaluation of direct costs/charges, healthcare resource utilization, and indirect/societal costs, and 7 publications reported economic evaluations of interventions. The populations, methods and outcomes used across these studies varied widely. CONCLUSIONS: CMV and cCMV infections impose a considerable economic impact on different countries, populations, and outcomes. There are substantial evidence gaps where further research is warranted.

Cytomegalovirus in donors for fecal microbiota transplantation, the phantom menace?

BACKGROUND: Fecal Microbiota Transplantation (FMT) has become the preferred treatment for recurrent Clostridioides difficile Infections (CDI). However, donor screening is a complex process that varies between countries. The primary objective of screening is to prevent the transfer of potential pathogens from the donor to the recipient via feces. Many guidelines recommend Cytomegalovirus (CMV) testing as part of donor screening, but is the risk of CMV transmission well supported by evidence? MATERIALS/METHODS: A French prospective cross-sectional multicenter single-arm study estimated the frequency of detection of CMV in the stool of voluntary healthy donors selected for FMT. All preselected donors were tested for CMV antibodies in blood, and if positive, CMV DNA PCR was performed on whole blood and stool. For samples CMV positive in stool PCR, or case of serological markers positive for IgM, we planned isolation of CMV in cell culture. RESULTS: From June 1, 2016, to July 31, 2017, 500 healthy donors (250 per center) were recruited and 483 included. Of these, 301 were CMV seronegative, and 182 tested positive for CMV IgM and/or IgG. Stool CMV PCR was performed in 162 donors. In two cases, the initial analysis was positive, but below the limit of quantification. Repeated PCR tests using Siemens and Altostar assays were negative. No infectious CMV could be detected in cell culture of these two samples and in the stool of 6 CMV IgM-positive donors. CONCLUSIONS: Our study shows that healthy volunteers with positive CMV serology do not shed CMV DNA in their stool, as detected by PCR or cell culture. This study provides another argument to remove CMV screening for FMT donors.

Prospects of Cytomegalovirus-Specific T-Cell Receptors in Clinical Diagnosis and Therapy.

Human cytomegalovirus (HCMV) is responsible for widespread infections worldwide. In immunocompetent individuals it is typically latent, while infection or reactivation in immunocompromised individuals can result in severe clinical symptoms or even death. Although there has been significant progress in the treatment and diagnosis of HCMV infection in recent years, numerous shortcomings and developmental limitations persist. There is an urgent need to develop innovative, safe, and effective treatments, as well as to explore early and timely diagnostic strategies for HCMV infection. Cell-mediated immune responses are the primary factor controlling HCMV infection and replication, but the protective role of humoral immune responses remains controversial. T-cells, key effector cells of the cellular immune system, are critical for clearing and preventing HCMV infection. The T-cell receptor (TCR) lies at the heart of T-cell immune responses, and its diversity enables the immune system to differentiate between self and non-self. Given the significant influence of cellular immunity on human health and the indispensable role of the TCR in T-cell immune responses, we posit that the impact of TCR on the development of novel diagnostic and prognostic methods, as well as on patient monitoring and management of clinical HCMV infection, will be far-reaching and profound. High-throughput and single-cell sequencing technologies have facilitated unprecedented quantitative detection of TCR diversity. With these current sequencing technologies, researchers have already obtained a vast number of TCR sequences. It is plausible that in the near future studies on TCR repertoires will be instrumental in assessing vaccine efficacy, immunotherapeutic strategies, and the early diagnosis of HCMV infection.

Another tool against cytomegalovirus after allogeneic hematopoietic cell transplantation.

Cytomegalovirus (CMV) viremia from reactivation of latent infection is a common complication after allogeneic hematopoietic cell transplantation (HCT). Untreated, CMV viremia can progress to affect other organs, resulting in organ dysfunction with high morbidity and mortality. In this issue of the JCI, Prockop and authors demonstrate that third-party donor T cells sensitized ex vivo to CMV pp65-derived overlapping pentadecapeptides are safe and effective for the treatment of CMV reactivation or CMV disease refractory to first-line pharmacotherapies occurring after HCT. They also provide insight into the biological differences between responders and nonresponders. This work confirms the utility of third-party CMV pp65 VSTs and suggests strategies for further improving the efficacy of this cell-therapy approach.

