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Cutting Edge: Increased Autoimmunity Risk
in Glycogen Storage Disease Type 1b Is
Associated with a Reduced Engagement of
Glycolysis in T Cells and an Impaired
Regulatory T Cell Function
Daniela Melis, Fortunata Carbone, Giorgia Minopoli,
Claudia La Rocca, Francesco Perna, Veronica De Rosa,
Mario Galgani, Generoso Andria, Giancarlo Parenti and
Giuseppe Matarese
Supplementary
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http://www.jimmunol.org/content/suppl/2017/04/06/jimmunol.160194
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J Immunol published online 7 April 2017
http://www.jimmunol.org/content/early/2017/04/06/jimmun
ol.1601946
Published April 7, 2017, doi:10.4049/jimmunol.1601946
Cutting Edge
The
Journal of
Immunology
Cutting Edge: Increased Autoimmunity Risk in Glycogen
Storage Disease Type 1b Is Associated with a Reduced
Engagement of Glycolysis in T Cells and an Impaired
Regulatory T Cell Function
Daniela Melis,*,1 Fortunata Carbone,†,1 Giorgia Minopoli,* Claudia La Rocca,†
Francesco Perna,‡ Veronica De Rosa,† Mario Galgani,† Generoso Andria,*
Giancarlo Parenti,*,x and Giuseppe Matarese †,{
G
lucose-6–phosphatase (G6Pase) is a functional complex system of proteins located in the endoplasmic
reticulum (ER) that catalyzes the hydrolysis of glucose6–phosphate (G6P) to glucose and inorganic phosphate. The
G6Pase system consists of G6P transporter (G6PT) and G6P
*Sezione di Pediatria, Dipartimento di Scienze Mediche Traslazionali, Università degli
Studi di Napoli “Federico II,” 80131 Naples, Italy; †Laboratorio di Immunologia, Istituto
di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, 80131
Naples, Italy; ‡Dipartimento di Medicina Clinica e Chirurgia, Università degli Studi di
Napoli “Federico II,” 80131 Naples, Italy; xIstituto Telethon di Genetica e Medicina,
80078 Pozzuoli, Naples, Italy; and {Laboratorio delle Cellule T Regolatorie, Dipartimento
di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli “Federico II,” 80131 Naples, Italy
1
D.M. and F.C. contributed equally to this work.
ORCIDs: 0000-0002-9458-3926 (D.M.); 0000-0001-5319-0977 (F.P.); 0000-0002-94770991 (V.D.R.); 0000-0001-8414-1676 (M.G.); 0000-0001-9429-0616 (G. Matarese).
Received for publication November 17, 2016. Accepted for publication March 13, 2017.
This work was supported by grants from the European Research Council (menTORingTregs;
310496), European Foundation for the Study of Diabetes/Juvenile Diabetes Research
Foundation/Lilly Programme 2015, and the Fondazione Italiana Sclerosi Multipla
(2016/R/18) (all to G. Matarese), the Fondazione Italiana Sclerosi Multipla (2014/R/21)
(to V.D.R.), and European Foundation for the Study of Diabetes/Juvenile Diabetes
Research Foundation/Lilly Programme 2016 (to M.G.).
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601946
catalytic subunit (G6PC). The primary role of G6PT (encoded
by the SLC37A4 gene) is to translocate G6P, the product of
gluconeogenesis and glycogenolysis, from the cytoplasm to
the lumen of the ER, where it is converted into glucose and
phosphate by G6PC (1). G6PT is a ubiquitously expressed
protein, and its mutations cause glycogen storage disease type
1b (GSD-1b; MIM23.2220). In contrast, G6PC (or G6Pase-a)
is expressed primarily in the liver, kidney, and intestine, and
its mutations result in the metabolic disorder GSD type 1a
(GSD-1a; MIM23.2200) (1). Because the G6Pase complex
has a key role in glycogenolysis and gluconeogenesis, both
disorders are characterized by a typical metabolic profile
with fasting hypoglycemia, hepatomegaly, nephromegaly,
hyperlacticacidemia, hyperlipidemia, hyperuricemia, and overweight (2). Recent studies showed that the loss of G6PT activity in GSD-1b results in impaired energy homeostasis and
functionality of neutrophils, higher oxidative stress, and apoptosis, leading to neutropenia (3). In addition, GSD-1b
patients manifest neutrophil dysfunctions, such as impairment in respiratory burst, chemotaxis, and calcium mobilization (4). As a result, GSD-1b patients show susceptibility to
recurrent bacterial infections (5). In contrast to the neutropenia observed in GSD-1b patients and mice, GSD-1a
mice show elevated peripheral blood neutrophil counts (6).
