Developmental induction of human T-cell responses against Candida albicans and Aspergillus fumigatus

Abstract

The origin of human T-cell responses against fungal pathogens early in life is not clearly understood. Here, we show that antifungal T-cell responses are vigorously initiated within the first years of life against lysates and peptides of Candida albicans or Aspergillus fumigatus, presented by autologous monocytes. The neonatal responding T-cell pool consists of 20 different TCR-V_β families, whereas infant and adult pools display dramatically less variability. Although we demonstrate no bias for anti-fungal IL-4 expression early in life, there was a strong bias for anti-fungal IL-17 production. Of note, only T-cells from neonates and infants show an immediate co-expression of multiple cytokines. In addition, only their T-cells co-express simultaneously transcription factors T-bet and RORγt in response to fungi and subsequently their target genes IL-17 and IFNγ. Thus, T-cells of neonates and infants are predetermined to respond quickly with high plasticity to fungal pathogens, which might give an excellent opportunity for therapeutic interventions.

Figure 1

Fungi-specific T cell proliferation. (A–C) Purified CD4⁺CD45RA⁺ T cells were labelled with CFSE and cultured with monocytes matured with heat-inactivated C. albicans or A. fumigatus at a ratio of 2:1. CFSE dilution profiles and the frequency of proliferating (CFSE^lo) T cells from neonates (A), infants and children (C) or adults (B) on day 3 and day 6 after stimulation. Data are representative of 5 donors. (D) Frequency of proliferating (CFSE^lo) T cells from neonates, infants, children, and adults stimulated with C. albicans (orange), A. fumigatus (blue) or anti-CD3/CD28 (black) determined by flow cytometry are plotted against age. The dotted lines represent the 95% confidence interval. The coefficient of determination (R²) according to the one-phase decay exponential model in response to C. albicans-antigen is 0,9209. (E) Bar graphs showing frequency of naïve (CD4⁺CD45RA⁺CD31⁺) or memory (CD4⁺CD45RO⁺) T cells of children, expressing CD25 upon stimulation with Tetanus Toxoid for 3 days. Cumulative results are shown and each dot represents a different donor. The error bars in figures denote ± SD. ****p < 0.0001, as determined by one-way Anova with Tukey post hoc test.

Figure 2

Fungi–specific up-regulation of activation markers. (A–C) Representative dot plots of flow cytometric analysis showing the frequency of CD4⁺CD45RA⁺ T cells of neonates (A), infants and children (C) or adults (B) expressing CD25 or CD69 after 3-day stimulation with monocytes matured with heat-inactivated C. albicans or A. fumigatus. Data are representative of at least 5 donors. (D) Bar graph showing CD25 expression of CD4⁺CD45RA⁺CD31⁺ T cells isolated form neonates, infants, children and adults in response to C. albicans PepMix (brown) as in (A–C). T cells were stimulated for 3 days and determined using flow cytometry. Cumulative results are shown and each dot represents a different donor. The error bars in figures denote ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by one-way Anova with Tukey post hoc test.

Figure 3

Fungi-induced T cell receptor V_ß family response. CD4⁺CD45RA⁺ T cells from neonates, infants, children, and adults were co-cultured with monocytes pulsed with C. albicans- or A. fumigatus-lysates and expression of different V_ß repertoire on T cells was measured by flow cytometry. (A–C) Frequency of V_ß9 (left panel), V_ß16 (middle panel) or V_ß17 (right panel) expressing unstimulated T cells (A), T cells stimulated for 3 days either with C. albicans (B) or A. fumigatus (C) are shown. Each dot represents a different donor. (D,E) Bar graph showing TCR-V_ß expression either by CD4⁺CD45RA⁺CD25⁺ T cells from neonates, infants, children, and adults or by CD4⁺CD45RO⁺CD25⁺ T cells from adults. T cells are stimulated for 3 days either with C. albicans (orange) or A. fumigatus (blue). Cumulative results are shown and each dot represents a different donor. The error bars in figures denote ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001, as determined by one-way Anova with Tukey post hoc test (A–C) or Kruskal Wallis with Dunn’s post hoc test (D and E) (F) TCR-Vß repertoire usage in CD4 + CD45RA + T cells of infants and children as well as adults after co-culturing with monocytes pulsed with C. albicans- or A. fumigatus-lysates. The allocation of the groups took place if the expression of TCR-Vß family in the stimulated samples was higher than in unstimulated controls.

