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Multiplexed combinatorial tetramer staining in a mouse model of virus infection
Journal of Immunological Methods 360 (2010) 157–161
Contents lists available at ScienceDirect
Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Technical Note
Multiplexed combinatorial tetramer staining in a mouse model of virus infection Tania Cukalac a,1, Sophie A. Valkenburg a,1, Nicole L. La Gruta a, Stephen J. Turner a, Peter C. Doherty a,b, Katherine Kedzierska a,⁎ a b
Department of Microbiology and Immunology, University of Melbourne, Parkville 3010, Melbourne, Australia Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA
a r t i c l e
i n f o
Article history: Received 12 April 2010 Received in revised form 2 June 2010 Accepted 8 June 2010 Available online 15 June 2010 Keywords: T cells Cytotoxic T cells pMHC tetramers Viral infection Mouse B6 model
a b s t r a c t Use of fluorescently labelled multimers, particularly tetramers of peptide and MHC class I glycoprotein (pMHC-I) complexes, is essential for the analysis of CD8+ T cell immunity in basic research and clinical settings. A recently described combinatorial approach using pMHC-I multimers coupled to a unique combination of distinct fluorochromes has facilitated the simultaneous screening of multiple T cell specificities within a single human blood sample. The present analysis establishes that this multiplexed tetramer staining protocol can also be applied in mouse models of a disease to detect multiple subdominant CD8+ T cell specificities in the presence of prominent immunodominant T cell sets at different stages of infection. We have established a modified protocol that concurrently identified influenza-specific CD8+ T cells at the acute and long-term + memory phases of influenza virus infection in B6 mice. Highly dominant (DbNP+ and 366CD8 + + + + b + b + b + b + D PA224CD8 ) and subdominant (K PB1703CD8 , D PB1-F262CD8 and K NS2114CD8 ) T cell responses can be detected simultaneously at levels comparable to the conventional tetramer staining with this combinatorial approach. The technique proved particularly useful with aged mice, where we used 5-fold fewer animals, making the detection of multiple T cell specificities more costeffective and less time-consuming. Overall, our study establishes that this comprehensive concurrent analysis of multiple T cell specificities is of value for analysing mouse models of disease, especially in situations where sample size and/or response magnitude is limiting. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Virus-specific CD8+ T cells play an essential part in limiting infectious process by killing virus-infected cells and by producing pro-inflammatory cytokines (Doherty et al., 2006). CD8+ T cell recognition of viral peptides is mediated through T cell receptor (TCR) interactions with viral peptide (p) bound to host Major Histocompatibility Complex class I molecules (pMHC-I epitope). In general, such CD8+ T cell-mediated immunity tends to be
Abbreviations: H, viral hemagglutinin molecule; N, viral neuraminidase; NP, influenza nucleoprotein; PA, influenza acid polymerase; NS, nonstructural; PB1, polymerase, i.n., intranasally; HK-X31 H3N2, influenza A virus. ⁎ Corresponding author. Tel.: + 61 3 8344 7062; fax: + 61 3 8344 7990. E-mail address: [email protected] (K. Kedzierska). 1 Authors contributed equally to the study. 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.06.003
directed at one or two prominent (immunodominant) epitopes and a greater number of subdominant determinants (Yewdell and Bennink, 1999). Understanding protective efficacy, homeostasis and memory for both immunodominant and subdominant T cell immunity is critical for the rational design of T cellmediated vaccines and immunotherapies. Historically, a number of assays have been used to detect and enumerate CD8+ T cell populations. Early experiments utilised Chromium51 release assay that provided a semi-quantitative measurement of CD8+ cytotoxic lymphocyte (CTL) activity, while the micro-cloning (limiting dilution assay) was a labour intensive, technically challenging technique that measured only some CTL precursors and underestimated the overall number of antigen-specific T cells by up to 10-fold. More accurate quantitation was achieved with short-term peptide re-stimulation assays (ICS and ELISPOT) to measure IFN-γ production by
158
T. Cukalac et al. / Journal of Immunological Methods 360 (2010) 157–161
antigen-specific T cells, though this approach does not allow to probe the functional status of cells recovered directly ex vivo (Doherty and Christensen, 2000). The revolution in quantification and characterisation of epitope-specific CD8+ T cell responses came with the development use of pMHC-I tetramer approach (Altman et al., 1996) that allowed immunologists to analyse viable antigen-specific CD8+ T cells directly ex vivo, a technical advance that has transformed the field of T cell immunology. The multimerization of biotinylated MHC-I molecules loaded with peptide was accomplished using fluorescently labelled streptavidin and allowed both qualitative and quantitative analysis of viable T cell populations at a phenotypic and functional level. However, despite spectacular advances in fluorochrome technology and flow cytometric detection, multimers have been used for the simultaneous detection of relatively few T cell specificities. Recently, two groups