Tissue distribution and clonal diversity of the T and B cell repertoire in type 1 diabetes
Howard R. Seay, Erik Yusko, Stephanie J. Rothweiler, Lin Zhang, Amanda L. Posgai, Martha Campbell-Thompson, Marissa Vignali, Ryan O. Emerson, John S. Kaddis, Dave Ko, Maki Nakayama, Mia J. Smith, John C. Cambier, Alberto Pugliese, Mark A. Atkinson, Harlan S. Robins, Todd M. Brusko
Howard R. Seay, Erik Yusko, Stephanie J. Rothweiler, Lin Zhang, Amanda L. Posgai, Martha Campbell-Thompson, Marissa Vignali, Ryan O. Emerson, John S. Kaddis, Dave Ko, Maki Nakayama, Mia J. Smith, John C. Cambier, Alberto Pugliese, Mark A. Atkinson, Harlan S. Robins, Todd M. Brusko
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Research Article Immunology

Tissue distribution and clonal diversity of the T and B cell repertoire in type 1 diabetes

Abstract

The adaptive immune repertoire plays a critical role in type 1 diabetes (T1D) pathogenesis. However, efforts to characterize B cell and T cell receptor (TCR) profiles in T1D subjects have been largely limited to peripheral blood sampling and restricted to known antigens. To address this, we collected pancreatic draining lymph nodes (pLN), “irrelevant” nonpancreatic draining lymph nodes, peripheral blood mononuclear cells (PBMC), and splenocytes from T1D subjects (n = 18) and control donors (n = 9) as well as pancreatic islets from 1 T1D patient; from these tissues, we collected purified CD4+ conventional T cells (Tconv), CD4+ Treg, CD8+ T cells, and B cells. By conducting high-throughput immunosequencing of the TCR β chain (TRB) and B cell receptor (BCR) immunoglobulin heavy chain (IGH) on these samples, we sought to analyze the molecular signature of the lymphocyte populations within these tissues and of T1D. Ultimately, we observed a highly tissue-restricted CD4+ repertoire, while up to 24% of CD8+ clones were shared among tissues. We surveyed our data set for previously described proinsulin- and glutamic acid decarboxylase 65–reactive (GAD65-reactive) receptors, and interestingly, we observed a TRB with homology to a known GAD65-reactive TCR (clone GAD4.13) present in 7 T1D donors (38.9%), representing >25% of all productive TRB within Tconv isolated from the pLN of 1 T1D subject. These data demonstrate diverse receptor signatures at the nucleotide level and enriched autoreactive clones at the amino acid level, supporting the utility of coupling immunosequencing data with knowledge of characterized autoreactive receptors.

Authors

Howard R. Seay, Erik Yusko, Stephanie J. Rothweiler, Lin Zhang, Amanda L. Posgai, Martha Campbell-Thompson, Marissa Vignali, Ryan O. Emerson, John S. Kaddis, Dave Ko, Maki Nakayama, Mia J. Smith, John C. Cambier, Alberto Pugliese, Mark A. Atkinson, Harlan S. Robins, Todd M. Brusko

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Figure 7

T cell receptor (TCR) sharing among intraislet and peripheral tissues.

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T cell receptor (TCR) sharing among intraislet and peripheral tissues. S...
Spleen, peripheral blood mononuclear cells (PBMC), pancreatic draining lymph node (pLN), and pancreatic islets harbor distinct adaptive immune repertoires in a 22-year-old organ donor (nPOD 6323) with type 1 diabetes (T1D) for 6 years. (A) The CD3+ insulitis lesion is evident by histology of insulin– islets within the pancreas tail region (CD3, brown; glucagon, red; scale bar: 100 μm). Serial sections of the pancreatic tail region show two islets with (B) insulitis (CD3, brown; glucagon, red) and (C) residual insulin+ β cells (Ki67, brown; insulin, red; scale bar: 200 μm). (D) CD19+ B cells were also observed surrounding an islet within the pancreatic head region (CD19, brown, glucagon, red; scale bar: 100 μm). The Venn diagrams show the absolute number of unique (E) CD8+ T cells, (F) CD4+ conventional T cells (Tconv), and (G) Treg complementarity determining region 3 β chain (CDR3β) amino acid (AA) sequences shared across PBMC (blue), intraislet (green), pLN (red), and spleen (orange) samples. Within the intraislet region, no Treg were isolated, so only total CD4+ or CD8+ sequences were available for comparison. The numbers show the CDR3β AA sequence overlap between the indicated tissue and the islet sample calculated for each clone that is detected in both samples: number of shared templates/total templates in both samples. Additional histology is freely available for review at the nPOD Aperio database with login credentials (http://ahc-path-apr01.ahc.ufl.edu/Login.php, for which the password request form can be found at http://www.jdrfnpod.org/for-investigators/password-request-form/).