Multiplex peptide-MHC tetramer staining using mass cytometry for deep analysis of the influenza-specific T-cell response in mice

Accepted Manuscript Multiplex peptide-MHC tetramer staining using mass cytometry for deep analysis of the influenza-specific T-cell response in mice

M. Fehlings, S. Chakarov, Y. Simoni, B. Shivshankar, F. Ginhoux, E.W. Newell PII: DOI: Reference:

S0022-1759(17)30426-X doi:10.1016/j.jim.2017.09.010 JIM 12374

To appear in:

Journal of Immunological Methods

Received date: Revised date: Accepted date:

2 February 2017 19 August 2017 29 September 2017

Please cite this article as: M. Fehlings, S. Chakarov, Y. Simoni, B. Shivshankar, F. Ginhoux, E.W. Newell , Multiplex peptide-MHC tetramer staining using mass cytometry for deep analysis of the influenza-specific T-cell response in mice. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jim(2017), doi:10.1016/j.jim.2017.09.010

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ACCEPTED MANUSCRIPT Multiplex peptide-MHC tetramer staining using mass cytometry for deep analysis of the influenza-specific T-cell response in mice M. Fehlings1,2*, S.Chakarov1, Y. Simoni1, B. Shivshankar3, F. Ginhoux1, E.W. Newell1* 1

Agency for Science, Technology and Research (A*STAR), Singapore Immunology Network (SIgN), Singapore.

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2

ImmunoSCAPE Pte. Ltd., Immunos Building, Singapore.

3

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Indoor Biotechnologies India Pvt. Ltd., Bangalore Bioinnovation Centre, India.

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*Corresponding authors:

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Michael Fehlings [email protected] 8A Biomedical Grove Immunos, #04-00 Singapore 138648

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or

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Evan W. Newell [email protected] 8A Biomedical Grove Immunos, #03-06 Singapore 138648 Tel: (+65) 6407-0641

Deep profiling of influenza-specific T cells in lymph nodes and lung

ACCEPTED MANUSCRIPT Abstract Antigen-specific T cells play a crucial role for the host protective immunity against viruses and other diseases. The combination of mass cytometry together with a combinatorial multiplex tetramer staining has successfully been applied for probing and characterization of multiple antigen-specific CD8+ T cells in human blood samples. The present study shows that this

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approach can also be used to rapidly identify the magnitude of influenzaspecific CD8+ T cell peptide dominance across lymph nodes and lungs in a murine model of a highly pathological influenza infection. Moreover, we also prove feasibility of this approach to be expandable for the concurrent identification of virus specific CD4+ T cells. By using a double coding approach, we probed for five influenza-specific MHCI-peptide complexes as

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well as one influenza-specific MHCII-peptide complex in the presence of irrelevant control peptides and show that this approach is capable of tracking

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antigen-specific T cells across individual lymph nodes and lungs. The simultaneous staining with 26 surface maker molecules further facilitated an in-depth characterization of T cells reacting with influenza epitopes and

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revealed tissue specific phenotypic differences between CD4+ T cells targeting the same pathogenic epitope. In conclusion, this approach provides

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the possibility for a rapid and comprehensive analysis of antigen-specific CD8+ and CD4+ T cells in different disease settings that might be

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advantageous for subsequent vaccine formulation strategies.

ACCEPTED MANUSCRIPT Introduction Cytotoxic CD8+ T lymphocytes (CTLs) are important immune-determinants that can be utilized to induce efficacious immunity against infectious pathogens as well as other diseases such as cancer. Through the detection of foreign (e.g., pathogen-derived or mutated) peptide sequences in the context of host derived major histocompatibility complex-I molecules (MHCI), CD8+ T

virus

spread

or

tumor

outgrowth.

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cells are capable of eliminating their specific target cells thereby controlling Consequently,

identification

and

characterization of antigen-specific T cells are of enormous importance for therapeutic disease management.