developed a combinatorial approach to facilitate the application of tetramer technology for the concurrent dissection of multiple human T cell specificities in a single blood sample (Hadrup et al., 2009; Newell et al., 2009; Kedzierska et al., 2009). The technique uses different pMHC-I multimers coupled to a unique combination of distinct fluorochromes, with the same fluorochrome being used on different tetramers in the presence (or absence) of additional fluorochrome “signatures”. Such multiplexed tetramer staining offers clear advantages for human immunology, as it allows the detection of multiple distinct T cell specificities where specimen size is limited (∼10 to 50 ml of blood). Many of the advances in our understanding of T cellmediated immunity have come from mouse disease models. Any defect in sample size is generally overcome by using multiple mice. However in case of rare strains of mice, particularly those that breed poorly, or aged animals where few are available, sample size or mouse numbers can be a limiting factor. Thus, a combinatorial tetramer staining approach for detecting multiple T cell specificities simultaneously would be a great advantage in such situations. However, there could be a problem if immunodominant T cell responses, especially those at acute phases of infection (sometimes N20% of all CD8+ T cells) overwhelm the subdominant T cell sets. In the present study, we have used a protocol modified from the human combinatorial approach (Hadrup et al., 2009; Newell et al., 2009) to detect multiple influenza-specific CD8+ T cells at the acute and long-term memory phases of infection in B6 mice. + We have shown that both highly dominant (DbNP+ 366CD8 and + b + b + DbPA+ CD8 ) and subdominant (K PB1 CD8 , D PB1224 703 + b + + + F262CD8 and K NS2114CD8 ) T cell responses can be detected simultaneously using this combinatorial approach at levels comparable to those detected by conventional tetramer staining. The technique provides a particular advantage for aged mice, where a comprehensive analysis utilised 5 animals instead of 25, making the detection of multiple T cell specificities more cost-effective and less time-consuming. 2. Materials and methods 2.1. Mice and virus infections The female C57BL/6J (H-2b) mice used in this study were bred and housed in the animal facility of the Department of Microbiology and Immunology at the University of Melbourne
(Parkville, Australia). Naïve 6- to 8-week-old mice were anesthetized by methoxyfluorane inhalation and infected intranasally (i.n.) with 1 × 104 PFU of the HK× 31 (x31, H3N2) influenza A virus in 30 μl of PBS. All animal studies were in compliance with the guidelines set out by the University of Melbourne Animal Experimental Ethics Committee. 2.2. Tissue sampling and preparation The spleen and bronchoalveolar lavage (BAL) (from 4 ml washing of the lungs in HBSS) was sampled at acute time points of infection (10 days post infection), while only the spleen was isolated at the memory time point. Spleens were homogenised using a plunger and 70 μm sieves and washed twice in HBSS to give single cell suspensions. Splenocytes were depleted of B cells by incubation on αIgG/IgM coated plates (Jackson ImmunoResearch Labs) for 45 min at 37 °C, and the BAL was depleted of macrophages by adherence to plastic for 45 min at 37 °C. 2.3. Combinatorial tetramerisation Monomers of DbNP366-374 (ASNENMETM), DbPA224-233 (SSLENFRAYV), KbPB1703-711 (SSYRRPVGI), DbPB1-F262-70 (LSLRNPILV), and KbNS2114-121 (RTFSFQLI) (purchased from the Monomer Facility, University of Melbourne) were conjugated to combinations of fluorescently labelled Streptavidins (SA) (eBiosciences) at equimolar ratios according to calculations from Newell et al. (Newell et al., 2009). Briefly, fluorescently labelled SA were mixed (according to the desired combinations), the volume adjusted with PBS to a total volume of 90 μl, then slowly added (a tenth of the SA mix volume added every 10 min) to 10 μg (1 mg/ml) monomer at room temperature (refer to Supplementary Table 1 for volumes). To quench any unconjugated SA, D-Biotin (Sigma) was added to give a final concentration of 26.4 μM for each tetramer, incubated on ice for 20 min and centrifuged at 17,000 ×g for 2 min, then the tetramer supernatant used for staining as described previously (Hadrup et al., 2009). 2.4. Tetramer and antibody surface stain Lymphocytes from BAL and spleen taken at acute and memory time points were stained with a cocktail of tetramers (DbNP366-APC, DbPA224-APC/PECy7, KbPB1703-APC/PE, DbPB1F262-PECy7 and KbNS2114-PE), with each at 3.5 μg/ml based on the original monomer concentration, that is, each tetramer was used at a 1:100 dilution (a saturating concentration) in FACS buffer (1% BSA, 0.02% Sodium Azide in PBS) for 1 h at room temperature in the dark. To determine the efficiency of the combinatorial tetramer staining protocol, splenocytes taken at day 10 after primary infection were also separately stained with each tetramer labelled with PE (at 1.75 μg/ml based on original monomer concentration). Each tetramer was used at concentration determined to be a saturating concentration by titration. Following the tetramer stain, cells were washed twice in FACS buffer and stained with antiCD8α-PerCP-Cy5.5 (1 μg/ml) in FACS buffer for 20 min on ice. Cells were then washed twice with FACS buffer and acquired by flow cytometry on a LSRII (BD Biosciences) and analysed by Flowjo software (Treestar).
