Absence of functional fetal regulatory T cells in humans causes in utero organ-specific autoimmunity

Abstract

Regulatory T cells play a critical role in preventing fetal organ-specific autoimmunity in humans. Autopsies of neonatal IPEX patients shortly after birth demonstrate chronic exocrine-dominant pancreatitis with tertiary lymphoid structures containing expanded oligoclonal T/B lymphocytes.

Figure E1.. Pedigree and Clinical Data.

Family pedigree and clinical information for IPEX Patients 1 and 2 (top), as well relevant clinical information regarding the five control patients used for comparison (bottom). (PNA: pneumonia; GBS: group B streptococcal disease; SMA: spinal muscular atrophy). The familial FOXP3 gene mutations are referenced as both are previously published to be associated with IPEX disease^E2,E3.

Figure E2.. Inflamed and noninflamed tissues from neonatal IPEX Patient 1.

Mononuclear cell infiltrates were evident in the (A) pancreas, (B) stomach and (C) thyroid while no inflammation was found in other organs commonly affected in IPEX, including the (D)adrenal, (E) pituitary, or (F) uninflamed testis (Hematoxylin and eosin, 100X [A-C, F], 200X [D,E]).

Figure E3.. RNA-Seq data from IPEX patient 1.

RNA-seq data comparing mRNA transcripts in head and tail of pancreas compared to the uninflamed testes from the extracted fetal autopsy frozen tissue. Gene counts were filtered for fold change >5 or transcript difference >500 when compared to uninflamed testes sample. Heatmaps were generated for: (A) Genes involved in tertiary lymphoid structure and/or germinal center formation; (B) Genes known to be upregulated in T1D; or (C) Interferon-driven chemokine and cytokine genes. Gene expression levels are shown as row normalized Z-scores with blue reflecting low relative expression and orange representing high relative expression.

Figure E4.. Chronic histologic changes and preserved insulin staining.

(A) Pancreas tissue sections from Patient 1 showed acinar dropout and fibrous inflammatory damage of exocrine ducts that was associated with squamous metaplasia (boxes). Islets of Langerhans were numerous (*) and lacked inflammation or cell destruction. Tertiary lymphoid structures including germinal center formation (arrowheads) were present (40X; all images 100X). (B) Percentage of insulin positive surface area compared among pancreatic tissue from Patient 1 (head and tail included), Patient 2 and five age-matched controls. Each mark represents the average of 10 random, non-overlapping 100X fields.

Figure E5.. Oligoclonal enrichment of unique TCR and BCR clones in pancreas compared to cord blood sample.

(A-B) Differential abundance scatter plots were generated comparing pancreas to cord blood to illustrate rearrangements that demonstrate expansion or enrichment based upon nucleotide identity. Testing was excluded on infrequent clones (light grey) determined by minimum threshold (n>10) given the sample size. Pair-wise scatter plots for productive frequency for each rearrangement were plotted between pancreas versus cord blood. Dots displayed on the axis were unique to each tissue. Clones with significant (p<0.01) enrichment in pancreas tissue (red) versus clones enriched in cord blood (blue) and clones lacking significance were also determined (dark grey). Analysis was performed separately for (D) IGH productive rearrangements and (E) TCRB productive rearrangements. The Frequency Equality line represents productive frequency equivalence between samples given total sample size of each. Total productive clonal templates analyzed: IGH cord blood (n=65,025), IGH pancreas (n=77,179), TCRB pancreas (n=193,719), and TCRB cord blood (n=70,668).

Figure 1.. Exocrine-predominant pancreatitis in neonatal IPEX patient.

Pancreas tissue from Patient 1 at age 29 hours (columns 1-2), Patient 2 at 19 days (column 3) and representative control patient (column 4) were serial sectioned and stained with H&E (row 1) and antibodies against CD3 (row 2), CD20 (row 3) and insulin (row 4, columns 2-4). Islets of Langerhans (*) were essentially devoid of lymphocytic infiltrates, best appreciated on high power images (CD3/CD20 – column 1, row 4 [400X]). Insulin staining highlights equivalent quantities of endocrine elements in all three samples (row 4, columns 2-4). Dense infiltrates permeated acinar compartments in IPEX cases, where tertiary lymphoid structures were evident, while lymphocytes were rare in control samples. [(*) Islet of Langerhans; (arrowhead) ectopic germinal center].

Figure 2.. Tamoxifen-inducible exocrine-dominant pancreatitis in the absence of functional regulatory T cells in mouse pancreas.

Representative H&E stained pancreas tissue from (A,B) Cdc42^flox/floxFoxp3^ER-Cre mouse (n=2) and (C, D) control C57Bl/6 mouse (n=2) receiving tamoxifen intraperitoneal injections for 19 days or from (E,F) an untreated control C57Bl/6 mouse. (A, B) Cdc42^flox/floxFoxp3^ER-Cre animals exhibited perivascular and exocrine inflammation (arrowheads) associated with acinar destruction despite unperturbed islets. (*) Islet of Langerhans. There was no inflammation in the pancreas of (D, F) either control mouse. Only moderately intense mononuclear cell infiltrates were restricted to the mesentery (arrowhead) in the control C57BI/6 mouse. (H&E, 40X, A, C, E; 200X, B, D, F).