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3986.Full.Pdf Characterizing the Impact of CD8 Antibodies on Class I MHC Multimer Binding Philmore O. Holman, Elizabeth R. Walsh and Stephen C. Jameson This information is current as of October 8, 2021. J Immunol 2005; 174:3986-3991; ; doi: 10.4049/jimmunol.174.7.3986 http://www.jimmunol.org/content/174/7/3986 Downloaded from References This article cites 25 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/174/7/3986.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 October 8, 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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Characterizing the Impact of CD8 Antibodies on Class I MHC Multimer Binding1 Philmore O. Holman, Elizabeth R. Walsh, and Stephen C. Jameson2 Many studies have suggested that CD8 Abs affect the binding of class I MHC tetramers/multimers to CD8؉ T cells, which has led to the interpretation that CD8 participates directly in multimer binding. In contrast, a recent publication has argued that CD8 Abs instead cause reorganization of TCR distribution and hence have an indirect effect on multimer binding to the TCR alone. We address these issues by testing the role of CD8 and the impact of CD8 Abs on the binding of normal and mutant multimers to Ag-specific mouse T cells. Our data suggest that, in this system, CD8 Abs act directly on CD8 and only mediate their effects on multimer binding when CD8 is capable of binding to the multimer. These data reinforce the paradigm that CD8 plays an active and direct role in binding of class I MHC multimers. The Journal of Immunology, 2005, 174: 3986–3991. eptide/MHC multimers have revolutionized the capacity to I multimer binding, CD8 Ab binding was influencing the ability of Downloaded from identify Ag-specific T cells. Because of their high valency, the TCR to engage the multimer. They went on to propose that, P multimers can compensate for the inherent low affinity of although some of these effects could be mediated by steric hin- the TCR-peptide/MHC interactions and allow detection of specific drance, a more likely model was that anti-CD8 Abs were causing T cells by flow cytometry and other techniques. changes in TCR distribution on the cell surface, leading to altered Initial studies on CD4 T cells indicated that peptide/MHC class multimer binding (12). These data raised serious doubts about the II tetramer binding occurred independently of the coreceptor. Al- proposed role for direct CD8 participation in multimer binding and http://www.jimmunol.org/ though CD4 was found to be critical for responses induced by also the validity of using anti-CD8 Abs to explore such a role. tetramers, the tetramer binding per se was equally efficient whether In this study, we revisited the role of CD8 in multimer binding, or not CD4 was available (1, 2). In contrast, we and others (3–9) using the 2C TCR-transgenic system, in which both CD8-depen- argued that binding of class I MHC multimers was influenced dent and -independent multimer binding can be analyzed. We stud- strongly by the participation of CD8, consistent with earlier data ied CD8ϩ and CD8Ϫ 2C T cells and the effects of various CD8 using monomeric class I MHC ligands (10). The requirement for Abs on binding of specific class I MHC tetramers possessing nor- CD8 in multimer binding varied with the particular TCR-peptide/ mal vs mutant CD8 binding sites. These data indicate that CD8 MHC combination from mild to absolute, in keeping with previous actively participates in tetramer binding and that CD8 Abs only functional studies, which have suggested CD8-dependent and -in- impact tetramer binding when CD8 is capable of engaging the by guest on October 8, 2021 dependent T cell interactions. However, in nearly all cases, some class I ligand. These findings were consistent over various multi- role for CD8 in multimer binding could be observed. mer staining conditions and held for both naive T cells and CTL For the most part, the role for CD8 in multimer binding was lines. Hence, these data support the model that CD8 binding di- tested using Abs to CD8. Intriguingly, some Abs were found to rectly contributes to class I multimer binding. inhibit or enhance multimer binding (3–9). Inhibitory Abs might be expected to occlude class I MHC binding sites, although it has Materials and Methods been proposed that enhancing Abs mediate their effects by stabi- Mice lizing higher affinity conformations of CD8 (11). Ϫ Ϫ 2C TCR-transgenic