RNAseq of tolerized auroreactive RBC-specific CD4 T cells, HODxOTII murine model of autoimmune hemolytic anemia (AIHA)

Published Jul 24, 2024 on Dryad. https://doi.org/10.5061/dryad.5qfttdzdf

Abstract

We have previously identified that T cell tolerance is the stopgap to red blood cell (RBC)-specific autoimmunity. We have also shown that while tolerance is robust when mice are young, it fails upon age and leads to development of RBC autoantibodies. Herein, we set out to determine which transcripts were associated with tolerization of RBC-autoreative CD4 T cells. For these studies, we used a murine model of autoimmune hemolytic anemia (AIHA), whereby HOD mice (expressing a known RBC-specific antigen) were bred with OTII animals (which react to the ovalbumin epitope contained within teh HOD antigen) [see PMCID PMC8634489]. To that end, splenocytes were collected from 10-12 week old HODxOTII F1 mice (n=4 each genotype) and stained with antibodies against Thy1.2, Ter119, CD19, and CD4. CD19+ B cells and Ter119+ RBCs cells were excluded from live singlets and CD4+Thy1.2+ T cells were sorted. Total RNA was harvested using RNeasy mini plus kit (Qiagen). Samples were sent to the Genome Technology Access Center in the Department of Genetics at Washington University School of Medicine for library preparation and RNAseq. Each sample was run on 3 lanes, with paired-end reads, and 2x150nts.

https://doi.org/10.5061/dryad.5qfttdzdf

CD4+ T cells were sorted from HODxOTII F1 mice on a C57Bl/6J background. HOD is a model antigen containing hen egg lysosome, ovalbumin, and human blood group molecule Duffy. HOD is expressed in a red blood cell (RBC)-specific fashion. CD4 OTII T cells react to an epitope within ovalbumin. Thus, HOD+OTII+ mice have RBC autoreactive CD4 T cells. For these studies, CD4 T cells were sorted from autoreactive HOD+OTII+ and littermate HOD-OTII+ negative controls. The goal of the RNAseq was to determine which genes were associated with CD4 T cells tolerance to RBC-specific autoantigens.

There are 8 total samples: HOD+OTII+ (n=4) and HOD-OTII+ (n=4). Each sample was run in 3 lanes, with paired-end reads. As such, there are 6 Fastq files (where 5_1/5_4, 6_1/6_4, 7_1/7_4 are paired reads). Each raw data file has been uploaded with the genotype and mouse number in the file name.

Individual fq.gz files are uploaded.

