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CD27 Mediates Interleukin-2-Independent Clonal Expansion of the CD8؉ T Cell Without Effector Differentiation

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CD27 Mediates Interleukin-2-Independent Clonal Expansion of the CD8؉ T Cell Without Effector Differentiation CD27 mediates interleukin-2-independent clonal expansion of the CD8؉ T cell without effector differentiation James M. Carr, Marlene J. Carrasco, James E. D. Thaventhiran, Paul J. Bambrough, Matthew Kraman, Alexander D. Edwards, Aymen Al-Shamkhani*, and Douglas T. Fearon† Wellcome Trust Immunology Unit, Department of Medicine, University of Cambridge, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom Contributed by Douglas T. Fearon, November 1, 2006 (sent for review September 5, 2006) The clonal expansion of antigen-specific CD8؉ T cells in response to and its ligation by CD70 promotes proliferation in vitro of microbial infections is essential for adaptive immunity. Although TCR-stimulated CD8ϩ T cells (12, 15). CD27-deficient mice IL-2 has been considered to be primarily responsible for this have impaired clonal expansion of virus-specific CD8ϩ T cells process, quantitatively normal expansion occurs in the absence of during primary infection with influenza (16) and secondary ؉ IL-2 receptor signaling. Here, we show that ligating CD27 on CD8 infection with lymphocytic choriomeningitis virus (17). Mice T cells that have been stimulated through the T cell receptor causes constitutively expressing CD70 on B cells develop a T cell their expansion in the absence of IL-2 by mediating two distinct proliferative disease in the absence of CD95 (18). As for CD137, cellular processes: enhancing cell cycling and promoting cell sur- it is expressed by activated but not naı¨ve CD8ϩ T cells and is vival by maintaining the expression of IL-7 receptor ␣. This path- ϩ ؉ required for normal expansion of CD8 T cells during secondary way for clonal expansion of the CD8 T cell is not associated with infections (19). Therefore, we have evaluated these two members ␥ the development of a capacity either for production of IFN- or for of the TNF receptor superfamily for their potential roles in cytotoxic T lymphocyte function and, therefore, is uncoupled from IL-2-independent CD8ϩ T cell expansion. differentiation. Furthermore, ligating CD27 increases the threshold concentration at which IL-2 induces IFN-␥-producing capability by Results the CD8؉ T cell, suggesting that CD27 signaling may suppress Loss of Function Studies Identify a CD70/CD27-Dependent Pathway of -effector differentiation. Finally, CD8؉ T cells that have been stim CD8؉ T Cell Proliferation Without Effector Differentiation. For a ulated by the TCR/CD27 pathway maintain their capacity for subsequent expansion and effector differentiation in response to model antigen-presenting cell, we took advantage of the A20 B cell lymphoma line that expresses CD70, CD137 ligand a viral challenge in vivo. Thus, the TCR/CD27 pathway enables the ␥ CD8؉ T cell to replicate by a process of self-renewal, which may (CD137L), CD80, CD86, and CD32, the Fc RII, which enables -؉ cross-linking of TCR-bound, agonistic antibody. For the re contribute to the continuous generation of new effector CD8 T ϩ cells in persistent viral infections. sponding CD8 T cell, we used transgenic OT-I cells (20) from RAGϪ/Ϫ mice that are specific for the ovalbumin-derived pep- b CD70 ͉ granzyme B ͉ Interferon-␥ ͉ TNF receptor superfamily tide, OVA257–264 (SIINFEKL) complexed to H-2K (pMHC). We cultured purified, CD62Lhigh OT-I cells with mitomycin ␧ he clonal distribution of antigen receptors, which is the C-treated A20 cells in the presence of anti-CD3 , IL-7, and Thallmark of the adaptive immune system, must be linked to blocking antibodies to IL-2 and CD25 to suppress IL-2R signal- a mechanism for rapid clonal expansion in response to microbial ing. The roles of CD27, CD137, and CD28 were assessed by the antigens. Although this requirement relates to all types of addition of blocking antibodies to their ligands. After 45 h, viable lymphocytes, it is perhaps most stringent for the CD8ϩ T cell OT-I cells were counted and cultured for an additional 45 h with because one of its antimicrobial functions, the killing of infected fresh mitomycin C-treated A20 cells and the original conditions. cells, requires direct cell-cell contact. For three decades, IL-2 has In the absence of ligand-blocking antibodies, the number of OT-I been considered to be primarily responsible for the clonal cells expanded 16-fold over 90 h in a manner that depended on expansion of the CD8ϩ T cell (1–3). However, over the past 10 anti-CD3␧ (Fig. 1). Blocking antibodies to CD137L, CD80, and years, studies have shown that expansion of CD8ϩ T cells in CD86 were without effect, whereas anti-CD70 inhibited expan- primary viral infections can occur in the absence of IL-2 receptor sion by 70%. In the absence of anti-CD3␧, no cells were (IL-2R) signaling (4–8). Furthermore, prior IL-2R stimulation recovered at 90 h. Therefore, ligation of CD27, but not CD137 of CD8ϩ T cells has been shown to be necessary (8), unnecessary (7), or even detrimental (9) for their subsequent in vivo expan- sion in response to viral challenge. These different experimental Author contributions: J.M.C. and M.J.C. contributed equally to this work; J.M.C., M.J.C., outcomes reflect different experimental protocols and indicate J.E.D.T., P.J.B., and D.T.F. designed research; J.M.C., M.J.C., J.E.D.T., P.J.B., M.K., and A.D.E. ϩ performed research; A.A.