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Volume 19, Issue 3, Pages 569-583 (April 2017) Sequence and Structural Analyses Reveal Distinct and Highly Diverse Human CD8+ TCR Repertoires to Immunodominant Viral Antigens  Guobing Chen, Xinbo Yang, Annette Ko, Xiaoping Sun, Mingming Gao, Yongqing Zhang, Alvin Shi, Roy A. Mariuzza, Nan-ping Weng  Cell Reports  Volume 19, Issue 3, Pages 569-583 (April 2017) DOI: 10.1016/j.celrep.2017.03.072 Copyright © 2017 Terms and Conditions

Cell Reports 2017 19, 569-583DOI: (10.1016/j.celrep.2017.03.072) Copyright © 2017 Terms and Conditions

Figure 1 NLV- and GIL-Specific CD8+ T Cell Repertoires (A) Isolation of NLV- and GIL-specific CD8+ T cells from the blood of healthy adults. CD8+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) via immunomagnetic separation and stained with NLV or GIL dextramer for enrichment using AutoMacs, followed by cell sorting and phenotyping. The purities of sorted NLV- and GIL-specific CD8+ T cells were over 95%. (B) Estimated α and β TCRs using DivE (Laydon et al., 2015) (projected to 107) of NLV- and GIL-specific CD8+ T cells of each study subject of both freshly isolated (n = 7 for both NLV- and GIL-TCR) and in vitro-stimulated (n = 8 for NLV-TCR and n = 6 for GIL-TCR) cells. IgG− and IgG+ represent anti-CMV IgG-negative and -positive subjects, respectively. Mean and SEM are indicated. (C) Diversity index of the NLV- and GIL-specific TCR repertoire by Simpson index (Magurran, 2004) using the UMI counts for each TCR. Mean and SEM are indicated. (D) Correlation of NLV- and GIL-specific α and β chains. Projected unique NLV- and GIL-specific TCRα and TCRβ in each of the freshly isolated and in vitro-stimulated subjects are presented. (E) Numbers of unique NLV- and GIL-specific TCRα and TCRβ based on reported and this study. The numbers indicate the unique TCR clonotype. (F) Occupancy of NLV- and GIL-specific TCRβ repertoires in total CD8+ TCRβ repertoires. The data are presented as the percentage of the combined projected NLV- and GIL-specific TCRβ repertoires in total CD8+ TCRβ repertoires (105–108) of each study subject (ND1,2,3,4,5,7,8, and 9). Cell Reports 2017 19, 569-583DOI: (10.1016/j.celrep.2017.03.072) Copyright © 2017 Terms and Conditions

Figure 2 Selective Expansion of Public NLV- and GIL-Specific TCR Clonotypes (A) TCR clonotypes shared among the study subjects and literature. A private TCR is defined as only existing in a single subject (indicates as 0) and a public TCR is defined as existing in at least two subjects in this study or one subject in this study and reported. The number on the y axis reflects the total unique TCRs; each TCR was assigned an arbitrary number. Regarding the number on the x axis, 1 indicates that the TCR was only found in one subject in this study but was shared with a published TCR, and 2–10 represent the shared number for ten subjects. The circle size represents the percentage of the UMIs of the TCR in the total UMIs of the subject. Black and red circles represent newly identified and reported TCR clonotypes, respectively. (B) The numbers and percentages of private and public NLV- and GIL-specific TCRs. The numbers inside the bars indicate the percentages of private clonotypes in total TCRs. (C) The percentages of public and private TCRs in weighted total TCR sequences based on the UMI counts from the combined data of eight subjects. The numbers inside the bars indicate the percentages of private clonotypes in weighted total TCRs. Cell Reports 2017 19, 569-583DOI: (10.1016/j.celrep.2017.03.072) Copyright © 2017 Terms and Conditions

Figure 3 Distinct V-J Usage and Dominant CDR3 Length of NLV- and GIL-Specific TCRs (A) V and J gene combinations in NLV- and GIL-specific TCRs. The V-J combinations were collected from all eight subjects and are presented using chord diagrams. The upper and lower parts of each semicircle represent J and V gene segments, respectively. n indicates the total number of V-J combinations. Low-abundance V and J gene segments are combined and labeled as the others. The percentage of UMI counts for dominant combinations is indicated inside the lower semicircle. The percentages indicate the top five most abundant V-J combinations. (B) The relationship between V-J combination and CDR3 length. The CDR3 amino acid length of each V-J combination is summarized and shows the V-J combination with the number of CDR3 lengths as 1, 2, 3, or > 3. The numbers inside the bar indicate the percentages of a single CDR3 length of a given V-J combinations of NLV- and GIL-specific TCRs. (C) The CDR3 length of all TCRs from the top ten most abundant V-J combinations. The percentages of each length of CDR3 in the weighted total TCR sequences are listed. Each rectangular bar represents one unique TCR, and the color map indicates the abundance of UMI in percentages. Cell Reports 2017 19, 569-583DOI: (10.1016/j.celrep.2017.03.072) Copyright © 2017 Terms and Conditions