T. Cukalac et al. / Journal of Immunological Methods 360 (2010) 157–161
3. Results and discussion In vivo infection of mice can result in immunodominant responses comprising as many as ∼20% of CD8+ T cells at acute time points after infection. Concurrently, much smaller CD8+ T cell responses are generated toward a spectrum of subdominant determinants. Recent studies have established that the simultaneous detection of multiple T cell specificities is feasible for human peripheral blood lymphocytes (PBL) (Hadrup et al.,
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2009; Newell et al., 2009), so we investigated whether this approach might also be used to detect multiple murine CD8+ T cells. The concern in this case was that the presence of highly immunodominant CD8+ T cell sets would overwhelm the system and thereby prevent detection of the subdominant specificities. The present study thus analysed whether the multiplexed combinatorial technique could be applied for the simultaneous detection of influenza-specific immunodominant + + (D b NP + and D b PA + 366 CD8 224 CD8 ) and subdominant
Fig. 1. Flow cytometry-gating scheme for three-color combinatorial pMHC tetramer staining. Representative flow cytometry dot plots of the gating scheme for CD8+ T cells from the spleens of B6 mice infected i.n. 10 days previously with the x31 influenza A virus are shown. The splenocytes were stained with a cocktail of tetramers specific for DbNP366-APC, DbPA224-APC/PECy7, KbPB1703-APC/PE, DbPB1-F262-PECy7, and KbNS2114-PE and a CD8 mAb, anti-CD8α-PerCP-Cy5.5. Cells were first gated based on forward versus side scatter (A), before doublets were removed using the width vs. area signal of forward scatter (B) and the CD8+ T cells were selected (C). The CD8+ T cells were then gated on each of the three tetramer-conjugated fluorochromes separately (D, E, F) to allow for subsequent analysis of tetramer staining. Gating of cells double positive for PerCP-Cy5.5 and APC (D) followed by separation into four populations based on PECy7 and PE staining (G) allowed the detection of DbNP366-, DbPA224- and KbPB1703-specific CD8+ sets. To detect KbNS2114- and KbPB1703-specific populations, cells double positive for PerCP-Cy5.5 and PE were first gated (E), and were then separated into four populations based on PECy7 and APC staining (H). Cells double positive for PerCP-Cy5.5 and PECy7 (F) then separated into four sets based on APC and PE staining (I), allowing the detection of DbPA224- and DbPB1-F262-specific CD8+ T cells. (J) Epitope-specific “signatures” defines by different staining combinations are shown.
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+ b + + + (KbPB1+ and KbNS2+ 703CD8 , D PB1-F262CD8 114CD8 ) at the peak (d10) of the response or in long-term memory (d210) after influenza virus infection. Our approach combined two recently described methods (Hadrup et al., 2009; Newell et al., 2009). Monomers expressing DbNP366–374, DbPA224–233, KbPB1703–711, DbPB1F262–70 and KbNS2114–121 were conjugated to various fluorescently labelled Streptavidins (SA) at equimolar ratios (Newell et al., 2009) (refer to Supplementary Table 1 for fluorophores and volumes). D-Biotin was used to quench any unconjugated streptavidin (Hadrup et al., 2009). This method combined an easily accessible streptavidin conjugated system for pMHC-I tetramerisation together with a rapid technique for removal of any unbound pMHC-I complexes. The validity of the combinatorial technique and representative flow cytometry dot plots of the gating scheme for CD8+ T cells isolated from spleen of mice infected i.n. with influenza virus is shown in Fig. 1. Splenocytes were stained with a cocktail of pMHC-I tetramers specific for DbNP366-APC, DbPA224-APC/ PECy7, KbPB1703-APC/PE, DbPB1-F262-PECy7 and KbNS2114-PE, followed by anti-CD8α-PerCP-Cy5.5. Single CD8+ T lymphocytes were identified by FSC versus SSC, FSC-A versus FSC-W characteristics and anti-CD8 mAb attaining (Fig. 1A–C). Each epitope-specific CD8+ T cell population was then identified based on the characteristic-staining signature assigned (Fig. 1G–J). CD8+ T cells that were positive for APC (Fig. 1D), followed by separation into four populations based on PECy7 and PE staining (Fig. 1G), which allowed detection of the DbNP366-, DbPA224- and KbPB1703-specific CD8+ T cells. To identify KbNS2114- and KbPB1703-specific CD8+ T cells, CD8+ T cells positive cells for PE were first gated on PE (Fig. 1E), then
separated into four populations based on PECy7 and APC staining (Fig. 1H). CD8+ T cells positive PECy7 (Fig. 1F) were separated into four populations based on APC and PE staining (Fig. 1I) to detect DbPA224- and DbPB1-F262-specific CD8+ T cells. Analysis of two immunodominant and three subdominant CD8+ T cell responses at the peak of influenza virus infection using the conventional and combinatorial tetramer method showed equivalent frequency of tetramer staining at the site of infection (BAL; Fig. 2A) and spleen (Fig. 2B). Importantly, both dominant and subdominant epitope-specific CD8+ T cell sets were detected with equivalent efficiency despite the 7– 8-fold difference between the largest and smallest responses detected within the one sample (Fig. 2AB). Mean fluorescence intensity (MFI) of tetramer staining was of lower intensity for tetramers that were conjugated to N1 fluorochrome (e.g. KbPB1703–APC/PE combination) in comparison to the singletetramer staining (KbNS2114-PE). This was evident in both the BAL and in the spleen (Fig. 2C) and reflects the fact that only half as much of any one tetramer can bind to its epitopespecific population. Importantly, this anticipated reduction in MFI had no effect on the ability to detect all of the epitope-specific populations (Fig. 2AB). Similar findings were made for combinatorial tetramer staining within human PBL (Hadrup et al., 2009; Newell et al., 2009). Our study is based on a mouse model of viral infection that uses relatively high affinity TCRs. As the weaker affinity TCRs might ‘smear’ when combining fluorochromes, we suggest that single-signature stains are used for staining the weak affinity repertoires, whereas double-signature stains are used for staining of high affinity repertoires.
