PD-1/PD-L1 Control of Antigen-Specifically Activated CD4 T-Cells of Neonates

Abstract

Newborns are highly susceptible to infections; however, the underlying mechanisms that regulate the anti-microbial T-helper cells shortly after birth remain incompletely understood. To address neonatal antigen-specific human T-cell responses against bacteria, Staphylococcus aureus (S. aureus) was used as a model pathogen and comparatively analyzed in terms of the polyclonal staphylococcal enterotoxin B (SEB) superantigen responses. Here, we report that neonatal CD4 T-cells perform activation-induced events upon S. aureus/APC-encounter including the expression of CD40L and PD-1, as well as the production of Th1 cytokines, concomitant to T-cell proliferation. The application of a multiple regression analysis revealed that the proliferation of neonatal T-helper cells was determined by sex, IL-2 receptor expression and the impact of the PD-1/PD-L1 blockade. Indeed, the treatment of S. aureus-activated neonatal T-helper cells with PD-1 and PD-L1 blocking antibodies revealed the specific regulation of the immediate neonatal T-cell responses with respect to the proliferation and frequencies of IFNγ producers, which resembled in part the response of adults' memory T-cells. Intriguingly, the generation of multifunctional T-helper cells was regulated by the PD-1/PD-L1 axis exclusively in the neonatal CD4 T-cell lineage. Together, albeit missing memory T-cells in neonates, their unexperienced CD4 T-cells are well adapted to mount immediate and strong anti-bacterial responses that are tightly controlled by the PD-1/PD-L1 axis, thereby resembling the regulation of recalled memory T-cells of adults.

Keywords: CD4 T cell; PD-1/PD-L1; Staphylococcus aureus (S. aureus); T-helper cells; bacteria; immune checkpoint molecule; newborn; pediatric immunology; staphylococcus enterotoxin B (SEB).

Figure 1

Neonatal CD4 T-cells up-regulate PD-1 and control strong responses against SEB via the PD-1/PD-L1 axis. (a) Scheme of activation of neonatal naïve (nT_N) or adult naïve (T_N) and memory (T_M) CD4 T-cells by S. aureus or SEB-loaded autologous monocytes (MC). Cells were isolated from neonatal umbilical cord blood mononuclear cells (CBMCs) or adult peripheral blood mononuclear cells (PBMCs), respectively. (b,c) CD69 expression (day 3) on nT_N, T_N or T_M in response to SEB (b) or to S. aureus vs. resting (c). (d) Viability of nT_N, T_N or T_M under resting conditions (left) or S. aureus-induced activation (right). (e,f) PD-1 expression of nT_N (e), T_N or T_M (f) on day 5. (g) Ratios of CD69 expression of S. aureus or SEB-stimulated PD-1/PD-L1-blocked CD4 T-cells to their isotype controls. The significance between blockade and isotype control is depicted above the respective box plot. Data points represent donors in box plots with median, interquartile, and range. Numbers indicate percentages of PD-1-expressing T-cells or p-values; * p < 0.05, ** p < 0.01, **** p < 0.0001; p-values were calculated using the Kruskal–Wallis test (b,d), one-way ANOVA with Sidak’s multiple comparison test (c), the Wilcoxon test or the paired t-test (g).

Figure 2

PD-1/PD-L1 blockade enhances early anti-S. aureus-initiated proliferation in neonatal naïve and adult memory CD4 T-cells. (a) Representative histograms of the proliferation of neonatal naïve (nT_N) or adult naïve (T_N) and memory (T_M) CD4 T-cells in resting conditions or in response to SEB on day 3 (b) Frequencies of proliferated nT_N, T_N or T_M on day 3 after SEB activation. (c) Representative proliferation histograms of nT_N, T_N or T_M in resting conditions and in response to S. aureus on day 5 (d,e) Frequencies of proliferated nT_N, T_N or T_M in resting or S. aureus-mediated activating conditions on day 3 (d) or day 5 (e). (f) Ratios of the proliferation of S. aureus-stimulated PD-1/PD-L1-blocked CD4 T-cells to their isotype controls. The significance between the blockade and the isotype control is depicted above the respective box plot. (g) Path diagram of a linear regression analysis of factors (coefficients) regulating anti-S. aureus-specific proliferation in nT_N (sex: female was coded 1, male 2). (h) Frequencies of CD25⁺ nT_N on day 3 after activation with S. aureus under PD-1/PD-L1 blockade or isotype control conditions. Data points represent donors in box plots with median, interquartile, and range. Numbers indicate the percentage of proliferated T-cells, p-values, adjusted R², or standardized estimates; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; p-values were calculated using the Kruskal–Wallis test (b,d,e), paired t-test, Wilcoxon test (f,h), or linear regression analysis (g).

Figure 3

PD-1/PD-L1 signals regulate antigen-specific CD4 T-cell responses of neonatal naïve and adult memory T-helper cells. (a) Representative histograms of the CD40L expression of S. aureus and SEB-activated, or resting nT_N, T_N or T_M on d3. (b) Frequencies of CD40L⁺ nT_N, T_N or T_M on day 3 after SEB-activation. (c) CD40L expression of resting or S. aureus-activated nT_N (left), T_N (middle) or T_M (right) on day 3, treated with PD-1/PD-L1-blocking or isotype control antibodies. (d) Frequencies of IL-17A or IFNγ producers of S. aureus-activated CD40L⁺ nT_N, T_N or T_M on day 3, treated with PD-1/PD-L1-blocking or isotype control antibodies. Data points represent donors in box plots with median, interquartile, and range. Numbers indicate the percentage of CD40L⁺ cells; * p < 0.05, *** p < 0.001, **** p < 0.0001; p-values were calculated using two-way ANOVA (Fisher’s LSD) (c) or the Wilcoxon test (d).

Figure 4

PD-1/PD-L1 axis regulates the early IFNγ response of neonatal CD4 T-cells. Frequencies of TNFα (a–c) or IFNγ (d–f) producers of resting or S. aureus-activated nT_N (a,d), T_N (b,e) or T_M (c,f) on day 3 and day 5, treated with PD-1/PD-L1-blocking or isotype control antibodies as indicated. Data points represent donors in box plots with median, interquartile, and range. * p < 0.05, ** p < 0.01; p-values were calculated using two-way ANOVA (Fisher’s LSD).

Figure 5

Multi-functionality of neonatal CD4 T-cells is controlled by PD-1/PD-L1. (a–c) IFNγ single or IFNγ co-expression along TNFα and/or IL-2 in nT_N (a), T_N (b) or T_M (c) on day 3 in response to S. aureus. (d) Summary of PD-1/PD-L1-regulated functions in S. aureus-activated neonatal naïve (nT_N, left) or naïve (T_N) and memory (T_M) CD4 T-cells from adults (right). Data points represent donors in bar graphs with mean and SD. * p < 0.05, ** p < 0.01; p-values were calculated using two-way ANOVA (Fisher’s LSD).