Human neonatal thymectomy induces altered B-cell responses and autoreactivity

Abstract

An association between T-cell lymphopenia and autoimmunity has long been proposed, but it remains to be elucidated whether T-cell lymphopenia affects B-cell responses to autoantigens. Human neonatal thymectomy (Tx) results in a decrease in T-cell numbers and we used this model to study the development of autoreactivity. Two cohorts of neonatally thymectomized individuals were examined, a cohort of young (1-5 years post-Tx, n = 10-27) and older children (>10 years, n = 26), and compared to healthy age-matched controls. T-cell and B-cell subsets were assessed and autoantibody profiling performed. Early post-Tx, a decrease in T-cell numbers (2.75 × 10⁹ /L vs. 0.71 × 10⁹ /L) and an increased proportion of memory T cells (19.72 vs. 57.43%) were observed. The presence of autoantibodies was correlated with an increased proportion of memory T cells in thymectomized children. No differences were seen in percentages of different B-cell subsets between the groups. The autoantigen microarray showed a skewed autoantibody response after Tx. In the cohort of older individuals, autoantibodies were present in 62% of the thymectomized children, while they were found in only 33% of the healthy controls. Overall, our data suggest that neonatal Tx skews the autoantibody profile. Preferential expansion and preservation of Treg (regulatory T) cell stability and function, may contribute to preventing autoimmune disease development after Tx.

Keywords: Autoimmunity; B cells; Homeostatic proliferation; Lymphopenia; Regulatory T cell; Thymectomy.

Figure 1

Neonatal Tx results in lymphopenia‐induced T‐cell proliferation. PBMCs and plasma were isolated from heparinized blood samples and analyzed by flow cytometry and Luminex, respectively. (A) T Lymphocyte count in healthy controls (young HC, n = 9) and thymectomized (young Tx, n = 10) children. (B) Proportion and numbers of naïve (CD45RA⁺CCR7⁺) CD3⁺ T cells in HC (n = 9) and Tx (n = 11) (left and right panel). (C) Proportion and numbers of memory (CD45RO⁺) CD3⁺ T cells in HC (n = 9) and Tx (n = 11) (left and right panel) (D) Percentage of ki‐67⁺ in CD3⁺ T cells of HC (n = 9) and Tx (n = 11) (E) CD4:CD8 (CD3⁺) ratio in HC (n = 9) and Tx (n = 10) (F) Tfh (CXCR5⁺PD‐1⁺) in CD4⁺ T cells in HC (n = 9) and Tx (n = 11). (G) Plasma CXCL13 levels (pg/mL) of healthy control (young HC, n = 19) and thymectomized (young Tx, n = 11) children. (H). IL‐21 expression in Tfh‐like cells in HC (n = 9) and Tx (n = 11). Panels A–F and H: Data are pooled from three independent experiments with three to five samples per experiment. Mean shown, error bars indicate SD. Panel G, data are pooled from one experiment with 30 samples. Median is shown and error bars indicate SD. Statistical analysis: Mann–Whitney U test. ^* p < 0.05.

Figure 2

Memory T‐cell expansion precedes development of autoreactivity. PBMCs and plasma were isolated from heparinized blood samples and analyzed by flow cytometry and autoantibody screening, respectively. Percentage memory (CD45RO⁺) CD4 T cells in HC (n = 9), autoantibody negative (n = 8) and positive (n = 6) young Tx patients. (Line indicates same patient (patients 3 and 9), but at different time). Data are pooled from three independent experiments with three to five samples per experiment. Statistical analysis: Mann–Whitney U test. ^* p < 0.05.

Figure 3

Thymectomized children manifest an altered autoantibody reactivity profile. Plasma was isolated from heparinized blood samples and analyzed by antigen microarray. IgM and IgG self‐antibody profile analysis of 68 differentially expressed antigens of HC (n = 10, red) and Tx (n = 15, pink). Data are representative of one experiment with 25 samples.

Figure 4

Autoantibodies persist later in life independent of T‐cell number, thymic output, and memory CD4 T cells. PBMCs and plasma were isolated from heparinized blood samples and analyzed by flow cytometry and autoantibody screening, respectively. (A) Lymphocyte count in healthy control (older HC, n = 10) and thymectomized (older Tx, n = 24) adolescents. Mean shown, error bars indicate SD. (B) Percentage of proliferation (ki‐67+) in CD4⁺ T cells of HC (n = 10) and Tx (n = 26). (C) Percentage memory (CD45RO⁺) CD4 T cells in autoantibody positive (ANA and ANCA) and negative older Tx patients. (D) Lymphocyte count in autoantibody positive and negative Tx patients. (E) Recent thymic emigrants (RTE, %CD31⁺ in CD45RA⁺CD4⁺ T cells) in autoantibody positive (n = 15) and negative (n = 10) Tx patients. Panels A–E, data are pooled from five independent experiments with three to five samples per experiment; Panels B–E, median is shown. Statistical analysis: Mann–Whitney U test used.

Figure 5

Preferential Treg‐cell proliferation during the first years after Tx. PBMCs were isolated from heparinized blood samples and analyzed by flow cytometry. (A) Treg cell (CD4⁺Foxp3⁺ T cells) count in “young HC” (n = 8), “young Tx” (n = 10), “older HC” (n = 10) and “older Tx” (n = 25) patients. (B) Percentage of Foxp3‐positive cells in CD4⁺ T cells in “young HC” (n = 9), “young Tx” (n = 11), “older HC” (n = 10), and “older Tx” (n = 25) patients. (C) Proliferation (%Ki‐67⁺) of Treg cells in “young HC” (n = 9), “young Tx” (n = 11), “older HC” (n = 10), and “older T” (n = 25) patients. (D) Percentage of Foxp3⁺CD45RA⁺ (nTreg, left panel), Foxp3⁺CD45RA⁻ (aTreg, middle panel), and Foxp3^dimCD45RA⁻ (cTreg, right panel) in CD4⁺ T cells of young children (HC 1–5 years, n = 9 black circles), young thymectomized (Tx 1–5 years, n = 11, gray circles), older children (Tx > 10 years, n = 11, black squares), and older thymectomized children (Tx > 10 years, n = 26, gray squares, and gray open squares for individuals who lack thymic regeneration). Panels A–D, data are pooled from three independent experiments with three to five samples per experiment. Mean shown, error bars indicate SD. Statistical analysis: Mann—Whitney U test. ^* p < 0.05.