Influenza virus infection in mice has extensively been used as a disease model to study virus-specific T cells (1,2). In contrast to humans where little

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precise information about the cellular immune response of influenza-specific T cells is available, influenza infection in mice is well-characterized by the

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emergence of an immunodominant CTL hierarchy (3,4) and by adapting the principles of combinatorial tetramer multiplexing (5,6) the range of the CD8+ T-cell magnitude in influenza-infected mice has previously accurately been

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described by flow cytometry (3). Limitations in the availability of parameters, however, have hampered a detailed phenotypic characterization of dominant

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and subdominant influenza-specific CD8+ T cell responses as well as other cells in this system. Besides CTLs, antigen-specific CD4+ T helper are

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critically involved in viral immunity (7) and in influenza infection these cells contribute to the protective immune response by inducing memory virus-

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specific CD8+ T cells (8) as well as by promoting high quality antibody responses (9).

Fluorescently labeled MHC-multimers are widely used to probe multiple CD8+ T-cell antigen-specific T cells. However, even with combinatorial MHC staining approaches (5,6), the number of epitopes and tissue compartments that can be assessed simultaneously at the single-cell level remains limited due to the restrictions on the number of useful fluorophores that are inherent with current flow cytometry devices. Mass cytometry (a.k.a. cytometry by time of flight, CyTOF) has tremendously advanced this approach. By using rare heavy metal isotopes with minimal signal spill over into neighboring channels

ACCEPTED MANUSCRIPT due to the unique flight of time resolution that is inherent with a specific isotope (10) currently >40 parameters can be interrogated simultaneously. This facilitates screening for hundreds of antigen-specificities in a single sample while retaining the possibility to further address the phenotypes of these cells at an unprecedented depth (11). By using a mass cytometry based combinatorial tetramer approach, we present a technique that facilitates a rapid and concurrent identification of

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antigen-specific CD4+ and CD8+ T cells in a murine model of influenza infection. By the incorporation of irrelevant MHC class I (MHCI) and MHC Class II (MHCII)-restricted peptides into the screening approach we demonstrate that this approach is feasible to clearly detect dominant as well as subdominant T-cell responses in a model of high pathological acute

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influenza A virus infection which is generally characterized by low T cell responses with a well-described epitope hierarchy (4,12). In addition, by including 26 metal-conjugated antibodies against relevant surface marker

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molecules, this approach enabled a comprehensive phenotypic profiling of antigen-specific T cells across lymph nodes and lungs at an unprecedented

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depth. To our knowledge, this is the first combinatorial tetramer approach based on mass cytometry that demonstrates the feasibility of combining MHCI-peptide- as well as MHCII-peptide-complexes for a simultaneous

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probing of influenza-specific CD8+ and CD4+ T cells at a single cell level

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across individual tissues and mice.

ACCEPTED MANUSCRIPT Material and Methods

Mice

C57BL/6 mice were obtained from the Biological Resource Center (BRC), Agency for Science, Technology and Research (A-Star), Singapore. All mice

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were bred and maintained in the Singapore Immunology Network (SIgN) animal facility before use at 6–8 weeks of age. Only healthy male mice were used in this study. All experiments and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of BRC (A-Star, Singapore) in accordance with the guidelines of the Agri-Food and Veterinary

Research (NACLAR) of Singapore.

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Influenza infection

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Authority (AVA) and the National Advisory Committee for Laboratory Animal

study

was

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The influenza virus strain A/Puerto Rico/8/1934 (H1N1) (PR8) used in this provided

by

Baalasubramanian

Shivshankar

(Indoor

Biotechnologies India, Bangalore, India). Mice were first anaesthetized with

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isoflurane and were then inoculated intranasally with the influenza strains at 25 plaque-forming units (PFU) in a total of 20 µl volume per mouse. The

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influenza dose was optimized to give ∼20% weight loss in wild type mice during the peak of infection and recovery without incidence of mortality. Illness

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was monitored daily by weighting and nine days post infection mice were euthanized and lung and lymph nodes were harvested.

Tissue harvesting and processing

Lungs were isolated and processed as previously described (13). Briefly, tissues were homogenized, incubated for 30 min in 10% FBS HBSS containing 0.2 mg/ml collagenase type IV (SIGMA) (working activity of 770 U/mg), and passed trough a 19G syringe to obtain a homogeneous cell

ACCEPTED MANUSCRIPT suspension. Cell suspensions were then passed through 70 µm cell strainers (Fisherbrand) and leukocytes were isolated using CD45 cell enrichment beads (Miltenyi Biotec) together with an autoMACS® Pro Separator (Miltenyi Biotec). Cell suspensions from lung draining lymph nodes were prepared in PBS 2% FCS by homogenizing them through 70 µm cell strainers. Cells were

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counted and adjusted to 1x107 cells/ ml.