Antigen-specific γδ T cells contribute to cytomegalovirus control after stem cell transplantation.

γδ T cells support the immunological control of viral infections, in particular during cytomegalovirus (CMV) reactivation in immunocompromised patients after allogeneic hematopoietic stem cell transplantation. It is unclear how γδ T cells sense CMV-infection and whether this involves specific T cell receptor (TCR)-ligand interaction. Here we summarize recent findings that revealed an adaptive-like anti-CMV immune response of γδ T cells, characterized by acquisition of effector functions and long-lasting clonal expansion. We propose that rather CMV-induced self-antigen than viral antigens trigger γδ TCRs during CMV reactivation. Given that the TCRs of CMV-activated γδ T cells are often cross-reactive to tumor cells, these findings pinpoint γδ T cells and their γδ TCRs as attractive multipurpose tools for antiviral and antitumor therapy.

A systematic literature review on the humanistic burden of cytomegalovirus.

OBJECTIVE: Cytomegalovirus (CMV) infection is typically asymptomatic in healthy individuals; however, certain populations are vulnerable to infection and may develop serious sequelae. CMV infection may also have a broad impact on humanistic outcomes, including patient health status and quality of life (QoL). We conducted a systematic literature review (SLR) to describe the global humanistic burden of CMV and congenital CMV (cCMV) infections across all age groups. METHODS: Medline, Embase, and LILACS were searched to identify studies on humanistic outcomes following CMV infection, including health status/QoL and any outcomes in domains such as auditory performance, cognitive ability, developmental status, intelligence, language, memory, mental health, motor performance, social communication, speech, and vocabulary. The SLR included articles published from 2000 to 2020 and focused geographically on Australia, Europe, Israel, Japan, Latin America, and North America. RESULTS: Sixty-three studies met the inclusion criteria. In general, individuals with symptomatic cCMV infection experience a greater burden of disease and more substantial impact on QoL versus those with asymptomatic cCMV infection. Children with hearing loss due to cCMV infection, both symptomatic and asymptomatic, showed improved auditory outcomes following cochlear implantation. Newborns, infants, and children with cCMV infections had worse cognitive outcomes in psychological development, sequential and simultaneous processing, phonological working memory, and attention control versus age-matched controls without cCMV infection. CMV infection was also associated with cognitive decline in elderly populations. CONCLUSIONS: CMV infection can have substantial, lifelong, heterogenous impacts on humanistic outcomes, including health status and QoL, which should be considered when developing and implementing treatment and prevention strategies.

Third-party cytomegalovirus-specific T cells improved survival in refractory cytomegalovirus viremia after hematopoietic transplant.

BackgroundRefractory CMV viremia and disease are associated with significant morbidity and mortality in recipients of hematopoietic stem cell transplant (HCT).MethodsIn phase I/II trials, we treated 67 subjects for CMV viremia or disease arising after HCT with adoptive transfer of banked, third-party, CMVpp65-sensitized T cells (CMVpp65-VSTs). All were evaluable for toxicity and 59 for response. Evaluable subjects had CMV disease or persisting viremia that had failed at least 2 weeks of induction therapy with a median of 3 antiviral drugs; 84.7% had more than 3 of 11 high-risk features. CMVpp65-VSTs were specific for 1 to 3 CMVpp65 epitopes, presented by a limited set of HLA class I or II alleles, and were selected based on high-resolution HLA matching at 2 of 10 HLA alleles and matching for subject and subject's HCT donor for 1 or more alleles through which the CMVpp65-VSTs were restricted.ResultsT cell infusions were well tolerated. Of 59 subjects evaluable for response, 38 (64%) achieved complete or durable partial responses.ConclusionsRecipients responding to CMVpp65VSTs experienced an improved overall survival. Of the risk factors evaluated, transplant type, recipient CD4+ and CD8+ T cell levels prior to adoptive therapy, and the HLA restriction of CMVpp65-VSTs infused each significantly affected responses. In addition, CMVpp65-specific T cells of HCT donor or recipient origin contributed to the durability of both complete and partial responses.Trial RegistrationNCT00674648; NCT01646645; NCT02136797 (NIH).FundingNIH (P01 CA23766, R21 CA162002 and P30 CA008748); Aubrey Fund; Claire Tow Foundation; Major Family Foundation; "Rick" Eisemann Pediatric Research Fund; Banbury Foundation; Edith Robertson Foundation; Larry Smead Foundation.

