Center for Cell and Gene Therapy, Baylor College of Medicine
Gerdemann, U., Vera, J. F., Rooney, C. M., Leen, A. M. Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant. J. Vis. Exp. (51), e2736, doi:10.3791/2736 (2011).
Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture – total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by >90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.
1. DC nucleofection
2. T cell stimulation
3. T cell expansion
4. Representative Results:
A schematic of our FDA-approved multivirus-specific CTL generation process is shown in Figure 1. In contrast to convention multivirus CTL protocols which use adenovectors and EBV-LCL to stimulate virus-reactive T cells2 we have replaced infectious virus material with DNA plasmids that encode multiple antigens derived from each of the viruses3. To stimulate trivirus CTL we designed three multicistronic plasmids encoding Hexon and Penton of adenovirus, IE1 and pp65 of CMV, and EBNA1, LMP2, and BZLF1 of EBV. These antigens were chosen based on encouraging clinical results of our own and other groups showing that T cells directed against Adv-hexon and penton2,4-6, and to CMV-1E1 and to CMV-pp65 are protective in vivo7. For EBV, EBNA1 is an immunodominant CD4+ T cell target antigen expressed in all EBV-associated malignancies and in normal EBV-infected B cells8,9, LMP2 is immunogenic across multiple HLA types and expressed in most EBV malignancies,10,11 while BZLF1 encodes an immunodominant, immediate early lytic cycle antigen that stimulates both CD4+ and CD8+ T cells from most individuals and is likely important for the control of cells replicating virus12. To further optimize our manufacturing methods we collaborated with Nature Technology who generated minimalized, antibiotic-free (FDA-compliant) plasmids for CTL stimulation13,14. Using this strategy we consistently achieve nucleofection efficiencies of >35% while maintaining high cell viability (data not shown)3. Figure 2 shows that the frequency of virus-specific T cells in response to optimized DNA plasmids as measured by IFNγ ELIspot, was greater than in response to conventional pShuttle-based expression plasmids expressing the same antigens (n=8 Adenovirus, n=4 CMV, and n=2 EBV). The optimal ratio of DC:PBMC was important for potent T cell stimulation as shown in Figure 3 where a ratio of 1:50 produced sub-optimal activation compared to a 1:20 S:R ratio (n=2 donors). Production of sufficient CTL numbers with broad antigen specificity is a pre-requisite for clinical efficacy against all three viruses. This is achieved by CTL culture in the G-Rex, which supports superior T cell expansion compared with conventional 24-well plates (Figure 4A)15, while the addition of IL4 and IL7 to cultures increases the repertoire and specificity as shown in Figure 4B where the frequency of T cells reactive against the CMV-pp65-derived HLA-A2 resticted NLV peptide was assessed in cultures generated in the presence or absence of IL4 and/or IL716,17. To assess the phenotype and functional capacity of the expanded cells we perform flow cytometric analysis, intracellular cytokine staining/IFNγ ELIspot, and Cr51 release assays on the final product for cryopreservation/infusion. Typically the generated cells are polyclonal with a mixed population of CD4+ and CD8+ T cells with antigen-specificity detectable in both T cell compartments. The CTL are able to kill viral antigen-expressing target cells but not virus negative partially-HLA matched targets, indicating that they should not induce graft-versus-host disease (GvHD) in vivo (Figure 5).
Figure 1. rCTL generation protocol. First, DCs are nucleofected with the viral antigen-encoding plasmids and then mixed with autologous PBMCs at an R:S of 10 or 20:1. Cells are expanded in the G-Rex for 10-14 days in the presence of IL4 and IL7, then harvested, counted, tested for function, identity and sterility, and then cryopreserved for clinical use.
Figure 2. Optimized DNA plasmids induce superior T cell activation in vitro. DCs were nucleofected with optimized, FDA-compliant plasmids encoding Hexon and Penton (Adv), IE1 and pp65 (CMV), and EBNA1, LMP2, and BZLF1 (EBV) or conventional pShuttle plasmids encoding the same antigens. These were used to stimulate T cells and specificity was analyzed by IFNγ ELIspot 10 days post-stimulation.