-S. contributed new reagents/analytic tools; J.M.C., M.J.C., that the regulation of CD8 T cell clonal expansion and differ- J.E.D.T., P.J.B., M.K., A.D.E., and D.T.F. analyzed data; and J.M.C., M.J.C., and D.T.F. wrote entiation is still incompletely understood. the paper. In contrast to the many studies of the role of IL-2 in mediating The authors declare no conflict of interest. ϩ the proliferation and effector differentiation of the CD8 T cell, Freely available online through the PNAS open access option. little is known of the mechanism or biological role of the Abbreviations: CFSE, carboxyfluorescein succinimidyl ester; IL-2R, IL-2 receptor. IL-2-independent pathway. In an attempt to identify this path- *Present address: Tenovus Research Laboratory, Cancer Sciences Division, University of way, we focused our attention on the TNF receptor superfamily Southampton School of Medicine, Southampton General Hospital, Tremona Road, because of the example of cytokine-independent proliferation of Southampton SO15 5PA, United Kingdom. B cells stimulated through CD40 (10). Of the several members †To whom correspondence should be addressed. E-mail: [email protected]. ϩ of the TNF receptor superfamily expressed by CD8 T cells, This article contains supporting information online at www.pnas.org/cgi/content/full/ CD27 (11, 12) and CD137 (13, 14) are involved in cellular 0609706103/DC1. ϩ proliferation. CD27 is expressed by resting, naı¨ve CD8 T cells, © 2006 by The National Academy of Sciences of the USA 19454–19459 ͉ PNAS ͉ December 19, 2006 ͉ vol. 103 ͉ no. 51 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609706104 Downloaded by guest on September 26, 2021 blocked required ligation of both TCR by anti-CD3␧ and CD27 by CD70 (Fig. 2 a and b). The absence of IL-7 caused a decrease in expansion of the TCR/CD27-stimulated OT-I cells (Fig. 2a, isotype Ϯ IL-7) but not of the TCR-stimulated cells (Fig. 2a, anti-CD70 Ϯ IL-7). IL-7 did not affect cell cycling in either stimulatory condition (Fig. 2b). Therefore, IL-7 enhances the expansion of TCR/CD27-stimulated CD8ϩ T cells by promoting their viability, not by driving cell cycling. An explanation for this selective effect of IL-7 was suggested by the finding that coli- gating CD27 maintained the expression of IL-7R␣, even in the presence of IL-7, whereas activation through the TCR alone down-regulated IL-7R␣ (Fig. 2c). Therefore, costimulation by CD27 at least partially counteracts the known suppressive effects ␣ ϩ of IL-7 and TCR stimulation on IL-7R expression (21, 22). This Fig. 1. The TNF receptor superfamily members, CD27 and CD137, and CD8 enables IL-7 to promote the viability, but not proliferation, of the T cell expansion in the presence of blocking antibodies to IL-2 and CD25. OT-I ϩ ϩ TCR/CD27-stimulated CD8 T cell. Without being able to CD8 T cells were stimulated with mitomycin C-treated A20 cells in the presence or absence of 20 ng/ml anti-CD3␧, IL-7, and anti-IL-2/anti-CD25. respond to IL-7, the OT-I cells activated solely through the TCR Cultures contained the indicated blocking or isotype control antibodies. After cycle but die. ϩ 45 h, CD8ϩ T cells were restimulated by using the original culture conditions We compared the effector differentiation of OT-I CD8 T and then assessed for expansion at 90 h. Comparable results were obtained in cells that had been stimulated for 6 days with TCR ligation in the three similar experiments. presence of CD70 or IL-2. Relative to naı¨ve, unstimulated OT-I cells, cells that had proliferated in response to the TCR/IL-2R ϩ pathway contained increased granzyme B, had down-regulated or CD28, promotes TCR-dependent expansion of CD8 T cells CD62L, were capable of synthesizing IFN-␥, and had CTL in the apparent absence of IL-2R stimulation. activity. In contrast, stimulation through the TCR/CD27 path- The role of IL-7 in this TCR- and CD27-dependent response way enhanced expression of CD62L, did not induce granzyme B, was examined by stimulating carboxyfluorescein succinimidyl caused only 25% of the cells to be capable of IFN-␥ production, ϩ ␧ ester (CFSE)-labeled OT-I CD8 T cells with anti-CD3 and and did not stimulate CTL differentiation [supporting informa- A20 cells in the presence or absence of this cytokine.
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