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Vivo Model CD4/CD8 Lineage Choice in a Physiologic in Positive Coreceptor Signal Strength Regulates Positive Selection but Does Not Determine CD4/CD8 Lineage Choice in a Physiologic In Vivo Model This information is current as of September 27, 2021. Batu Erman, Amala S. Alag, Oyvind Dahle, François van Laethem, Sophia D. Sarafova, Terry I. Guinter, Susan O. Sharrow, Alexander Grinberg, Paul E. Love and Alfred Singer J Immunol 2006; 177:6613-6625; ; Downloaded from doi: 10.4049/jimmunol.177.10.6613 http://www.jimmunol.org/content/177/10/6613 http://www.jimmunol.org/ References This article cites 50 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/177/10/6613.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Coreceptor Signal Strength Regulates Positive Selection but Does Not Determine CD4/CD8 Lineage Choice in a Physiologic In Vivo Model1 Batu Erman,*† Amala S. Alag,* Oyvind Dahle,* Franc¸ois van Laethem,* Sophia D. Sarafova,* Terry I. Guinter,* Susan O. Sharrow,* Alexander Grinberg,‡ Paul E. Love,‡ and Alfred Singer2* TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8␣ gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8␣ gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased Downloaded from -the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8؉ T cells, even for thymocytes ex pressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice. The Journal of Immunology, 2006, 177: 6613–6625. http://www.jimmunol.org/ cell immunocompetence requires CD4ϩ T cells to express intracellular Lck with TCR components, initiating TCR signal trans- Ag receptors specific for MHC class II (MHC-II)3 deter- duction (12, 13). However, the CD4 cytosolic tail associates with ϩ T minants and requires CD8 T cells to express Ag recep- intracellular Lck more efficiently than does the CD8 cytosolic tail, tors specific for MHC class I (MHC-I) determinants (1–4). Such with the result that CD4 coreceptor signaling intensity is greater than concordance between TCR MHC specificity and either CD4 or that of CD8 (14–17). The difference in coreceptor signaling intensity CD8 coreceptor expression is established during differentiation in between CD4 and CD8 is thought to have an important impact on the thymus (4), but the mechanism by which TCR MHC specificity positive selection, although there is significant disagreement as to the by guest on September 27, 2021 determines lineage fate remains highly controversial despite sig- nature of the impact that coreceptor signaling intensity has on positive nificant progress in understanding CD4 and CD8 gene regulation selection. The original “strength-of-signal” hypothesis remains highly (5–10). In addition, it is not known why the thymus normally gen- ϩ ϩ popular (18) and proposes that the intensity of coreceptor signals qual- erates more CD4 than CD8 T cells with the result that the itatively affects positive selection by dictating CD4/CD8 lineage Ͼ CD4:CD8 ratio is 1 in nearly all mammalian species. choice, with strong coreceptor signals from CD4 directing thymocytes Double-positive (DP) thymocytes expressing TCR with appropri- into the CD4 lineage and weak coreceptor signals from CD8 directing ate affinity for intrathymic self-ligands are signaled to undergo posi- thymocytes into the CD8 lineage (19–23). A more recent modifica- tive selection and to further differentiate into single-positive (SP) T tion of the strength-of-signal model proposes that strong coreceptor cells (4, 11). As both CD4 and CD8 coreceptors are associated with signals from CD4 result in long duration TCR signals that direct thy- the nonreceptor protein tyrosine kinase Lck, ligand-induced coen- mocytes into the CD4 lineage, whereas weak coreceptor signals from gagement of either CD4 or CD8 with TCR physically approximates CD8 result in short duration TCR signals that direct thymocytes into the CD8 lineage (23). In contrast, we have proposed that signal strength does not affect CD4/CD8 lineage choice but only has a quan- *Experimental Immunology Branch, National Cancer Institute, Bethesda, MD 20892; †Biological Sciences and Bioengineering Program, Faculty of Engineering and Nat- titative effect on positive selection in that it influences the number of ural Sciences, Sabanci University, Istanbul, Turkey; and ‡Laboratory of Mammalian DP thymocytes signaled to undergo positive selection, with stronger Genes and Development, National Institute of Child Health and Human Development, Bethesda, MD 20892 CD4 signals stimulating more DP thymocytes to undergo positive selection than weaker CD8 signals (24), a perspective supported by Received for publication June 15, 2006. Accepted for publication August 25, 2006. findings from two laboratories (25, 26). