T Cells of Infants Are Mature, but Hyporeactive Due to Limited Ca2+ Influx

Abstract

CD4 T cells in human infants and adults differ in the initiation and strength of their responses. The molecular basis for these differences is not yet understood. To address this the principle key molecular events of TCR- and CD28-induced signaling in naive CD4 T cells, such as Ca2+ influx, NFAT expression, phosphorylation and translocation into the nucleus, ERK activation and IL-2 response, were analyzed over at least the first 3 years of life. We report dramatically reduced IL-2 and TNFα responses in naive CD31+ T cells during infancy. Looking at the obligatory Ca2+ influx required to induce T cell activation and proliferation, we demonstrate characteristic patterns of impairment for each stage of infancy that are partly due to the differential usage of Ca2+ stores. Consistent with those findings, translocation of NFATc2 is limited, but still dependent on Ca2+ influx as demonstrated by sensitivity to cyclosporin A (CsA) treatment. Thus weak Ca2+ influx functions as a catalyst for the implementation of restricted IL-2 response in T cells during infancy. Our studies also define limited mobilization of Ca2+ ions as a characteristic property of T cells during infancy. This work adds to our understanding of infants' poor T cell responsiveness against pathogens.

Fig 1. Frequencies of naive CD31⁺ T cells within the CD4⁺CD45RA⁺ compartment remain constant over ages.

(A) T cell subsets of CB or PBMCs analyzed by flow cytometry. Representative flow cytometry dot plots and histograms are shown with the percentage of CD4⁺CD45RA⁺ T cell subsets for CB (top), infant (middle; 2.8 months), and adult (bottom) samples. (B) Frequencies of CD4⁺ among lymphocytes, (C) of CD45RA⁺ among CD4⁺ T cells, and (D) CD31⁺ among peripheral CD4⁺CD45⁺ cells are shown (* P<0.05; two-sided ANOVA Tukey-Kramer post-hoc test). The mean value and standard deviations (SD) are indicated. Age of infants and children is indicated in months. n = number of individuals.

Fig 2. Ca²⁺ influx is a key event for IL-2 in adult CD31⁺ naive T cells.

(A) No Ca²⁺ influx signal with GAMIg alone (black curve; anti-CD28 isotype Ab were substituted for anti-CD28 Ab) or anti-CD28 Ab plus GAMIg alone (red curve) in T cells stained for anti-CD4, anti-CD45, and anti-CD31. Results representative of at least 3 experiments are shown. (B-C) Ca²⁺ influx into adult CD31⁺ naive T cells stimulated using anti CD3 Ab and anti CD28 Ab. (B) A representative Ca²⁺ influx experiment and (C) box plots for the different T cell subsets showing scatter plots of mean value and SD of Ca²⁺ influx experiments by normalizing maximal Ca²⁺ signals to the maximal Ca²⁺ influx ionomycin are shown (* P<0.05; two-tailed ANOVA of differences). (D) Increased Ca²⁺ influx in adult CD31⁺ naive T cells compared with anti-CD3/anti-CD28 Ab (red) in response to anti-CD3 Ab alone (blue, with anti-CD28 isotype Ab) (* P<0.05; two-tailed ANOVA of differences) (E) Increased levels of NFATc2 protein expression in response to anti-CD3/anti-CD28 Ab stimulation in naive T cells. The immunoblot detection of the relative protein expression level by ratio of NFATc2 or pNFATc2 to αTubulin is shown for naive CD31⁺ or CD31^- T cells of adults stimulated with anti-CD3 Ab in combination with soluble anti-CD28 Ab (dark gray) or anti-CD28 Ab isotype (light gray). Laminin and αTubulin were used as loading controls. Results are representative of at least two experiments. unstim. = unstimulated. (F) IL-2 cytokine in supernatants does not differ in cultures under TCR/CD3 stimulation between subtypes of naive CD4 T cells (* P<0.05; two-tailed ANOVA of differences). ns = not significant.

Fig 3. Key signaling pathways for IL-2 transcription are activated in TCR/CD3 stimulated naive CD4⁺ T cells of CB.

