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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

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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

(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-conjugated 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, allophycocyanin-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. Mul- Results timer staining on non-T cells (Thy1.2Ϫ, CD4Ϫ) is shown as a dotted his- The 2C TCR system exhibits CD8-dependent and -independent togram. For the cells stained with A6/Kb multimer in the absence of anti- multimer binding CD8 Abs (gray line, D), a marker (arrow) was introduced to determine the b b To test the role of CD8 in class I MHC multimer binding, we frequency of A6/K multimer low and high populations. A6/K multimer- http://www.jimmunol.org/ positive cells represented 66% of the bulk 2C (Thy1.2ϩ, CD4Ϫ) pool, selected the 2C TCR-transgenic system. These mice generate both ϩ Ϫ ϩ Ϫ Ϫ closely matching the frequency of CD8 2C cells in this sample (70%, data a CD4 CD8 and a CD4 CD8 population of mature T cells not shown). Multimer binding is also shown for 2C cells stained in the bearing the transgenic TCR, allowing for analysis of the role of presence of the anti-CD8 Abs 53-6.7 (B and E) or CT-CD8␣ (C and F), CD8 in the absence of anti-CD8 Abs. Previous reports (3, 6) have gating on the CD8ϩ (open histogram) and CD8Ϫ (filled histogram) popu- b suggested 2C T cells can bind some ligands, including SIY/K lations. B and C, The mean fluorescent intensity for the CD8ϩ and CD8Ϫ multimers, even in the absence of CD8. Accordingly, we find populations is given for comparison. SIY/Kb multimer binds CD8ϩ 2C cells only slightly (but repro- ducibly) more intensely than CD8Ϫ 2C cells (Fig. 1, A–C). SIY/Kb by guest on October 8, 2021 binding to CD8ϩ 2C cells can be enhanced or diminished by anti- CD8 Abs (Fig. 1, A–C), but again, the effects are moderate com- Staining with class I multimers carrying a mutant CD8 binding pared with other CD8 T cells, including the OT-I TCR-transgenic site is not influenced by anti-CD8 Abs model (3, 6). In contrast, binding of SIY/Kb monomers to CD8ϩ vs A central conclusion by Wooldridge et al. (12) was that CD8 Abs CD8Ϫ 2C cell lines was shown to be strongly CD8 dependent (13), might influence multimer binding beyond their direct effects on indicating that our use of SIY/Kb multimers may partially over- CD8-class I interactions. This model was based in part on the come the requirement for CD8. Furthermore, we found that 2C T finding that binding of class I ligands that cannot engage CD8 was cell binding to different peptide/MHC multimers was highly influ- still influenced by anti-CD8 Abs. To test this in our model, we enced by the presence of CD8. An alanine substitution at position constructed multimers in which the Kb molecule was mutated from 6 of the SIY peptide (here called “A6”) does not influence peptide D3K at residue 227. This mutation influences a key binding re- binding to Kb (14) but does impact 2C recognition, the A6/Kb gion between CD8 and class I alleles, and such mutants have been ligand acting as a TCR antagonist for 2C cells (15). Multimers shown to cripple CD8-class I interactions (10, 16, 17). The mutant prepared with this ligand bound to 2C cells in a strictly CD8- SIY/Kb 227K multimers could stain 2C T cells efficiently, but dependent way (Fig. 1, D–F). First, this multimer bound only de- changes in binding were observed (Fig. 2). First, binding of tectably to CD8ϩ 2C cells (Fig. 1, D and E). Although staining in SIY/Kb 227K multimers was virtually identical on both CD8ϩ and the absence of CD8 Abs (gray histogram, Fig. 1D) precludes iden- CD8Ϫ 2C cells. Second, and more importantly for this discussion, tification of the CD8ϩ/CD8Ϫ pools, we note that the frequency of binding of SIY/Kb 227K multimers to CD8ϩ 2C cells was not A6/Kb multimer-binding cells matches the frequency of CD8ϩ 2C influenced by anti-CD8 Abs (Fig. 2). So, although binding of wild- cells (Fig. 1 legend). Second, A6/Kb binding to 2C cells was in- type SIY/Kb multimers to 2C cells was reduced (to a similar ex- fluenced strongly by anti-CD8 Abs, which were enhanced by 53- tent) by