PMC5425576_01
PMC5425576_02
Major CD4 T-cell depletion in a chronic granulomatous disease (CGD) patient. (A) Longitudinal CD4 T-cell counts. (B) Proportion of naive (CD45RA+CD27+) CD4 T-cells in the CGD patient; open bar represents mean +- SEM of healthy subjects (n = 25), and dot plots illustrate CCR7/CD45RA expression within CD4 T-cells.
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F1
T-cell depletion, the phenotypic analysis revealed marked loss of naive cells within both CD4 (Figure 1B) and CD8 T-cells (6.2% of total CD8 T-cells, 100 cells/mul at 36 years of age). This was in agreement with an impaired replenishment of the T-cell pool by recently produced cells.
Impaired T-cell production despite increased circulating interleukin (IL)-7 levels in a chronic granulomatous disease (CGD) patient. (A) Signal joint T-cell receptor rearrangement excision circle (sjTREC) frequency and sj/betaTREC ratio in total T-cells in relation to age in the CGD patient and healthy subjects. (B) IL-7 levels and CD4 counts in the CGD patient (blue), healthy subjects (black, n = 47), and untreated HIV-1-infected individuals (green, n = 61); each dot represents one individual/time point. P-value and Spearman's correlation coefficient are shown.
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F2
Both sjTREC frequency and the sj/betaTREC ratio, which are considered to reflect intra-thymic precursor T-cell proliferation and directly correlate with thymic output, were markedly low for the patient's age (Figure 2A). Of note, reduced thymic activity was observed despite the levels of IL-7, an essential homeostatic cytokine, being highly enhanced (Figure 2B), even in comparison with those of untreated HIV-1-infected individuals.
T-cell activation and terminal differentiation in a chronic granulomatous disease (CGD) patient. (A) Representative dot plots of the analysis of cytokine production and chemokine expression within CD4 T-cells, regulatory T-cell-associated markers within CD4+FoxP3+ cells, as well as activation and cycling markers within memory-effector CD4 T-cells from the CGD patient at 36 years of age and a representative age-matched healthy control. (B) Frequency of interleukin (IL)-17-, IL-22-, IFN-gamma-, or TNF-alpha-producing cells, as well as the frequency of FoxP3+, HLA-DR+CD38+, or Ki-67+ cells within the CD4 subset, in the peripheral blood of the CGD patient and healthy subjects. (C) Representative dot plots showing the co-expression of IFN-gamma and perforin within CD8 T-cells in the CGD patient and an age-matched control, and a graph showing the frequency of IFN-gamma-producing cells within CD8 T-cells of the CGD patient at different ages and of healthy adults (bar represents mean +- SEM, n = 24). (D) Telomere length of naive (CD45RA+) and memory (CD45RA-) CD4 T-cells as well as CD8 T-cells defined according to CD27 expression from the CGD patient and controls.
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F3
T-cell loss was accompanied by upregulation of activation markers and by increased frequency of cycling cells (Figure 3A). The analysis of cytokine production by T-cells revealed an effector differentiation profile with a significant production of pro-inflammatory cytokines, particularly IL-17 (Figure 3B), as has been previously reported. T-cells were CD4+ (96%). There was also an increased frequency of CD4 T-cells producing IL-22 (Figure 3B), of which 42% concomitantly produced IL-17. CD25 and CD39, markers associated with suppressive function (Figures 3A,B). CD8 T-cells featured an activated and terminally differentiated phenotype, as illustrated by their high levels of perforin and IFN-gamma production (Figure 3C). In agreement with these findings, both CD4 and CD8 T-cells, irrespective of their degree of differentiation, featured markedly reduced telomere length, further supporting persistent immune stimulation and increased cell cycling in parallel with reduced de novo T-cell replenishment (Figure 3D).
PMC5425576_03
T-cell subsets in the gut mucosa of the chronic granulomatous disease (CGD) patient. (A) Frequency of CD4 and CD8 T-cells within CD3+ lymphocytes in cell suspensions of sigmoid biopsies from the CGD patient at 34 years of age and from healthy subjects (n = 12). (B) Representative dot plots of the expression of IFN-gamma, interleukin (IL)-17, IL-22, and FoxP3 within sigmoid CD4+ cells from the CGD patient and an age-matched control; the graph indicates the frequency of IFN-gamma-, IL-17-, and IL-22-producing cells, as well as FoxP3+ cells within sigmoid CD4+ cells from the CGD patient and healthy subjects. (C)
IL-17, IL-22, IL-23, IDO1, and AHR mRNA levels of expression; total RNA extracted from sigmoid biopsies of the CGD patient and healthy controls and mRNA levels expressed in relative units, normalized to the mean Ct levels of GAPDH and r18S. Each dot represents one individual. Bars represent mean of values obtained in healthy controls.
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F4
The CGD patient featured a decreased frequency of CD4+ cells within total T-lymphocytes in the sigmoid mucosa (Figure 4A) and a very low CD4/CD8 ratio (0.14), although we were unable to quantify the absolute counts in the histological material to confirm the CD4 loss. Importantly, in contrast to the circulating CD4 subset, the relative proportions of cells producing IL-17, IL-22, or IFN-gamma, as well as FoxP3-expressing Treg, within sigmoid CD4 cells were within the range of healthy subjects (Figure 4B). Moreover, there were no alterations in the levels of expression of genes implicated in the regulation of these populations, namely, IL-17, IL-22, IL-23, IDO1, and AHR, in the sigmoid biopsies (Figure 4C).