Immunology and Inflammation

H2-O deficiency promotes regulatory T cell differentiation and CD4 hyperactivity

Robin A. Welsh
Nianbin Song
Chan-su Park
J. David Peske
Scheherazade Sadegh-Nasseri author has email address

Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD, USA
Department of Pharmaceutics, College of Pharmacy, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju 28644, Korea

https://doi.org/10.7554/eLife.90845.1

Open access
Copyright information

Figures and data

Loss of H2-O function supports increased thymic Regulatory T cell development
A. Left: representative gating showing how “Auditing” vs “Clonal Deleted” CCR7+CD4+ T cells are defined. Middle: Frequency of the activation marker CD69 in either “auditing” medulla CD4+ T cells or (Right) “clonally deleted” medulla CD4+ T cells.
B. Subdivision of auditing CD4 T cells into three maturation stages: Semi-Mature (Left), Mature 1 (Middle), and Mature 2 (Right). Expression has been normalized to the average H2-O WT levels within each experiment to allow for comparison across experiments. Data from 5 replicate experiments.
C. Frequency of medulla specific (CCR7+) CD4 T cells selected for clonal deleted (Caspase-3+) ns = not significant
* = <0.05
** = <0.001
***= <0.0001
**** = <0.00001
Statistics: simple student T-test

DO-KO thymi have an increased proportion of medulla CD4 T cells at the Mature 1 stage
A. The percentage of Foxp3+ CD25+ cells within the CD4 single-positive thymus population in H2-O WT (white) or H2-O KO (red) cells. Data from 3 replicate experiments
B. Geometric mean fluorescence intensity (gMFI) of CD25 (left), CD5 (right) and Nur77 (Bottom) expressed by Foxp3+ CD4+ T cells in the thymus of H2-O WT (white) or H2-O KO (red) mice.
C. Subdivision of Treg cells into three maturation stages: Semi-Mature (Left), Mature 1 (Middle), and Mature 2 (Right). Expression has been normalized to the average H2-O WT levels within each experiment to allow for comparison across experiments. Data from 2 replicate experiments.

H2-O KO mice have an increased activated peripheral CD4 T cell population
A. Percentage of CD4+ T cells within the splenic CD3+ population of unimmunized 6-10-week-old H2-O WT (white) and H2-O KO (red) mice. Representative of >4 independent experiments, N = 16 mice per group.
B. Percentage of CD4+ T cells expressing the lymphoid tissue homing receptor CCR7
C. CCR7 and CD62L expression levels in unimmunized splenic CD4 T cells
D. Unimmunized levels of CD44 and CD69 expressed by splenic CD4 T cells. Increased CD69 expression, a marker of recent activation showed increased levels on H2-O KO CD4 T cells
* = <0.05
** = <0.001
*** = <0.0001
**** = <0.00001

Peripheral H2-O KO Tregs have increased TCR stimulation
A. Frequency of Foxp3+CD25+ cells in splenic CD4+ T cell population of unimmunized mice
B. H2-O KO peripheral Tregs express decreased levels of naïve (CD44+ CD62L+) expressing cells
C. Normalized Nur77 gMFI levels in Foxp3+CD25+ Treg cells in H2-O WT (white) and H2-O KO (red) cells. To account for experimental variation the average Nur77 gMFI level in H2-O WT samples was calculated. gMFI levels in both H2-O WT and H2-O KO samples were then divided by the calculated H2-O WT average. An increased Nur77 ratio indicates increased Nur77 gMFI levels. Summary of 3 repeat experiments.

Loss of H2-O function causes increased basal CD4 cell activation
A. scRNA-seq clustering of CD4 T cells after Seurat analysis. Data represents the average of of 3 biological replicates per genotype.
B. Breakdown of clusters in H2-O WT (Left) or H2-O KO (Right) samples. Clusters are grouped based upon, (1) known CD4 T cell subset markers and (2) gene comparison to published CD4 T cell data sets available on the Immunological Genome Project (www.immgen.org). Identified CD4 Cell phenotypes were: Non-activated, Activated, and Regulatory T cells. “Other” refers to a minor macrophage and NKT cell contamination from the sorting process.
C. Distribution of the for the Non-activated and Activated phenotypes across the H2-O WT and H2-O KO biological replicates (N = 3 mice per genotype).
D. In vivo proliferation of adoptively transferred naïve Thy1.1+ CD4 T cells (Thy1.1+ CD3+ CD4+ CD44-CD25-) after 7 days in either Thy1.2 H2-O WT (white) or Thy1.2 H2-O KO (red) hosts. Pooled data from 2 independent experiments (N= 6 H2-O WT, 5 H2-O KO)

The H2-O KO Treg population has a more effector like phenotype
A. (Left) distribution of the two Treg populations identified by scRNA-seq clustering in either H2-O WT (left) or H2-O KO (right) samples. (Right) Expression of key Treg phenotypic markers: Foxp3 (Top) and CD25 [Il2ra] (Bottom)
B. Subdivision of the splenic CD4+ T cell population by Foxp3 and Helios expression
C. Comparison of cluster 2 upregulated genes (Log₂Avg FC >0.5) in H2-O WT and H2-KO samples to a published (Miragaia et al.) splenic effector Treg genetic profile

A. Representative plots showing CD4, CD8 and Double Positive (DP) percentages in H2-O WT (Top) and H2-O KO (Bottom)
B. Summary plots of the single-positive CD4 (Top) and CD8 (Bottom) percentages from 5 repeat experiments.
C. (Left) representative flow plots showing the “Signaled” vs “Non-signaled” thymocytes in H2-O WT (Top) or H2-O KO (Bottom).
(Right) Summary plots of >5 repeat experiments.
D. Frequency of signaled CCR7+ medulla CD4 T cells in H2-O WT (white) and H2-O KO (red) mice

Top: breakdown of H2-O KO (right) and H2-O WT (Left) Seurat clustering
Bottom: Seurat clustering for each individual biological replicate

Sign up for email alerts