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An atlas of B-cell clonal distribution in the human body

Abstract

B-cell responses result in clonal expansion, and can occur in a variety of tissues. To define how B-cell clones are distributed in the body, we sequenced 933,427 B-cell clonal lineages and mapped them to eight different anatomic compartments in six human organ donors. We show that large B-cell clones partition into two broad networks—one spans the blood, bone marrow, spleen and lung, while the other is restricted to tissues within the gastrointestinal (GI) tract (jejunum, ileum and colon). Notably, GI tract clones display extensive sharing of sequence variants among different portions of the tract and have higher frequencies of somatic hypermutation, suggesting extensive and serial rounds of clonal expansion and selection. Our findings provide an anatomic atlas of B-cell clonal lineages, their properties and tissue connections. This resource serves as a foundation for studies of tissue-based immunity, including vaccine responses, infections, autoimmunity and cancer.

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Figure 1: Diversity, similarity and networks of large clones.
Figure 2: Tissue distributions of large clones.
Figure 3: Analysis of sequence variants in clonal lineages.
Figure 4: Somatic hypermutation in different tissues.

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Acknowledgements

We thank the Human Immunology Core facility at the University of Pennsylvania for assistance with cell preparations and sequencing support. We thank Y. Louzoun, M. Cancro and R. Thomas Jr. for discussions. We thank the organ donor families and the LiveOnNY transplant staff and coordinators. This work was supported by NIH P01 AI106697 (D.L.F., U.H., M.J.S., W.M., B.Z., A.M.R., D.R., D.J.C., N.M., T.G. and E.T.L.P.), P30-CA016520 (E.T.L.P.) and F31AG047003 (J.J.C.T.). G.W.S. was funded by the US Department of Education Graduate Assistance in Areas of National Need (GAANN) program, CFDA Number: 84.200.

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Authors

Contributions

W.M. designed and performed experiments, developed methods, analyzed data, prepared figures and helped write the manuscript. B.Z., G.W.S. and A.M.R. developed methods for data analysis and visualization, analyzed data, prepared figures and helped revise the manuscript. D.R. performed experiments and helped revise the manuscript. J.J.C.T., D.J.C., N.M., H.L., A.L.F. and T.G. were involved in donor recruitment, organ recovery and helped revise the manuscript. D.L.F. directs the organ donor tissue resource for acquisition of tissues and helped write the manuscript. M.J.S. designed experiments, contributed ideas and helped write the manuscript. E.T.L.P. and U.H. planned the study. U.H. developed methods for data analysis and visualization, designed the data analysis and helped write the manuscript. E.T.L.P. designed experiments, contributed ideas to the data analysis, oversaw the overall study and wrote the manuscript.

Corresponding authors

Correspondence to Uri Hershberg or Eline T Luning Prak.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Rarefaction Analysis and Sampling.

a, Rarefaction plots show the expected levels of coverage for different subsamples of sequencing libraries for repertoires of clone size of at least 2 (darkest blue line), 10 (second darkest blue), 20 (red, the cut-off chosen for the overlap analysis), 30 (light blue), 40 (lighter blue) or 50 and greater (light grey) in the repertoire of D207. Separate plots are shown for each tissue. b, Sampling needed for clones of different sizes and coverage thresholds in D207. Bars indicate the number of additional libraries needed to sample 0.5,0.75,0.9 and 0.95 (fractions) of all clones at different clone size cut-offs. Colors of the bars indicate the tissue. PBL = peripheral blood; BM = bone marrow; SPL = spleen; MLN = mesenteric lymph node.

Supplementary Figure 2 Cosine Similarity Analysis.

a, Box plots of distribution of cosine similarity between all sample pairs within a tissue. Similarity is assessed for C20 clones, C20 clones without the top 10 clones in each tissue, all clones, or all clones without the top 10 copy number clones in each tissue. Boxes represent the first and third quartiles bisected by the median. Whiskers represent the most extreme data excluding outliers, where outliers (dots) are data beyond the third or first quartile by a distance exceeding 1.5 times the inter-quartile interval. The C20 clone panel for D207 corresponds to Fig. 1b in the main manuscript. b, The cosine similarity of clonal populations between tissue pairs in D181 and D207 with and without blood clones. Each section represents a tissue and each arrow within each section represents the level of overlap (cosine similarity) between that tissue and clones from other tissues. Longer arrows indicate higher value of cosine similarity indicating more overlap of clonal populations between the tissues. Top: all C20 clones (same as Fig. 1c). Bottom: C20 overlap without blood clones; clones that have sequences in the blood were computationally removed from all tissues.

Supplementary Figure 3 Analysis of Clonal Networks in D207 Using Different Clone Size Definitions.

Clonal overlap was evaluated using cosine similarities between different tissue pairs, as described in the Online Methods. Each wedge represents a tissue and each arrow represents the cosine similarity between the tissues (arrow colors are indicated in the legend, on the left). Three different definitions of clone size are used for the analysis: a, the copy number of all sequences in a clone in a given tissue; b, the total number of unique sequence instances in a clone in a given tissue; and c, the definition that we used in the manuscript (same image as Fig. 1c) - the number of independent sequencing library samples in which the clone appears, in a tissue.

Supplementary Figure 4 Clone Sizes Grouped by Clone Category.

Sizes of large clones (in binned clone sizes of sequence instances, see Online Methods), grouped by tissue distribution category in D181 (left) and D207 (right).

Supplementary Figure 5 Large Clone Tissue Distribution Types in D207.

