PMC4289093_01
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 beta-actin as a
positive control.
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F1
The patient suffered a homozygous c.2032 C>T substitution in exon 16 of LRBA, which encodes the third armadillo domain of LRBA, resulting in the replacement of amino acid glutamine 678 with a stop codon (Q678X) and in absent protein expression (Fig. 1A-D). The mutation was confirmed by Sanger sequencing, and both parents were heterozygous carriers of the mutation (Figure E1 in the Online Repository). It was confirmed by Sanger sequencing, and resulted in absent protein expression (Figure 1A-D). Both parents and the patients' two clinically unaffected siblings (II.3, and II.4; Figure 1A) were heterozygous carriers of the mutation (Figure E2 in the Online Repository). We also studied three previously described Saudi Arabian siblings (P4, P5 and P6; Family C) with LRBA deficiency due to a homozygous deletion in the BEACH domain of LRBA that abolished protein expression (Figure 1A, B, D) .
LRBA deficiency leads to defect in TR 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 TR cell marker in patient and control subjects. ** p<0.01,
*** p<0.001 by unpaired two-tailed Student's t-test.
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F2
Both parents and the patients' two clinically unaffected siblings (II.3, and II.4; Figure 1A) were heterozygous carriers of the mutation (Figure E2 in the Online Repository). Flow cytometric analysis of peripheral blood TR cells of patient P1 demonstrated a markedly reduced number of CD4+FOXP3+ TR cells (Figure 2A). Analysis of the five other patients with LRBA deficiency revealed that they all share a profound decrease in TR cell frequency in the peripheral blood (controls: 7.54+-0.64% vs. patients: 2.45+-0.29%) (Figure 2B). TR cell markers, including FOXP3, CD25 (IL-2RA), CTLA-4, and Helios, was profoundly decreased in LRBA-deficient subjects relative to controls (Figure 2C). Thus LRBA deficiency was associated with decreased numbers and aberrant phenotype of TR cells.
LRBA-deficient TR cells have decreased in vitro suppressive
ability. A. Flow cytometric analysis of CellTrace Violet proliferation dye
dilution in Teff (CD4+CD25-) cells stimulated
with anti-CD2/CD3/CD28 mAb-coated bead in the absence or presence of TR cells
(CD4+CD25+CD127low) 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 TR 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.
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LRBA-deficient TR cells manifested decreased suppression of T cell proliferation, measured by tracer dye dilution in target Teff cells, as compared to control TR cells, indicative of their impaired function (Figure 3A, B). IL-10 production, whose deficiency in TR cells is associated with colitis . IL-10 production was increased in patient as compared to control TR cells (Figure 3C). Similar to IPEX patients and unlike SLE patients, they were negative for anti-nuclear and anti-dsDNA antibodies (Figure 5A; Figure E3 in the Online Repository and data not shown).
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 naive
CD45RA+CCR7+ CD8 T cells in an LRBA-deficient and a control
subject. F. Frequency of circulating TEMRA
CD45RA+CCR7- and naive
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.
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F4
LRBA deficient subjects: 59.5%+-2.8 vs. 86%+-2.1 for CD4 T cells, and 29.4%+-4.2 vs. 50.3%+-6.2 for CD8 T cells, respectively) (Figure 4A-D). The frequency of naive CD45RA+CCR7+ CD8+T cells was decreased in LRBA-deficient subjects (control vs. LRBA deficient subjects: 10.4%+-3 vs. 3%+-0.7). However, there was no difference in the frequency of CD8+CD45RA+CCR7- [(CD45RA+ effector memory (TEMRA)] T cells as compared to control subjects (Figure 4E-F). The gating strategy for detecting circulating TFH and TFR cells and the specificity of the staining are illustrated in Figures E4 and E5 in the Online Repository, respectively. There was a striking increase in the frequency of TFH cells in the peripheral blood of LRBA-deficient patients (controls: 4.9%+-0.6 vs. patients: 31.2%+-5.3) (Figure 5B, C). The patient CXCR5+PD-1hi cells expressed high levels of B cell lymphoma 6 protein (BCL6), inducible co-stimulator protein (ICOS) and CD40 ligand (CD40L), consistent with their TFH phenotype (Figure 5D).
Increased autoantibodies and dysregulated TFH 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 TFH
(CD4+CXCR5+PD-1hi) cells in patient and control
subjects. C. Frequency of TFH cells within the peripheral blood
CD4+ T cells of LRBA-deficient and control subjects. D. Flow
cytometric overlays of the TFH 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 TFR cells. T cells within the
CD4+CXCR5+PD-1hi gate were analyzed for Foxp3
expression. F. Frequencies of TFR 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.
