AKT Hyperphosphorylation and T Cell Exhaustion in Down Syndrome

Abstract

Down syndrome (DS) is associated with increased susceptibility to infections, auto-immunity, immunodeficiency and haematological malignancies. The exact underlying immunological pathophysiology is still unclear. The immunophenotype and clinical characteristics of DS resemble those of Activated PI3K Delta Syndrome (APDS), in which the PI3K/AKT/mTOR pathway is overactivated. We hypothesized that T cell exhaustion and the hyperactivation of the AKT signalling pathway is also present in immune cells of children with DS. In this observational non-interventional cohort study we collected blood samples of children with DS (n=22) and healthy age-matched controls (n=21) for flowcytometric immunophenotyping, phospho-flow AKT analysis and exhaustion analysis of T cells. The median age was 5 years (range 1-12y). Total T and NK cells were similar for both groups, but absolute values and transitional B cells, naive memory B cells and naive CD4+ and CD8+ T cells were lower in DS. pAKT and AKT were increased for CD3+ and CD4+ T cells and CD20+ B cells in children with DS. Total AKT was also increased in CD8+ T cells. Children with DS showed increased expression of inhibitory markers Programmed cell dealth-1 (PD-1), CD244 and CD160 on CD8+ T cells and increased PD-1 and CD244+ expression on CD4+ T cells, suggesting T cell exhaustion. Children with DS show increased pAKT and AKT and increased T cell exhaustion, which might contribute to their increased susceptibility to infections, auto immunity and haematological malignancies.

Keywords: AKT pathway; T cell exhaustion; activated PI3 kinase delta syndrome; down syndrome; immunodeficiencies; immunophenotyping.

Figure 1

Gating strategy of B cells and T cell subset for the determination of baseline AKT and pAKT expression in DS and healthy controls. (A) Gating strategy of CD3+CD4+ T cells, CD3+CD8+ T cells and CD20+ B-cells. (B) Representative phospho-flow plots for AKT and pAKT in unstimulated CD3+ T cells in a DS patient and age matched healthy control.

Figure 2

Immunophenotyping of B cells in 22 children with Down syndrome and 20 healthy age-matched controls. (A) Absolute values of transitional B cells (CD38high/CD24high) and naive mature B cells (CD38dim/CD24dim/IgD+/CD27-) did not differ between children with Down syndrome and healthy controls, whereas natural effector B cells (CD38dim/IgD+/CD27+) and memory B cells (CD28dim/IgD-/CD27+) were significantly decreased in children with Down syndrome (resp. p=0.0002 and p=0.0004). Median and interquartile ranges are indicated (red lines represent the median). (B) In the subsets of memory B cells, children with Down syndrome have increased IgM memory B cells (p<0.0001) and decreased IgA (p=0.0003) and IgG (p<0.0001) memory B cells. ***p < 0.001; ns p > 0.05.

Figure 3

Immunophenotyping of the T cell compartment of 21 children with Down syndrome and 21 healthy age-matched controls. Children with Down syndrome have significantly lower naive CD4+ T cells (CD4+CD45RA+CCR7+) (A) and lower naive CD8+ T cells (CD8+CD45RA+CCR7+) (B) compared to healthy controls. (C) Within the CD4+ T cells, the proportion of CM and EM cells is increased in children with Down syndrome. (D) For CD8+ T cell distribution, there is no significant difference in the percentage CM cells, however, children with Down syndrome have increased EM and EMRA. Median and interquartile ranges are indicated. *p <0.05; **p < 0.01; ****p < 0.0001; ns p > 0.05.

Figure 4

Phospho-flow analysis in 21 children with Down syndrome and 21 healthy age-matched controls. (A) The total AKT levels were elevated in all different cells for children with Down syndrome. (B) In children with Down syndrome, pAKT levels were increased in CD3+, CD4+ and CD20+ cells, but not in CD8+ T cells. Median and interquartile ranges are indicated. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns p > 0.05.

Figure 5

T cell exhaustion of CD4+ and CD8+ cells in 21 children with Down syndrome and 21 healthy age-matched controls. (A) Increased expression of PD-1+ and CD244+ and similar levels of CD160+ and PD-1+CD160+CD244+ co-expression in CD4+ T cells. (B) Elevated levels of all exhaustion markers (PD-1, CD160 and CD244) in CD8+ T cells. Median and interquartile ranges are indicated. *p <0.05; **p < 0.01; ****p < 0.0001; ns p > 0.05.