It is interesting to point out that, in addition to abnormalities in neutrophil count and function, GSD-1b patients
are characterized by an increased risk for developing autoimmune disorders. Indeed, several reports have described the
Address correspondence and reprint requests to Prof. Giuseppe Matarese or Dr. Daniela
Melis, Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli
Studi di Napoli “Federico II,” via S. Pansini 5, 80131 Napoli, Italy (G. Matarese) or
Dipartimento di Scienze Mediche Traslazionali, Sezione di Pediatria, Università degli
Studi di Napoli “Federico II,” via S. Pansini, 5, 80131 Napoli, Italy (D.M.). E-mail
addresses: [email protected] (G.M.) or [email protected] (D.M.)
The online version of this article contains supplemental material.
Abbreviations used in this article: ECAR, extracellular acidification rate; ER, endoplasmic reticulum; FOXP3-E2, FOXP3 containing exon 2; G6P, glucose-6–phosphate;
G6Pase, glucose-6–phosphatase; G6PC, G6P catalytic subunit; G6PT, G6P transporter;
GSD-1a, glycogen storage disease type 1a; GSD-1b, glycogen storage disease type 1b;
OCR, oxygen consumption rate; pTreg, peripheral Treg; Tconv, conventional T cell;
Treg, regulatory T cell.
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Glycogen storage disease type 1b (GSD-1b) is an
autosomal-recessive disease caused by mutation of glucose-6–phosphate transporter and characterized by altered
glycogen/glucose homeostasis. A higher frequency of autoimmune diseases has been observed in GSD-1b patients, but the molecular determinants leading to this
phenomenon remain unknown. To address this question, we investigated the effect of glucose-6–phosphate
transporter mutation on immune cell homeostasis and
CD4+ T cell functions. In GSD-1b subjects, we found
lymphopenia and a reduced capacity of T cells to engage
glycolysis upon TCR stimulation. These phenomena
associated with reduced expression of the FOXP3 transcription factor, lower suppressive function in peripheral
CD4+CD25+FOXP3+ regulatory T cells, and an impaired capacity of CD4+CD252 conventional T cells
to induce expression of FOXP3 after suboptimal TCR
stimulation. These data unveil the metabolic determinant leading to an increased autoimmunity risk in
GSD-1b patients. The Journal of Immunology, 2017,
198: 000–000.
2
CUTTING EDGE: REDUCED GLYCOLYSIS AND Treg FUNCTION IN GSD-1b
Materials and Methods
Subjects
All enrolled subjects or their parents or legal guardians gave informed consent
to the study that was approved by the Ethical Committee of the Università di
Napoli “Federico II.” We enrolled 8 GSD-1b patients (three males and five
females, mean age 21 y, range 4.6–31 y) and 10 GSD-1a patients (four males
and six females, mean age 22 y, range 1–28.2 y), all of whom were clinically
followed at the Dipartimento di Scienze Mediche Traslazionali, Sezione di
Pediatria, Università degli Studi di Napoli “Federico II” (Supplemental Fig.
1A). Fifty-seven sex-, age-, body mass index–, and pubertal stage–matched
healthy controls also were included in the study (Supplemental Fig. 1A). The
diagnosis of GSD-1a and GSD-1b was based on mutation analysis of the
G6PC and SLC37A4 gene, respectively. The presence of autoimmune disorders and other complications in GSD-1b patients is summarized in
Supplemental Fig. 1B.