Antibody labeling

Purified antibodies (lacking carrier proteins) were labeled 100 ug at a time with heavy metal loaded maleimide conjugated DN3 MAXPAR chelating polymers (Fluidigm) according to the recommendations provided by Fluidigm.

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Antibody clones and providers are listed in Supplementary Table 1.

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Peptides

Influenza A specific CD8+ T-cell epitopes (H2Db-NP366-374 (ASNENMETM),

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H2Db-PA224-233 (SSLENFRAYV), H2Db-PB1-F262-70 (LSLRNPILV), H2KbNS2114-121 (RTFSFQLI), H2Kb-PB1703-711 (SSYRRPVGI)) as well as CD8+ T

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cell control peptides (H2Db-HYsmcy (KCSRNRQYL), H2Db-LCMV-GP33-41 (KAVYNFATM), and H2Kb-OVA257-264 (SIINFEKL)) were purchased from

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Mimotopes with a purity of at least 80%. Influenza A specific CD4+ T-cell epitope (I-Ab-NP311-325 (QVYSLIRPNENPAHK) and control peptide (I-Ab-

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human CLIP87-101 (PVSKMRMATPLLMQA) in the form of biotinylated peptideMHC monomers were obtained through the NIH tetramer facility (Emory University, Atlanta, USA).

Peptide-MHC production and tetramerization

Production and preparation of peptide-MHC molecules was performed in house as described elsewhere (11,14). Briefly, recombinant H-2Kb and H-2Db heavy chains and human β2 microglobulin light chains were refolded in the presence of a UV-cleavable linker (J) (H2-Kb SIINFEJL and H2-Db

ACCEPTED MANUSCRIPT FAPGNYJAL, Mimotopes) and subsequently biotinylated. After purification, peptide-specific MHC complexes were generated by a 15-min exposure to 365 nm UV irradiation in the presence of a single target candidate peptide. Peptide exchange reactions were setup in 96 well plates at least 12 h before tetramerization with up to 100 ul of 100 ug/ml H2-Kb or H2-Db monomers and 50 uM of single target peptides in PBS. Streptavidin metal labeling was prepared at least 24 h before usage.

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Therefore, 50 ug of protein at a time were conjugated with the respective heavy metal loaded DN3 polymer as previously described (11,14) and finally diluted to a concentration of 200 ug/ml. For combinatorial peptide-MHC tetramerization, two out of five differently metal-labeled streptavidins were pre-mixed at equimolar ratios accordingly to generate peptide specific

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combinations (H2-Db-NP366 (Tb-159 and Ho-165), H2-Db-PA224 (Tb-159 and ER-168), H2-Db-PB1F262 (Tb-159 and Yb-173), H2-Kb-NS2114 (Ho-165 and ER-168), H2-Kb-PB1703 (Ho-165 and Yb-173),, I-Ab-NP311-325 (Tb-159 and Ho-

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165) as well as the MHC control peptides H2-Kb-SIINFEKL (Ho-165 and Lu175) H2Db-HYsmcy (Tb-159 and Lu—175), H2Db-LCMV-GP33 (Yb-173 and

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Lu-175), and I-Ab-hCLIP87 (Er-168 and Yb-173). The individual streptavidin mixtures were then added in four steps (10 min incubation at RT per addition) to the respective peptide-MHC complexes in the 96 well plates to achieve a

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total final molar ratio of 1 (streptavidin) : 4 (peptide-MHC) followed by a final addition of 10uM free biotin (Sigma) to quench any remaining unreacted

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streptavidin molecules. The tetramerized peptide-MHC-complexes were then combined and concentrated by using a 10 kDa (Merck Millipore) cut-off filter.

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After exchanging the buffer into cytometry buffer (PBS, 2% FCS, 2mM EDTA, 0.05% sodium azide) the tetramer cocktail was filtered through a 1 um centrifugal filter (Merck Millipore) and used to stain the cells with each peptide-MHC tetramer at a concentration of ~ 500nM.