anti-CD8 blockade and by lack of CD8 expression, bind- 6.7 and inhibited by CT-CD8␣ (Fig. 1, E and F), in keeping with ing to SIY/Kb 227K multimers was not influenced by either previously reported effects of these Abs (3, 6). Thus, these data expression or blockade of CD8. suggested considerable variation in the contribution of CD8 to It was possible that the CD8 dependence of multimer binding multimer staining of 2C T cells depending on the peptide/MHC was influenced by the dose of multimer used. Use of saturating ligands involved, offering an opportunity to additionally define the doses of multimers might minimize the contribution of CD8 and role of CD8 in multimer binding. mask an effect of the anti-CD8 Abs. To address this, we titrated both wild-type and 227K mutant SIY/Kb multimers on 2C cells in the presence or absence of anti-CD8 Abs. As expected, titration of 3 Abbreviations used in this paper: PE, phycoerythrin; LN, lymph node; FRET, flu- both ligands leads to decreased multimer staining, yet the qualita- orescence resonance energy transfer. tive impact of anti-CD8 Abs was preserved (Fig. 3). Hence, for 3988 CD8 CONTRIBUTION TO CLASS I MULTIMER BINDING

FIGURE 3. Multimer titration does not change the effects of CD8 Abs in SIY/Kb and SIY/Kb 227K multimer binding. 2C cells were stained with SIY/Kb (A–C) or SIY/Kb 227K (D–F) multimers in the presence of 53-6.7 (open histogram) or CT-CD8␣ (shaded histogram). Multimer staining on FIGURE 2. Binding of CD8-null multimer on 2C T cells is not affected non-T cells (Thy1.2Ϫ, CD4Ϫ) is shown as a dotted histogram. Multimers Downloaded from b by anti-CD8 Abs. The SIY/K 227K multimer used to stain 2C LN cells were diluted 1/4 (A and D), 1/32 (B and E), or 1/512 (C and F) relative to binds equally well in the presence or absence of CD8 molecules. A, Mul- the usual concentration (10 ␮g/ml). timer 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 Ϫ Ϫ Abs (gray histogram). Multimer staining on non-T cells (Thy1.2 , CD4 ) of PE and allophycocyanin are in close proximity and suitable is shown as a dotted histogram. Multimer staining in the presence of 53-6.7 ϩ Ϫ orientation, excitation of PE will produce FRET in the allophyco- http://www.jimmunol.org/ (B) or CT-CD8␣ (C) is shown for the CD8 (open histogram) and CD8 (filled histogram) populations. cyanin molecule, which can be detected by flow cytometry as a FL-3 signal, and this approach has been used previously to study peptide/MHC binding to TCR and CD8 (6, 7, 18). As expected, ϩ based on previous reports (6, 7), we could detect a FRET signal SIY/Kb staining 53-6.7 enhanced multimer binding to CD8 2C when PE-labeled SIY/Kb multimers were allowed to bind to 2C cells, whereas multimer staining in the presence of the blocking ϩ Ϫ cells in the presence of allophycocyanin-conjugated 53-6.7 but not CT-CD8␣ Ab was similar on CD8 and CD8 2C cells (Fig. 3, in the presence of allophycocyanin-conjugated CT-CD8␣ (Fig. 4, A–C, and data not shown). In contrast, SIY/Kb 227K multimer top panels), despite the fact that multimer staining of 2C cells was binding to 2C cells was similar regardless of CD8 expression or evident in the presence of either CD8 Ab. The FRET signal was by guest on October 8, 2021 the presence of either CD8 Ab (Fig. 3, D–F, and data not shown). specific to the fluorochrome combination, in that using FITC-con- Thus, these data argue that the dose of multimer does not influence jugated anti-CD8 Abs with PE-multimers did not yield a FL-3 the overall pattern of CD8 requirement in binding to 2C cells. ϩ Ϫ (FRET) signal (data not shown). In contrast to these effects, neither CD8 nor CD8 2C cells Using this system, we could further explore the role of CD8 and were capable of binding to the A6/Kb 227 mutant multimers (data anti-CD8 Abs on multimer binding. Notably, although SIY/Kb not shown). Furthermore, anti-CD8 Abs had no effect on binding 227K multimers bind 2C T cells efficiently (Fig. 2), no FRET of this multimer, consistent with the idea that its engagement was completely dependent on an intact CD8 binding site. Thus, in aggregate, these data are contrary to the model of Wooldridge et al. (12), which predicts that anti-CD8 Abs would influence binding of both CD8-dependent and CD8-independent multimers.

