Thymic Epithelium Abnormalities in DiGeorge and Down Syndrome Patients Contribute to Dysregulation in T Cell Development - PubMed (original) (raw)
Clinical Trial
doi: 10.3389/fimmu.2019.00447. eCollection 2019.
Ileana Bortolomai 1 3, Francesca Ferrua 1 4 5, Elena Fontana 3 6, Luisa Imberti 7, Erika Conforti 8, Donato Amodio 2 9, Sonia Bergante 10, Giulia Macchiarulo 2 9, Veronica D'Oria 8, Francesca Conti 9, Silvia Di Cesare 2, Georgia Fousteri 11, Adriano Carotti 12, Alessandro Giamberti 13, Pietro Luigi Poliani 14, Luigi D Notarangelo 15, Caterina Cancrini 2 9, Anna Villa 1 3, Marita Bosticardo 1 15
Affiliations
- PMID: 30949166
- PMCID: PMC6436073
- DOI: 10.3389/fimmu.2019.00447
Clinical Trial
Thymic Epithelium Abnormalities in DiGeorge and Down Syndrome Patients Contribute to Dysregulation in T Cell Development
Genni Enza Marcovecchio et al. Front Immunol. 2019.
Abstract
The thymus plays a fundamental role in establishing and maintaining central and peripheral tolerance and defects in thymic architecture or AIRE expression result in the development of autoreactive lymphocytes. Patients with partial DiGeorge Syndrome (pDGS) and Down Syndrome (DS) present alterations in size and architecture of the thymus and higher risk to develop autoimmunity. We sought to evaluate thymic architecture and thymocyte development in DGS and DS patients and to determine the extent to which thymic defects result in immune dysregulation and T cell homeostasis perturbation in these patients. Thymi from pediatric patients and age-matched controls were obtained to evaluate cortex and medullary compartments, AIRE expression and thymocyte development. In the same patients we also characterized immunophenotype of peripheral T cells. Phenotypic and functional characterization of thymic and peripheral regulatory T (Treg) cells was finally assessed. Histologic analysis revealed peculiar alterations in thymic medulla size and maturation in DGS and DS patients. Perturbed distribution of thymocytes and altered thymic output was also observed. DGS patients showed lower mature CD4+ and CD8+ T cell frequency, associated with reduced proportion and function of Tregs both in thymus and peripheral blood. DS patients showed increased frequency of single positive (SP) thymocytes and thymic Treg cells. However, Tregs isolated both from thymus and peripheral blood of DS patients showed reduced suppressive ability. Our results provide novel insights on thymic defects associated with DGS and DS and their impact on peripheral immune dysregulation. Indeed, thymic abnormalities and defect in thymocyte development, in particular in Treg cell number and function could contribute in the pathogenesis of the immunodysregulation present in pDGS and in DS patients.
Keywords: DiGeorge syndrome; Down syndrome; regulatory T (Treg) cells; thymocytes; thymus.
Figures
Figure 1
Analyses of thymic size and architecture. (A) Thymic size, in terms of grams, in patients as compared to HDs. Mean ± SEM are represented (HD, n = 26; DGS, n = 5; DS, n = 8) (Mann-Whitney test, *_p_-value < 0.05; **_p_-value < 0.002); (B) Representative H&E staining of thymic cortex (C) and medulla (M) of HDs, DGS and DS patients belonging to the 2–5 months, 5–9 months, and 2–5 years age-groups. Scale bar = 500 μm (main figure) and 100 μm (inset); (C) Graphic representation of medullary area evaluated for our patients and compared to HDs (2–5 months: HD, n = 3, samples 9, 10, 13 in Supplemental Table 1; DGS, n = 1, patient 10 in Supplemental Table 2; DS, n = 4, patients 15, 16, 18, 20 in Supplemental Table 2. 5–9 months: HD, n = 3, samples 6, 17, 20 in Supplemental Table 1; DGS, n = 1, patient 9 in Supplemental Table 2; DS, n = 1, patient 17 in Supplemental Table 2. 2–5 years: HD, n = 3, samples 21, 23, 24 in Supplemental Table 1; DGS, n = 1, patient 8 in Supplemental Table 2; DS, n = 2, patients 14, 19 in Supplemental Table 2).
Figure 2
Analysis of thymic maturation and AIRE expression in thymic samples. (A) Involucrin immunohistochemical staining in HD, DGS and DS thymic samples. Scale bar = 500 μm (left) and 100 μm (right); (B) AIRE immunohistochemical staining in HDs, DGS, and DS thymic samples in different age-groups. Scale bar = 50 μm; (C) AIRE+ cell counts normalized on medullary area (mm2) (2–5 months: HD, n = 3, samples 9, 10, 13 in Supplemental Table 1; DGS, n = 1, patient 10 in Supplemental Table 2; DS, n = 4, patients 15, 16, 18, 20 in Supplemental Table 2. 5–9 months: HD, n = 3, samples 6, 17, 20 in Supplemental Table 1; DGS, n = 1, patient 9 in Supplemental Table 2; DS, n = 1, patient 17 in Supplemental Table 2. 2–5 years: HD, n = 3, samples 21, 23, 24 in Supplemental Table 1; DGS, n = 1, patient 8 in Supplemental Table 2; DS, n = 2, patients 14, 19 in Supplemental Table 2).
