Regulatory T cell homing and activation is a signature of neonatal sepsis

Abstract

Regulatory T cells (Treg) play a prominent role in utero tolerating non-inherited maternal antigens and in regulating immune responses against pathogens at birth. This study investigates Treg immunity in newborns in West Africa, where sepsis remains a major public health problem. Treg phenotypes on neonates subgroups with early-onset sepsis (EOS), presumed sepsis, and healthy newborn with and without prenatal risk factors were evaluated. Treg phenotypes varied according to prenatal conditions, with increase in Treg frequency and Foxp3 expression in healthy newborns with prenatal risk factors compared to those with none risk. Compared to healthy newborns with prenatal risk factors, EOS neonates had a significantly reduced frequency of Treg and Foxp3 expression. In the Treg pool, higher frequency of activated Treg was observed in EOS neonates, suggesting an in-utero activation upstream of the sepsis onset. Their migration to the infection site may explain the reduced frequency of circulating Integrin α4β1⁺ Treg suggestive of homing to the endothelial tissue. EOS neonates show increases expression of CTLA-4, PD-1 and CD39 on Treg, which negatively regulate the activation of effector T cells (Teff) corroborating by the lower frequency of Teff in EOS neonates. The higher frequency of CD39⁺ Treg and the lower frequency of integrinα4β1⁺ Treg in EOS non-survivor suggests that Treg exhaustement and endothelial homing are associated with outcome severity. Neonates developing EOS are born with an altered Treg phenotypic profile. Treg expression of CTLA-4, PD-1, CD39, and integrinα4β1 cell markers can be considered as early warning or diagnostic markers of EOS.

Keywords: Treg; immunity; malaria in pregnancy; newborn; prematurity; sepsis.

Figure 1

Flow-chart of study population.

Figure 2

Frequencies of regulatory T cells (Treg), activated Treg, effector T cells and FoxP3 expression cells from the cord blood of subgroups of study cohort. (A) Frequencies of CD4⁺CD25⁺CD127^-FoxP3⁺ regulatory T cells. (B) FOXP3 expression on Treg. geometric Mean Fluorescence Intensity (gMFI) of Forkhead box P3 (FoxP3) in CD4⁺CD25⁺CD127^-FoxP3⁺ regulatory T cells is shown. (C) Frequencies of CD4⁺CD127^-CD45RA^lowFoxP3^hi activated Treg cells are shown. (D) Frequencies of CD4⁺CD127^-CD45RA^hiFoxP3^hi resting Treg cells are shown. (E) Frequencies of CD4⁺CD25⁺CD127⁺ effector T cells are shown. EOS neonates (clinic (n=32) and presumed (n=13)) are compared to Hospital non sepsis neonates (n=41), as controls. Hospital non sepsis neonates is compared to sub-urban non sepsis neonates (n=12) to evaluate risk factors impact. The plots are shown with median and minimum/maximum values. p-values were calculated by the Mann–Whitney U-test. P<0.05 indicates a statistically significant difference. p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, no statistically significant difference.

Figure 3

Frequency and expression of CTLA-4, PD-1 and CD39 on Treg pool from the cord blood of subgroups of study cohort. (A, C, E) dot plots compare the frequencies of Treg CTLA-4⁺, Treg PD-1⁺, Tregs CD39⁺respectively between neonates’ subgroups: EOS clinic (n=32), EOS presumed (n=13), Hospital no sepsis (n=41), sub-urban no sepsis (n=12). (B, D, F) representative dot plots of the comparison of CTLA-4, PD-1, CD39 expression level on Treg surface between neonates’ subgroups. EOS neonates (clinic and presumed) are compared to Hospital non sepsis neonates, as controls. The plots are shown with median and minimum/maximum values. p-values were calculated by the Mann–Whitney U-test. P<0.05 indicates a statistically significant difference. p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, no statistically significant difference.

Figure 4

Frequency and expression of homing molecules (Integrin α4β1 & Integrin α4β7) Treg. (A, C) Frequency of Treg Integrin α4β1⁺ and Treg Integrin α4β7⁺cells are shown. (B, D) Integrin α4β1 and Integrin α4β7 expression on Treg. EOS clinic (n=32), EOS presumed (n=13), Hospital no sepsis (n=41), sub-urban no sepsis (n=12). geometric Mean Fluorescence Intensity (gMFI) of Integrin α4β1 and Integrin α4β7 in CD4⁺CD25⁺CD127^-FoxP3⁺ regulatory T cells is shown. EOS neonates (clinic and presumed) are compared to Hospital non sepsis neonates, as controls. The plots are shown with median and minimum/maximum values. p-values were calculated by the Mann–Whitney U-test. P<0.05 indicates a statistically significant difference. p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, no statistically significant difference.

Figure 5

Origin of Treg subpopulations in neonates. Tregs origin were examined for expression of the surface markers CD45RA and CD31. EOS clinic (n=32), EOS presumed (n=13), Hospital no sepsis (n=41), sub-urban no sepsis (n=12). (A) Representative dot plot comparing median percentage of CD3⁺CD4⁺CD25⁺FOXP3⁺CD45RA⁺CD31⁺ Recent thymic emigrant Treg from the cord blood of subgroups of study cohort. (B) Representative dot plot comparing median percentage of CD3⁺CD4⁺CD25⁺FOXP3⁺CD45RA⁺CD31^- Peripherally induced naïve Treg from the cord blood of subgroups of study cohort. EOS neonates (clinic and presumed) are compared to Hospital non sepsis neonates, as controls. The plots are shown with median and minimum/maximum values. p-values were calculated by the Mann–Whitney U-test. P<0.05 indicates a statistically significant difference. p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, no statistically significant difference.

Figure 6

Frequency of CTLA-4, PD-1, CD39 α4β1 and α4β7 on Treg, Comparison between survivors and non-survivors in EOS neonates. Mann–Whitney U-test was performed for comparison. (A) Percentage of Treg CD39⁺. (B) Percentage of Tregs CTLA-4⁺. (C) Percentage of Tregs PD-1⁺. (D) Percentage of Treg Integrin α4β1⁺. (E) The percentage of Treg Integrin α4β7⁺. The plots are shown with median and minimum/maximum values. p-values were calculated by the Mann–Whitney U-test. P<0.05 indicates a statistically significant difference. p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, no statistically significant difference.