Figure 3. Optimal DC:T cell ratios for CTL activation. DCs from 2 donors were nucleofected with all three optimized plasmids and then used to stimulate autologous PBMCs at 1:20 or 1:50 DC:PBMC ratio. The frequency of reactivated T cells was assessed on day 10 by IFNγ ELIspot.
Figure 4. T cell expansion in the G-Rex using enhancing cytokines. Panel A shows the G-Rex device as well as CTL appearance on the gas permeable membrane, evaluated by microscopy. A comparison between cell output achieved in convention tissue culture treated plates vs G-Rex is also shown. Panel B shows the frequency of CMV pentamer positive CTL achieved in cultures expanded in the presence of no cytokine, IL4 alone, IL7 alone and IL4+IL7.
Figure 5. Phenotype and function of expanded CTL. Panel A shows a representative example of the phenotype of the expanded multivirus CTL, which are polyclonal with a mixture of CD4+ (45% - helper) and CD8+ (42% - cytotoxic) T cells, of which the majority (95%) expressed the memory marker CD45RO+/CD62L+. Panel B shows that these cells are specific for all the stimulating antigens and are polyfunctional as assessed by intracellular cytokine staining to detect production of IFNΓ and TNFα after antigen stimulation. Panel C shows that the expanded CTL are functional as measured by Cr51 assay. Autologous LCL, either alone or transduced with a null vector or an adenoviral vector expressing CMV-pp65 were used as targets. Alloreactivity was assessed using allogeneic PHA blasts as a target.
Viral infections account for substantial morbidity and mortality in patients who are immunocompromised by their disease or its treatment. After HSCT, for example, infections caused by persistent herpesviruses such as EBV and CMV, as well as by respiratory viruses such as Respiratory Syncytial Virus (RSV), are well known, while the importance of infections caused by Adv, BK virus, and human herpesvirus (HHV)-6 have more recently been appreciated. While pharmacological agents are standard therapy for some infections, they have substantial toxicities, generate resistant variants, and are frequently ineffective. In contrast, virus-specific T cells derived from stem cell donors have proven safe and effective for the prevention and treatment of viral infection or disease in the hemopoietic stem cell transplant (HSCT) setting2,5,6,18-21. However, the broader implementation of T cell immunotherapy is ultimately limited by the cost, complexity and time required for CTL production.
Our novel and rapid approach to generate multivirus CTL, described in the current manuscript, should substantially increase the feasibility of cytotoxic T cell therapy for viral diseases, enabling the strategy to become a standard of care for the immunocompromised host. The use of plasmid nucleofected DCs as APCs enables antigen presentation on both MHC class I and II without competition from viral vectors or indeed from multiple viral antigens being expressed within a single cell since different DC populations are utilized for each plasmid3. The use of IL-4/7 increases T cell survival and proliferation, which correspondingly helps increase the frequency and repertoire of responding antigen-specific T cells16,17. Finally, culture in the G-Rex dramatically reduces T cell apoptosis during culture. Gas exchange (O2 in and CO2 out) occurs across a gas permeable silicon membrane at the base of the flask, preventing hypoxia while allowing a greater depth of medium above the cells, providing more nutrients and diluting waste products. This platform can also be extended to additional viruses as when protective antigens are identified.
Juan F. Vera is a consultant for Wilson Wolf Manufacturing. The other authors have no relevant disclosures.
This work is supported by a Production Assistance for Cellular Therapies (contract NIH-NHLBI (HB-10-03) HHSN26820100000C) (C.M.R.), a Specialized Centers for Cell-based Therapy Grant NIH-NHLBI 1 U54 HL081007 (C.M.R.), an ASBMT Young Investigator Award (U.G. and J.V.), a Leukemia and Lymphoma Society Special Fellow in Clinical Research Award (U.G.), and an Amy Strelzer Manasevit Scholar Award (A.M.L).
|Hyclone RPMI 1640||Thermo Fisher Scientific, Inc.||SH30096.01|
|Human AB serum||Valley Biomedical, Inc.||HP1022|
|Nucleofector||Amaxa||AAF-1001B & AAF-1001X|
|G-Rex||Wilson Wolf Manufacturing||AY11-00027|