Expression of immune checkpoint molecules on adult and neonatal T-cells

Abstract

Term and especially preterm neonates are much more susceptible to serious bacterial infections than adults. But not only the susceptibility to infection is increased in neonates, but also their risk for developing post-inflammatory diseases such as bronchopulmonary dysplasia (BPD) and periventricular leukomalacia (PVL). This may be due to an impaired ability to terminate inflammation. In the study presented here, we aimed to investigate the proliferative response and the expression of immune-checkpoint molecules (ICM) and activation markers on neonatal T-cells in comparison to adult T-cells with the hypothesis that an increased activation of neonatal T-cells may contribute to the failure of inflammation resolution observed in neonates. We show that neonatal CD4⁺ and CD8⁺ T-cells show an increased proliferative capacity and an increased expression of activation markers compared to adult T-cells upon stimulation with OKT3 as well as a decreased expression of ICM, especially PD-L1 on their surface. This decreased expression of PD-L1 by neonatal T-cells was also observed after stimulation with GBS, but not after stimulation with E. coli, the two most important pathogens in neonatal sepsis. Expression of the T-cell receptor CD3 and the co-stimulatory molecule CD28 did not differ between adult and neonatal T-cells upon bacterial stimulation. Decreased expression of ICM upon T-cell activation may be a reason for the increased risk of neonates to develop post-inflammatory diseases.

Keywords: Co-stimulatory molecules; Immune-checkpoint molecules; Neonatal sepsis; T-cell proliferation.

Fig. 1

Proliferation of cord blood and adult T-cells upon stimulation with OKT3. T-cells from CBMC or PBMC were enriched by MACS and CFSE-stained. MACS-isolated monocytes from PBMC (different donor) were added in a 1:2 ratio. Co-cultures were stimulated with 0.01 μg/ml OKT3 and after 96 h T-cell proliferation was assessed by flow cytometry. A, C Histograms show proliferation of CD4⁺ T-cells (A) and CD8⁺ T-cells (C) isolated from adult blood (left histograms) or cord blood (right histograms). B, D Scatter plots with connecting lines show proliferation of CD4⁺ (B) and CD8⁺ (D) T-cells in co-culture with adult monocytes. n = 6, *p < 0.05; Wilcoxon matched-pairs signed rank test

Fig. 2

Expression of activation markers on neonatal and adult T-cells upon stimulation with OKT3. Mononuclear cells from cord blood (CBMC) and peripheral blood of healthy adults (PBMC) were isolated and incubated with OKT3 overnight. Expression of surface activation markers on CD4⁺ and CD8⁺ T-cells was determined by flow cytometry. A, C, E, G, I, K, M, O Representative density plots show percentages of CD25- (A, C), CD38- (E, G), CD69- (I, K) and HLA-DR- (M, O) expressing CD4⁺ (A, E, I, M), and CD8⁺ (C, G, K, O) T-cells in adult (left plots) and cord blood (right plots). B, F, J, N, D, H, L, P Scatter plots with bars show percentages of CD25- (B, D), CD38- (F, H), CD69- (J, L), and HLA-DR-expressing (N, P) cells on adult (plain bars) and cord blood T-cells (checked bars) after stimulation with OKT3. Bars represent pooled data from 5 independent experiments and each point represents an individual sample. *p < 0.05; ns, not significant; Wilcoxon matched-pairs signed rank test

Fig. 3

Expression of ICM on neonatal and adult T-cells upon stimulation with OKT3. Mononuclear cells from cord blood (CBMC) and peripheral blood of healthy adults (PBMC) were isolated and incubated with OKT3 overnight. Expression of surface ICM on CD4⁺ and CD8⁺ T-cells was determined by flow cytometry. A, C, E, G, I, K, M, O Representative density plots show percentages of PD-1- (A, C), PD-L1- (E, G), PD-L2- (I, K), and CTLA-4- (M, O) expressing CD4⁺ (A, E, I, M) and CD8⁺ (C, G, K, O) T-cells in adult (left plots) and cord blood (right plots). B, F, J, N, D, H, L, P Scatter plots with bars show percentages of PD-1- (B, D), PD-L1- (F, H), PD-L2- (J, L), and CTLA-4-expressing (N, P) cells on adult (plain bars) and cord blood T-cells (checked bars) after stimulation with OKT3. Bars represent pooled data from 6 independent experiments and each point represents an individual sample. *p < 0.05; **p < 0.01; ns, not significant; Wilcoxon matched-pairs signed rank test

Fig. 4

Expression of ICM on neonatal and adult T-cells upon stimulation with GBS and E. coli. Mononuclear cells from cord blood (CBMC) and peripheral blood of healthy adults (PBMC) were isolated and incubated with GBS or E. coli overnight. Expression of surface ICM on CD4⁺ and CD8⁺ T-cells was determined by flow cytometry. Scatter plots with bars show percentages of PD-1- (A, B, I, J), PD-L1- (C, D, K, L), PD-L2- (E, F, M, N), and CTLA-4- (G, H, O, P) expressing CD4⁺ T-cells (A–G, I–O) and CD8⁺ T-cells (B–H, J–P) from adult blood (plain bars) and cord blood (checked bars) after stimulation with GBS (A–H) or E. coli (I–P). Bars represent pooled data from 8 to 11 independent experiments and each point represents an individual sample. *p < 0.05; ***p < 0.001; ns, not significant; Mann–Whitney test

Fig. 5

Expression of CD3 and CD28 on neonatal and adult T-cells upon stimulation with E. coli or GBS. Mononuclear cells from cord blood (CBMC) and peripheral blood of healthy adults (PBMC) were isolated and incubated with GBS or E. coli overnight. Expression of the T-cell receptor CD3 and the co-stimulatory molecule CD28 on CD4⁺ and CD8⁺ T-cells was determined by flow cytometry. A, D, G, J Representative density plots show expression of CD3 and CD28 on CD4⁺ (A, D) and CD8⁺ (G, J) T-cells in adult (left plots) and cord blood (right plots). B, C, E, F, H, I, K, L Scatter plots with bars show the mean fluorescent intensity (MFI) for expression of CD3 (B, E, H, K) and CD28 (C, F, I, L) on CD4⁺ T-cells (B, C, H, I), and CD8⁺ T-cells (E, F, K, L) after stimulation with GBS (B, C, E, F) or E. coli (H, I, K, L). Bars represent pooled data from 7 to 9 independent experiments, and each point represents an individual sample. ns, not significant; Mann–Whitney test