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance This study was undertaken to rigorously determine the impact of with 18 U.S.C. Section 1734 solely to indicate this fact. increased signal strength on positive selection by creating a new 1 This work was supported by the Intramural Research Program of the National In- gene knockin model in which the endogenous CD8␣ gene was stitutes of Health, National Cancer Institute, Center for Cancer Research and the Intramural Research Program of the National Institutes of Health, National Institute re-engineered to encode a chimeric CD8/CD4 protein consisting of for Child Health and Human Development. the extracellular and transmembrane domains of CD8␣ and the 2 Address correspondence and reprint requests to Dr. Alfred Singer, Experimental cytosolic tail of CD4 (“CD8.4”). Because expression of the CD8.4 Immunology Branch, National Cancer Institute, Building 10 Room 4B36, Bethesda, knockin allele was regulated by endogenous CD8␣ transcriptional MD 20892. E-mail address: [email protected] control elements, the timing and expression pattern of chimeric 3 Abbreviations used in this paper: MHC-II, MHC class II; MHC-I, MHC class I; DP, ␣ double positive; SP, single positive; WT, wild type; TK, thymidine kinase; NEO, CD8.4 proteins were identical with those of wild-type (WT) CD8 neomycin; ES, embryonic stem; LN, lymph node; Tk, T-killer. proteins. Consequently, CD8.4 mice provide a physiologic model Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 6614 POSITIVE SELECTION AND LINEAGE CHOICE in which to examine coreceptor signal strength without the poten- TCR-V␣3 (RR3-16), TCR-V␣8 (B21.14), TCR V␣11 (RR8-1), CD4 tial artifacts induced by aberrant CD8 coreceptor expression that (GK1.5), and Qa2 (1-1-2) were obtained from BD Pharmingen; mAbs spe- ␣ ␣ plagued CD8 coreceptor transgenic mice. Indeed, the results ob- cific for CD8 (CT-CD8 ) were obtained from Caltag Laboratories; and mAbs specific for granzyme B (clone 16G6) and CD154 (CD40L) were tained with CD8.4 knockin mice are remarkably unequivocal: in- obtained from eBioscience. creased CD8 coreceptor signal strength significantly increased the efficiency of MHC-I-specific positive selection and the number of Cell lysis and immunoprecipitations CD8ϩ T cells generated in the thymus, but it had no effect on DP thymocytes were lysed for 30 min at 4°C in buffer containing 1% CD4/CD8 lineage choice. Thus, coreceptor signaling intensity af- Nonidet P-40 (Pierce), 10 mM Tris-HCl (pH 7.2), 140 mM NaCl, 2 mM fects TCR signaling thresholds for positive selection and so is a EDTA, 1 mM NaF, 1 mM orthovanadate, and protease inhibitors. Insoluble material was removed by centrifugation for 10 min at 4°C. Lysates were key determinant of the CD4:CD8 ratio, but it does not influence incubated overnight with anti-CD8␣ mAbs coupled to protein G-Sepharose CD4/CD8 lineage choice. beads (Amersham Biosciences). Proteins were resolved by reducing SDS- PAGE and transferred to polyvinylidene difluoride membranes. Blots con- Materials and Methods taining anti-CD8 immunoprecipitates and total cell lysates were incubated Mice with anti-Lck (3A5 mAb; Santa Cruz Biotechnology) followed by HRP- conjugated protein A, with reactivity revealed by ECL (Pierce). C57BL/6 (B6) were purchased from The Jackson Laboratory; F5 TCR transgenic mice (27), CD8␣o mice (28), CD8␤o mice (29), MHC-IIo mice Intracellular calcium mobilization (30), and EIIa-Cre transgenic mice (31) were bred in our own animal col- Thymocytes were loaded with the calcium dye Indo-1 (Molecular Probes; ony.
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