Figure 4

Fungi specific Th1 cytokine expression by T cells of different age groups. CD4⁺CD45RA⁺ T cells from neonates, infants, children, and adults were stimulated with C. albicans (orange) or A. fumigatus (blue) (as in Fig. 1) for 3 and 6 days respectively (A) Frequency of T cells expressing intracellular IL-2 (left panel), TNFα (middle panel) or IFNγ (right panel) was determined by flow cytometry. (B) Determination of IL-2 (left panel), TNFα (middle panel) or IFNγ (right panel) cytokine release of CD4⁺CD45RA⁺ T cells of neonates, infants and children or adults by LegendPlex which were either stimulated or not for 3 days. (C,D) CD4⁺CD45RA⁺ T cells were stimulated with C. albicans (C) or A. fumigatus (D) as in A, and the cells expressing single or multiple cytokines IL-2, TNFα, and IFNγ were determined by flow cytometry and analysed by Boolean gating and shown as fraction of all CD4⁺ T cells in a pie chart. The subsets that simultaneously express no (grey), one (blue), two (yellow) or three (red) different cytokines are grouped by colour. The data are representative of at least 5 donors. Cumulative results are shown and each dot in (A) and (B) represent a different donor. The error bars in figures denote ± SD. ^*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by one-way Anova with Tukey post hoc test (A) or Kruskal Wallis with Dunn’s post hoc test (B).

Figure 5

Age-dependent IL-4 production by fungi specific T cells. (A,B) CD4⁺CD45RA⁺ T cells from neonates, infants, children, and adults were stimulated with C. albicans (orange) or A. fumigatus (blue) (as in Fig. 4) for 3 (left panel) and 6 days (right panel) respectively, and analysed for the expression of intracellular un-glycosylated IL-4 isoform (upper panel) and mature IL-4 (lower panel). Cumulative results are shown and each dot represents a different donor. The error bars in figures denote ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by one-way Anova with Tukey post hoc test.

Figure 6

Fungi specific T cells produce IL-17 in an age dependent manner. CD4⁺CD45RA⁺CD31⁺ T cells from neonates, infants, children, and adults were co-cultured with monocytes pulsed with C. albicans- or A. fumigatus-lysates. (A,B) The frequency of T cells expressing signature Th17 molecules IL-17 (A) and RORγt (B) were analysed by flow cytometry at day 3 (upper Panel) and day 6 after stimulation (lower panel) (C) Bar graph representing the ELISPOT analysis of the quantitative IL-17, produced by the T cells from neonates and adults which were either stimulated or not for 3 days as described in (A). (D) Determination of IL-17A cytokine release of CD4⁺CD45RA⁺ T cells of neonates, infants and children or adults by LegendPlex which were either stimulated or not for 3 days as described in (A). (E) Frequency of T cells expressing intracellular IL-17 (white), IL-4 (black) and both IL-17/IL-4 (grey) were measured by flow cytometry after 6 days of stimulation, analysed by boolean gating and shown as different fractions of cytokine expressing cells in a stacked bar chart. (F) Bar graph showing IL-17 expression by CD4⁺CD45RA⁺CD31⁺ T cells of neonates, adults, and infants of 0.5-2 years old, stimulated for 6 days as described in (A) in the presence or absence of neutralizing antibodies for IL-1ß, IL-6 or both. Cumulative results are shown and each dot in (A–D) and (F) represent a different donor. The error bars in figures denote ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by one-way Anova with Tukey post hoc test (A-C, F) or Kruskal Wallis with Dunn’s post hoc test (D).

Figure 7

Identification of age-dependent fungi specific multifunctional Th1/Th17 cells. (A) CD4⁺CD45RA⁺CD31⁺ T cells from neonates, infants and children of different age groups, and adults were co-cultured with monocytes pulsed with C. albicans (left panel) or A. fumigatus (right panel). The frequency of T cells expressing intracellular IL-17 (white), IFNγ (black) and both IL-17/IFNγ (grey) were determined by flow cytometry at the indicated time points, analysed by boolean gating and shown as different fractions of cytokine expressing cells in a stacked bar chart. (B) Frequency of naïve (CD4⁺CD45RA⁺CD31⁺) or memory (CD4⁺CD45RO⁺) T cells of adults expressing intracellular IL-17 (white), IFNγ (black) and both IL-17/IFNγ (grey) were determined by flow cytometry at the indicated time points and analysed as described in (A). (C) Frequency of CD4⁺CD45RA⁺CD31⁺ T cells from neonates and adults expressing Th1 and Th17 transcription factors T-bet, RORγt and both T-bet as well as RORγt following stimulation with C. albicans (orange bars) or A. fumigatus (blue bars) for 3 and 6 days as described in (A). Cumulative results are shown and each dot represents a different donor. The error bars in figures denote ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by Kruskal Wallis test with Dunn’s post hoc test.