Rationally designed synthetic vectors for therapeutic nucleic acid delivery against human cytomegalovirus infection.

RNA therapy represents a great way to precisely regulate cellular processes by modulating the gene expression. Despite this potential, a profound gap exists in our knowledge of how to subsequently deliver these RNAs into the specific target cells and turn therapeutically active RNAs into practical medicines. An advanced series of interlocked, thermodynamically self-regulated processes that enable the precise assembly of functional synthetic carriers of siRNA to the target cells in vivo was developed. To demonstrate the efficacy of this delivery system, we used it to treat human cytomegalovirus (HCMV) infection in a humanized mouse model. In this study, we use small interfering RNA (siRNA) and small complementary RNA (scRNA) to inhibit the expressions of two HCMV genes, IE1 and IE2. The auto-regulated nanocarrier polywraplex with core-shell structure was designed to condense and package these RNAs for delivering. To allow these particles recognize the HCMV-infected cells, a ligand was coupled on the surface of nanoparticle, which would specifically target the HCMV-encoded CX3 CL1 chemokine receptor presented in the HCMV-infected cells. The results demonstrated that the polywraplex conjugated with the target molecule CX3 CL1 effectively and specifically delivered the siRNA/scRNA to HCMV infected cells and inhibited virus growth in vitro and in vivo.

Current management of CMV infection in cancer patients (solid tumors). Epidemiology and therapeutic strategies

Little evidence is available regarding the incidence of CMV disease in patients with solid cancers. Latest data show that approximately 50 % of these patients with CMV PCR positivity developed clinically relevant CMV-viremia, and would require specific therapy. In the clinical arena, CMV reactivation is an important differential diagnosis in the infectological work up of these patients, but guidelines of management on this subject are not yet available. CMV reactivation should be considered during differential diagnosis for patients with a severe decline in lymphocyte counts when receiving chemoradiotherapy or immunochemotherapy with lymphocyte-depleting or blocking agents. Monitoring of CMV reactivation followed by the implementation of preemptive strategies or the establishment of early antiviral treatment improves the prognosis and reduces the morbidity and mortality of these patients. (AU)

Efficacy of pp65-specific TCR-T cell therapy in treating cytomegalovirus infection after hematopoietic stem cell transplantation.

Cytomegalovirus (CMV) infection remains a major cause of mortality after hematopoietic stem cell transplantation (HSCT). Current treatments, including antiviral drugs and adoptive cell therapy with CMV-specific cytotoxic T lymphocytes (CTLs), only show limited benefits in patients. T-cell receptor (TCR)-T cell therapy offers a promising option to treat CMV infections. Here, using tetramer-based screening and single-cell TCR cloning technologies, we identified various CMV antigen-specific TCRs from healthy donors, and generated TCR-T cells targeting multiple pp65 epitopes corresponding to three major HLA-A alleles. The TCR-T cells showed efficient cytotoxicity toward epitope-expressing target cells in vitro. After transfer into immune-deficient mice bearing pp65+ HLA+ tumor cells, TCR-T cells induced dramatic tumor regression and exhibited long-term persistence. In a phase I clinical trial (NCT04153279), CMV TCR-T cells were applied to treat patients with CMV reactivation after HSCT. Except one patient who withdrew at early treatment stage, all other six patients were well-tolerated and achieved complete response (CR), no more than grade 2 cytokine release syndrome (CRS) and other adverse events were observed. CMV TCR-T cells persisted up to 3 months. Among them, two patients have survived for more than 1 year. This study demonstrates the great potential in the treatment and prevention of CMV infection following HSCT or other organ transplantation.

