Regulatory T-cell deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like disorder caused by loss-of-function mutations in LRBA

Abstract

Background: A number of heritable immune dysregulatory diseases result from defects affecting regulatory T (Treg) cell development, function, or both. They include immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, which is caused by mutations in forkhead box P3 (FOXP3), and IPEX-like disorders caused by mutations in IL-2 receptor α (IL2RA), signal transducer and activator of transcription 5b (STAT5b), and signal transducer and activator of transcription 1 (STAT1). However, the genetic defects underlying many cases of IPEX-like disorders remain unknown.

Objective: We sought to identify the genetic abnormalities in patients with idiopathic IPEX-like disorders.

Methods: We performed whole-exome and targeted gene sequencing and phenotypic and functional analyses of Treg cells.

Results: A child who presented with an IPEX-like syndrome and severe Treg cell deficiency was found to harbor a nonsense mutation in the gene encoding LPS-responsive beige-like anchor (LRBA), which was previously implicated as a cause of common variable immunodeficiency with autoimmunity. Analysis of subjects with LRBA deficiency revealed marked Treg cell depletion; profoundly decreased expression of canonical Treg cell markers, including FOXP3, CD25, Helios, and cytotoxic T lymphocyte-associated antigen 4; and impaired Treg cell-mediated suppression. There was skewing in favor of memory T cells and intense autoantibody production, with marked expansion of T follicular helper and contraction of T follicular regulatory cells. Whereas the frequency of recent thymic emigrants and the differentiation of induced Treg cells were normal, LRBA-deficient T cells exhibited increased apoptosis and reduced activities of the metabolic sensors mammalian target of rapamycin complexes 1 and 2.

Conclusion: LRBA deficiency is a novel cause of IPEX-like syndrome and Treg cell deficiency associated with metabolic dysfunction and increased apoptosis of Treg cells.

Keywords: Autoantibodies; LPS-responsive beige-like anchor; T follicular helper cells; T follicular regulatory cells; X-linked syndrome; autoimmunity; enteropathy; forkhead box P3; immune dysregulation; mammalian target of rapamycin complex; polyendocrinopathy; regulatory T cells.

Figure 1

A. Pedigrees of the LRBA-deficient families. Generations are designated by Roman numerals (I–II) and individuals by Arabic numerals. Double lines connecting parents indicate consanguinity. Probands in the respective families are indicated by P1-P6 labeling. Squares, males; circles, females; filled symbols, patients. B. Schematic representation of LRBA protein showing the respective domains and the location of patient mutations. C. Sanger sequencing flurograms showing genomic DNA mutations of patient P1 and P2 as compared to the equivalent DNA sequences of healthy controls (Ctrl). D. Western blot analysis of LRBA protein expression in fibroblasts of patient P1 and lymphocytes of patients P2, P3, P5, and P6 as compared to those of controls. The blots were reprobed for β-actin as a positive control.

Figure 2. LRBA deficiency leads to defect in T_R cell frequency and phenotype

A. Representative dot plot analysis of FOXP3 expression in patient P1 vs. a control subject. B. Percent CD4⁺FOXP3⁺ T cells in the peripheral blood of healthy controls (n=8; open circles) and LRBA deficient patients (n=6; closed circles). C. Representative flow cytometric histogram plots of FOXP3, CD25, CTLA4 and Helios expression in FOXP3⁺CD4⁺ T cells from controls (dotted line) and LRBA deficient patients (solid line). Gray area represents control isotype staining. D. Mean fluorescence intensity of the respective T_R cell marker in patient and control subjects. ** p<0.01, *** p<0.001 by unpaired two-tailed Student's t-test.

Figure 3. LRBA-deficient T_R cells have decreased in vitro suppressive ability

A. Flow cytometric analysis of CellTrace Violet proliferation dye dilution in T_eff (CD4⁺CD25⁻) cells stimulated with anti-CD2/CD3/CD28 mAb-coated bead in the absence or presence of T_R cells (CD4⁺CD25⁺CD127^low) from healthy controls or LRBA deficient patients. B. Cumulative Results of in vitro suppression assays in patient and control subjects. N=6 individuals per group, ****p<0.0001 by unpaired two-tailed Student's t-test. C. IL-10 production in patient and control T_R cells at baseline and following stimulation with the aCD2/CD3/CD28 or PMA + Ionomycin (Iono) for 4 days. N.D.: Not detected. *P<0.05 by 2-way ANOVA with post-test analysis.

