Association of intrahepatic baseline immunophenotyping and subsequent treatment response.

<p>Association of intrahepatic baseline immunophenotyping and subsequent treatment response.</p

Pediatric autoimmune hepatitis shows a disproportionate decline of regulatory T cells in the liver and of IL-2 in the blood of patients undergoing therapy

<div><p>Background & Aims</p><p>The autoimmune hepatitis (AIH) is a chronic hepatitis driven by the adaptive immunity that affects all age groups. A functional and numerical regulatory T cell (Treg) defect has been reported in pediatric AIH (pAIH), while an intrahepatic increase in adult AIH (aAIH) patients has been detected in current research findings.</p><p>Methods</p><p>Therefore, we quantified the intrahepatic numbers of Treg, T and B cells, as well as serum cytokine levels before and during therapy in pAIH.</p><p>Results</p><p>We found a disproportional intrahepatic enrichment of Tregs in untreated pAIH compared to pediatric non-alcoholic fatty liver disease. The increase of Treg/total T cells was even more pronounced than in aAIH due to fewer infiltrating T and B cells. Portal densities of Treg, as well as total T and B cells, declined significantly during therapy. However, portal Treg densities decreased disproportionately, leading to even decreasing ratios of Treg to T and B cells during therapy. Out of 28 serum cytokines IL-2 showed the strongest (10fold) decrease under therapy. This decline of IL-2 was associated with decreasing intrahepatic Treg numbers under therapy. None of the baseline T and B cell infiltration parameters were associated with the subsequent treatment response in pAIH.</p><p>Conclusions</p><p>Intrahepatic Tregs are rather enriched in untreated pAIH. The disproportional decrease of Tregs during therapy may be caused by a decrease of IL-2 levels. New therapies should, therefore, aim in strengthening intrahepatic immune regulation.</p></div

Patient data.

<p>Patient data.</p

Multicolor immunofluorescence of formalin-fixed and paraffin embedded liver biopsies from pAIH.

<p><b>(A)</b> T cell staining with CD4 (red), CD8 (green), FOXP3 (blue) in a single formalin fixed and paraffin embedded liver biopsy section with pediatric AIH and <b>(B)</b> B cell staining with CD79a (red) and CD4 (green, autofluorescence in blue) in subsequent liver biopsy sections. The white lines surround the evaluated area of the portal infiltrates and exclude lumen of veins, arteries and bile ducts. White bars represent 100 μm. <b>(C)</b> T cell staining of comparator liver biopsies with pediatric non-alcoholic fatty liver disease as in A. <b>(D)</b> Surface expression of CD4 (red) and CD8 (green) in comparison to nuclear colocalization of FOXP3 (blue) and DAPI (white) in pediatric AIH. White bars represent 50 μm.</p

IL-2 associated decline of portal Treg infiltration under therapy for pAIH.

<p><b>(A)</b> Comparison of portal cell densities and portal cell ratios of CD4⁺FOXP3⁺ Treg in untreated pediatric AIH (n = 40) and under ongoing therapy (n = 13). <b>(B)</b> Fold changes of CD4⁺FOXP3⁺ (Treg; n = 11), CD4⁺+CD8⁺ (T cells; n = 11) and CD79a⁺ cells (B cells; n = 9) under therapy in paired samples. <b>(C)</b> Comparison of CD4⁺FOXP3⁺ Treg and total T cells (CD4⁺+CD8⁺) in treated pediatric AIH (n = 13) and treated adult AIH matched for the treatment response (n = 16). <b>(D)</b> Quantification of serum cytokine levels in untreated pAIH at diagnosis (D; n = 43) and during follow-up (FU; n = 28) under therapy. Depicted are all cytokine with significant changes in the follow-up in paired and unpaired non-parametric comparisons. The horizontal line and error bars represent the median and the interquartile range. The seventeen cytokines without significant changes are not shown. Cytokine levels below the detection threshold were set to the lowest detected concentration. <b>(E)</b> Serum IL-2 levels in pediatric AIH similar to (D) and adult AIH (diagnosis: n = 88; under therapy: n = 34), as well as age and gender, matched pediatric controls for cytokines (n = 34). (n.s.: not significant; * p<0.05; ** p<0.01; *** p<0.001)</p

Portal T cell infiltration pattern in untreated AIH-1.