Staining and data acquisition

Three to five million cells/tissue were transferred into 96 well plates and washed once with cytometry buffer followed by 5 min staining with 100 ul of 200 uM cisplatin (Sigma) for the discrimination of dead cells. Cells were then

ACCEPTED MANUSCRIPT washed twice and each sample was stained with 50 ul of tetramer cocktail for 1 h at RT. Following two washing steps the cell were then stained with 50 ul of heavy metal-labeled antibody cocktail (Supplementary Table 1) in cytometry buffer for 30 min. Subsequently, the cells were washed twice in cytometry buffer and once with PBS (GIBCO) followed by fixing the cells in 2 % paraformaldehyde (Electron Microscopy Sciences) in PBS for a minimum of 12 h at 4°C. The cells were then pelleted, incubated for 5min on ice in 200 ul

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of 1X permeabilization buffer (Biolegend) and washed once before barcoding. Each sample per tissue was stained for 30 min on ice with a unique dual combination

out

of

six

metal

linked

barcodes

consisting

of

Bromoacetamidobenzyl-EDTA (BABE, Dojindo) or DOTA-maleimide (DM, Macrocyclics) (DM-Rh-102, BABE-Pd-104, BABE-Pd-106, BABE-Pd-108,

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BABE-Pd-110, and DM-Ln-113) as recently described (15). The cells were subsequently washed once with perm buffer, incubated with cytometry buffer for 5 min on ice, and labeled at room temperature with 250 nM (1:2000)

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iridium DNA nucleic acid intercalator (Fluidigm) in 2% PFA/PBS at room temperature for 20 minutes. The cells were washed twice with cytometry

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buffer and twice in distilled water before all samples per tissue were combined and adjusted to 0.5 million cells/ml in distilled water for the acquisition on a

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Data analysis

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mass cytometer.

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Mass cytometry data were exported as .fcs files and all zero values of the .fcs files were randomized by a custom R-script that uniformly distributes values between minus-one and zero. Cells for each dual barcode combination were de-barcoded manually by selecting cells that were positive for only two barcode channels using FlowJo software (Treestar, Inc). T cells were identified by gating on live, cisplatin–, single cell DNA+, CD45+ immune cells and were further specified as CD19- TCR beta+ and CD90+ cells before distinguishing into CD8+ and CD4+ T cells. Antigen-specific T cells were further identified by manual gating on CD8+ and CD4+ T ells that were only positive for the dual metal combinations as defined in the coding scheme

ACCEPTED MANUSCRIPT described above. t-SNE analysis was carried out by using an R-package including the “flowCore” and “Rtsne” CRAN R packages for an efficient implementation of t-SNE via the Barnes-Hut approximations) (Amir el et al., 2013; Van der Maaten and Hinton, 2008). In R, all data were transformed using the “logicleTransform” function by using the “flowCore” package (parameters: w=0.25, t=16409, m=4.5, a=0). For t-SNE dimensionality reduction the cell events per single sample were down-sampled to a

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maximum number of 10000 CD8+ T cells/tissue. Percentages of T cells specific for a particular antigen and median intensity values for each marker assessed were calculated and summarized in scatter plots and pie charts using Graphpad Prism and histograms using Flowjo software. t-SNE map derived heat maps were generated by using customized R-scripts. Statistical

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analysis was done using Mann-Whitney test or ANOVA followed by Tukey’s

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multiple comparisons test. p<0,05 was considered significant.

ACCEPTED MANUSCRIPT Results and discussion

The primary immune response in the murine model of acute influenza infection is reflected by a clear hierarchy of T cells reactive with dominant and subdominant epitopes that is established eight days post infection and differs

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from the early days of infection and the long-term antigen-specific T-cell prevalence (3,4). To facilitate tracking of T cells with different influenza virus antigen specificities in lymph nodes and lungs from acutely virus infected mice and to simultaneously define cellular characteristics, we used a combinatorial

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tetramer staining approach (5,6,11) and encoded each target peptide with a unique dual heavy metal combination. Lymph nodes and lungs from individual

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mice infected with influenza virus PR8 were harvested and processed nine days post infection and single cell suspensions were stained with a cocktail

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consisting of five influenza-specific peptide-MHCI tetramer complexes (H2DbNP366-374, H2Db-PA224-233, H2Db-PB1-F262-70, H2Kb-NS2114-121, H2Kb-PB1703as well as one influenza-specific peptide-MHCII tetramer (I-Ab-NP311-325).