Figure 4. Increased frequency of memory T cells in LRBA-deficient subjects

A. Flow cytometric analysis of circulating CD45RO⁻CD45RA⁺ and CD45RO+CD45RA⁻ CD4⁺ T cells in an LRBA-deficient and a control subject. B. Frequency of activated CD45RO⁺CD45RA⁻ within the peripheral blood CD4⁺ T cell pool of LRBA-deficient and control subjects. C. Flow cytometric analysis of circulating CD45RO⁻CD45RA⁺ and CD45RO+CD45RA⁻ CD8 T cells in an LRBA-deficient and a control subject. D. Frequency of activated CD45RO+CD45RA⁻ within the peripheral blood CD8⁺ T cell pool of LRBA-deficient and control subjects. E. Flow cytometric analysis of circulating TEMRA CD45RA⁺CCR7⁻ and naïve CD45RA⁺CCR7⁺ CD8 T cells in an LRBA-deficient and a control subject. F. Frequency of circulating TEMRA CD45RA⁺CCR7⁻ and naïve CD45RA⁺CCR7⁺ within the peripheral blood CD8⁺ T cell pool of LRBA-deficient and control subjects. *p<0.05, ***p<0.001 by Student’s unpaired two tailed t test.

Figure 5. Increased autoantibodies and dysregulated T_FH cell response in LRBA-deficient patients

A. Heat map display of autoantibody reactivity against self-antigens in sera of LRBA-deficient and control subjects, a patient with IPEX and a patient with SLE. A value of 1 (black) is equal to the control average + 1 standard deviation (SD). B. Flow cytometric analysis of circulating T_FH (CD4⁺CXCR5⁺PD-1^hi) cells in patient and control subjects. C. Frequency of T_FH cells within the peripheral blood CD4⁺ T cells of LRBA-deficient and control subjects. D. Flow cytometric overlays of the T_FH markers BCL6, ICOS and CD40L within the peripheral blood CD4⁺CXCR5⁺PD-1⁺ cell pool of LRBA-deficient and control subjects. E. Flow cytometric analysis of circulating T_FR cells. T cells within the CD4⁺CXCR5⁺PD-1^hi gate were analyzed for Foxp3 expression. F. Frequencies of T_FR cells within the peripheral blood CD4⁺CXCR5⁺PD-1⁺ cell pool. *p<0.05, ***p<0.001 by Student’s unpaired two tailed t test.

Figure 6. LRBA deficiency does not affect the in vitro generation of FOXP3⁺ iT_R cells

A. Representative flow cytometric analyses of CD45RA/RO markers in purified CD4⁺ T cells (upper panel) and in cell sorted CD3⁺CD4⁺CD25⁻CD45RO⁻CD45RA⁺ T cells (lower panels) of a control and an LRBA-deficient subject. B. Representative flow cytometric analyses of Foxp3 expression in T cells sorted as in panel A and stimulated in culture with anti-CD2/3/28 mAbs in the absence or presence of TGF-β1. C. Collated frequencies of FOXP3⁺ iT_R cells in control and LRBA-deficient subjects (N=4–5/group). Results are representative of 2 independent experiments.

Figure 7. Increased turnover of LRBA-deficient T cells

A, C E. Flow cytometric analysis of AnnexinV (A), Ki67 (B) and CD31 (C) staining in ex-vivo CD4⁺ T_conv and T_R cells from LRBA-deficient and control subjects. B, D, F. Frequencies of AnnexinV⁺ (B), Ki67⁺ (D) and CD31⁺ (F) cells within T_conv and T_R cell populations of LRBA-deficient and control subjects. N=4–5 patients and 5–8 controls/group . *p<0.05, **p<0.01 by 2-way ANOVA with post-test analysis.

Figure 8. Impaired mTORC1 and mTORC2 activities in LRBA-deficient T_conv and T_R cells

A. Flow cytometric analysis of pS6 of conventional (T_conv) and regulatory (T_R) T cells from LRBA-deficient and control subjects before after anti-CD2/3/28 stimulation. B. Mean Fluorescence Intensity (MFI) of pS6 within T_conv and T_R cells of LRBA-deficient and control subjects before and after anti-CD2/3/28 stimulation. C. Flow cytometric analysis of p4E-BP1 of T_conv and T_R cells from LRBA-deficient and control subjects after anti-CD2/3/28 stimulation. D. MFI of p4E-BP1 within T_conv and T_R cells of LRBA-deficient and control subjects before and after anti-CD2/3/28 stimulation. E. Flow cytometric analysis of p-AKT (S473) of T_conv and T_R cells from LRBA-deficient and control subjects after anti-CD2/3/28 stimulation. F. MFI of p-AKT (S473) within T_conv and T_R cells of LRBA-deficient and control subjects before and after anti-CD2/3/28 stimulation. *p<0.05, **p<0.01 by 2-way ANOVA with post-test analysis.