Human neonatal naive CD4+ T cells have enhanced activation-dependent signaling regulated by the microRNA miR-181a

Abstract

Compared with older children and adults, human neonates have reduced and delayed CD4(+) T cell immunity to certain pathogens, but the mechanisms for these developmental differences in immune function remain poorly understood. We investigated the hypothesis that impaired human neonatal CD4(+) T cell immunity was due to reduced signaling by naive CD4(+) T cells following engagement of the αβ-TCR/CD3 complex and CD28. Surprisingly, calcium flux following engagement of CD3 was significantly higher in neonatal naive CD4(+) T cells from umbilical cord blood (CB) compared with naive CD4(+) T cells from adult peripheral blood. Enhanced calcium flux was also observed in adult CD4(+) recent thymic emigrants. Neonatal naive CD4(+) T cells also had higher activation-induced Erk phosphorylation. The microRNA miR-181a, which enhances activation-induced calcium flux in murine thymocytes, was expressed at significantly higher levels in CB naive CD4(+) T cells compared with adult cells. Overexpression of miR-181a in adult naive CD4(+) T cells increased activation-induced calcium flux, implying that the increased miR-181a levels of CB naive CD4(+) T cells contributed to their enhanced signaling. In contrast, AP-1-dependent transcription, which is downstream of Erk and required for full T cell activation, was decreased in CB naive CD4(+) T cells compared with adult cells. Thus, CB naive CD4(+) T cells have enhanced activation-dependent calcium flux, indicative of the retention of a thymocyte-like phenotype. Enhanced calcium signaling and Erk phosphorylation are decoupled from downstream AP-1-dependent transcription, which is reduced and likely contributes to limitations of human fetal and neonatal CD4(+) T cell immunity.

FIGURE 1

Naive CD4⁺ T cells of CB have a higher calcium flux than APB naive CD4⁺ T cells after CD3 mAb cross-linking using anti-mouse IgG (αmIg). (A) Representative flow cytometry plots of Alexa488 labeled and unlabeled naive CD4⁺ T-cell cell populations (left panels) and their median indo-1 ratio of Alexa488 labeled CB (solid black line) and unlabeled APB (dashed gray line) naive CD4⁺ T cells (right top panel) versus unlabeled CB and Alexa488 labeled APB CD4⁺ T cells (right bottom panel). (B) Mean [Ca²⁺]_i after CD3 mAb cross-linking is significantly higher in CB naive CD4⁺ T cells compared to those of APB (n=3). (C) Alexa488 cell labeling does not have a significant effect on a peak calcium flux of a cell population (n=6). Individual donors are connected by lines. (D) Mean [Ca²⁺]_i following ionomycin addition in CB and APB naive CD4⁺ T cells (n=3). p-values were calculated using a paired, two-way ANOVA to account for both labeling and sample source. All samples were gated on live, CD8⁻ CD19⁻ CD45RO⁻ MACS-purified naive CD4⁺ T cells.

FIGURE 2

CD4⁺ PTK7⁺ RTEs of APB have higher calcium flux after CD3 mAb cross-linking than more mature naive CD4⁺ T cells. (A) Representative flow cytometry plot of PTK7 staining (black line) overlaid on an isotype control stain (filled gray histogram), and kinetics plot of calcium flux after CD3 mAb crosslinking, ionomycin addition, and EGTA addition in samples from an individual adult donor. (B) Peak [Ca²⁺]_i and (C) mean [Ca²⁺]_i of PTK7⁺ naive CD4⁺ T cells (RTE) and PTK7⁻ naive CD4⁺ T cells after CD3 mAb cross-linking (n=4). PTK7⁺ and PTK7⁻ cell populations from the same individual are connected by lines. p-values were calculated using a two-tailed, paired student’s t-test. All samples were gated on live, CD8⁻ CD19⁻ CD45RO⁻ purified naive CD4⁺ T cells.

FIGURE 3

Erk phosphorylation is higher in CB naive CD4⁺ T cells than APB cells. (A) %pErk⁺ after stimulation with plate-bound anti-CD3 and anti-CD28 is significantly higher in CB naive CD4⁺ T cells after 15 (p=0.01), and 30 (p=0.03) minutes of stimulation. (B) Change in MFI (ΔMFI) is significantly higher (p=0.04) in CB naive CD4⁺ T cells after 15 minutes of stimulation with plate-bound anti-CD3 and anti-CD28. (C) %pErk⁺ is not significantly different between CB and APB naive CD4⁺ T cells after stimulation with ionomycin and PMA. (D) ΔMFI is not significantly different between CB and APB (p>0.05) after stimulation with ionomycin and PMA. (E) Representative plot of pErk staining in one APB sample stimulated with anti-CD3 and anti-CD28. Time points are alternately filled or white, for clarity. Line indicates position of positive gate. (F) Representative plot of pErk staining in one CB sample stimulated with anti-CD3 and anti-CD28. Cells are gated on CD3⁺ CD4⁺ CD45RA⁺ lymphocytes.

FIGURE 4

Higher expression of miR-181a in CB naive CD4⁺ T cells regulates calcium flux. (A) CB naive CD4⁺ T cells (n=5) have significantly higher levels of miR-181a expression than those of APB (n=7). (B) miR-181a expression is significantly higher in PTK7⁺ RTEs than in PTK7- naive CD4⁺ T cells of APB (n=6). Lines indicate samples from individual donors. (C) Transfection with pre-miR-181a RNA results in increased expression of mature miR-181a. (D) Representative [Ca²⁺]_i plot of replicates of pre-miR-181a transfected (solid black line) and control pre-miRNA transfected (dashed gray line) APB naive CD4⁺ T cells. (E) Mean and (F) peak [Ca²⁺]_i following CD3 mAb cross-linking from all APB donors was higher after transfection with the pre-miRNA control or pre-miR181a. (G) Peak [Ca²⁺]_i in response to addition of ionomycin of CB and APB naive CD4⁺ cells was not different after transfection with a pre-miRNA control or pre-miR-181a RNA.

FIGURE 5

AP-1-dependent transcription is reduced in activated CB naive CD4⁺ T cells compared to those of APB. (A) AP-1 reporter luciferase activity in CB (filled circles, n=4) and APB (open squares, n=4) either unstimulated or after 4 hours of stimulation with CD3 and CD28 mAbs. (B) AP-1 reporter activity after stimulation with ionomycin and PMA for 4 hours was not significantly different. (C) Luciferase activity driven by a β-actin promoter reporter was not significantly different between CB and APB naive CD4⁺ T cells. (D) Cbl-b mRNA was upregulated in CB naive CD4⁺ T (n=3) cells and downregulated in APB naive CD4⁺ T cells (n=4) after 4h stimulation with anti-CD3 and anti-CD28 (p=0.025, by a paired Student’s t-test).