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711)

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To assess possible non-specific peptide-MHC tetramer staining we also included three irrelevant peptide-MHCI tetramers (H2Db-HYsmcy, H2Db-

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LCMV-GP33-41, H2Kb-OVA

257-264

(SIINFEKL)) as well as one peptide-MHCII

tetramer complex (I-Ab-human CLIP87-101) as controls. In parallel, we stained all cells with a cocktail of 26 differently labeled antibodies specific for relevant surface molecules at the same time, which allowed for the identification and phenotypic profiling of CD8+ and CD4+ T cells. Additionally, the usage of a binary barcoding approach facilitated the simultaneous probing of different antigen-specificities and phenotypes across individual tissue samples.

ACCEPTED MANUSCRIPT We gated on CD8+ and CD4+ T cells by selecting live (cisplatin–, DNA+) CD45+, CD19- TCR-beta+ CD90+ cells and identified antigen-specific T cells by standard manual biaxial gating (Fig. 1A). We found substantial numbers of influenza-specific T cells specific for all influenza virus epitopes tested across the different tissues (Fig. 1 B). In lymph nodes from virus infected mice we

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detected comparable frequencies for CD8+ T cells targeting four different antigens (H2Db-NP366, H2Db-PA224, H2Kb-PB1703, and H2Db-PB1-F262) whereas we found only a small number of CD8+ T cells to be specific for H2Kb-NS2114. Within the same tissues we identified equal percentages of

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influenza-specific CD4+ T cells (I-Ab-NP311) as have been observed for the dominant CD8+ T-cell responses. In contrast to lymph nodes, we found

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significantly larger numbers of antigen-specific T-cell counterparts in the lungs from mice infected with influenza (Fig. 1B). Moreover, there were also

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remarkable differences in the percentages of CD8+ T cells targeting different epitopes and we identified a dominant CD8+ T cell response that was directed

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towards H2Kb-PB1703, whereas the frequencies of T cells specific for H2Db-

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NP366, H2Db-PA224, and H2Db-PB1-F262 varied at different degrees. Similar to lymph nodes we found the lowest percentages of tetramer-positive T cells in

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the lungs to be reactive for H2Kb-NS2114. Interestingly, whereas in lymph nodes the numbers of antigen-specific CD4+ T cells were comparable to the numbers of dominant antigen-specific CD8+ T cells, this could not be observed for the lung specific counterparts where we detected lower frequencies of such specific CD4+ T cells as compared to the dominant CD8+ T-cell responses.

ACCEPTED MANUSCRIPT Having the possibility to probe a large number of relevant surface marker molecules simultaneously in addition to the tetramer complexes, our mass

cytometry

based

staining

approach

allowed

for

the

further

characterization of lymph node and lung-derived influenza-specific T cells and their comparison at an unprecedented depth. To facilitate the interpretation of

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such high dimensional immune profiles we applied the t-distributed stochastic neighbor embedding (t-SNE) algorithm (16,17) that reduces complex high dimensional data into two parameters (t-SNE1 and t-SNE2) and plots cell with similar phenotypes to nearby points onto a two-dimensional space. t-SNE was

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performed on a merged dataset concatenated from lymph node and lung derived CD4+ and CD8+ T-cells and then manually gated antigen-specific T

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cells were overlaid onto the two-dimensional t-SNE density plot (Fig. 2A). According to the t-SNE based CD8+ and CD4+ T-cell cluster segregation we

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localized lymph node and lung derived influenza-specific T cells in the corresponding CD8+ and CD4+ T cell compartments. The majority of CD8+ T

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cells specific for a certain antigen from both tissues occupied the same cell

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cluster on the t-SNE map, except for lymph node derived H2Kb-NS2114-specifc T cells that could not be assigned to a certain cluster which was likely due to

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the low frequencies of these cells detected. In contrast, t-SNE segregated CD4+ T cells targeting I-Ab-NP311 into two main clusters, depending on whether the cells derived from either lymph nodes or lungs from virus infected mice (Fig. 2A). We used heat plots based on the t-SNE map to summarize the phenotypic profiles these antigen-specific CD8+ and CD4+ T-cell clusters and to provide a visualization of the different marker combinations expressed by these cells found in the individual tissues (Fig 2.B). We detected a

