Enzyme replacement therapy (ERT) combined with transient low-dose methotrexate (TLD-MTX) results in age- and disease-dependent immune profile changes in Infantile- vs. late-onset Pompe disease patients.

BACKGROUND: Transient low-dose methotrexate (MTX) induces long-term immune tolerance to enzyme replacement therapy (ERT) in Pompe disease; however, the underlying immunological mechanisms remain unclear. We hypothesized that TLD-MTX would reduce immune activation and induce a regulatory phenotype in patients with Infantile-Onset Pompe Disease (IOPD) and Late-Onset Pompe Disease (LOPD) treated with ERT.

METHODS: We evaluated immune cell profile changes in patients treated with ERT+MTX using multiparameter flow cytometry on peripheral blood mononuclear cells collected at baseline and six months post-treatment. We analyzed 40 immune parameters in (1) thirteen unpaired samples from ERT+MTX-untreated (n=10) and -treated (n=3) IOPD patients and (2) six paired pre- and post-treatment samples from LOPD (n=6) patients. Linear regression analyses were used to assess correlations between immune parameters and disease activity measured by urinary glucose tetrasaccharide (Glc4; a breakdown product of glycogen).

RESULTS: In IOPD, ERT+MTX-treated samples (median age: 12.3 months) showed reduced % CD16+ monocytes and increased classical CD14+CD16- monocytes, suggesting a shift toward a less activated immune profile. Untreated samples had higher % mature memory B cells and lower % plasmablasts, central memory CD4+ and CD8+ T cells, and TIGIT+ CD8+ T cells. In contrast, LOPD patients (median age: 51.5 years) exhibited decreased % B cells and increased % TIGIT+ CD4+ T cells after treatment. Baseline Glc4 levels were higher in IOPD than LOPD (42.5 vs. 7) and decreased in both groups following ERT+MTX. In untreated IOPD patients, Glc4 levels positively correlated with CD16+ monocytes, mature memory B cells, and terminally differentiated CD8+ T cells; these associations were not observed after treatment. In LOPD, T follicular regulatory cells correlated with Glc4 prior to treatment but not after, and no additional significant correlations were identified.

DISCUSSION: These findings demonstrate distinct immune profiles before and after ERT+MTX treatment in IOPD and LOPD patients. In IOPD, reduced pro-inflammatory monocytes and memory T cells provide evidence to suggest decreased immune activation. In LOPD, increased TIGIT+ CD4+ T cells may reflect a regulatory mechanism contributing to immune tolerance.