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Vitro Generated to Ex Vivo CD8 T Cells Comparative and Functional Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021 is online at: average * The Journal of Immunology published online 27 August 2012 from submission to initial decision 4 weeks from acceptance to publication Comparative and Functional Evaluation of In Vitro Generated to Ex Vivo CD8 T Cells Dzana D. Dervovic, Maria Ciofani, Korosh Kianizad and Juan Carlos Zúñiga-Pflücker J Immunol http://www.jimmunol.org/content/early/2012/08/26/jimmun ol.1200979 Submit online. Every submission reviewed by practicing scientists ? is published twice each month by http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://www.jimmunol.org/content/suppl/2012/08/28/jimmunol.120097 9.DC1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 24, 2021. Published August 27, 2012, doi:10.4049/jimmunol.1200979 The Journal of Immunology Comparative and Functional Evaluation of In Vitro Generated to Ex Vivo CD8 T Cells Dzˇana D. Dervovic´,*,† Maria Ciofani,*,†,1 Korosh Kianizad,*,† and Juan Carlos Zu´n˜iga-Pflu¨cker*,† The generation of the cytotoxic CD8 T cell response is dependent on the functional outcomes imposed by the intrathymic constraints of differentiation and self-tolerance. Although thymic function can be partly replicated in vitro using OP9-DL1 cell cultures to yield CD8 ab TCR-bearing cells from hematopoietic progenitor cells, a comprehensive and functional assessment of entirely in vitro generated CD8 T cells derived from bone marrow hematopoietic stem cells has not been established and remains controversial. In this study, we demonstrate that a phenotypic, molecular, and functional signature of in vitro derived CD8 T cells is akin to that of ex vivo CD8 T cells, although several significant differences were also observed. Transfer of in vitro derived CD8 T cells into syngeneic and immunodeficient host mice showed no graft-versus-host response, whereas a robust homeostatic proliferation was Downloaded from observed, respectively. These findings, along with a diverse and broad TCR repertoire expressed by the in vitro derived CD8 T cells, allowed for the successful generation of Ag-specific T cells to be obtained from an entirely in vitro generated CD8 T cell pool. These findings support the use of Ag-specific in vitro derived effector CD8 T cells for immune reconstitution approaches, which would be amenable to further tailoring for their use against viral infections or malignancies. The Journal of Immunology, 2012, 189: 000–000. http://www.jimmunol.org/ cells are generated in the thymus after colonization from CD8 T cells is thought to be achieved through a neglect, select, the blood by bone marrow (BM)-derived progenitors (1– eliminate process that is partly dependent on the strength of signal T 6). In the thymus, T cell development can be character- induced by the self-peptide MHC (pMHC) complex. Taken to- ized as a series of distinct subsets based on the expression of cell gether, these processes determine not only survival, but also the surface proteins. The earliest T cell progenitors, double-negative appropriate lineage specification of ab T cells in the periphery, (DN) cells, lack expression of the TCR, CD4, or CD8. Subsequent which in general correlates with their phenotypic and functional signaling through the rearranged pre-TCR and growth factor specialization into MHCII-restricted CD4 Th cells or into MHCI- receptors induces cell proliferation and differentiation into double- restricted CD8 CTLs (11–19). positive (DP) cells that express both CD4 and CD8 coreceptors, as Moreover, the development of mature, functional, and self- by guest on September 24, 2021 well as the mature ab-TCR (7–9). Further differentiation is ini- restricted T cells, expressing a diverse Ag-specific repertoire, tiated by interaction of the TCR with the self-peptides presented which is essential for an efficient immune response, is achieved by MHC proteins, MHCII or MHCI, to generate mature CD4 and through signals induced via reciprocal interactions between T cell CD8 T cells, respectively (10). The generation of mature CD4 and precursors and multiple soluble as well as membrane-bound factors provided by cortical and medullary thymic stromal cells. Among these signals, Notch–ligand interactions are compulsory for T *Department of Immunology, University of Toronto, Toronto, Ontario M4N 3M5, lymphopoiesis (20–25). Canada; and †Sunnybrook Research Institute, Toronto, Ontario M4N 3M5, Canada With regard to T lymphopoiesis, Notch signaling has been 1Current address: Molecular Pathogenesis Program, The Kimmel Center for Biology implicated in B versus T, and ab versus gd T cell lineage com- and Medicine of the Skirball Institute, New York University School of Medicine, mitment, differentiation through early DN stages of thymocyte New York, NY. development, progression from DN to DP stages, CD4 Th1 versus Received for publication April 4, 2012. Accepted for publication July 25, 2012. Th2 cell specification, as well as regulation of cytolytic effector This work was supported by funds from the Canadian Institutes of Health Research function in CD8 T cells (22, 26–37). Similarly, ectopic expression (MOP-12927 and MOP-119538). D.D.D. was supported by a scholarship from the Lady Tata Memorial Fund; K.K. was supported by a Natural Sciences and Engineer- of the Notch ligand Delta-like 1 by mouse OP9 BM cells supports ing Research Council studentship; and J.C.Z.