Fig. 2. Comparable staining with single pMHC tetramer complex vs. combinatorial pMHC tetramer complexes for influenza-specific epitopes. Naïve B6 mice were infected i.n. with x31 influenza virus and BAL (A, C) and spleen (B, C) were harvested 10 days later for analysis of CD8+ DbNP366, DbPA224, KbPB1703, DbPB1-F262, and KbNS2114-specific T cell responses. Shown are (A, B) the mean percentage of CD8+tetramer+ cells ± SD; (C) the mean fluorescence intensity (MFI) ±SD of PEtetramer staining for a single-stained tetramer (KbNS2114-PE, no difference in MFI) versus doubly-labelled tetramer (KbPB1703-PE/APC, decreased MFI) (n = 5 mice per tetramer at each time point).
T. Cukalac et al. / Journal of Immunological Methods 360 (2010) 157–161
Having verified the combinatorial tetramer staining technique, we assessed the sensitivity of detection for five different specificities of influenza-specific CD8+ T cells in acutelyinfected (d10; Fig. 3A) mice and aged (long-term memory, 7 months after influenza infection; Fig. 3B) animals known to contain very small populations of epitope-specific CD8+ T cells. Looking at CD8+ populations detected by the combinatorial tetramer approach at the acute (d10) phase of primary + + influenza virus infection, the DbNP+ and DbPA+ 366CD8 224CD8 populations dominated in a numerical sense and were markedly b higher than the expansions of subdominant KbPB1+ 703, D PB1b + + F2+ and K NS2 CD8 populations (Fig. 3A). Simultaneous 62 114 detection of mouse influenza-specific CD8+ T cell specificities in the long-term memory (∼d210) showed ∼10–20-fold decrease in population size when compared to the acute response. Interestingly, memory profiles showed comparable numbers of + b + b + b + + + DbPA+ 224CD8 , K PB1703CD8 , D PB1-F262CD8 and K NS2114 + CD8+ T cells, although DbNP+ set remained at ∼2× 366CD8 higher frequency (Fig. 3B). This lower proportions of memory CD8+ T cells recognising immunodominant responses have been noted frequently for a lesser spectrum of epitopes (Kedzierska et al., 2006), but the use of the combinatorial approach has now allowed us now to extend this observation for a much broader spectrum of epitopes (Fig. 3CD). A possible explanation for such enhanced survival capacity of subdominant CD8+ T cell populations could be related to the lower activation level and/or expansion of those cells consequent to their incomplete recruitment into the immune response (La Gruta et al., 2010).
+ Fig. 3. Immunodominance of influenza-specific DbNP+ and DbPA+ 366CD8 224 CD8+ T cell populations decreases with progression to long-term memory. Following primary infection with x31 influenza virus, splenocytes were stained with a cocktail of tetramers specific for DbNP366, DbPA224, KbPB1703, DbPB1-F262, and KbNS2114, and anti-CD8 mAb at 10 days (A, C), or 7 months after infection (B, D). Data represent (A, B) the absolute number of tetramer positive lymphocytes mean ± SD (n = 5) and (C, D) the average proportion tetramer+ of CD8+ T cells.
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Taken together, our study has established that the multiplexed combinatorial tetramer staining is feasible in a mouse model of a disease in which the range of CD8+ T cell response magnitude is relatively broad. The technique was subsequently used to assess the relationship between the immunodominant and subdominant influenza-specific CD8+ T cell sets at acute and long-term memory. Acknowledgments We thank Drs Mark Davis and Evan Newell for helpful suggestions and University of Melbourne 526-326 students of 2009 for technical assistance. This work was supported by an Australian National Health and Medical Research Council (NHMRC) Project Grant 454312 awarded to KK. KK and NLG are NHMRC RD Wright Research Fellows and SJT is an Australian Pfizer Research Fellow. SAV is a recipient of an Australian Postgraduate Award, TC is a recipient of the NHMRC Biomedical Postgraduate Scholarship (ID 520643). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jim.2010.06.003. References Altman, J.D., Moss, P.A., Goulder, P.J., Barouch, D.H., McHeyzer-Williams, M.G., Bell, J.I., McMichael, A.J., Davis, M.M., 1996. Phenotypic analysis of antigenspecific T lymphocytes. Science 274, 94. Doherty, P.C., Christensen, J.P., 2000. Accessing complexity: the dynamics of virus-specific T cell responses. Annu. Rev. Immunol. 18, 561. Doherty, P.C., Turner, S.J., Webby, R.G., Thomas, P.G., 2006. Influenza and the challenge for immunology. Nat. Immunol. 7, 449. Hadrup, S.R., Bakker, A.H., Shu, C.J., Andersen, R.S., van Veluw, J., Hombrink, P., Castermans, E., Thor Straten, P., Blank, C., Haanen, J.B., Heemskerk, M.H., Schumacher, T.N., 2009. Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers. Nat. Meth. 6, 520. Kedzierska, K., Venturi, V., Field, K., Davenport, M.P., Turner, S.J., Doherty, P.C., 2006. Early establishment of diverse TCR profiles for influenza-specific CD62Lhi CD8+ memory T cells. Proc. Natl. Acad. Sci. U. S. A. 103, 9184. Kedzierska, K., Stambas, J., Doherty, P.C., 2009. Finding multiple needles in one immune haystack. Nat. Meth. 6, 489. La Gruta, N., Rothwell, W., Cukalac, T., Swan, N., Valkenburg, S., Kedzierska, K., Thomas, P., Doherty, P., Turner, S., 2010. Primary CTL response magnitude in mice is determined by the extent of naïve T cell recruitment and subsequent clonal expansion. J. Clin. Invest. 120, 1885. Newell, E.W., Klein, L.O., Yu, W., Davis, M.M., 2009. Simultaneous detection of many T-cell specificities using combinatorial tetramer staining. Nat. Meth. 6, 497. Yewdell, J.W., Bennink, J.R., 1999. Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu. Rev. Immunol. 17, 51.