(A) Protein expression by Western blot of ERK1/2 and phosphorylated ERK1/2Tyr202/ Tyr204 (pERK1/2) in naive CD4⁺ T cells of CB as well as for naive CD31⁺ adult T cells. The ratios of relative protein expression levels are indicated below the respective bands. Data are representative of two independent experiments. (B) Whole cell protein extract of NFATc2 and phosphorylated NFATc2 (pNFATc2) in CB naive CD4⁺ T cells and adult naive CD31⁺ T cells under different stimulation conditions. The densitometric analyses of the immunoblot detection for the relative protein expression level are shown as ratio of NFATc2 or pNFATc2 to αTubulin. Lysates from three different donors were pooled. Data are representative of at least three independent experiments. αTubulin was used as a loading control. (C-D). The NFATc2 protein expression in cytoplasm or nucleoplasm (separated through a dashed line) in (C) naive CD4⁺ T cells of CB or (D) CD31⁺ naive T cells of adult were detected and the phosphorylated (pNFATc2) and dephosphorylated (NFATc2) forms quantified. Cells were stimulated as indicated in the presence or absence of cyclosporin A (CsA). One representative experiment out of two comparable experiments is shown. unstim. = unstimulated.

Fig 4. Different Ca²⁺ responses of CB and adult CD31⁺ naive T cells.

(A-B) Ca²⁺ mobilization in CD4⁺CD45RA⁺CD31⁺ T cells of one healthy donor (representative of at least eight healthy individuals) of PBMCs (A, adult) or CB (B) were performed in response to 0.05 μg/ml anti-CD3 Ab plus 0.5 μg/ml soluble anti-CD28 Ab (red curve) or only 0.05 μg/ml of anti-CD3 Ab alone (black curve, with anti-CD28 isotype Ab) in combination with GAMIg. The blue dotted line displays the maximum Ca²⁺ response of adult CD31⁺ naive T cells for anti-CD3 Ab stimulation alone. (C) Box plot with scatter plots representing means and SD of Ca²⁺ influx response normalized by maximal Ca²⁺ influx response to ionomycin of adult (gray circle) and CB (black circle) and their dependency on anti-CD28 Ab costimulation (anti-CD3 Ab plus anti-CD28 Ab (red box) or anti-CD3 Ab with anti-CD28 Ab isotype (blue box)). Statistical significance between groups * P<0.05 was determined by two-tailed ANOVA with Tukey-Kramer post-hoc test. n = number of individuals. (D) Comparison of different CD4⁺ T cell subset stimulated with anti-CD3 Ab plus anti-CD28 Ab (red box) or with anti-CD3 Ab alone (blue box). Box plot with scatter plots representing means and standard deviations of Ca²⁺ influx response normalized by maximal Ca²⁺ influx response to ionomycin. Statistical significance of differences between anti-CD3/anti-CD28 Ab or anti-CD3 Ab stimulation at concentration 0.05 μg/ml of anti-CD3 Ab CD31⁺ between groups of different T cell subsets was determined by two-tailed ANOVA P = 0.7745, CD45RA⁺ P = 0.8195, CD4⁺ P = 0.9926. ns = not significant. n = number of individuals. (E) CD4⁺CD45RA⁺CD31⁺ T cells of CB (filled line), and adults (dashed line) treated with 0.5 μg/ml soluble anti-CD3 Ab and anti-CD28 Ab cross-linked with GAMIg in the presence (red) or absence (black) of 2 mM EGTA. One representative experiment out of three comparable experiments is shown. (F) STIM1 protein expression in CD4⁺ T cells of CB (black) and in naive CD31⁺ T cells from adults (gray) after stimulation using anti-CD3/anti-CD28 Ab. The densitometric analyses of the ratio of STIM1/α Tubulin are shown. Results are representative of at least two experiments.

Fig 5. Dose response curves of the Ca²⁺ influx after TCR ligation in CB, infant/children, and adult for CD31⁺ and CD31^- naive T cells.

