PMC

Nodes (CDR3 AA sequences) were connected by edges defined by a Levenshtein distance of 1.
DOI: http://dx.doi.org/10.7554/eLife.22057.005

CD4⁺ T cell networks formed by the thousand most frequent CDR3 AA segments expressed in two mice. Nodes (CDR3 AA sequences) were connected by edges defined by a Levenshtein distance of 1.
DOI: http://dx.doi.org/10.7554/eLife.22057.006

Frequency of the top 1000 most frequent CDR3 sequences by sharing level for young (6–8 weeks, n = 3) and aged (17–20 months, n = 3) mice.
DOI: http://dx.doi.org/10.7554/eLife.22057.022

Shown is a representative network of the 1000 most frequent sequences from a mouse. Nodes are labeled according to 4 categories: CDR3 sequences that are not public; CDR3 sequences shared by all 11 human samples; CDR3 sequences shared by at least 25 mice; CDR3 sequences shared by at least 25 mice and all 11 humans.
DOI: http://dx.doi.org/10.7554/eLife.22057.012

A comparison of network connectivity formed by the thousand most frequent CDR3 AA segments expressed in 39 humans at different ages (data from . The Mean degree was calculated for each human sample and colored according to 4 age groups: 6–25, 34–43, 61–66, and 71–90 years.
DOI: http://dx.doi.org/10.7554/eLife.22057.021

The mean betweenness centrality is presented as a function of the sharing level in the dataset of 28 mice, for networks composed of the 1000 most frequent CDR3 AA sequences and for networks composed of 1000 randomly selected CDR3 AA sequences from the dataset. Error bars indicate standard error (SE) across the 12 mice used in this study.
DOI: http://dx.doi.org/10.7554/eLife.22057.007

A comparison of network clusters in young and aged mice. Network representations of the 1000 most frequent clones in (A) young and (B) aged mice. The networks composed of the 1000 most frequent clones in the young mice (n = 3) manifested 590.3 ± 61.9 clustered nodes with 992.7 ± 147.4 edges. In contrast, networks composed of the 1000 most frequent clones in the aged mice (n = 3) had 334.7 ± 63.5 clustered nodes with 362.3 ± 153.8 edges. Nodes are colored according to the sharing level of their corresponding CDR3 sequence in the 28 mice reference dataset.
DOI: http://dx.doi.org/10.7554/eLife.22057.020

Each dot represents one CS-public sequence that is found among the most abundant 1000 sequences in at least one mouse and at least one human subject (n = 45 sequences). There is a significant correlation between the degree of CS-public sequences in the two species (R = 0.65, spearman); Sequences that are more connected in one species are typically more connected in the other as well.
DOI: http://dx.doi.org/10.7554/eLife.22057.013

Comparison of the number of nt sequences encoding, on average, an AA CDR3 sequence, for public CDR3 AA sequences, found to be shared by more than 25 out of 28 mice in the reference dataset. Public CDR3 sequences coming from DN thymocytes were encoded on average by a lower number of nucleotide (nt) sequences compared to those from CD4⁺ splenic T cells (p<2.2e-16 for each of these top sharing levels).
DOI: http://dx.doi.org/10.7554/eLife.22057.017

TCRβ repertoires of 11 healthy young human subjects previously investigated by . Shown is the mean degree of nodes as a function of their sharing level in the dataset, for networks composed of the 1000 most frequent CDR3 aa sequences and for networks composed of 1000 randomly selected sequences. Note that public human TCRs manifest a higher degree of connectivity than do private TCRs.
DOI: http://dx.doi.org/10.7554/eLife.22057.008

We generated 100 datasets of simulated human and mouse repertoires, with number of individuals (11 humans, 28 mice) and repertoire sizes as in the experimental data. For each of the 86 observed CS-public sequences, we plot its mean sharing level in the simulations, for human repertoires (red) and mouse (blue) repertoires . The top panel shows 54 sequences that are CS-public in both experiment and simulations. The lower panel shows 32 sequences that are CS-public in the experimental data but not in the simulations. Note that there were additionally about 200 CS-public sequences in the simulations which were not CS-public in the data.
DOI: http://dx.doi.org/10.7554/eLife.22057.011

An example of a network constructed from the 1000 most abundant CDR3β AA sequences from a single mouse. Both panels show the same network. In the left panel, nodes are colored by the dominating J-gene; in the right panel color indicates the dominating V-gene for each AA sequence. Network clusters mostly consist of a single J-gene, with only a few clusters featuring two or three primary J-genes (left). In contrast, V-gene usage in clusters is heterogeneous, with no obvious dominating gene segment (right). This pattern of clusters with homogenous J-gene and heterogeneous V-gene usage was consistent in all top 1000 CDR3β AA sequence networks we examined.
DOI: http://dx.doi.org/10.7554/eLife.22057.004

(A) All CDR3β sequences in the 28 mouse dataset were categorized according to their sharing level, from private (found in only one mouse), to public (found in all 28 mice). The graph presents the percent of sequences within each category that were also found in the human dataset (in at least 1 of 11 young subjects). (B) All CDR3β sequences in the 11 young human subjects were categorized according to their sharing level, from private (found in only one subject), to public (found in all subjects). The graph presents the percent of sequences within each group that were also found in at least one of the 28 mice. In both cases, the fraction of cross-species sequences increases with the sharing level; sequences that are more public in one species are more frequently found in the other species.
DOI: http://dx.doi.org/10.7554/eLife.22057.010