Contents lists available at ScienceDirect
Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Technical Note
Multiplexed combinatorial tetramer staining in a mouse model of virus infection Tania Cukalac a,1, Sophie A. Valkenburg a,1, Nicole L. La Gruta a, Stephen J. Turner a, Peter C. Doherty a,b, Katherine Kedzierska a,⁎ a b
Department of Microbiology and Immunology, University of Melbourne, Parkville 3010, Melbourne, Australia Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA
a r t i c l e
i n f o
Article history: Received 12 April 2010 Received in revised form 2 June 2010 Accepted 8 June 2010 Available online 15 June 2010 Keywords: T cells Cytotoxic T cells pMHC tetramers Viral infection Mouse B6 model
a b s t r a c t Use of fluorescently labelled multimers, particularly tetramers of peptide and MHC class I glycoprotein (pMHC-I) complexes, is essential for the analysis of CD8+ T cell immunity in basic research and clinical settings. A recently described combinatorial approach using pMHC-I multimers coupled to a unique combination of distinct fluorochromes has facilitated the simultaneous screening of multiple T cell specificities within a single human blood sample. The present analysis establishes that this multiplexed tetramer staining protocol can also be applied in mouse models of a disease to detect multiple subdominant CD8+ T cell specificities in the presence of prominent immunodominant T cell sets at different stages of infection. We have established a modified protocol that concurrently identified influenza-specific CD8+ T cells at the acute and long-term + memory phases of influenza virus infection in B6 mice. Highly dominant (DbNP+ and 366CD8 + + + + b + b + b + b + D PA224CD8 ) and subdominant (K PB1703CD8 , D PB1-F262CD8 and K NS2114CD8 ) T cell responses can be detected simultaneously at levels comparable to the conventional tetramer staining with this combinatorial approach. The technique proved particularly useful with aged mice, where we used 5-fold fewer animals, making the detection of multiple T cell specificities more costeffective and less time-consuming. Overall, our study establishes that this comprehensive concurrent analysis of multiple T cell specificities is of value for analysing mouse models of disease, especially in situations where sample size and/or response magnitude is limiting. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Virus-specific CD8+ T cells play an essential part in limiting infectious process by killing virus-infected cells and by producing pro-inflammatory cytokines (Doherty et al., 2006). CD8+ T cell recognition of viral peptides is mediated through T cell receptor (TCR) interactions with viral peptide (p) bound to host Major Histocompatibility Complex class I molecules (pMHC-I epitope). In general, such CD8+ T cell-mediated immunity tends to be
Abbreviations: H, viral hemagglutinin molecule; N, viral neuraminidase; NP, influenza nucleoprotein; PA, influenza acid polymerase; NS, nonstructural; PB1, polymerase, i.n., intranasally; HK-X31 H3N2, influenza A virus. ⁎ Corresponding author. Tel.: + 61 3 8344 7062; fax: + 61 3 8344 7990. E-mail address: [email protected] (K. Kedzierska). 1 Authors contributed equally to the study. 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.06.003
directed at one or two prominent (immunodominant) epitopes and a greater number of subdominant determinants (Yewdell and Bennink, 1999). Understanding protective efficacy, homeostasis and memory for both immunodominant and subdominant T cell immunity is critical for the rational design of T cellmediated vaccines and immunotherapies. Historically, a number of assays have been used to detect and enumerate CD8+ T cell populations. Early experiments utilised Chromium51 release assay that provided a semi-quantitative measurement of CD8+ cytotoxic lymphocyte (CTL) activity, while the micro-cloning (limiting dilution assay) was a labour intensive, technically challenging technique that measured only some CTL precursors and underestimated the overall number of antigen-specific T cells by up to 10-fold. More accurate quantitation was achieved with short-term peptide re-stimulation assays (ICS and ELISPOT) to measure IFN-γ production by
158
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antigen-specific T cells, though this approach does not allow to probe the functional status of cells recovered directly ex vivo (Doherty and Christensen, 2000). The revolution in quantification and characterisation of epitope-specific CD8+ T cell responses came with the development use of pMHC-I tetramer approach (Altman et al., 1996) that allowed immunologists to analyse viable antigen-specific CD8+ T cells directly ex vivo, a technical advance that has transformed the field of T cell immunology. The multimerization of biotinylated MHC-I molecules loaded with peptide was accomplished using fluorescently labelled streptavidin and allowed both qualitative and quantitative analysis of viable T cell populations at a phenotypic and functional level. However, despite spectacular advances in fluorochrome technology and flow cytometric detection, multimers have been used for the simultaneous detection of relatively few T cell specificities. Recently, two groups developed a combinatorial approach to facilitate the application of tetramer technology for the concurrent