Immune Repertoire Profiling Reveals that Clonally Expanded B and T Cells Infiltrating Diseased Human Kidneys Can Also Be Tracked in Blood

<div><p>Recent advances in high-throughput sequencing allow for the competitive analysis of the human B and T cell immune repertoire. In this study we compared Immunoglobulin and T cell receptor repertoires of lymphocytes found in kidney and blood samples of 10 patients with various renal diseases based on next-generation sequencing data. We used Biomed-2 primer panels and ImmunExplorer software to sequence, analyze and compare complementarity determining regions and V-(D)-J elements. While generally an individual’s renal receptor repertoire is different from the repertoire present in blood, 94% (30/32) of the lymphocytes with clonal expansion in kidney can also be traced in blood however, not all of these clonotypes are equally abundant. Summarizing the data of all analyzed patients, 68% of highly expanded T cell clonotypes and 30% of the highly expanded B cell clonotypes that have infiltrated the kidney can be found amongst the five most abundant clonotypes in blood. In addition, complementarity determining region 3 sequences of the immunoglobulin heavy chains are on average more diverse than T cell receptor beta chains. Immune repertoire analysis of tissue infiltrating B and T cells adds new approaches to the assessment of adaptive immune response in kidney diseases. Our data suggest that expanded clonotypes in the tissues might be traceable in blood samples in the course of treatment or the natural history of the disease.</p></div

CDR3 sequences, V-(D)-J elements and functionality of highly expanded clonotypes in kidney and blood of all patients.

<p>Percent values for expanded clonotypes in kidney and blood samples are highlighted in bold. Expanded clonotypes that could not be detected in the other compartment were marked as not detected, n.d. Clonotypes are also categorized according to primer sets regarding Biomed-2 primer panel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143125#pone.0143125.ref015" target="_blank">15</a>]. B cell results from patient 6 were discarded due to low amount of DNA input leading to inaccurate clonotype proportions. T cell results from blood sample of patient 10 were sorted in CD4⁺/CD8^- and CD4^-/CD8⁺ subpopulations. The T helper cell subpopulation showed no expansions and is therefore not listed (we found a very low abundance of the expanded clonotype in the kidney in this sample and we assume that these are traces from the CD4^-/CD8⁺ population and therefore this sample was discarded). Functionality of the rearranged chain is marked P for productive and UP for unproductive.</p><p>CDR3 sequences, V-(D)-J elements and functionality of highly expanded clonotypes in kidney and blood of all patients.</p

Inverse Simpson’s diversity index and cell input.

<p>(a) IGH and TRB diversity of the average of all patients and healthy volunteers based on the inverse Simpson’s diversity index. Independent two-sided t test were performed with a level of significance of 0.05. (b) Average cell input of all patients and healthy volunteers for B and T cells. Paired two-sided t test were performed with a level of significance of 0.05. Error bars represent the standard deviation between samples of the same group.</p

Quantification of element frequency (%) in highly expanded clonotypes.

<p>(A) VH, DH and JH element distribution of expanded B cell clonotypes and (B) Vß, Dß and Jß element distribution of expanded T cell clonotypes. We would appreciate the creation of a publicly available database with common V-(D)-J element frequency distribution in human populations to identify potential shifts in the course of diseases as to our knowledge this has not been provided to the community until this date.</p

Average percentage of blood and kidney lymphocyte subpopulations in all patients.

<p>T cells were separately analyzed for CD4⁺ and CD8⁺ subpopulations. NKT cells were gated using CD3⁺/CD56⁺ and NK cells, by CD3^-/CD56⁺. Cells presenting CD19⁺ and/or CD20⁺ surface markers were categorized as B cells. Significant differences were marked by a connecting line and the corresponding P-value (paired two-sided t-test).</p