T Cells + Mature CD8 Positive Selection by the Pre-TCR Yields

Positive Selection by the Pre-TCR Yields Mature CD8 + T Cells Yuriko Ito, Satoko Arai, Nicolai S. C. van Oers, Iannis Aifantis, Harald von Boehmer and Toru Miyazaki This information is current as of September 29, 2021. J Immunol 2002; 169:4913-4919; ; doi: 10.4049/jimmunol.169.9.4913 http://www.jimmunol.org/content/169/9/4913 Downloaded from References This article cites 39 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/169/9/4913.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 29, 2021 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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Positive Selection by the Pre-TCR Yields Mature CD8؉ T Cells1 Yuriko Ito,2* Satoko Arai,2*† Nicolai S. C. van Oers,* Iannis Aifantis,‡ Harald von Boehmer,‡ and Toru Miyazaki3* It has been of much interest whether there is functional redundancy between the constitutively signaling pre-T␣/TCR␤ (pre-TCR) and ligated TCR␣␤ complexes, which independently operate the two distinct checkpoints during thymocyte development, i.e., the pre-TCR involved in ␤-selection at the CD4؊CD8؊ double-negative stage and the TCR␣␤ being crucial for positive/negative -␣selection at the CD4؉CD8؉ double-positive stage. We found that the pre-TCR expressed on double-positive cells in TCR ,deficient (TCR␣؊/؊) mice produced a small number of mature CD8؉ T cells. Surprisingly, when pre-T␣ was overexpressed resulting in augmentation of pre-TCR expression, there was a striking increase of the CD8؉ T cells. In addition, even in the ؉ ؊ ؊ absence of up-regulation of pre-TCR expression, a similar increase of CD8 T cells was also observed in TCR␣ / mice over- Downloaded from expressing Egr-1, which lowers the threshold of signal strength required for positive selection. In sharp contrast, the CD8؉ T cells drastically decreased in the absence of pre-T␣ on a TCR␣؊/؊ background. Thus, the pre-TCR appears to functionally promote positive selection of CD8؉ T cells. The biased production of CD8؉ T cells via the pre-TCR might also support the potential involvement of signal strength in CD4/CD8 lineage commitment. The Journal of Immunology, 2002, 169: 4913–4919. he development of T cells in the thymus is controlled at phosphorylation of ZAP-70 and syk protein kinases, activation of http://www.jimmunol.org/ two consecutive checkpoints (1, 2). At the first check- the ras/mitogen-activated protein kinase pathway, and rapid in- T point, termed ␤-selection, CD4ϪCD8Ϫ double-negative crease in intracellular calcium levels (5–9). Because of these sim- thymocytes with a productive TCR␤ rearrangement are rescued ilarities, we asked the question whether in certain situations the from programmed cell death by the pre-TCR consisting of the pre-TCR can partially or even entirely substitute for the ligated varied TCR␤ and the invariant pre-TCR␣ (pT␣)4 chain. As a re- TCR␣␤, resulting in the generation of “␣␤-less” mature T cells. In sult, the cells proliferate, and begin to coexpress CD4 and CD8 and this study, we approached this question by qualifying different to rearrange the TCR␣ locus. Some evidence suggests that the strains of mice expressing the pre-TCR on the DP precursor cells. pre-TCR is also involved in channeling cells into the ␣␤-lineage because it reduces the number of ␥␦ T cells with productive TCR␤ by guest on September 29, 2021 rearrangements (3, 4). At the second checkpoint, the engagement Materials and Methods of the TCR␣␤ by appropriate MHC ligands rescues CD4ϩCD8ϩ Generation of transgenic mice double-positive (DP) thymocytes from apoptotic death, resulting in A 1.3-kb fragment of pT␣ cDNA (kindly provided by H. J. Fehling, Ulm, the generation of mature CD4ϩCD8Ϫ or CD4ϪCD8ϩ single-pos- Germany) was subcloned into the blunt-ended BamHI site of the expres- itive (SP) thymocytes. Very interestingly, there exists an obvious sion vector controlled by the lck-proximal promoter (10). The complete plasmid (plck-pT␣) was digested with NotI, and the transgene fragment no similarity of proximal signaling events between the constitutively longer containing vector sequence was purified by Geneclean II kit (Bio signaling pre-TCR and the ligated TCR␣␤, resulting in tyrosine 101, Vista, CA). DNA was microinjected into fertilized eggs of C57BL/6 phosphorylation of CD3␨ and CD3⑀ subunits, recruitment and mice. Resulting founders were screened for transgene by PCR and South- ern