ACCEPTED MANUSCRIPT remarkable variation in the expression of markers across the antigen-specific and bulk (tetramer-negative) T-cell clusters. The functionality of our staining approach was confirmed by comparing the phenotypes of antigen-specific and bulk T cells from the different tissues (Fig 2C). For instance, we found higher intensities of CD62L expression (naïve) on bulk CD8+ T cells in the lymph

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nodes whereas we observed a higher expression of CD44 (antigenexperienced) on lung-derived as well antigen-specific T cells. In addition, while these cells reflected an activated status of virus specific T cells by upregulation of Sca-1 and ICOS (18,19) the expression intensities for these

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markers were remarkably lower on tetramer-negative CD8+ T cells found in the lymph nodes (Fig 2C).

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t-SNE was able to accurately delineate two different clusters from lymph node or lung-derived antigen-specific CD4+ T cells, which were

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validated using comparative statistical analyses. Whilst sharing similar expression intensities for the majority of markers, we detected significant

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differences in the median expression intensities for a few surface proteins

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expressed by cells from either compartment and these results were consistent across all individual tissues tested (Fig 3A). For instance, whereas antigen-

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specific CD4+ T cells from lymph nodes displayed higher expression levels of markers associated with T-cell differentiation (CD24) and recruitment (CXCR3) their lung-derived counterparts were identified by a higher Ly6C expression that has been shown to be associated with higher anti-viral activities but lesser potential for memory cell differentiation (20). This is also in line with the cluster specific expression intensities of these markers that are visible on the two-dimensional t-SNE heat map (refer to Fig 2B). Although we found that

ACCEPTED MANUSCRIPT CD8+ T cells targeting different influenza-specific epitopes clustered together, we observed different expression intensities for some of the makers within this cluster (i.e., CD25, KLRG-1, Lag-3, and ICOS) that also differed from the bulk CD8+ T-cell clusters that were segregated by t-SNE (refer to Fig 2B). When we compared the frequencies of cells expressing these markers, we found

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differences between single T-cell specificities and tetramer negative cells at varying degrees (Fig. 3B). For instance, whereas we were not able to detect differences in cell numbers for the expression of CD25 or KLRG-1, we found higher percentages of all antigen-specific CD8+ T cells expressing ICOS when

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comparing to the tetramer negative T-cell fraction. In contrast, although there was a trend towards higher percentages of antigen-specific T cells expressing

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Lag-3, we only found significant higher percentages for H2Kb-PB1703 specific T cells as compared to tetramer-negative CD8+ T cells (p<0.05 ANOVA with

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Tukey’s test for multiple comparisons).

In summary, our data has demonstrated that a mass cytometry based

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combinatorial tetramer staining approach is capable of simultaneously

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identifying and quantifying influenza-specific CD4+ and CD8+ T cells with different epitope usage across secondary lymphoid tissues (lymph nodes) and

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the site of infection (lung) in virus influenza-infected mice at an individual animal level. The additional capability of including a broad spectrum of surface marker molecules at the same time facilitates an in-depth profiling of such virus relevant cells within the same sample and shows that a broad combination of markers provides a better insight into tissue and cell type characteristics than single marker molecules. We believe that this methodology has a great potential for the investigation of antigen-specific T

ACCEPTED MANUSCRIPT cells in acute infections as well as the long-term prevalence of these cells in infected individuals. This will be of particular importance for the generation of novel vaccines that induce robust cellular immune responses to subsequent

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infections with alternate virus strains.

ACCEPTED MANUSCRIPT Acknowledgments This study was funded by A-Star/SIgN core funding (E.W.N. and F.G.) and AStar/SIgN immunomonitoring platform funding (E.W.N).

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Competing financial interests

E.W.N is a board director and shareholder of immunoSCAPE Pte. Ltd. M.F. is

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Director, Scientific Affairs and shareholder of immunoSCAPE Pte. Ltd.

