Transcriptome profiling of regulatory T cells from children with transient hypogammaglobulinemia of infancy

Abstract

Transient hypogammaglobulinemia of infancy (THI) is one of the most common forms of hypogammaglobulinemia in the early childhood. THI is usually associated with chronic, recurrent bacterial and viral infections, life-threatening in some cases, yet its pathogenesis is still largely unknown. As our previous findings indicated the possible role of Treg cells in the pathomechanism of THI, the aim of the current study was to investigate gene expression profile of Treg cells isolated from THI patients. The transcriptome-wide gene profiling was performed using microarray technology on THI patients in two time-points: during (THI-1), and in resolution phase (THI-2) of hypogammaglobulinemia. As a result, a total of 1086 genes were differentially expressed in THI-1 patients, when compared to THI-2 as well as control group. Among them, 931 were up- and 155 downregulated, and part of them encodes genes important for Treg lymphocyte biology and function, i.e. transcription factors/cofactors that regulate FOXP3 expression. Thus, we postulate that Treg cells isolated from THI patients during hypogammaglobulinemia display enhanced suppressor transcriptome signature. Treg expression profile of THI children after normalization of Ig levels largely resembles the results obtained in healthy control group, suggesting THI Treg transcriptome seems to return to that observed in healthy children. Taken together, we suggest that THI pathomechanism is associated not only with transiently elevated Treg cell numbers, but also with their enhanced regulatory/inhibitory functions. These findings expand our knowledge of human Treg cells and may be useful for the future diagnosis or management of THI.

Keywords: long non-coding RNA; microRNA precursors; microarray analysis; regulatory T cells; transcriptome profiling; transient hypogammaglobulinemia of infancy.

Graphical Abstract

Figure 1.

Study design. Two-step protocol was performed: (a) First T_reg cells number was assessed using flow cytometry (FACS) and next (b) T_regs were isolated using magnetic-activated cell sorting (MACS) technology and then frozen at −80°C until RNA isolation. The group of children definitely diagnosed as THI (n = 10), on the basis of diagnosis criteria, was formed from children initially diagnosed as THI (n = 30). RNA isolation followed by microarray analysis was performed in children with definitive THI diagnosis in two time-points: during hypogammaglobinemia (THI-1) and after Ig levels normalization (THI-2). Twenty-two healthy children were evaluated in this study in whom T_reg cells number was assessed. In nine of them, microarray analysis was performed.

Figure 2.

The absolute number of circulating T_reg cells and its percentage among CD3⁺CD4⁺ cells in all analyzed groups (a and b, respectively) and the changes of T_regs among the same THI patients in two time-points (d and e, respectively). The percentage of CD4⁺Foxp3⁻ cells in all analyzed groups (c) and their changes in THI patients in two time-points (f). The differences between studied groups were analyzed using Kruskal–Wallis (Figure a–c) or Wilcoxon paired t-test (Figure d–f). In the first case, median with interquartile range is shown. Asterisks mark significant differences: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3.

Results of microarray analysis of T_reg cells’ profile in all analyzed comparisons: THI-1 vs. THI-2, THI-1 vs. control, and THI-2 vs. control. Volcano plot showing differentially expressed transcripts (P-values < 0.05) with red spots representing upregulated and the green ones representing the downregulated genes. The grey colored region represents the non-significantly differentially expressed gene. The pie graphs, situated below each volcano plot, show upregulated (top) and downregulated (bottom) genes, according to their affiliation to one of nine groups of transcripts.

Figure 4.

Microarray validation results. Relative expression of FOXP3, IL2RA, CCR7, and LEF1 validated by qPCR, presented as fold change of each DEG relative expression normalized to S18 expression and children from THI-2 group (2^−ΔΔCT). The results were obtained from individual real-time PCR reactions performed in seven THI patients during (THI-1) and after resolution of hypogammaglobulinemia (THI-2). Dashed line was set on value 1 as it signifies the THI-2 group. Data were analyzed using non-parametric Kruskal–Wallis test with Dunn’s post hoc test. Median with interquartile range is shown. Asterisks mark significant differences: *P < 0.05, ****P < 0.0001.

Figure 5.

Venn’s diagram illustrating the overlap of the analyzed pairs of groups (a) and the pie graphs showing 1086 genes from the hatched field, divided into upregulated (top) and downregulated (bottom) genes, according to their affiliation to one of nine groups of transcripts.

Figure 6.

The bar chart depicts the top 15 Gene Ontology (GO) annotation categories including biological process, cellular component, and molecular function.

Figure 7.

Top enriched KEGG pathways of DEGs, demonstrated by no. of genes and P-value.

Figure 8.

Heatmaps of core T_reg genes expression results. Color scale depicts minimum (blue) and maximum (red) normalized expression values compared across all samples between THI-1 and healthy controls (a) and THI-1 and THI-2 group (b).