For autoreactive, tolerized, CD4+ T cells

HOD+OTII+ Mouse #1

Paired read #1

run_2895_s_5_1_withindex_HODposOTIIpos__1.fq.gz

run_2895_s_5_4_withindex_HODposOTIIpos__1.fq.gz

Paired read #2

run_2895_s_6_1_withindex_HODposOTIIpos__1.fq.gz

run_2895_s_6_4_withindex_HODposOTIIpos__1.fq.gz

Paired read #3

run_2895_s_7_1_withindex_HODposOTIIpos__1.fq.gz

run_2895_s_7_4_withindex_HODposOTIIpos__1.fq.gz

HOD+OTII+ Mouse #2

Paired read #1

run_2895_s_5_1_withindex_HODposOTIIpos__2.fq.gz

run_2895_s_5_4_withindex_HODposOTIIpos__2.fq.gz

Paired read #2

run_2895_s_6_1_withindex_HODposOTIIpos__2.fq.gz

run_2895_s_6_4_withindex_HODposOTIIpos__2.fq.gz

Paired read #3

run_2895_s_7_1_withindex_HODposOTIIpos__2.fq.gz

run_2895_s_7_4_withindex_HODposOTIIpos__2.fq.gz

HOD+OTII+ Mouse #3

Paired read #1

run_2895_s_5_1_withindex_HODposOTIIpos__3.fq.gz

run_2895_s_5_4_withindex_HODposOTIIpos__3.fq.gz

Paired read #2

run_2895_s_6_1_withindex_HODposOTIIpos__3.fq.gz

run_2895_s_6_4_withindex_HODposOTIIpos__3.fq.gz

Paired read #3

run_2895_s_7_1_withindex_HODposOTIIpos__3.fq.gz

run_2895_s_7_4_withindex_HODposOTIIpos__3.fq.gz

HOD+OTII+ Mouse #4

Paired read #1

run_2895_s_5_1_withindex_HODposOTIIpos__4.fq.gz

run_2895_s_5_4_withindex_HODposOTIIpos__4.fq.gz

Paired read #2

run_2895_s_6_1_withindex_HODposOTIIpos__4.fq.gz

run_2895_s_6_4_withindex_HODposOTIIpos__4.fq.gz

Paired read #3

run_2895_s_7_1_withindex_HODposOTIIpos__4.fq.gz

run_2895_s_7_4_withindex_HODposOTIIpos__4.fq.gz

For littermate negative control CD4+ T cells

HOD-OTII+ Mouse #1

Paired read #1

run_2895_s_5_1_withindex_HODnegOTIIpos__1.fq.gz

run_2895_s_5_4_withindex_HODnegOTIIpos__1.fq.gz

Paired read #2

run_2895_s_6_1_withindex_HODnegOTIIpos__1.fq.gz

run_2895_s_6_4_withindex_HODnegOTIIpos__1.fq.gz

Paired read #3

run_2895_s_7_1_withindex_HODnegOTIIpos__1.fq.gz

run_2895_s_7_4_withindex_HODnegOTIIpos__1.fq.gz

HOD-OTII+ Mouse #2

Paired read #1

run_2895_s_5_1_withindex_HODnegOTIIpos__2.fq.gz

run_2895_s_5_4_withindex_HODnegOTIIpos__2.fq.gz

Paired read #2

run_2895_s_6_1_withindex_HODnegOTIIpos__2.fq.gz

run_2895_s_6_4_withindex_HODnegOTIIpos__2.fq.gz

Paired read #3

run_2895_s_7_1_withindex_HODnegOTIIpos__2.fq.gz

run_2895_s_7_4_withindex_HODnegOTIIpos__2.fq.gz

HOD-OTII+ Mouse #3

Paired read #1

run_2895_s_5_1_withindex_HODnegOTIIpos__3.fq.gz

run_2895_s_5_4_withindex_HODnegOTIIpos__3.fq.gz

Paired read #2

run_2895_s_6_1_withindex_HODnegOTIIpos__3.fq.gz

run_2895_s_6_4_withindex_HODnegOTIIpos__3.fq.gz

Paired read #3

run_2895_s_7_1_withindex_HODnegOTIIpos__3.fq.gz

run_2895_s_7_4_withindex_HODnegOTIIpos__3.fq.gz

HOD-OTII+ Mouse #4

Paired read #1

run_2895_s_5_1_withindex_HODnegOTIIpos__4.fq.gz

run_2895_s_5_4_withindex_HODnegOTIIpos__4.fq.gz

Paired read #2

run_2895_s_6_1_withindex_HODnegOTIIpos__4.fq.gz

run_2895_s_6_4_withindex_HODnegOTIIpos__4.fq.gz

Paired read #3

run_2895_s_7_1_withindex_HODnegOTIIpos__4.fq.gz

run_2895_s_7_4_withindex_HODnegOTIIpos__4.fq.gz

Three excel files are also provided.

This file contains the normalized read counts per million:

HODposOTIIpos_v_HODnegOTIIpos_CD4_T_cells_TMM_normalized_reads_count_per_million.xls

This file contains the analyzed fold change, p-values, and false discover rate of all genes/transcripts, comparing HOD+OTII+ and HOD-OTII+ CD4 T cells:

HODposOTIIpos_v_HODnegOTIIpos_CD4T_cells_all_detectable_genes.xls

GO Pathway analysis between HOD+OTII+ and HOD-OTII+ CD4+ T cells

HODposOTIIpos_vs_HODnegOTIIpos_GO_enrichment.xls

Missing data code: NA

RNAseq of tolerized auroreactive RBC-specific CD4 T cells, HODxOTII murine model of autoimmune hemolytic anemia (AIHA)

Data files

Abstract

README: HODxOTII CD4 T cell RNAseq

Methods