dissection of multiple human T cell specificities in a single blood sample (Hadrup et al., 2009; Newell et al., 2009; Kedzierska et al., 2009). The technique uses different pMHC-I multimers coupled to a unique combination of distinct fluorochromes, with the same fluorochrome being used on different tetramers in the presence (or absence) of additional fluorochrome “signatures”. Such multiplexed tetramer staining offers clear advantages for human immunology, as it allows the detection of multiple distinct T cell specificities where specimen size is limited (∼10 to 50 ml of blood). Many of the advances in our understanding of T cellmediated immunity have come from mouse disease models. Any defect in sample size is generally overcome by using multiple mice. However in case of rare strains of mice, particularly those that breed poorly, or aged animals where few are available, sample size or mouse numbers can be a limiting factor. Thus, a combinatorial tetramer staining approach for detecting multiple T cell specificities simultaneously would be a great advantage in such situations. However, there could be a problem if immunodominant T cell responses, especially those at acute phases of infection (sometimes N20% of all CD8+ T cells) overwhelm the subdominant T cell sets. In the present study, we have used a protocol modified from the human combinatorial approach (Hadrup et al., 2009; Newell et al., 2009) to detect multiple influenza-specific CD8+ T cells at the acute and long-term memory phases of infection in B6 mice. + We have shown that both highly dominant (DbNP+ 366CD8 and + b + b + DbPA+ CD8 ) and subdominant (K PB1 CD8 , D PB1224 703 + b + + + F262CD8 and K NS2114CD8 ) T cell responses can be detected simultaneously using this combinatorial approach at levels comparable to those detected by conventional tetramer staining. The technique provides a particular advantage for aged mice, where a comprehensive analysis utilised 5 animals instead of 25, making the detection of multiple T cell specificities more cost-effective and less time-consuming. 2. Materials and methods 2.1. Mice and virus infections The female C57BL/6J (H-2b) mice used in this study were bred and housed in the animal facility of the Department of Microbiology and Immunology at the University of Melbourne
(Parkville, Australia). Naïve 6- to 8-week-old mice were anesthetized by methoxyfluorane inhalation and infected intranasally (i.n.) with 1 × 104 PFU of the HK× 31 (x31, H3N2) influenza A virus in 30 μl of PBS. All animal studies were in compliance with the guidelines set out by the University of Melbourne Animal Experimental Ethics Committee. 2.2. Tissue sampling and preparation The spleen and bronchoalveolar lavage (BAL) (from 4 ml washing of the lungs in HBSS) was sampled at acute time points of infection (10 days post infection), while only the spleen was isolated at the memory time point. Spleens were homogenised using a plunger and 70 μm sieves and washed twice in HBSS to give single cell suspensions. Splenocytes were depleted of B cells by incubation on αIgG/IgM coated plates (Jackson ImmunoResearch Labs) for 45 min at 37 °C, and the BAL was depleted of macrophages by adherence to plastic for 45 min at 37 °C. 2.3. Combinatorial tetramerisation Monomers of DbNP366-374 (ASNENMETM), DbPA224-233 (SSLENFRAYV), KbPB1703-711 (SSYRRPVGI), DbPB1-F262-70 (LSLRNPILV), and KbNS2114-121 (RTFSFQLI) (purchased from the Monomer Facility, University of Melbourne) were conjugated to combinations of fluorescently labelled Streptavidins (SA) (eBiosciences) at equimolar ratios according to calculations from Newell et al. (Newell et al., 2009). Briefly, fluorescently labelled SA were mixed (according to the desired combinations), the volume adjusted with PBS to a total volume of 90 μl, then slowly added (a tenth of the SA mix volume added every 10 min) to 10 μg (1 mg/ml) monomer at room temperature (refer to Supplementary Table 1 for volumes). To quench any unconjugated SA, D-Biotin (Sigma) was added to give a final concentration of 26.4 μM for each tetramer, incubated on ice for 20 min and centrifuged at 17,000 ×g for 2 min, then the tetramer supernatant used for staining as described previously (Hadrup et al., 2009). 2.4. Tetramer and antibody surface stain Lymphocytes from BAL and spleen taken at acute and memory time points were stained with a cocktail of tetramers (DbNP366-APC, DbPA224-APC/PECy7, KbPB1703-APC/PE, DbPB1F262-PECy7 and KbNS2114-PE), with each at 3.5 μg/ml based on the original monomer concentration, that is, each tetramer was used at a 1:100 dilution (a saturating concentration) in FACS buffer (1% BSA, 0.02% Sodium Azide in PBS) for 1 h at room temperature in the dark. To determine the efficiency of the combinatorial tetramer staining protocol, splenocytes taken at day 10 after primary infection were also separately stained with each tetramer labelled with PE (at 1.75 μg/ml based on original monomer concentration). Each tetramer was used at concentration determined to be a saturating concentration by titration. Following the tetramer stain, cells were washed twice in FACS buffer and stained with antiCD8α-PerCP-Cy5.5 (1 μg/ml) in FACS buffer for 20 min on ice. Cells were then washed twice with FACS buffer and acquired by flow cytometry on a LSRII (BD Biosciences) and analysed by Flowjo software (Treestar).