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Figures legends. Figure 1. Frequencies of influenza-specific CD8+ and CD4+ T cells in lymph nodes and lungs determined by multiplexed tetramer staining and mass cytometry. (A) Staining example for a double-coded peptide-MHC tetramer staining from lymph nodes and lungs to identify antigen-specific

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CD8+ and CD4+ T cells nine days post influenza virus PR8 infection. Live T cells were defined as cisplatin–, DNA+ CD45+ cells and were further specified as CD19- TCR beta+ and CD90+ cells before distinguishing into CD8+ and CD4+ T cells. H2Kb-NP366-specific CD8+ T cells were identified as Ho-165 and Tb-159 double positive cells whereas CD4+ T cells specific for I-Ab-NP311 were identified by tetramers labeled with ER-168 and Lu-175 elements. Tetramers

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loaded with H2Kb-NP-SIINFEKL (Ho-165 and Yb-76 elements) or I-Ab-hCLIP87 (ER-168 and Yb-173 elements) served as negative controls. Data shown are

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combined from five individual tissues (mice). (B) Percentages of CD8+ and CD4+ tetramer positive T cells from lymph nodes and lungs nine days post influenza virus PR8 infection. By using a double coding tetramer staining

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approach the frequencies of T cells specific for six influenza relevant epitopes (Db-NP366, Db-PA224, Db-PB1F262, Kb-NS2114, Kb-PB1703, I-Ab-NP311) tested

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were simultaneously identified in lymph nodes and lungs. Tetramers loaded with Kb-SIINFEKL, KB-LCMV-GP33, Db-HY-smcy, and I-Ab-hCLIP87 served

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as negative controls. Each dot per epitope represents data obtained from one individual tissue (mouse). Horizontal lines indicate the mean ± SD of the

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absolute number of tetramer-positive T cells (n=5 individual mice). Pie charts display the average proportion of T cells specific for each epitope.

Figure 2. High-dimensional phenotypic analysis of CD8+ and CD4+ T cells derived from lymph nodes and lungs of influenza-infected mice. Analysis was carried out on gated CD8+ and CD4+ T cells from lymph nodes and lungs of mice infected with influenza virus PR8 nine days post infection (n=5). t-SNE was performed on a total of 1x105 CD8+ and CD4+ T cells and antigen-specific T cells were manually gated and then overlaid onto the total t-

ACCEPTED MANUSCRIPT SNE map. (A) Distribution of lymph node and lung derived influenza-specific CD8+ (blue; Db-NP366, Db-PA224, Db-PB1F262, Kb-NS2114, Kb-PB1703) and CD4+ (red; I-Ab-NP311) T cells on a two-dimensional dot plot within the bulk CD8+ and CD4+ T-cell clusters delineated by t-SNE. (B) Heat maps representation of the normalized expression intensities for each of the marker molecules probed on the two-dimensional t-SNE map. (C) Histograms showing differential expression levels of relevant marker molecules on lymph

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node and lung-derived bulk CD8+ T cells, influenza-specific CD8+ (Db-NP366), and influenza-specific CD4+ (I-Ab-NP311) T cells.

Figure 3. Analysis of phenotypic differences of influenza-specific CD4+

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and CD8+ T-cells derived from lymph nodes and lungs based on the cluster segregation by t-SNE. (A) Comparison of differentially expressed markers on influenza-specific CD4+ T cells from lymph nodes and lungs

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represented by histograms (combined data, n=5 individual mice) and scatter plots (horizontal lines indicate the mean ± SD of the median expression intensity, n=5; p*<0.05, Mann-Whitney test). (B) Comparison of the

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percentages of tetramer positive influenza-specific CD8+ T cells (Db-NP366, Db-PA224, Db-PB1F262, Kb-NS2114, Kb-PB1703) and tetramer negative bulk

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CD8+ T cells (tetneg) expressing markers with different intensities based on

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the two-dimensional t-SNE map representation.

ACCEPTED MANUSCRIPT

Olson M, Russ B, Doherty P, Turner S, Stambas J. Influenza A virusspecific CD8 T-cell responses: from induction to function. Future Virol. 1. März 2010;5(2):175–83.

2.

Doherty PC, Turner SJ, Webby RG, Thomas PG. Influenza and the challenge for immunology. Nat Immunol. Mai 2006;7(5):449–55.

3.

