Dataset Information

Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4⁺ T Cells in Peripheral Blood of Subjects with Type 1 Diabetes.

ABSTRACT: The significance of islet Ag-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear, partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior Ag exposure, inferred from expanded TCR clonotypes, and essential phenotypic properties in their transcriptomes. To test this, we developed single-cell RNA sequencing procedures for identifying TCR clonotypes and transcript phenotypes in individual T cells. We applied these procedures to analysis of islet Ag-reactive CD4⁺ memory T cells from the blood of T1D and HC individuals after activation with pooled immunodominant islet peptides. We found extensive TCR clonotype sharing in Ag-activated cells, especially from individual T1D subjects, consistent with in vivo T cell expansion during disease progression. The expanded clonotype from one T1D subject was detected at repeat visits spanning >15 mo, demonstrating clonotype stability. Notably, we found no clonotype sharing between subjects, indicating a predominance of "private" TCR specificities. Expanded clones from two T1D subjects recognized distinct IGRP peptides, implicating this molecule as a trigger for CD4⁺ T cell expansion. Although overall transcript profiles of cells from HC and T1D subjects were similar, profiles from the most expanded clones were distinctive. Our findings demonstrate that islet Ag-reactive CD4⁺ memory T cells with unique Ag specificities and phenotypes are expanded during disease progression and can be detected by single-cell analysis of peripheral blood.

SUBMITTER: Cerosaletti K

PROVIDER: S-EPMC5499675 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4<sup>+</sup> T Cells in Peripheral Blood of Subjects with Type 1 Diabetes.

Cerosaletti Karen K Barahmand-Pour-Whitman Fariba F Yang Junbao J DeBerg Hannah A HA Dufort Matthew J MJ Murray Sara A SA Israelsson Elisabeth E Speake Cate C Gersuk Vivian H VH Eddy James A JA Reijonen Helena H Greenbaum Carla J CJ Kwok William W WW Wambre Erik E Prlic Martin M Gottardo Raphael R Nepom Gerald T GT Linsley Peter S PS

Journal of immunology (Baltimore, Md. : 1950) 20170531 1

The significance of islet Ag-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear, partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior Ag exposure, inferred from expanded TCR clonotypes, and essential phenotypic properties in their transcriptomes. To test this, we developed single-cell RNA sequencing procedures for identifying TCR clonotypes and transcript p ...[more]

PMID: 28566371

Similar Datasets

Project description:ObjectivesWe sought to develop medium throughput standard operating procedures for screening cryopreserved human peripheral blood mononuclear cells (PBMCs) for CD4+ and CD8+ T cell responses to potential autoantigens.MethodsDendritic cells were loaded with a peptide cocktail from ubiquitous viruses or full-length viral protein antigens and cocultured with autologous T cells. We measured expression of surface activation markers on T cells by flow cytometry and cytometry by time of flight 24-72 h later. We tested responses among T cells freshly isolated from healthy control PBMCs, cryopreserved T cells, and T cells derived from a variety of T cell expansion protocols. We also compared the transcriptional profile of CD8+ T cells rested with interleukin (IL)7 for 48 h after 1) initial thawing, 2) expansion, and 3) secondary cryopreservation/thawing of expanded cells. To generate competent antigen presenting cells from PBMCs, we promoted differentiation of PBMCs into dendritic cells with granulocyte macrophage colony stimulating factor and IL-4.ResultsWe observed robust dendritic cell differentiation from human PBMCs treated with 50 ng/mL GM-CSF and 20 ng/mL IL-4 in as little as 3 days. Dendritic cell purity was substantially increased by magnetically enriching for CD14+ monocytes prior to differentiation. We also measured antigen-dependent T cell activation in DC-T cell cocultures. However, polyclonal expansion of T cells with anti-CD3/antiCD28 abolished antigen-dependent upregulation of CD69 in our assay despite minimal transcriptional differences between rested CD8+ T cells before and after expansion. Furthermore, resting these expanded T cells in IL-2, IL-7 or IL-15 did not restore the antigen dependent responses. In contrast, T cells that were initially expanded with IL-2 + IL-7 rather than plate bound anti-CD3 + anti-CD28 retained responsiveness to antigen stimulation and these responses strongly correlated with responses measured at initial thawing.SignificanceWhile screening techniques for potential pathological autoantibodies have come a long way, comparable full-length protein target assays for screening patient T cells at medium throughput are noticeably lacking due to technical hurdles. Here we advance techniques that should have broad applicability to translational studies investigating cell mediated immunity in infectious or autoimmune diseases. Future studies are aimed at investigating possible CD8+ T cell autoantigens in MS and other CNS autoimmune diseases.

Dataset Information

Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4⁺ T Cells in Peripheral Blood of Subjects with Type 1 Diabetes.

Publications

Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4<sup>+</sup> T Cells in Peripheral Blood of Subjects with Type 1 Diabetes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4+ T Cells in Peripheral Blood of Subjects with Type 1 Diabetes.

Publications

Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4<sup>+</sup> T Cells in Peripheral Blood of Subjects with Type 1 Diabetes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4⁺ T Cells in Peripheral Blood of Subjects with Type 1 Diabetes.