Ca²⁺ mobilization in response to different anti-CD3 Ab concentrations plus 0.5 μg/ml soluble anti-CD28 Ab or with anti-CD3 alone (anti-CD28 Ab isotype) in combination with GAMIg measured using Indo-1AM staining and flow cytometry. Maximal Ca²⁺ influx response was normalized to the maximal Ca²⁺ influx of ionomycin treated samples and displayed as dose response curves for CD4⁺CD45RA⁺CD31⁺ and CD4⁺CD45RA⁺CD31^- naive T cells in (A) CB, (B) infant aged 1–2 months, (C) infant aged 3–5 months, (D) infant and children aged 6–66 months, and (E) adult. (F) Dose response curves by maximal Ca²⁺ influx normalized to the maximal Ca²⁺ influx ionomycin after anti-CD3 Ab TCR ligation and with anti-CD28 Ab stimulation displayed for CB, infant/children, and adult. The anti-CD3 Ab concentration of 0.05 μg/ml is marked with a gray bar. CD31⁺ = CD4⁺CD45RA⁺CD31⁺; CD31^- = CD4⁺CD45RA⁺CD31^-.

Fig 6. Age-dependent signatory Ca²⁺ influx and cytokine concentrations in supernatants of naive CD4⁺ T cells.

(A-B) CD4⁺CD45RA⁺CD31⁺ T cells stimulated with anti-CD3 Ab as indicated either with costimulation by 0.5 μg/ml soluble anti-CD28 Ab (A, dark gray) or with the CD28 isotype control (B, light gray). Compiled data of box plots with scatter plots represent the Ca²⁺ influx response normalized to the maximal Ca²⁺ influx ionomycin response. Statistical significance between groups was determined by two tailed ANOVA Tukey-Kramer post-hoc test * P<0.05. n = number of individuals. (C) NFATc2 expression after anti-CD3 Ab plus anti-CD28 Ab engagement. The densitometric analyses of the immunoblots for the relative protein expression levels are shown as ratios of NFATc2 or pNFATc2 to αTubulin. Lysates from three different donors were pooled. Results are representative of at least two independent experiments. IFNγ (D), IL-2 (E), and TNFα (F) concentrations in the supernatants of unstimulated (white), of soluble anti-CD3 Ab plus anti-CD28 Ab stimulated (dark gray), and of TCR/CD3 stimulated alone (light gray, with anti-CD28 Ab isotype) of naive T cells using a Bio-Plex cytokine assay (Bio-Rad). The mean value and SD are indicated for five independent experiments (two tailed ANOVA Tukey-Kramer post-hoc test * P<0.05). n = number of individuals.

Fig 7. Visualizing the occurrence of age-dependent characteristics of T cell activation.

The requirement of activation by the TCR/CD3 complex is less dependent on costimulation by CD28 in CB naive CD4⁺ T cells compared to that seen in adult cells. The intensity of Ca²⁺ influx, NFATc2 expression and IL-2 response are all age-dependent. A dramatic shift is seen in the naive T cell response at the age of 2 months. The cells’ capacity to produce high amounts of IL-2 is suddenly abrogated. Ca²⁺ influx declines to the lowest values observed in life. At 6 months of age, the IL-2 response starts to improve slowly. This “reprogramming” of T cells takes place as the passively transferred maternal Abs in the infant are beginning to decline. Limited T-cell responses likely contribute to the high risk of infants to suffer from infections and infection-related pathologies such as Sepsis and SIDS [43,44] during the first months of life.

Fig 8. Schematic model for the molecular basis of age-dependent T cell activation.

Engagement of T cell receptors (TCR) and costimulatory CD28 receptors (blue) by soluble anti-CD3 Ab and/or anti-CD28 Ab stimulation promotes signaling cascades of adaptor proteins (LAT) and kinases (ERK). These trigger signal transduction pathways resulting in the activation of the transcription factors NFATc (red), NF-κB (white) and the association of AP-1 with cFos/cJun (green). These transcription factors cooperate with each other during the activation of the IL-2 promoter. Shown are the schematic molecular responses of (A) CB and (B) adult with soluble anti-CD3 Ab stimulation alone or together with costimulation by anti-CD28 Ab (blue) for (C) CB and (D) adult. A hypothetical molecular mechanism for infants aged 1–2 months (E) or 3–5 months (F) after TCR/CD3 engagement plus CD28 costimulation with unknown pErk response.