Figure 4 CDR3 Motifs and TCR α/β Pairing of NLV- and GIL-Specific TCRs (A) Phylogenetic groups of CDR3 sequences. CDR3 sequences from this study and reported previously were aligned with MUSCLE and phylogenetically grouped with the Phylip neighbor joining method. The numbers of groups are listed. (B) The top five highest ranked CDR3 motifs of NLV- or GIL-specific TCRs. Motif analysis is described in Experimental Procedures. All the CDR3 motifs were ranked based on the number of TCRs, studies, subjects, and UMI counts in the current study and the average scores of motif analysis in each group. The consensus sequences of the top five highest-ranked motifs from each category are presented. (C and D) CDR3 motif pairing between TCRα and TCRβ of NLV- (C) and of GIL- (D) specific TCRs. All paired TCR sequences collected either from single-cell analysis in this study or reported previously were mapped to motif types and are presented using chord diagrams. The upper and lower parts of the semicircle represent CDR3β and CDR3α motif types, respectively. (E and F) The top five CDR3α/CDR3β motif pairs of NLV- (E) or GIL- (F) specific TCRs are listed. The numbers represent the percentages of each pair in total pairs. Cell Reports 2017 19, 569-583DOI: (10.1016/j.celrep.2017.03.072) Copyright © 2017 Terms and Conditions

Figure 5 Structure of TCR–GIL–HLA-A2 Complexes and Comparison of TCR Footprints on GIL–HLA-A2 (A) Side view of the F6–GIL–HLA-A2 complex (ribbon diagram). Cyan, TCR α chain; green, TCRβ chain; orange, HLA-A2 heavy chain; gray, β2 microglobulin (β2m); magenta, GIL peptide. (B) Positions of CDR loops of TCRs F6 and JM22 (PDB: 1OGA) (Stewart-Jones et al., 2003) on GIL–HLA-A2 (top view). CDRs of F6 are shown as numbered red loops. CDRs of JM22 are shown in green. HLA-A2 is shown in gray. The GIL peptide is shown in blue. (C) Footprint of TCR F6 on GIL–HLA-A2. The top of the MHC molecule is depicted as a gray surface. The areas contacted by individual CDR loops are color-coded: green, CDR1α; red, CDR2α; blue, CDR3α; magenta, CDR1β; orange, CDR2β; cyan, CDR3β. (D) Side view of the F8–GIL–HLA-A2 complex. (E) Positions of CDR loops of TCRs F8 and JM22 on GIL–HLA-A2. CDRs of F8 are shown in orange. CDRs of JM22 are shown in green. (F) Footprint of TCR F8 on GIL–HLA-A2. Cell Reports 2017 19, 569-583DOI: (10.1016/j.celrep.2017.03.072) Copyright © 2017 Terms and Conditions

Figure 6 Interactions of TCRs F6 and F8 with the GIL Peptide and HLA-A2 (A) Direct and water-mediated interactions between TCR F6 (green) and the GIL peptide (magenta). The HLA-A2 α1 helix is shown in orange. The side chains of contacting residues are drawn in stick representation with carbon atoms in CDR1β and CDR2β or GIL; nitrogen atoms are shown in blue, and oxygen atoms are shown in red. Water molecules are depicted as red spheres. Hydrogen bonds are shown as red dashed lines. Peptide residues are identified by a one-letter amino acid designation followed by position (P). (B) Interactions between CDR3β of F6 and GIL–HLA-A2, showing the hydrogen bond network mediated by Arg98β and Ser99β. The guanidinium group of Arg98β inserts into a shallow pocket between the GIL peptide and the HLA-A2 α2 helix. (C) Interactions between CDR3α (cyan) of F6 and GIL–HLA-A2. (D) Interactions between CDR3α of JM22 and GIL–HLA-A2 (Stewart-Jones et al., 2003). (E) Electron density in the interface of the F8–GIL–HLA-A2 complex. Density from the final 2Fo – Fc map at 4.0-Å resolution is contoured at 1σ. (F) Interactions between the F8 α chain and HLA-A2 α2 helix. The side chains of contacting residues are drawn in stick representation, with carbon atoms shown in cyan (F8). (G) Interactions between the F8 β chain (green) and the GIL peptide. (H) Interactions between CDR3β of F8 and GIL–HLA-A2. The pocket between the GIL peptide and the HLA-A2 α2 helix is marked by a red star. It is not filled by any residue of F8 CDR3β. Cell Reports 2017 19, 569-583DOI: (10.1016/j.celrep.2017.03.072) Copyright © 2017 Terms and Conditions