Insights Through Detailed Immunophenotyping into The Immune Etiology Of Indeterminate Pediatric Acute Liver Failure (IND-PALF): An Immune Dysregulatory Disease

Abstract

Approximately one-third of acute pediatric liver failure may be due to indeterminate etiology (IND-PALF). Liver histopathology has shown dense infiltrates of CD8+CD103+ (tissue-resident memory T cell phenotype) Perforin+ T cells in most of these patients suggesting underlying immune dysregulation, which may concurrently evolve to severe aplastic anemia (SAA). A viral etiology has been postulated, and management may require liver transplant and/or HCT. Given the high likelihood of immune dysregulation contributing to the severity of the disease and lack of detailed immunological investigation, we undertook cellular and molecular studies in a small cohort of IND-PALF patients. Four patients (3–8y, 3 males, 1 female) presented with PALF and histopathology consistent with IND-PALF. We performed detailed blood immunophenotyping on 3 of the 4 patients (Table). Key immune anomalies included pan-lymphopenia in 2/3 patients, expansion of activated, exhausted and senescent CD8+ T cells, decreased switched memory B cells and CD19+27+ (total memory + plasmablasts) B cells. To facilitate selection of immunomodulatory therapy best suited for disease control, the ability of FOXP3+Tregs to upregulate CTLA4 on activation was assessed and significantly reduced in 2 of the 3 patients. All patients displayed increased sIL-2R, CXCL9, IL-18 and IL-6 levels compared to healthy controls, with CXCL9 higher than IL-18 indicating T cell activation. Genomic analyses including bulk RNA-seq and pathogen-seeking metagenomics was performed on liver tissue and PBMCs on all 3 patients with appropriate controls. A preliminary analysis of metagenomics data did not reveal evidence of viral RNA in tissue. A monogenic immune etiology was not identified in any of the patients. One patient was initiated on abatacept (P1) and has remained clinically stable without liver transplant or HCT and evidence of normalization of bone marrow cellularity; one patient required a liver transplant (P3) and two patients required allogeneic HCT (P2 and P4). All patients are currently on immunosuppressive therapy. In summary, severe CD8+ T cell dysregulation exemplifies patients diagnosed with IND-PALF, and detailed blood immunophenotyping performed early in the disease course can help identify these patients and enable prompt initiation of targeted immunomodulation, which can mitigate end-stage liver and bone marrow failure.TableImmune Profile of IND-PALF patients at diagnosisP1P2P3Reference Interval/Cut-offAge at Presentation/Dx3 yrs.8 yrs.3 yrs.SexFMMCD45+ALC (cells/µL)23219248191695–5226 cells/µLCD3+Tcell (cells/µL)1281523746969–3471 cells/µLCD4+Tcell (cells/µL)7143121513–2081 cells/µLCD8+Tcell (cells/µL)1071066568336–1355 cells/µLCD19+20+ B cell (cells/µL)487249165–1154 cells/µLCD16/56+ NK cell (cells/µL)632217116–1075 cells/µLT cell Activation MarkersCD4+ HLA DR+ (%CD4)40.1420.676.3610.67%CD8+ HLA DR+ (%CD8)47.4254.5038.3814.60%CD4+CD38+DR+ (%CD4)30.3812.604.245.50%CD8+CD38+DR+ (%CD8)42.9350.3436.347.21%T cell Senescence/Exhaustion MarkersCD4+CD57+ (%CD4)51.3724.054.445.55%CD4+CD28-(%CD4)28.8514.443.402.49%CD8+CD57+ (%CD8)76.6246.4522.9622.43%CD8+CD28-(%CD8)56.1312.4811.7018.53%CD4+PD-1+DR+ (%CD4)24.077.780.651.10%CD8+PD-1+DR+ (%CD8)26.7425.0423.782.28%CTLA4 expression on Activated Tregs (ratio of stimulated/unstimulated)1.111.775.624.22%B cell SubsetsCD19+CD27+ Total memory + plasmablasts (%CD19)6.084.588.356–34%CD19+27+IgM-IgD- (sm B cells) (%CD19)4.120.983.102–18%CD19+CD24++CD38++Transitional B cells (%CD19)39.1937.387.413–23%Cytokines (pg/mL)sIL-2R5987.502986.253741.5144–1329 pg/mLCXCL913926.506224.083582<647 pg/mLIL-181968.041152.733819.5089–540 pg/mLIL-621.03106.7021.8<10 pg/mLFeatures of aplastic anemia were observed in all 4 patients though only 2 required HCT. Abnormal values indicated in red font.