Assessing direct interactions between CD8 and class I MHC multimers The experiments described above show that class I multimer binding is influenced by CD8 expression and anti-CD8 Abs, consistent with a direct role for CD8 in multimer binding. However, additional experiments were needed to define whether CD8 itself engages the multimers. This was a particular concern for the experiments using SIY-containing multimers because SIY/Kb binds only slightly better to CD8ϩ vs CD8Ϫ 2C cells and because SIY/Kb 227 mutant multimers stain 2C cells almost as efficiently as wild-type multimers. Such data raised the question of whether CD8 actually engages SIY/Kb multimers when they are bound to the 2C TCR. FIGURE 4. FRET is observed only between multimers that can associ- Initially, we determined peptide/MHC multimer colocalization ate with CD8. LN cells from a 2C RagϪ/Ϫ mouse were stained with PE- with CD8 using fluorescence resonance energy transfer (FRET) (6, labeled multimers as indicated in the presence of allophycocyanin-labeled 7). The emission spectrum of the fluorochrome PE overlaps the anti-CD8␣ Abs 53-6.7 or CT-CD8␣ as indicated. The y-axis shows the excitation wavelengths for allophycocyanin. Hence, if molecules FRET between PE and allophycocyanin, as detected by a signal in FL3. The Journal of Immunology 3989 signals were induced by this interaction, even in the presence of Participation of CD8 in multimer binding to CTL lines 53-6.7 (Fig. 4, middle panels). It is most likely that the absence of The experiments described above and in our previous studies in- a FRET signal in this case indicates that the multimer and CD8 volved naive T cells, whereas those of Wooldridge et al. (12) used (bound to Ab) are not in close proximity, although the same result long-term CTL lines. It was possible that the role of CD8 in mul- could potentially occur if the orientation of the two molecules was timer binding was altered in naive vs effector CTL, which might changed significantly. These data then suggest that the FRET sig- lead to altered sensitivity to anti-CD8 Abs. nal requires direct interaction between CD8 and the multimer, To test this in our model, we developed long-term 2C T cell rather than reflecting a constitutive bystander association of CD8 lines by regular Ag stimulation in vitro. These cells maintained with the TCR. A weak but reproducible FRET signal was also b expression of CD8 and the 2C TCR, although CD8 expression produced when A6/K multimers were bound to 2C cells in the levels broadened compared with naive 2C T cells (data not shown). presence of 53-6.7, again reflecting the key involvement of CD8 in Importantly, multimer binding to the 2C line showed the same binding to this ligand (Fig. 4, bottom panels, and data not shown). characteristics as we observed for naive 2C cells; binding of Next, we used another approach to test the impact on 2C binding b b b SIY/K and A6/K multimers was enhanced or blocked by anti- of mutating the CD8 binding site on SIY/K multimers. Although b b b CD8 Abs as expected, whereas binding of SIY/K 227K multimers 2C T cells can bind both SIY/K and SIY/K 227K multimers, if was not influenced by anti-CD8 Abs (Fig. 6). Similar results were CD8 bound directly to the multimer, we would expect that the b found when 2C cells were analyzed after one primary in vitro presence of the CD8 binding site on the wild-type K multimers stimulation compared with five rounds of restimulation, suggesting would offer these multimers a competitive advantage in binding to the longevity of the line did not influence the pattern of multimer CD8ϩ 2C cells. Hence, we developed a competition assay, in b b staining (data not shown). Hence, similar to naive 2C T cells, we Downloaded from which PE-SIY/K and allophycocyanin-SIY/K 227K were mixed conclude that multimer binding on the 2C CTL lines was influ- and used to stain 2C T cells. In the presence of the enhancing CD8 enced only by anti-CD8 Abs when the multimer possessed an in- Ab (53-6.7), two populations of multimer-binding cells were ob- tact CD8 binding site. served: one pool (CD8Ϫ 2C cells) bound both multimers, whereas the other population (CD8ϩ 2C cells) bound preferentially to the SIY/Kb multimers (Fig. 5, A and B). A similar result was seen Discussion when no anti-CD8 Ab was used in the staining; although we can- This study was designed to test the role of CD8 in binding to class http://www.jimmunol.org/ not be certain which cells are CD8ϩ vs CD8Ϫ in this case, the I MHC multimers. Our previous reports and those of others (3–6, frequency of cells bound to one vs both multimers indicate a sim- 10, 19) were interpreted to mean that enhancement or blockade of ilar pattern of staining