(A) The number of edges in networks formed by the 1000 most abundant CDR3 sequences in three TCR datasets: 12 naïve mice; 5 mice immunized with peptide p277 (HSP60 437–460 VLGGGCALLRCIPALDSLTPANED) emulsified in Complete Freund’s Adjuvant (CFA); and 5 mice immunized with p277+CFA whose splenic T cells were stimulated in-vitro with peptide p277. (B) The number of edges in networks formed by the 1000 most abundant CDR3 sequences in four TCR datasets: 12 naïve mice; 5 mice immunized with OVA 323–339 peptide (ISQAVHAAHAEINEAGR) in CFA; 3 mice immunized with OVA+CFA whose splenic T cells were stimulated in-vitro with the same OVA peptide; and 5 mice immunized with OVA+CFA whose splenic T cells were analyzed 2 months post-immunization.
DOI: http://dx.doi.org/10.7554/eLife.22057.019

Sequence visualization of the red (top right) cluster in the mouse CDR3 sequences network shown in . The original full network is formed by the 1000 most shared mouse CDR3 sequences (found in >25 of 28 mice). 124 CDR3 sequences that were previously annotated (see []) were added to the network and are presented as red arrowheads. 13 annotated sequences were either identical to, or at a Levenshtein distance of 1 from one of the nodes in this cluster, and their associated pathology/antigen is listed next to the corresponding node.
DOI: http://dx.doi.org/10.7554/eLife.22057.015

The cumulative frequency (in %) of relatively private CDR3 sequences from the top 1000 most frequent sequences in the repertoires of patients pre and post CTLA4 blockade treatment with tremelimumab (). Sharing was defined by comparison with a reference dataset of CDR3 sequences from 11 young healthy individuals (): Relatively private sequences were defined as CDR3 sequences shared by 0–5 individuals out of 11 in the reference dataset, where 0 indicates a sequence not found in any of the 11 individuals in the reference cohort. There is a significant increase in the frequency of relatively private sequences (p-value=0.01947, ranked Wilcox paired test).
DOI: http://dx.doi.org/10.7554/eLife.22057.023

(A) Mean number of clustered nodes in networks formed by the top 1000 CDR3 sequences from the following repertoires: DN thymocytes (CD4⁻CD8⁻) (n = 3), CD4⁺ spleen T cells (n = 3), Quad-KO mice() (lack MHC-I, MHC–II, CD4 and CD8) (n = 4), and their WT controls (C57BL/6) (n = 4). Error bars signify standard error. (B) Cumulative frequency of the 86 CS-public CDR3 sequences (observed in the reference datasets of 28 WT mice and 11 healthy humans) is shown for: DN thymocytes (CD4^-CD8^-) (n = 3), CD4⁺ spleen T cells (n = 3) (left), Quad-KO mice (n = 4), and their WT controls (C57BL/6) (n = 4). Error bars signify standard error. (C) Cumulative frequency of nucleotide sequences coding for two annotated (C9 and COPD, top) and two unknown (bottom) public AA CDR3 sequences from repertoires of DN thymocytes and CD4⁺ spleen T cells (sequences from 3 mice are shown). Each color represents a different nucleotide sequence.
DOI: http://dx.doi.org/10.7554/eLife.22057.016

(Right panel is a zoomed-in version of the left panel). Results are shown for 4 representative conditions, with different levels of observed network connectivity, as expressed by the number of clustered nodes (degree >0). These graphs show that regardless of sample size, (A, B) networks from a naïve mouse are the most connected, followed by those of immunized (p277), aged mice, and lastly p277 in vitro stimulation, which is the least connected. (C, D) networks for 39 human samples () divided into 4 age groups. Above ~1000 sequences, the trend is linear; hence the relative fraction of clustered nodes is not sensitive to sample size. Thus, our analysis of network connectivity is not sensitive to the number of sequences used.
DOI: http://dx.doi.org/10.7554/eLife.22057.003

(A) Human (left) or mouse (right) CDR3 sequences are grouped according to their sharing level in the corresponding dataset. For each sharing group, we plotted the percentage of sequences that were shared by at least one subject of the other species. (B) Examples of CS-Public CDR3 sequences, and their V and J segments in mouse and human repertoires. (C) A network formed by the top 1000 CDR3 sequences of a single human subject. Node color represents its sharing within or between species: Pink - shared by all 11 human subjects; Green - shared by at least 25 of the 28 mice; Black – CS-public nodes shared by all 11 humans and at least 25 mice; Blue - not shared. (D) The mean number of edges per node (degree) in the 11 human and 28 mouse networks, subdivided into the four categories as in C. Error bars mark SE.
DOI: http://dx.doi.org/10.7554/eLife.22057.009

(A) A network formed by the 1000 most shared mouse CDR3 sequences (found in >25 of 28 mice). Node size corresponds to the mean abundance of the sequence. Nodes are colored according to their cluster association. 124 CDR3 sequences that were previously annotated (see []) were added to the network and are presented as arrowheads. 63 annotated sequences were either identical to, or at a Levenshtein distance of 1 from one of the nodes, and are listed next to each cluster (with the corresponding color). Annotations of 61 un-clustered sequences are also listed. (B) A network formed by the 1000 most frequent public CDR3 sequences in humans (found in all 11 subjects). Previously annotated mouse (n = 124) and human (n = 30) CDR3 sequences were added to the network as in A (arrowheads). The clusters were distinctly colored in order to visually match between clusters and their annotated sequences, not to define antigen specificity of a cluster. A list of linked annotated CDR3 sequences is shown next to each cluster (11 of 30 human and 23 of 124 mouse annotated CDR3 sequences), together with a list of unclustered annotated human sequences.
DOI: http://dx.doi.org/10.7554/eLife.22057.014