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3. Results and discussion In vivo infection of mice can result in immunodominant responses comprising as many as ∼20% of CD8+ T cells at acute time points after infection. Concurrently, much smaller CD8+ T cell responses are generated toward a spectrum of subdominant determinants. Recent studies have established that the simultaneous detection of multiple T cell specificities is feasible for human peripheral blood lymphocytes (PBL) (Hadrup et al.,
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2009; Newell et al., 2009), so we investigated whether this approach might also be used to detect multiple murine CD8+ T cells. The concern in this case was that the presence of highly immunodominant CD8+ T cell sets would overwhelm the system and thereby prevent detection of the subdominant specificities. The present study thus analysed whether the multiplexed combinatorial technique could be applied for the simultaneous detection of influenza-specific immunodominant + + (D b NP + and D b PA + 366 CD8 224 CD8 ) and subdominant
Fig. 1. Flow cytometry-gating scheme for three-color combinatorial pMHC tetramer staining. Representative flow cytometry dot plots of the gating scheme for CD8+ T cells from the spleens of B6 mice infected i.n. 10 days previously with the x31 influenza A virus are shown. The splenocytes were stained with a cocktail of tetramers specific for DbNP366-APC, DbPA224-APC/PECy7, KbPB1703-APC/PE, DbPB1-F262-PECy7, and KbNS2114-PE and a CD8 mAb, anti-CD8α-PerCP-Cy5.5. Cells were first gated based on forward versus side scatter (A), before doublets were removed using the width vs. area signal of forward scatter (B) and the CD8+ T cells were selected (C). The CD8+ T cells were then gated on each of the three tetramer-conjugated fluorochromes separately (D, E, F) to allow for subsequent analysis of tetramer staining. Gating of cells double positive for PerCP-Cy5.5 and APC (D) followed by separation into four populations based on PECy7 and PE staining (G) allowed the detection of DbNP366-, DbPA224- and KbPB1703-specific CD8+ sets. To detect KbNS2114- and KbPB1703-specific populations, cells double positive for PerCP-Cy5.5 and PE were first gated (E), and were then separated into four populations based on PECy7 and APC staining (H). Cells double positive for PerCP-Cy5.5 and PECy7 (F) then separated into four sets based on APC and PE staining (I), allowing the detection of DbPA224- and DbPB1-F262-specific CD8+ T cells. (J) Epitope-specific “signatures” defines by different staining combinations are shown.
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+ b + + + (KbPB1+ and KbNS2+ 703CD8 , D PB1-F262CD8 114CD8 ) at the peak (d10) of the response or in long-term memory (d210) after influenza virus infection. Our approach combined two recently described methods (Hadrup et al., 2009; Newell et al., 2009). Monomers expressing DbNP366–374, DbPA224–233, KbPB1703–711, DbPB1F262–70 and KbNS2114–121 were conjugated to various fluorescently labelled Streptavidins (SA) at equimolar ratios (Newell et al., 2009) (refer to Supplementary Table 1 for fluorophores and volumes). D-Biotin was used to quench any unconjugated streptavidin (Hadrup et al., 2009). This method combined an easily accessible streptavidin conjugated system for pMHC-I tetramerisation together with a rapid technique for removal of any unbound pMHC-I complexes. The validity of the combinatorial technique and representative flow cytometry dot plots of the gating scheme for CD8+ T cells isolated from spleen of mice infected i.n. with influenza virus is shown in Fig. 1. Splenocytes were stained with a cocktail of pMHC-I tetramers specific for DbNP366-APC, DbPA224-APC/ PECy7, KbPB1703-APC/PE, DbPB1-F262-PECy7 and KbNS2114-PE, followed by anti-CD8α-PerCP-Cy5.5. Single CD8+ T lymphocytes were identified by FSC versus SSC, FSC-A versus FSC-W characteristics and anti-CD8 mAb attaining (Fig. 1A–C). Each epitope-specific CD8+ T cell population was then identified based on the characteristic-staining signature assigned (Fig. 1G–J). CD8+ T cells that were positive for APC (Fig. 1D), followed by separation into four populations based on PECy7 and PE staining (Fig. 1G), which allowed detection of the DbNP366-, DbPA224- and KbPB1703-specific CD8+ T cells. To identify KbNS2114- and KbPB1703-specific CD8+ T cells, CD8+ T cells positive cells for PE were first gated on PE (Fig. 1E), then
separated into four populations based on PECy7 and APC staining (Fig. 1H). CD8+ T cells positive PECy7 (Fig. 1F) were separated into four populations based on APC and PE staining (Fig. 1I) to detect DbPA224- and DbPB1-F262-specific CD8+ T cells. Analysis of two immunodominant and three subdominant CD8+ T cell responses at the peak of influenza virus infection using the conventional and combinatorial tetramer method showed equivalent frequency of tetramer staining at the site of infection (BAL; Fig. 2A) and spleen (Fig. 2B). Importantly, both dominant and subdominant epitope-specific CD8+ T cell sets were detected with equivalent efficiency despite the 7– 8-fold difference between the largest and smallest responses detected within the one sample (Fig. 2AB). Mean fluorescence intensity (MFI) of tetramer staining was of lower intensity for tetramers that were conjugated to N1 fluorochrome (e.g. KbPB1703–APC/PE combination) in comparison to the singletetramer staining (KbNS2114-PE). This was evident in both the BAL and in the spleen (Fig. 2C) and reflects the fact that only half as much of any one tetramer can bind to its epitopespecific population. Importantly, this anticipated reduction in MFI had no effect on the ability to detect all of the epitope-specific populations (Fig. 2AB). Similar findings were made for combinatorial tetramer staining within human PBL (Hadrup et al., 2009; Newell et al., 2009). Our study is based on a mouse model of viral infection that uses relatively high affinity TCRs. As the weaker affinity TCRs might ‘smear’ when combining fluorochromes, we suggest that single-signature stains are used for staining the weak affinity repertoires, whereas double-signature stains are used for staining of high affinity repertoires.
Fig. 2. Comparable staining with single pMHC tetramer complex vs. combinatorial pMHC tetramer complexes for influenza-specific epitopes. Naïve B6 mice were infected i.n. with x31 influenza virus and BAL (A, C) and spleen (B, C) were harvested 10 days later for analysis of CD8+ DbNP366, DbPA224, KbPB1703, DbPB1-F262, and KbNS2114-specific T cell responses. Shown are (A, B) the mean percentage of CD8+tetramer+ cells ± SD; (C) the mean fluorescence intensity (MFI) ±SD of PEtetramer staining for a single-stained tetramer (KbNS2114-PE, no difference in MFI) versus doubly-labelled tetramer (KbPB1703-PE/APC, decreased MFI) (n = 5 mice per tetramer at each time point).