in the presence or absence of the 53-6.7 Ab. multimer binding by CD8 Abs implied participation of CD8 in the These data suggest that, for CD8Ϫ 2C cells, both wild-type and binding. In contrast, a recent report (12) reached the surprising 227K mutant multimers had equal opportunity to stably bind, conclusion that CD8 Abs may be indirectly influencing the ability whereas for CD8ϩ 2C cells, the presence of a CD8 binding site on of TCR to engage peptide/MHC multimers and that CD8-class I wild-type multimers gave them a selective competitive advantage. interactions per se were not required for multimer binding. When blocking CD8 Abs were used in the staining, both CD8ϩ Our findings support the former conclusions, i.e., that CD8 Ab by guest on October 8, 2021 and CD8Ϫ 2C cells bound both multimers equally well (Fig. 5C). effects reflect participation of CD8 in binding to the multimer. We These data suggest that the CD8 interaction directly participates in show this in several ways. First, in our hands, binding of specific stabilizing the SIY/Kb multimer binding to the 2C TCR and that peptide/MHC multimers to CD8ϩ 2C cells in the presence of CD8Ϫ 2C cells behave similarly to CD8ϩ T cells treated with blocking CD8 Abs mimics the binding of these multimers to blocking CD8 Abs. CD8Ϫ T cells. Second, binding of multimers carrying the D227K

FIGURE 5. Competition between SIY/Kb and SIY/Kb 227K multimers for binding to 2C T cells. 2C LN cells were incubated simultaneously with PE-conjugated SIY/Kb 227K and allophycocyanin- conjugated SIY/Kb multimers in the absence of anti- CD8 Abs (A) or presence of FITC-conjugated 53-6.7 (B) or FITC-conjugated CT-CD8␣ (C). The dot plots show staining for both multimers. Cells binding either one or both multimers were gated, and the histograms in B and C indicate multimer staining on CD8ϩ cells (open histogram) or CD8Ϫ cells (shaded histogram). Staining of multimer-negative cells is shown as a dotted histogram. 3990 CD8 CONTRIBUTION TO CLASS I MULTIMER BINDING

also suggested by reports from our group and others describing noncognate binding of CD8 to class I multimers (i.e., interactions in which CD8 but not the TCR bind the peptide/MHC ligand). Such noncognate interactions are strongly influenced by anti-CD8 Abs, and the patterns of enhancement or inhibition by specific anti-CD8 Ab clones are identical for both cognate and noncognate binding (Refs. 3, 11, 21, 22 and our unpublished observations). For noncognate multimer binding, it has been shown that the presence or absence of the TCR is irrelevant (11, 21, 22). Hence, these data additionally support the direct interpretation of how anti-CD8 Abs mediate their effects. In aggregate, our studies were unable to confirm the model proposed by Wooldridge et al. (12), suggesting additional effects of anti-CD8 Abs (beyond the direct effect on CD8-class I MHC interactions) on multimer binding. The basis for the discrepancy between our results and those of Wooldridge et al. (12) is currently unclear. The most obvious concern is that the CD8-null multimers used in the earlier study were, in fact, still able to bind CD8 to

some extent. Functional and biophysical studies argue that the mu- Downloaded from tants used in that (and in this) study show compromised CD8 binding, but the magnitude of the loss in binding is difﬁcult to deter- FIGURE 6. Impact of anti-CD8 Abs on multimer binding to a 2C T cell mine because the afﬁnity of CD8 binding to wild-type class I MHC line. A 2C CTL line was generated and maintained by restimulation for 6 is itself very low. However, such a model is hard to sustain with wk in culture. The cells were stained with SIY/Kb (A), SIY/Kb 227K (B), and A6/Kb (C) multimers in the absence of anti-CD8 Abs (gray histogram) the range of CD8-null multimers used by Wooldridge et al. (12)

or in the presence of 53-6.7 (open histogram) or CT-CD8␣ (filled and is difficult to reconcile with the greater resistance of the CD8- http://www.jimmunol.org/ histogram). null multimers to anti-CD8 Ab blockade reported by that group. The majority of studies reported by Wooldridge et al. (12) involved CTL lines or clones, whereas we focused primarily on naive T cells. As the association between CD8 and TCR could alter mutation in the CD8 binding site is equivalent on both CD8ϩ and with activation, this might be a source of the discrepancy. How- CD8Ϫ T cells and is not affected by anti-CD8 Abs. Third, we ever, we also analyzed long-term 2C CTL lines and concluded observed a FRET signal between anti-CD8 and peptide/MHC mul- that, as for naive cells, CD8-class I interactions contributed di- timers only when the latter was capable of binding to CD8. Fourth, rectly to class I multimer