mice were maintained on a B6 or a Thy1.1,Rag-1 / However, these interpretations have been challenged by recent background under specific pathogen-free conditions at the University of data from Wooldridge et al. (12). These authors presented intrigu- Minnesota and were used in accordance with Institutional Animal Care and ing data that argued that CD8 Abs have dramatic effects on pep- Use Committee guidelines. tide/MHC multimer binding even when the multimer was not ex- 2C cell line pected to engage CD8. Thus, using class I MHC multimers bearing crippling mutations in key CD8 binding sites, they were still able 2C splenocytes were stimulated with irradiated (1500 cGy) BALB/c to observe enhancement and inhibition of tetramer binding by anti- splenocytes in 24-well tissue culture plates. Recombinant human IL-2 (Te- cin, supplied by the Biological Resources Facility, National Cancer Insti- CD8 Abs when tested on human and mouse CTL lines. These tute/National Institutes of Health) was added every 3–4 days to a final authors suggested that, rather than directly influencing CD8-class concentration of 500 U/ml. The line was restimulated weekly with fresh irradiated BALB/c splenocytes and IL-2. Peptides and multimers Center for Immunology and Department of Laboratory Medicine and Pathology, Uni- versity of Minnesota, Minneapolis, MN 55455 The peptides SIY (SIYRYYGL) and A6 (SIYRYAGL) were obtained from b Received for publication October 19, 2004. Accepted for publication January 7, 2005. Research Genetics and Invitrogen Life Technologies. The K 227K mutant was generated by site directed mutagenesis of the Kb-bsp plasmid (a kind The costs of publication of this article were defrayed in part by the payment of page gift from J. Altman, Emory University (Atlanta, GA)) using the Quick charges. This article must therefore be hereby marked advertisement in accordance change kit according to the manufacturer’s protocol (Stratagene). Primers with 18 U.S.C. Section 1734 solely to indicate this fact. usedwere5Ј-AATGGGGAGGAGCTGATCCAGAAGATGGAGCTTGTG 1 This work was supported by National Institutes of Health Grant AI52163 (to S.C.J.). GAGACC-3Ј and 5Ј-GGTCTCCACAAGCTCCATCTTCTGGATCAGCT 2 Address correspondence and reprint requests to Dr. Stephen C. Jameson, MMC 334, CCTCCCC-3Ј (nucleotides changed are italicized). Monomers containing 420 Delaware Street SE, Minneapolis, MN 55455. E-mail address: [email protected] either SIY or A6 were generated and biotinylated as described previously Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 3987 (3). Refolded MHC monomers were purified by size exclusion, concen- trated to 1 mg/ml, dialyzed against water, and stored in 25 ␮l of aliquot at Ϫ70°C. Multimers were made fresh before use by mixing an equal concentration of purified monomers and streptavidin:phycoerythrin (PE)3 or streptavidin:allophycocyanin (Molecular Probes), followed by incubation at room temperature for at least 1 h. Multimers were used individually at a final concentration of 10 ␮g/ml or as indicated in the figure legend. Flow cytometry Naive lymph node (LN) cells (0.15–1 ϫ 106) and a 2C line (0.15 ϫ 106) were preincubated on ice with an anti-CD16/32 Ab mixture (Fc block) (eBioscience) for 10 min in FACS buffer (PBS, 1% FCS, 0.02% sodium azide) before costaining with multimers and Abs for 1 h. The FITC-con- jugated anti-CD8␣ Ab 53-6.7 (BD Biosciences or BioLegend) and CT- CD8␣ (Caltag Laboratories) were used at 5 ␮g/ml for enhancing and blocking multimer staining, respectively. In some experiments, allophyco- cyanin-conjugated anti-CD8␣ Abs were used for enhancing (53-6.7; eBio- science) and blocking (CT-CD8␣; Caltag Laboratories) multimer staining. FIGURE 1. CD8-dependent and -independent multimer binding on na- Allophycocyanin-conjugated Thy1.1 or Thy1.2, FITC-conjugated Thy1.2 ive 2C T cells. The impact of anti-CD8 Abs on binding of SIY/Kb (A–C) (eBioscience), and PerCP-CD4 (BD Biosciences) were used for identifying or A6/Kb (D–F) multimers to CD8ϩ and CD8Ϫ 2C T cells was determined. T cells. Data was collected on a BD Biosciences FACSCalibur or LSRII 2C LN cells were incubated with multimers alone or together with the Downloaded from instrument and analyzed using FlowJo (Tree Star). indicated anti-CD8 Abs. A and D, Multimer staining on bulk 2C T cells (Thy1.2ϩ, CD4Ϫ) in the presence of 53-6.7 (open histogram), CT-CD8␣ (filled histogram), or absence of CD8 Abs (gray histogram) is shown.
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