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Having verified the combinatorial tetramer staining technique, we assessed the sensitivity of detection for five different specificities of influenza-specific CD8+ T cells in acutelyinfected (d10; Fig. 3A) mice and aged (long-term memory, 7 months after influenza infection; Fig. 3B) animals known to contain very small populations of epitope-specific CD8+ T cells. Looking at CD8+ populations detected by the combinatorial tetramer approach at the acute (d10) phase of primary + + influenza virus infection, the DbNP+ and DbPA+ 366CD8 224CD8 populations dominated in a numerical sense and were markedly b higher than the expansions of subdominant KbPB1+ 703, D PB1b + + F2+ and K NS2 CD8 populations (Fig. 3A). Simultaneous 62 114 detection of mouse influenza-specific CD8+ T cell specificities in the long-term memory (∼d210) showed ∼10–20-fold decrease in population size when compared to the acute response. Interestingly, memory profiles showed comparable numbers of + b + b + b + + + DbPA+ 224CD8 , K PB1703CD8 , D PB1-F262CD8 and K NS2114 + CD8+ T cells, although DbNP+ set remained at ∼2× 366CD8 higher frequency (Fig. 3B). This lower proportions of memory CD8+ T cells recognising immunodominant responses have been noted frequently for a lesser spectrum of epitopes (Kedzierska et al., 2006), but the use of the combinatorial approach has now allowed us now to extend this observation for a much broader spectrum of epitopes (Fig. 3CD). A possible explanation for such enhanced survival capacity of subdominant CD8+ T cell populations could be related to the lower activation level and/or expansion of those cells consequent to their incomplete recruitment into the immune response (La Gruta et al., 2010).
+ Fig. 3. Immunodominance of influenza-specific DbNP+ and DbPA+ 366CD8 224 CD8+ T cell populations decreases with progression to long-term memory. Following primary infection with x31 influenza virus, splenocytes were stained with a cocktail of tetramers specific for DbNP366, DbPA224, KbPB1703, DbPB1-F262, and KbNS2114, and anti-CD8 mAb at 10 days (A, C), or 7 months after infection (B, D). Data represent (A, B) the absolute number of tetramer positive lymphocytes mean ± SD (n = 5) and (C, D) the average proportion tetramer+ of CD8+ T cells.
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Taken together, our study has established that the multiplexed combinatorial tetramer staining is feasible in a mouse model of a disease in which the range of CD8+ T cell response magnitude is relatively broad. The technique was subsequently used to assess the relationship between the immunodominant and subdominant influenza-specific CD8+ T cell sets at acute and long-term memory. Acknowledgments We thank Drs Mark Davis and Evan Newell for helpful suggestions and University of Melbourne 526-326 students of 2009 for technical assistance. This work was supported by an Australian National Health and Medical Research Council (NHMRC) Project Grant 454312 awarded to KK. KK and NLG are NHMRC RD Wright Research Fellows and SJT is an Australian Pfizer Research Fellow. SAV is a recipient of an Australian Postgraduate Award, TC is a recipient of the NHMRC Biomedical Postgraduate Scholarship (ID 520643). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jim.2010.06.003. References Altman, J.D., Moss, P.A., Goulder, P.J., Barouch, D.H., McHeyzer-Williams, M.G., Bell, J.I., McMichael, A.J., Davis, M.M., 1996. Phenotypic analysis of antigenspecific T lymphocytes. Science 274, 94. Doherty, P.C., Christensen, J.P., 2000. Accessing complexity: the dynamics of virus-specific T cell responses. Annu. Rev. Immunol. 18, 561. Doherty, P.C., Turner, S.J., Webby, R.G., Thomas, P.G., 2006. Influenza and the challenge for immunology. Nat. Immunol. 7, 449. Hadrup, S.R., Bakker, A.H., Shu, C.J., Andersen, R.S., van Veluw, J., Hombrink, P., Castermans, E., Thor Straten, P., Blank, C., Haanen, J.B., Heemskerk, M.H., Schumacher, T.N., 2009. Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers. Nat. Meth. 6, 520. Kedzierska, K., Venturi, V., Field, K., Davenport, M.P., Turner, S.J., Doherty, P.C., 2006. Early establishment of diverse TCR profiles for influenza-specific CD62Lhi CD8+ memory T cells. Proc. Natl. Acad. Sci. U. S. A. 103, 9184. Kedzierska, K., Stambas, J., Doherty, P.C., 2009. Finding multiple needles in one immune haystack. Nat. Meth. 6, 489. La Gruta, N., Rothwell, W., Cukalac, T., Swan, N., Valkenburg, S., Kedzierska, K., Thomas, P., Doherty, P., Turner, S., 2010. Primary CTL response magnitude in mice is determined by the extent of naïve T cell recruitment and subsequent clonal expansion. J. Clin. Invest. 120, 1885. Newell, E.W., Klein, L.O., Yu, W., Davis, M.M., 2009. Simultaneous detection of many T-cell specificities using combinatorial tetramer staining. Nat. Meth. 6, 497. Yewdell, J.W., Bennink, J.R., 1999. Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu. Rev. Immunol. 17, 51.