C20 clones (including clones in the blood): a, Single tissue clones; b, Two-tissue clones; c, Regional (found in 3-5 tissues) and d, Global clones (found in 6-8 tissues). Tissues that contain only a single instance of a clone are not counted towards the distribution pattern of that clone. Each line is a clone. The position along the line represents a tissue. The size of the circles at each position represents the total number of instances the clones have in each tissue. The colored wedges represent the fraction of sequencing libraries that contain at least one instance of the clone in each tissue. A sample clone is shown for each tissue distribution pattern. The number to the left of each line is that clone’s unique clone ID in http://immunedb.com/tissue-atlas. The number to the right is the number of different clones with this tissue distribution pattern. p = peripheral blood; b = bone marrow; s = spleen; l = lung; m = mesenteric lymph node; j = jejunum; i= ileum; c = colon.

Supplementary Figure 6 Large Clone Tissue Distribution Types in D181.

C20 clones (including clones in the blood): a, Single tissue clones; b, Two-tissue clones; c, Regional (found in 3-5 tissues) and d, Global clones (found in 6-8 tissues). Tissues that contain only a single instance of a clone are not counted towards the distribution pattern of that clone. Each line is a clone. The position along the line represents a tissue. The size of the circles at each position represents the total number of instances the clones have in each tissue. The colored wedges represent the fraction of sequencing libraries that contain at least one instance of the clone in each tissue. A sample clone is shown for each tissue distribution pattern. The number to the left of each line is that clone’s unique clone ID in http://immunedb.com/tissue-atlas. The number to the right is the number of different clones with this tissue distribution pattern. p = peripheral blood; b = bone marrow; s = spleen; l = lung; m = mesenteric lymph node; j = jejunum; i= ileum; c = colon.

Supplementary Figure 7 Effects of Clone Size Thresholding on Clone Tissue Membership.

a, Similar blood and GI tract networks are seen at different clone size cut-offs. Overlapping clones are plotted at different clone size cut-offs (greater than or equal to 0.01%, 0.005% and 0.001% of total copies of all of the sequencing libraries within each donor). Each horizontal line represents a clone. Networks of overlapping clones were visualized using MiXCR and VDJtools software (see Online Methods). Only clones from D207 that overlapped between two or more tissues are shown. PBL = peripheral blood; BM = bone marrow; SPL = spleen; Lun = lung; MLN = mesenteric lymph node; Jej = jejunum; Ile = ileum; Col = colon. b, Breadth of tissue membership using different CDR3 identity thresholds. The tissue distribution of C20 clones in the two most deeply sequenced donors was studied using: 85% amino acid identity in CDR3 (the cut-off used in the main manuscript), 90% amino acid identity and no requirement for any CDR3 identity (“bucket”). All of the sequences were collapsed at these different CDR3 similarity thresholds into clones. Then C20 clones were analyzed for their tissue representation: single tissue, double (2 tissues), regional (3-5 tissues) and global (6-8 tissues). The C20 clones were recalculated for each threshold. c, Comparable partitioning of clones from D207 into blood and GI tract networks at two different CDR3 amino acid similarity thresholds. Networks of C20 clones with 85% CDR3 identity (left image is from Fig. 1c in the main manuscript) and 90% CDR3 amino acid identity (right panel) were analyzed using the cosine statistic (see Online Methods). Cosine similarity for two-tissue comparisons is plotted using the color scheme defined in the legend.

Supplementary Figure 8 Blood and GI Tract Networks are Present in All Donors.

Clonal overlap was evaluated in all donors with clones that comprised 0.01% or more of total copies. Each horizontal line represents a clone. Networks of overlapping clones were visualized using MiXCR and VDJtools software (see Online Methods). Only clones that overlapped between two or more tissues are shown. The MLN sample in D168 was hypocellular. PBL = peripheral blood; BM = bone marrow; SPL = spleen; Lun = lung; MLN = mesenteric lymph node; Jej = jejunum; Ile = ileum; Col = colon.

Supplementary Figure 9 Clonal Lineage Networks in D207 with and without Blood Clones.

Each horizontal line represents a clone with at least 20 unique sequence instances in one tissue that overlaps with at least one other tissue, each column represents a tissue. All sub plots are identical, but are colored differently to highlight clones that reside in different tissues. In each instance, the clones that overlap in a specific tissue are colored as indicated by the tissue listed at the top of each panel. All other clones are denoted in black. a, Blood tissue clones highlighted; b, GI tract clones highlighted. Top row: all C20 clones. Bottom row: blood C20 clones computationally excluded (any clone with sequences found in the blood is removed from the analysis). Clones are ordered by their tissue membership.

Supplementary Figure 10 Clonal Lineage Networks in D181 with and without Blood Clones.

Each horizontal line represents a clone with at least 20 unique sequence instances in one tissue that overlaps with at least one other tissue, each column represents a tissue. All sub plots are identical, but are colored differently to highlight clones that reside in different tissues. In each instance, the clones that overlap in a specific tissue are colored as indicated by the tissue listed at the top of each panel. All other clones are denoted in black. a, Blood tissue clones highlighted; b, GI tract clones highlighted. Top row: all C20 clones. Bottom row: blood C20 clones computationally excluded (any clone with sequences found in the blood is removed from the analysis). Clones are ordered by their tissue membership.

Supplementary Figure 11 Sequence Mixing within Clonal Lineages.

a (inset), Numbers of clones used for the clumpiness analysis. Shown are the numbers of clones with nodes in each pair of tissues. This analysis is restricted to C20 clones in D207; these numbers correspond to the clones analyzed in Fig. 3b. b, Sequence mixing within clonal lineages varies between different tissue types. The frequency distributions of clumpiness values between two tissues for all clones with nodes in each tissue pair (combinations are listed at the top of each plot). This analysis was performed on C20 clones in D207.

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Meng, W., Zhang, B., Schwartz, G. et al. An atlas of B-cell clonal distribution in the human body. Nat Biotechnol 35, 879–884 (2017). https://doi.org/10.1038/nbt.3942

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