-P. is a recipient of a Canada Research efficient T lymphopoiesis from mouse fetal liver (FL) progenitors, Chair in Developmental Immunology. adult BM-derived hematopoietic stem cells (HSCs), embryonic The microarray data presented in this article have been submitted to the Gene Ex- stem cells, and cord blood-derived CD34+ into mature TCR ab+ pression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37669) + under accession number GSE37669. CD8 T cells (22, 29, 38). However, the functional assessment of + Address correspondence and reprint requests to Dr. Juan Carlos Zu´n˜iga-Pflu¨cker, entirely in vitro generated CD8 T cells remains ambiguous and Sunnybrook Research Institute, 2075 Bayview Avenue, Room A3-31, Toronto, ON not fully characterized. M4N 3M5, Canada. E-mail address: [email protected] In this study, we demonstrate that ab+ CD8 T cell development The online version of this article contains supplemental material. proceeds normally in HSC/OP9-DL1 cocultures, leading to their Abbreviations used in this article: BM, bone marrow; DC, dendritic cell; DN, double- differentiation into functionally mature cells. The maturation negative; DP, double-positive; FL, fetal liver; Gzm-B, granzyme-B; HSC, hemato- poietic stem cell; hTRP-2, human TRP-2; LCMV, lymphocytic choriomeningitis status of in vitro generated CD8 T cells showed similarities to that viral; LN, lymph node; MFI, mean fluorescence intensity; pMHC, peptide MHC; of ex vivo CD8 T cells as determined by the surface expression QRT-PCR, quantitative real-time PCR; SP, single-positive; TRP-2, tyrosinase-related of differentiation markers, with some significant differences also protein-2. noted. At the molecular level, in vitro derived CD8 T cells por- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 trayed predominantly a normal CD8-biased gene expression pro- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200979 2 IN VITRO GENERATED CD8 T CELLS file, albeit Notch-regulated genes showed elevated expression. In cytometric cell sorting of CD117+ Sca-1high expressing cells, referred to as addition, in vitro stimulation assays confirmed that these CD8 the HSC-enriched fraction. Thymus and spleen were isolated from 4- to 8- T cells are functionally mature cells that can proliferate and express wk-old mice. Single-cell suspensions were disaggregated through a 40 mM nylon cell strainer with a syringe plunger and were washed further with CD8-specific activation markers. Correspondingly, we found that OP9 medium. Spleens were depleted of RBCs with ammonium chloride these cells mature into effector cells as defined by their capacity to lysing reagent (BD Pharmingen), as recommended by the supplier. Prior to produce antiviral cytokines and cytolytic effector molecules. cell sorting, OP9-DL1 cocultures were passaged through 40 mM nylon cell We also demonstrate that CD8 T cells generated in HSC/OP9- strainer. For the generation of dendritic cells (DCs), BM cells were iso- lated, as described above, and 5 3 106 cells were placed in 10 ml DMEM, DL1 cocultures are broadly tolerant to self-pMHC Ags, as assayed containing 10% FBS, with the addition of 5 ng/ml each, mouse IL-4, and by in vitro and in vivo responses to allogeneic or syngeneic APCs. mouse GM-CSF (PeproTech). Fresh media and cytokines were added on In addition, we observed augmented lymphopenia-driven prolif- days 3 and 6. In the last 12–16 h, DCs were activated by addition of 0.1 + eration and expansion of in vitro derived CD8+ T cells when mg/ml LPS (Sigma-Aldrich). At day 7, CD11c cells were FACS sorted or transferred into RAG22/2 host mice. In addition, we revealed the directly purified with CD11c microbeads, according to the manufacturer’s protocol (Miltenyi Biotec). presence of a diverse and polyclonal TCR repertoire in coculture- derived CD8 T cells. Finally, we show that in vitro derived CD8+ BM HSC/OP9-DL1 cell cocultures T cells generated an enhanced primary Ag-specific response 5 BM-derived HSCs (10 cells) were cocultured on a monolayer of OP9-DL1 against tumor-associated Ag, tyrosinase-related protein-2 (TRP- cells in 10-cm plates, as previously described (39).
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