<p><b>(A-C)</b> Comparison of size of portal infiltrates, portal cell densities and portal cell ratios, as well as absolute numbers per portal tract of T cells (CD4⁺+CD8⁺), B cells (CD79a⁺) and CD4⁺FOXP3⁺ Treg in untreated pediatric AIH (pAIH; n = 40), pediatric non-alcoholic liver disease (pNAFLD; n = 12) and untreated adult AIH (aAIH; n = 45). <b>(D)</b> Correlation analysis (n = 38; SR: Spearman rank correlation coefficient) of the portal CD4⁺FOXP3⁺ Treg cell densities and cell ratios as well as the absolute number per portal tract with the hepatitis activity index (mHAI). Horizontal line represents the median and error bars the interquartile range. (* p<0.05; ** p<0.01; *** p<0.001; n.s.: not significant)</p

Iron homeostasis is potentially deregulated by HGF driven suppression of hepcidin-25 in untreated AIH-1.

<p>(A) Spearman rank correlation (SR) analysis of serum ferritin in AIH-1 with alanine aminotransferase (ALT) at diagnosis (Dx) in patients with subsequent biochemical remission (BR, left panel, N = 24) and incomplete biochemical response (IR, right panel, N = 24) matched for ALT, gender and age as far as possible. (B) SR analysis of serum ferritin and hepcidin-25 in patients with IR (N = 8; left panel) and with BR (N = 21; right panel) upon standard therapy and (C) in patients with achieved BR after 6–12 months of therapy (M 6–12; right; N = 7). (D) The hepatocyte growth factor (HGF, red; autofluorescence in green and blue) is expressed in (top) the portal tracts, endothelium and (bottom) liver sinusoids in a representative liver biopsy of untreated AIH-1. White bars represent 100 μm. (E) SR analysis of HGF and hepcidin-25 in untreated AIH-1 (N = 12) with subsequent BR and high ferritin (>2,09x ULN). (F) HGF in patients with high (hSF, N = 30) and low serum ferritin (lSF, N = 37). (* p<0.05; ** p<0.01; *** p<0.001; not significant, p≥0.05; ULN = upper limit of normal)</p

Reversible hyperferritinemia and mild iron deposition in untreated AIH-1.

<p>(A) Intrahepatic iron deposition (blue granula) in untreated AIH-1 in (left) hepatocytes, (middle) portal fields and (right) the sinusoidal compartment (white arrow). (B) The semi-quantitative histopathological iron deposition score (left) in untreated AIH-1 with subsequent biochemical remission (BR: N = 47) or incomplete response (IR: N = 10) and (right) under therapy (Tx; N = 24) compared to baseline at diagnosis (Dx; N = 61). (C) Longitudinal course (mean and standard deviation) under therapy (M = month; Y = year) in BR (black) and IR (grey). (* p<0.05; ** p<0.01; *** p<0.001; ULN = upper limit of normal).</p

Diagnostic performance of the treatment response score to predict incomplete treatment response in untreated AIH-1.

<p>Diagnostic performance of the treatment response score to predict incomplete treatment response in untreated AIH-1.</p

Hyperferritinemia and hypergammaglobulinemia were associated with the subsequent treatment response upon standard therapy in untreated AIH-1.

<p>(A) Left panel: Serum ferritin (SF) in untreated AIH-1 patients with subsequent biochemical remission (BR; N = 83) and incomplete biochemical response (IR; N = 26). Right panel: Histological treatment response with complete remission (CR: N = 16), BR with incomplete histological response (BR/IHR: N = 7) and IR (N = 26). (B) The same analysis for immunoglobulin G (IgG). (C) The AUROC analysis for the prediction of IR before the treatment initiation for SF (dashed line), IgG (dotted line) and their combined treatment response score (black solid line) in the retrospective trainings cohort (N = 76). (D) Rates of BR according to the treatment duration for the treatment response score<1 (solid line) and score≥1 (dashed line) for the training and (E) validation cohort. (* p<0.05; not significant, p≥0.05)</p