Third-party CMV- and EBV-specific T-cells for first viral reactivation after allogeneic stem cell transplant.

Virus-specific T-cells (VSTs) from third-party donors mediate short- and long-term antiviral effects in allogeneic hematopoietic stem cell transplant (HSCT) recipients with relapsed or refractory viral infections. We investigated early administration of third-party VSTs, together with antiviral therapy in patients requiring treatment for first cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. Thirty HSCT patients were treated with 1 to 4 VST infusions (2 × 107 cells/m2; CMV n=27, EBV n=3) at a median of 4 days after initiation of antiviral treatment. The overall viral response rate was 100%, with a complete response (CR) rate of 94%. Of the 28 patients who achieved a CR, 23 remained virus PCR negative (n=9) or below quantitation limit (n=14) for the duration of follow-up. Four patients had brief episodes of quantifiable reactivation not requiring additional therapy, and one required a second infusion after initial CR, remaining PCR negative thereafter. All 3 patients treated for EBV post-transplant lymphoproliferative disorder achieved sustained CR. Rates of aGVHD and cGVHD after infusion were 13% and 23%, respectively. There were no serious infusion-related adverse events. VST infusion was associated with rapid recovery of CD8+CD45RA-CD62L- and a slower recovery of CD4+CD45RA-CD62L- effector memory T-cells; CMV-specific T-cells comprised up to 13% of CD8+ cells. At 1 year post-transplant, non-relapse mortality was 10%, cumulative incidence of relapse was 7%, overall survival was 88% and 25 of 27 patients had ECOG status of 0 or 1. Early administration of third-party VSTs in conjunction with antiviral treatment appears safe and leads to excellent viral control and clinical outcomes. Registered on Australian New Zealand Clinical Trials Registry as #ACTRN12618000343202.

Rapid Generation of TCR and CD8αß Transgenic Virus Specific T Cells for Immunotherapy of Leukemia.

Background: Virus-specific T cells (VSTs) are an attractive cell therapy platform for the delivery of tumor-targeted transgenic receptors. However, manufacturing with conventional methods may require several weeks and intensive handling. Here we evaluated the feasibility and timelines when combining IFN-γ cytokine capture (CC) with retroviral transduction for the generation of T cell receptor (TCR) and CD8αß (TCR8) transgenic VSTs to simultaneously target several viral and tumor antigens in a single product. Methods: Healthy donor peripheral blood mononuclear cells were stimulated with cytomegalovirus (CMV) and Epstein-Barr-Virus (EBV) peptide mixtures derived from immunogenic viral proteins, followed by CC bead selection. After 3 days in culture, cells were transduced with a retroviral vector encoding four genes (a survivin-specific αßTCR and CD8αß). TCR8-transgenic or control VSTs were expanded and characterized for their phenotype, specificity and anti-viral and anti-tumor functions. Results: CC selected cells were efficiently transduced with TCR8. Average fold expansion was 269-fold in 10 days, and cells contained a high proportion of CD8+ T central memory cells. TCR8+ VSTs simultaneously expressed native anti-viral and transgenic anti-survivin TCRs on their cell surface. Both control and TCR8+ VSTs produced cytokines to and killed viral targets, while tumor targets were only recognized and killed by TCR8+ VSTs. Conclusions: IFN-γ cytokine capture selects and activates CMV and EBV-specific memory precursor CD8+ T cells that can be efficiently gene-modified by retroviral transduction and rapidly ex vivo expanded. Our multi-specific T cells are polyfunctional and recognize and kill viral and leukemic targets expressing the cognate antigens.