Group B streptococci infection model shows decreased regulatory capacity of cord blood cells

Abstract

Background: Sepsis is one of the leading causes of morbidity and mortality in the neonatal period. Compared to adults, neonates are more susceptible to infections, especially to systemic infections with Group B Streptococcus (GBS). Furthermore, neonates show defects in terminating inflammation. The immunological causes for the increased susceptibility to infection and the prolonged inflammatory response are still incompletely understood.

Methods: In the present study, we aimed to investigate the reaction of cord blood mononuclear cells (MNC) to stimulation with GBS in comparison to that of MNC from adult blood with focus on the proliferative response in an in vitro infection model with heat-inactivated GBS.

Results: We demonstrate that after stimulation with GBS the proliferation of T cells from adult blood strongly decreased, while the proliferation of cord blood T cells remained unchanged. This effect could be traced back to a transformation of adult monocytes, but not cord blood monocytes, to a suppressive phenotype with increased expression of the co-inhibitory molecule programmed death ligand 1 (PD-L1).

Conclusions: These results point towards an increased inflammatory capacity of neonatal MNC after stimulation with GBS. Targeting the prolonged inflammatory response of neonatal immune cells may be a strategy to prevent complications of neonatal infections.

Impact: Neonatal sepsis often leads to post-inflammatory complications. Causes for sustained inflammation in neonates are incompletely understood. We show that cord blood T cells exhibited increased proliferative capacity after stimulation with group B streptococci (GBS) in comparison to adult T cells. Adult monocytes but not cord blood monocytes acquired suppressive activity and expressed increased levels of PD-L1 after GBS stimulation. Increased proliferative capacity of neonatal T cells and decreased suppressive activity of neonatal monocytes during GBS infection may contribute to prolonged inflammation and development of post-inflammatory diseases in newborns.

Fig. 1. Proliferation of adult and cord blood mononuclear cells after stimulation with GBS.

CBMC and PBMC were isolated, stained with CFSE and stimulated with GBS overnight. The next day, cells were stimulated with OKT3. After another 3 days, T cell proliferation was assessed by CFSE dye dilution. T cells without pre-stimulation with GBS served as control. a, c Representative histogram plots show proliferation of CD4⁺-T cells (a) and CD8⁺-T cells (c) from adult (PB) and cord blood (CB) of unstimulated cells (ctrl), stimulated cells without (w/o GBS) and with additional stimulation with GBS (w GBS). b, d Scatter plots with bars show the percentage of proliferation of CD4⁺ (b) and CD8⁺ (d) T cells in PBMC (white) and CBMC (grey) without (clean) or with (checked) stimulation with GBS. n = 18, ***p < 0.001, ****p < 0.0001, ns: not significant; Kruskal–Wallis test and Dunn’s multiple comparison test.

Fig. 2. T cell proliferation in adult blood after pre-stimulation of monocytes and T cells with GBS separately.

Monocytes or T cells from PBMC were enriched by MACS and stimulated with GBS overnight. The next day, freshly isolated and CFSE-stained T cells from a different adult donor were added to pre-incubated monocytes (a, b) or pre-incubated, CFSE-stained T cells were added to freshly isolated monocytes from a different adult donor in a 2:1 ratio. Non-stimulated cells served as control. Co-cultures were stimulated with OKT3, and after 72 h, T cell proliferation was assessed by flow cytometry. a, b Scatter diagrams with bars show proliferation of CD4⁺ (a, c) and CD8⁺ (b, d) T cells in co-culture with monocytes without (clean) or with (checked) pre-stimulation with GBS of monocytes (a, b) or T cells (c, d). n = 6, *p < 0.05, ns: not significant; Wilcoxon matched-pairs signed rank test.

Fig. 3. Inhibition of T cell proliferation by GBS-stimulated monocytes.

Monocytes were enriched from CBMC/PBMC by MACS and stimulated with GBS overnight. Monocytes without GBS stimulation served as control. The next day, monocytes were added to freshly isolated, CFSE-stained and OKT3/IL-2-stimulated PBMC. After 4 days, T cell proliferation was assessed by CFSE dye dilution. Proliferation index was determined as the ratio of T cell proliferation with and without addition of monocytes. a, c Representative histogram plots show proliferation of CD4⁺ T cells (a) and CD8⁺ T cells (c) after addition of monocytes from PB and from CB of unstimulated cells (ctrl) and stimulated cells without (w/o GBS) and with additional stimulation with GBS (w GBS). b, d Bar graphs show the inhibitory effect of monocytes on proliferation of CD4⁺ (b) and CD8⁺ T cells (c). Dashed line shows proliferation of target PBMC without addition of monocytes. White/grey bars show T cell proliferation after addition of adult/cord blood monocytes. Clean bars show the effect of monocytes without and checked bars with pre-stimulation with GBS. n = 6, *p < 0.05, ns not significant; Friedman test and Dunn’s multiple comparison test.

Fig. 4. Expression of surface molecules and effector enzymes on monocytes after stimulation with GBS.

CBMC and PBMC were isolated and incubated with GBS overnight. Expression of surface molecules, intracellular effector enzymes, and reactive oxygen production was determined by flow cytometry. a Representative density plots show the expression of CD80, CD86, PD-L1, PD-L2, ArgI, iNOS, IDO and Rhodamine (y-axis) on CD14⁺ adult (PB) and cord blood (CB) monocytes (x-axis). b–i Scatter plots with bars show the mean fluorescent intensity (MFI) for the expression of CD80 (b), CD86 (c), PD-L1 (d), PD-L2 (e) ArgI (f), iNOS (g), IDO (h) on and the production of ROS (i) by adult (white) and cord blood (grey) monocytes after stimulation of GBS. n = 5–9, **p < 0.01, ****p < 0.0001, ns not significant; Mann–Whitney test.

Fig. 5. Expression of surface molecules on T cells after stimulation with GBS and inhibition of T cell proliferation by GBS-treated T cells.

CBMC and PBMC were isolated and incubated with GBS overnight. Cells without GBS stimulation served as control. Expression of surface molecules (a–f) or intracellular FoxP3 (g, h) on T cells was determined by flow cytometry. Pre-incubated T cells were added to freshly isolated, CFSE-stained and OKT3/IL-2-stimulated PBMC. After 4 days, T cell proliferation was assessed by CFSE dye dilution. Proliferation index was determined as the ratio of T cell proliferation with and without addition of T cells (i, j). Representative density plots show the expression of CD25 and CD69 on CD3⁺ T cells (a), the expression of CD25 on CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T cells (d), and the expression of CD25 and FoxP3 on CD3⁺/CD4⁺ T cells (g) from adult peripheral blood (PB) and cord blood (CB). Cells in d were pre-gated on CD3 and cells in panel g were pre-gated on CD3 and CD4. Scatter plots with bars show the percentage of CD3 T-cells expressing CD25 (b) or CD69 (c) percentage of CD25 on CD3⁺/CD4⁺ (e), CD3⁺/CD8⁺ (f) and percentage of CD25 and FoxP3 expresssion on CD3 and CD4 T-cells, adult (white) and cord blood (grey) T cells after stimulation of GBS. Scatter blot n = 6–8, **p < 0.01, ns not significant; Mann–Whitney test. Bar graphs show the inhibitory effect on proliferation of CD4⁺ (i) and CD8⁺ T cells (j). Dashed line shows the proliferation of target PBMC without addition of T cells. White/grey bars show T cell proliferation after addition of adult/cord blood T cells. Clean bars show the effect of T cells without and checked bars with pre-stimulation with GBS. n = 5, *p < 0.05, ns not significant; Friedman test and Dunn’s multiple comparison test.