Figure 3
Thymocyte absolute number and analysis of early thymocyte development. (A,B) Graphic representation of thymocyte absolute numbers normalized per gram of tissue (A) and per total thymus weight (B) (HD, n = 26; DGS, n = 4; DS, n = 8) (Mann-Whitney test; *_p_-value < 0.01. (C) Frequencies of Myeloid Progenitors (MP) (CD34− CD7−) and early Thymic Progenitors (ETP) (CD34+ CD7+ and CD34− CD7+) calculated on the DN gate (HD, n = 26; DGS, n = 4; DS, n = 8); (D) Frequencies of PRO-T1 (CD7+ CD5−), PRO-T2 (CD7+ CD5+) and PRE-T (CD1a+ CD7+ CD5+) calculated on the CD34− CD7+ gate (HD, n = 26; DGS, n = 4; DS, n = 8). Bars represent medians ± SEM [NPC test: *_T_-cell subset global distribution, *_T_-cell subset partial test (HD and DS); * and *_p_-value < 0.05].
Figure 4
Thymocyte composition and late stages of thymocyte development. (A) Frequencies of DN, DP, SP4, and SP8 composing the thymus (HD, n = 26; DGS, n = 4; DS, n = 8); (B,C) Frequencies of latest stages of thymocyte development for SP4 (B) and SP8 (C) with the dissection of their developmental stages starting from the Ist stage to the Vth (HD, n = 26; DGS, n = 4; DS, n = 8); Bars represent medians ± SEM [NPC test: *_T_-cell subset global distribution, *_T_-cell subset partial test (HD and DS); * and *_p_-value < 0.05; ** and **_p_-value < 0.002].
Figure 5
Thymic and peripheral Treg cell distribution and analysis of their suppressive function in DGS and DS patients. (A–D) Absolute counts (HD, n = 15; DGS, n = 3; DS, n = 8) (A), frequency (HD, n = 15; DGS, n = 3; DS, n = 8) (Mann-Whitney test; *_p_-value < 0.01; **_p_-value < 0.002; ***_p_-value < 0.0001) (B), and phenotype (C,D) of thymic Treg cells [NPC test: *_T_-cell subset global distribution, *_T_-cell subset partial test (HD and DS); * and *_p_-value < 0.05] (HD, n = 15; DGS, n = 3; DS, n = 8); (E–G) Absolute counts (E), frequency (F), and phenotype (G) of peripheral Treg cells (HD, n = 34; DGS, n = 10; DS, n = 8) (Mann-Whitney and NPC test; *_p_-value < 0.01; **_p_-value < 0.002; ***_p_-value < 0.0001). Mean ± SEM are represented; (H,I) Thymic (HD, n = 5; DGS, n = 4; DS, n = 4) (H) and peripheral (HD, n = 5; DGS, n = 4; DS, n = 4) (I) Treg suppressive ability tested at different Tconv:Treg ratios (Two-way ANOVA; ***_p_-value < 0.001; ****_p_-value < 0.0001). Mean ± SEM are represented. Suppressive ability of Tregs is calculated with the following formula: [(% of proliferation of Tconv alone – % of proliferation of Tconv and Treg at different ratios)/(% of proliferation on Tconv alone)]*100.
Figure 6
Evaluation of TRECs and RTEs in children with DGS and DS. (A) sj-TREC per million of thymocytes (HD, n = 26; DGS, n = 4; DS, n = 8); (B) _sj_-TREC per million of PBMCs (HD, n = 34; DGS, n = 10; DS, n = 8); (C,D) Frequencies of RTEs among thymocytes (HD, n = 26; DGS, n = 4; DS, n = 8) (C) and among PBMCs (HD, n = 34; DGS, n = 10; DS, n = 8) (D). Mean ± SEM are represented (Mann-Whitney test; *_p_-value < 0.05; **_p_-value < 0.01; ***_p_-value < 0.0001).
Figure 7
Total lymphocytes and distribution of CD4+ and CD8+ T-cell subsets. (A) Absolute counts per microliter of total blood of lymphocytes (HD, n = 34; DGS, n = 10; DS, n = 8); (B,D) Absolute counts and frequencies of CD4+ cells (HD, n = 34; DGS, n = 10; DS, n = 8); (C,E) Absolute counts and frequencies of CD8+ cells (HD, n = 34; DGS, n = 10; DS, n = 8); (F) CD4/CD8 T lymphocyte ratios of HD, DGS, and DS patients. Mean ± SEM are represented (Mann-Whitney test; *_p_-value < 0.01; **_p_-value < 0.002; ***_p_-value < 0.0001).
Figure 8
Analyses of peripheral T-cell immunophenotype and gating strategy. (A) Gating strategy used to analyze the maturation stages of CD4+ and CD8+ T cells; (B,C) Frequency of CD4+ (B) and CD8+ (C) T-cell subsets in all groups of patients and age-matched HDs (HD, n = 34; DGS, n = 10; DS, n = 8). Mean ± SEM are represented [NPC test: *_T_-cell subset global distribution, *_T_-cell subset partial test (HD and DS); * and *_p_-value < 0.05; ** and **_p_-value < 0.002; ***_p_-value < 0.0001).
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