The Journal of Immunology Preferential Expansion of Human Virus-Specific Multifunctional Central Memory T Cells by Partial Targeting of the IL-2 Receptor Signaling Pathway: The Key Role of CD4 + T Cells Michael Schmueck,* ,†,‡ Annika M. Fischer,* ,‡ Ben Hammoud,* Gordon Brestrich, †,‡ Henrike Fuehrer, †,‡ Si-Hong Luu, †,‡ Karin Mueller, †,‡ Nina Babel, †,‡ Hans-Dieter Volk,* ,‡,1 and Petra Reinke †,‡,1 Effector memory T cells are effective in controlling acute infections, but central memory T cells play a key role in long-lasting protection against viruses and tumors. In vivo/in vitro challenge by Ag commonly supports the generation of effector memory T cells with limited longevity. To our knowledge, this study demonstrates for the first time in the human system and under rechallenge conditions that targeting IL-2R by partial mammalian target of rapamycin inhibition or blocking IL-2Ra enriches human CD4 + /CD8 + central memory T cells within the virus-specific T cell product associated with enhanced functionality (i.e., multicytokine secretors, including IL-2; enhanced CD137 and CD107a expression on CD8 + and CD4 + T cells, respectively; and killing infected target cells). Remarkably, the effects on CD8 + T cells are mainly mediated via the enhancement of CD4 + T cell function. The data reveal new insights into the role of CD4 + T cell support for the quality of CD8 + T cell memory, even under rechallenge conditions. Moreover, our method offers a new approach to improve the long-lasting efficacy of adoptive T cell therapy in patients. The Journal of Immunology, 2012, 188: 5189–5198. P rimary/secondary T cell deficiency is associated with poor control of viruses, virus-associated tumors, and other in- tracellular pathogens. Adoptive T cell therapy is a novel therapeutic approach to restore immune competence (1–3), and it might be a particularly efficient tool for accelerating immune re- constitution following hematopoietic stem cell transplantation to control life-threatening viral and fungal infections in those pa- tients (4). Recent proof-of-concept studies demonstrated promis- ing data in EBV-related posttransplant lymphoproliferative disease and human CMV (HCMV) disease in permanently immunosup- pressed solid organ transplant (SOT) recipients as well (1, 5, 6). Virus-specific T cell lines with effector function can be success- fully generated in vitro, even from diseased and chronically im- munosuppressed patients. The infused T cells showed no adverse effects after adoptive transfer and induced rapid and effective control of viruses in almost all SOT patients (7, 8). However, the promising data on adoptive therapy in hematopoietic stem cell transplantation and SOT patients also revealed some limitations: the duration of efficacy is not sufficient in many patients (1, 7, 8). Our recent data on adoptive therapy of antiviral drug-resistant HCMV disease in SOT patients illustrate the problem: HCMV causes frequent and severe complications in SOT recipients (9). We developed a whole protein-spanning peptide pool-based protocol to generate HCMV-specific T cell lines to almost all CD4/8 epitopes in an HLA-independent manner (7). HCMV-specific CD4 + /CD8 + T cells were first infused as proof of concept to an antiviral drug-resistant lung transplant patient who suffered from severe chronic HCMV disease. The adoptively transferred T cells rapidly cleared the viral infection; within a few days, the patient no longer required ventilation, which had been necessary for months. After a full recovery, the patient was released from the clinic 3 wk later but relapsed 7 wk later (1). Similar data were seen with two other patients. This illustrates the strengths and limitations of this approach. The rapid relapse cannot simply be explained by the chronic immunosuppression in those patients, because we used a similar approach in SOT patients who were on comparable immunosuppression and suffered from EBV-associated posttransplant lymphoproliferative disease. As in HCMV disease, we observed a rapid reduction of viral load postinfusion without any side effects; however, in contrast to HCMV-infected patients, the EBV control was long-lasting (up to .10 y) in about the half of the SOT patients (6). Analysis of HCMV-specific T cells *Institute of Medical Immunology, Charite ´ University Medicine Berlin, 13353 Berlin, Germany; † Renal and Transplant Research Unit, Department of Nephrology and Internal Intensive Care, Charite ´ University Medicine Berlin, 13353 Berlin, Ger- many; and ‡ Berlin-Brandenburg Center for Regenerative Therapies, Charite ´ Univer- sity Medicine Berlin, 13353 Berlin, Germany 1 H.-D.V. and P.R. are to be considered cosenior authors. Received for publication December 23, 2011. Accepted for publication March 16, 2012. This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG- SFB-TR36 project A3), the Bundesministerium fuer Bildung und Forschung (STThera 01GU0802), and the Berlin-Brandenburg Center for Regenerative Therapies. The fun- ders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. M.S. led the project, performed most of the experiments, analyzed the data, and signifi- cantly contributed to writing of the manuscript. P.R. and H.-D.V. designed the research, assisted in interpreting the data, and significantly contributed to writing of the manu- script. H.F. performed some of the experiments. B.H., K.M., S.-H.L., and G.B. performed some of the experiments and helped to analyze data. A.M.F. and N.B. performed some of the experiments, helped to analyze data, and assisted with writing of the manuscript. Address correspondence and reprint requests to Michael Schmueck, Foehrer Straße. 15/Suedstraße. 2, Charite ´ University Medicine Berlin, 13353 Berlin, Germany. E-mail address: michael.schmueck@charite.de The online version of this article contains supplemental material. Abbreviations used in this article: FSC, forward light scatter; HCMV, human CMV; LCL, lymphoblastoid B cell line; LD, low dose; mTOR, mammalian target of rapa- mycin; rh, recombinant human; nTreg, natural regulatory T cell; SOT, solid organ transplantation; T CM , central memory T cell; T EM , effector memory T cell; T EMRA , terminally differentiated effector T cell; VLD, very low dose. Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103763