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F5
This was especially true for patient P1, whose phenotype and autoantibody profile were similar to those of the subject with true IPEX disease (Figure 5A and data not shown). Similar to IPEX patients and unlike SLE patients, they were negative for anti-nuclear and anti-dsDNA antibodies (Figure 5A; Figure E3 in the Online Repository and data not shown). The gating strategy for detecting circulating TFH and TFR cells and the specificity of the staining are illustrated in Figures E4 and E5 in the Online Repository, respectively. There was a striking increase in the frequency of TFH cells in the peripheral blood of LRBA-deficient patients (controls: 4.9%+-0.6 vs. patients: 31.2%+-5.3) (Figure 5B, C). The patient CXCR5+PD-1hi cells expressed high levels of B cell lymphoma 6 protein (BCL6), inducible co-stimulator protein (ICOS) and CD40 ligand (CD40L), consistent with their TFH phenotype (Figure 5D). TFH cell population, the frequency of FOXP3+ TFR cells was significantly decreased in patients relative to controls (controls: 10.3%+-2 vs. patients: 3.5%+-0.6) (Figure 5E, F). These results are consistent with dysregulated TFH and compromised TFR cell compartments in LRBA-deficient subjects.
LRBA deficiency does not affect the in vitro generation of
FOXP3+ iTR 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-beta1. C. Collated frequencies of FOXP3+ iTR
cells in control and LRBA-deficient subjects (N=4-5/group). Results are
representative of 2 independent experiments.
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CD4+ T cells to differentiate into induced (i)TR cells upon stimulation with anti-CD2/CD3/CD28 mAbs in presence of TGF-beta. CD4+ T cells of patient and control subjects were enriched by magnetic separation, and naive CD4+ T cells were purified >98% purity by cell sorting (Figure 6A and data not shown). LRBA sufficient and deficient T cells were found equivalent in their capacity to differentiate into iTR cells that equally expressed high levels of FOXP3 (Figure 6B, C).
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+
Tconv and TR cells from LRBA-deficient and control subjects.
B, D, F. Frequencies of AnnexinV+ (B),
Ki67+ (D) and CD31+ (F) cells within
Tconv and TR 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.
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LRBA deficient subjects: 6.3%+-0.6 vs. 13.7%+-3.2 for CD4+ Tconv cells and 15.7%+-1.3 vs. 22.6%+-0.3 for TR cells, respectively) (Figure 7A, B). LRBA-deficient Tconv but not TR cells also exhibited increased Ki67 staining, indicative of increased proliferation (control vs. LRBA deficient subjects: 4.7%+-0.5 vs. 9%+-0.7 for CD4+ Tconv cells and 13%+-1 vs. 14.4%+-1.7 for TR cells, respectively) (Figure 7C, D). T cells, a marker of recent thymic emigrant cells (RTEs), was similar in peripheral blood of patient and control subjects (Figure 7E, F). These results pointed to increased turnover (proliferation and apoptosis) of Tconv cells and increased attrition (apoptosis) of TR cells in the periphery of LRBA-deficient subjects in the face of apparently normal thymic efflux of the respective population.
Impaired mTORC1 and mTORC2 activities in LRBA-deficient Tconv and
TR cells. A. Flow cytometric analysis of pS6 of conventional
(Tconv) and regulatory (TR) T cells from LRBA-deficient and
control subjects before after anti-CD2/3/28 stimulation. B. Mean Fluorescence
Intensity (MFI) of pS6 within Tconv and TR cells of LRBA-deficient
and control subjects before and after anti-CD2/3/28 stimulation. C. Flow
cytometric analysis of p4E-BP1 of Tconv and TR cells from
LRBA-deficient and control subjects after anti-CD2/3/28 stimulation. D. MFI
of p4E-BP1 within Tconv and TR cells of LRBA-deficient and control
subjects before and after anti-CD2/3/28 stimulation. E. Flow cytometric
analysis of p-AKT (S473) of Tconv and TR cells from LRBA-deficient
and control subjects after anti-CD2/3/28 stimulation. F. MFI of p-AKT (S473)
within Tconv and TR 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.
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Stimulation of PBMCs of LRBA-deficient and control subjects with anti-CD2/CD3/CD28 mAbs revealed that phosphorylation of S6 and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), downstream substrates of mTORC1, was decreased in patient vs. control TR and Tconv cells (Figure 8A-D) . TR and Tconv cells (Figure 8E-F) . These findings indicated that LRBA deficiency was associated with impaired activation of mTORC1 and mTORC2 complexes, with potentially deleterious impact on TR cell function.