binding (Fig. 6). Another potential ex- a role for CD8 was manifested by the ability of SIY/Kb multimers ϩ perimental difference between our studies is the sequential order by guest on October 8, 2021 to outcompete SIY/Kb 227K multimers for binding to CD8 2C T and incubation temperatures used for staining with anti-CD8 Abs cells, but this competitive advantage was negated by the absence or and multimers. However, using the staining protocol described by blockade of CD8 on the 2C T cells. Wooldridge et al. (12) discuss our previous data in the 2C sys- Wooldridge et al. (12), we still did not reveal additional effects of tem, making the valid point that multimer binding to CD8Ϫ 2C anti-CD8 Abs on multimer binding (data not shown). cells may be difficult to interpret because of the possible impact of Importantly, in the report by Wooldridge et al. (12), the most CD8 on TCR distribution. However, we observed striking parallels clear-cut experiments demonstrating a disconnect between CD8- of binding for D227K mutant multimers binding to 2C cells com- class I binding and anti-CD8 Ab effects on multimer binding in- pared with wild-type multimers binding to either CD8Ϫ cells or volved analysis of human CTL lines. Although mouse CD8 T cells CD8ϩ T cells treated with blocking anti-CD8 Abs. Likewise, nei- were also studied, leading those authors to reach similar conclu- ther CD8Ϫ 2C T cells nor CD8ϩ 2C cells cultured in the presence sions about the indirect effects of anti-CD8 Abs on multimer bind- of the blocking CT-CD8␣ Ab could bind A6/Kb multimers. Hence, ing, Wooldridge pointed out that there are well-defined differences in our studies, analysis of CD8Ϫ 2C T cells was fully consistent in the affinity and dependency for CD8 in human vs mouse sys- with other methods used to test the role of CD8-class I interactions. tems. Therefore, it is certainly possible that studies in the two In additional experiments, we explored the role of CD8 in bind- species cannot be fully reconciled, either due to inherent differing to another TCR-transgenic model system, OT-I, reactive to ences in the nature of CD8-class I binding or of the specific prop- OVA/Kb. As in previous studies, binding of specific multimers to erties of the panels of anti-CD8 Abs available. Hence, our data these CD8ϩ T cells was highly influenced by anti-CD8 Abs (3, 6). should not be taken to contradict the conclusions of Wooldridge et The correlation for a direct role of CD8 binding to class I was al. (12) with respect to the human system. However, our findings supported by the inability of OVA/Kb227K to bind to OT-I T cells do reinforce the model that, in the mouse, the effect of anti-CD8 (our unpublished observations). Likewise, Schott and Ploegh (20) Abs can be interpreted as a direct role of CD8-class I MHC en- reported that OVA/Kb multimers carrying other CD8 binding site gagement. Furthermore, our data do not imply that anti-CD8 Abs mutants (E223K or a Q226L, D227N double mutation) were com- can have no effect other than to alter class I MHC binding. As with pletely unable to bind OT-I T cells. Although the overarching de- the CD4 coreceptor, several studies have demonstrated signaling pendence of CD8 availability for multimer binding in the OT-I events induced by Ab-mediated CD8 cross-linking (12, 23, 24), system ironically limits its usefulness for the current studies, these and recent data suggest such interactions can lead to premature observations are fully consistent with a direct role for CD8-class I death of immature thymocytes (25). Our current studies were de- binding in regulating multimer binding to OT-I T cells. signed to test what effect CD8 plays in multimer binding. How- That anti-CD8 Abs influence multimer binding to CD8 directly, ever, experimental approaches that permit signaling from anti-CD8 rather than mediating an indirect effect on TCR engagement, is Ab engagement may well alter multimer binding by additional The Journal of Immunology 3991 means, and this should be considered in design of experiments CD8␣-MHC class I interaction indicates antibody stabilization of a higher affinity using multimer staining approaches. CD8 conformation. Immunol. Lett. 93:123. 12. Wooldridge, L., S. L. Hutchinson, E. M. Choi, A. Lissina, E. Jones, F. Mirza, P. R. Dunbar, D. A. Price, V. Cerundolo, and A. K. Sewell. 2003. Anti-CD8 Acknowledgments antibodies can inhibit or enhance peptide-MHC class I (pMHCI) multimer binding: this is paralleled by their effects on CTL activation and occurs in the absence We thank the members of the Jameson/Hogquist labs for their valuable of an interaction between pMHCI and CD8 on the cell surface. J. 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