blotting. Five independent founders carried the transgene. Two of the five mice that expressed transgene at relatively higher levels, as assessed by Northern blotting using thymus RNA, were backcrossed within TCR␣Ϫ/Ϫ *Center for Immunology, University of Texas Southwestern Medical Center, Dallas, background. All animals used in the experiments were cared in accordance TX 75390; †Department of Applied Biological Chemistry, University of Tokyo, To- with institutional guidelines. kyo, Japan; and ‡Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115 Received for publication June 11, 2002. Accepted for publication August 26, 2002. RNA analysis The costs of publication of this article were defrayed in part by the payment of page Total RNA was isolated from thymus and lymph nodes (LNs) by using charges. This article must therefore be hereby marked advertisement in accordance RNAzolB (Tel-Test, Friendswood, TX) from various mice. A quantity with 18 U.S.C. Section 1734 solely to indicate this fact. amounting to 15 ␮g total RNA was denatured, electrophoresed in a 1% 32 1 This work was supported by research grant of Juvenile Diabetes Foundation (1- agarose gel, blotted on a nylon membrane, and then hybridized with P- 2001-581), American Liver Foundation Herman Lopata Memorial Liver Scholar labeled either pT␣ or ␤-actin cDNA fragment. Award, and Howard Hughes Medical Institute research support (to T.M.); National Institutes of Health Grant RO1AI42953 (to N.S.C.v.O.); National Institutes of Health Grants RO1A14728 and RO1A145846 (to H.v.B.); and the “Eugenia Spanopolou” Immunoprecipitation and Western blotting (to I.A.). Thymocytes from various mice (6.0 ϫ 107) were lysed at a concentration 2 Y.I. and S.A. contributed equally to this study. of 1 ϫ 108 cells/ml in a Triton X-100-containing lysis buffer (20 mM 3 Address correspondence and reprint requests to Dr. Toru Miyazaki, Center for Im- Tris-Cl, pH 7.6, 150 mM NaCl, 2.0 mM EDTA, and protease and phos- munology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard phatase inhibitors). The TCR␨ subunit was specifically immunoprecipi- NA 7200, Dallas, TX 75390-9093. E-mail address: [email protected] tated with an anti-␨ mAb (6B10.2). The immunoprecipitates were separated 4 ␣ ␣ ␤ ␤ on SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bed- Abbreviations used in this paper: pT , pre-TCR ; DP, double positive; 2m, 2- microglobulin; HSA, heat-stable Ag; int␤, intracellular TCR␤; LN, lymph node; SP, ford, MA). Membranes were immunoblotted with anti-phosphotyrosine or single positive; TG, transgenic; WT, wild type. anti-␨ mAb, as previously described (7). Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 4914 PRE-TCR-MEDIATED POSITIVE SELECTION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021 FIGURE 1. Positive selection of CD8ϩ T cells by the pre-TCR. A, The pT␣ expression was markedly enhanced in the thymus (Thy) of pT␣-TG/ TCR␣Ϫ/Ϫ mice, but not in the LN, as assessed by Northern blotting. B, Increased CD3␨ chain phosphorylation under the overexpression of pT␣ in TCR␣Ϫ/Ϫ mouse thymocytes. Lysates of thymocytes from WT, TCR␣Ϫ/Ϫ, and pT␣-TG/TCR␣Ϫ/Ϫ mouse thymi were immunoprecipitated with an anti- CD3␨ mAb and probed sequentially on Western blots with anti-phosphotyrosine mAb 4G10 (top panel) and anti-CD3␨ mAb 6B10.2 (bottom panel). C, The Journal of Immunology 4915 Antibodies to achieve efficiently increased signals through the pre-TCR in ␣ transgenic thymi. Abs used in this study: anti-mouse CD4 (GK1.5), anti-mouse CD8 (53- Ϫ/Ϫ 6.7), anti-mouse TCR␤ chain (H57-597), anti-mouse TCR␥␦ (GL-3), anti- Surprisingly, peripheral LNs of pT␣-TG/TCR␣ mice con- mouse CD8␤ (H35-17.2), anti-pan NK cells (DX-5), anti-Thy-1.2 tained a large number (2.7 ϫ 105 cells in all of collected mesen- (30-H12), anti-mouse CD25 (3C7), anti-mouse CD69 (H1.2F3), anti- teric and inguinal LNs: 4.5% of total LN cells) of CD8ϩ cells that mouse heat-stable Ag (HSA) (30-F ), anti-phosphotyrosine (4G10), and 1 harbored int␤ (Fig. 1C, right). A small, but clearly detectable num- anti-CD3␨ (6B10.2) were purchased from BD PharMingen (San Diego, ␤ϩ ϩ ␣Ϫ/Ϫ CA). The anti-CD3⑀ mAb (145-2C11) was purchased from eBioscience ber of the int CD8 cells also did exist in LNs from TCR 4 (San Diego, CA). Intracellular staining was performed by using the Cyto- mice (1.2 ϫ 10 cells in LNs, 0.2% of total LN cells; Fig. 1C, left).

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