Cukalac T, Valkenburg SA, La Gruta NL, Turner SJ, Doherty PC, Kedzierska K. Multiplexed combinatorial tetramer staining in a mouse model of virus infection. J Immunol Methods. 31. August 2010;360(1– 2):157–61.

4.

Cukalac T, Chadderton J, Zeng W, Cullen JG, Kan WT, Doherty PC, u. a. The influenza virus-specific CTL immunodominance hierarchy in mice is determined by the relative frequency of high-avidity T cells. J Immunol Baltim Md 1950. 1. Mai 2014;192(9):4061–8.

5.

Newell EW, Klein LO, Yu W, Davis MM. Simultaneous detection of many T-cell specificities using combinatorial tetramer staining. Nat Methods. Juli 2009;6(7):497–9.

6.

Hadrup SR, Bakker AH, Shu CJ, Andersen RS, van Veluw J, Hombrink P, u. a. Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers. Nat Methods. Juli 2009;6(7):520–6.

7.

Sant AJ, McMichael A. Revealing the role of CD4+ T cells in viral immunity. J Exp Med. 30. Juli 2012;209(8):1391–5.

8.

Olson MR, Seah SG, Edenborough K, Doherty PC, Lew AM, Turner SJ. CD154+ CD4+ T-cell dependence for effective memory influenza virusspecific CD8+ T-cell responses. Immunol Cell Biol. August 2014;92(7):605–11.

9.

Topham DJ, Doherty PC. Clearance of an influenza A virus by CD4+ T cells is inefficient in the absence of B cells. J Virol. Januar 1998;72(1):882–5.

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10. Bendall SC, Nolan GP, Roederer M, Chattopadhyay PK. A deep profiler’s guide to cytometry. Trends Immunol. Juli 2012;33(7):323–32. 11. Newell EW, Sigal N, Nair N, Kidd BA, Greenberg HB, Davis MM. Combinatorial tetramer staining and mass cytometry analysis facilitate Tcell epitope mapping and characterization. Nat Biotechnol. Juli 2013;31(7):623–9. 12. Rutigliano JA, Sharma S, Morris MY, Oguin TH, III, McClaren JL, u. a. Highly Pathological Influenza A Virus Infection Is Associated with Augmented Expression of PD-1 by Functionally Compromised VirusSpecific CD8+ T Cells. J Virol. Februar 2014;88(3):1636.

ACCEPTED MANUSCRIPT 13. Kandasamy M, Ying PC, Ho AWS, Sumatoh HR, Schlitzer A, Hughes TR, u. a. Complement Mediated Signaling on Pulmonary CD103+ Dendritic Cells Is Critical for Their Migratory Function in Response to Influenza Infection. PLOS Pathog. 10. Januar 2013;9(1):e1003115. 14. Leong ML, Newell EW. Multiplexed Peptide-MHC Tetramer Staining with Mass Cytometry. Methods Mol Biol Clifton NJ. 2015;1346:115–31.

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15. Sumatoh HR, Teng KWW, Cheng Y, Newell EW. Optimization of mass cytometry sample cryopreservation after staining. Cytom Part J Int Soc Anal Cytol. Januar 2017;91(1):48–61. 16. Amir ED, Davis KL, Tadmor MD, Simonds EF, Levine JH, Bendall SC, u. a. viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol. Juni 2013;31(6):545–52.

NU

17. Maaten L van der, Hinton G. Visualizing Data using t-SNE. J Mach Learn Res. 2008;9(Nov):2579–605.

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18. Humphreys IR, Edwards L, Snelgrove RJ, Rae AJ, Coyle AJ, Hussell T. A critical role for ICOS co-stimulation in immune containment of pulmonary influenza virus infection. Eur J Immunol. November 2006;36(11):2928–38.

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19. Whitmire JK, Eam B, Whitton JL. Mice deficient in stem cell antigen-1 (Sca1, Ly-6A/E) develop normal primary and memory CD4+ and CD8+ T cell responses to virus infection. Eur J Immunol. Juni 2009;39(6):1494– 504.

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20. Hu Z, Blackman MA, Kaye KM, Usherwood EJ. Functional heterogeneity in the CD4+ T cell response to murine γ-herpesvirus 68. J Immunol Baltim Md 1950. 15. März 2015;194(6):2746–56.

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