Immunophenotypic and flow cytometry analyses
Heparinized blood samples were obtained between 9 and 11 AM and processed
within 4 h. Immunophenotypic analysis of peripheral blood of healthy controls and GSD-1a and GSD-1b patients was performed with a COULTER
EPICS XL Flow Cytometer using SYSTEM II software (both from Beckman
Coulter), as previously described (17). The following mAbs were used for
staining and FACS analysis (FACSCanto II; BD Biosciences) of PBMCs:
CD4–allophycocyanin–H7 (RPA-T4) and CD25–PE–Cy7 (M-A251) (both
from BD Pharmingen). Thereafter, cells were washed, fixed, and permeabilized (Human FoxP3 Buffer Set; BD Pharmingen) and were stained
with the following mAbs: FOXP3-PE (150D/E4; eBioscience), FOXP3 All-
PE (259D/C7), Ki67-FITC (B56), and CD152-allophycocyanin (BNI3) (all
from BD Pharmingen). Analyses were performed with FACSDiva (BD) and
FlowJo (Tree Star) software.
T cell cultures, proliferation assays, and Treg/Tconv isolation
Human PBMCs were isolated by stratifying heparinized whole blood on FicollHypaque (GE Healthcare). PBMCs (2 3 105 per well) were cultured in
96-well round-bottom plates (Corning Falcon) in medium supplemented
with 5% autologous subject serum or 5% heterologous commercial pooled
AB human serum (EuroClone) and were stimulated or not for 60 h with antiCD3 mAb (OKT3). Human peripheral Tregs (pTregs) (CD4+CD25+) and
Tconvs (CD4+CD252) were purified (90–95% pure) from the PBMCs of
healthy controls or GSD-1a and GSD-1b patients, respectively, by magnetic
cell separation with a Regulatory CD4+CD25+ T Cell Kit (Thermo Fisher) or
by flow sorting with a BD FACSJazz (Becton-Dickinson). Autologous or
heterologous Tconvs were cultured (1 3 104 cells per well) in round-bottom
96-well plates (Corning Falcon) alone or in the presence of pTregs at various
ratios and were stimulated for 60 h with anti-CD3/anti-CD28–coated
Dynabeads (0.5 beads per cell; Thermo Fisher). After 48 h, [3H]thymidine
(0.5 mCi per well; Amersham-Pharmacia Biotech) was added to the cell
cultures, and cells were harvested 12 h later. Radioactivity was measured with
a b cell plate scintillation counter (Wallac).
Bioenergetics and metabolism of T lymphocytes
Real-time measurements of the extracellular acidification rate (ECAR) and the
oxygen consumption rate (OCR) were made using an XFe96 Analyzer (Seahorse Bioscience). PBMCs from GSD-1a patients, GSD-1b patients, and
control subjects were cultured in medium or stimulated with anti-CD3
(OKT3) (4 3 105 cells per well in 96-well culture plate) in 200 ml of
RPMI 1640 medium supplemented with 5% autologous subject serum or 5%
heterologous commercial pooled AB human serum (EuroClone) and incubated at 37˚C for 12 h. ECAR and OCR measurements were performed as
previously described (16).
Western blotting
Highly purified Tconvs and pTregs from subjects with GSD-1a or GSD-1b
and from healthy controls, respectively, were lysed soon after isolation or at
24 and 36 h after suboptimal stimulation with anti-CD3/anti-CD28–coated
Dynabeads (0.1 bead per cell; Thermo Fisher). Total cell lysates were obtained, and total protein was subjected to SDS-PAGE, as described (16). The
following Abs were used: anti-Erk1/2 (H72; Santa Cruz Biotechnology), Ab
to FOXP3 all (PCH101; eBioscience), and Ab to FOXP3-PE (150D/E4;
eBioscience). Results were calculated as the densitometry of protein normalized to that of total Erk1/2. We scanned at least three films with different
exposures from Western blotting, and averaged values were used as densitometry to reduce variations among samples.
Measurement of cytokine production
We measured cytokine levels in supernatants from cell cultures, collected 24 h
after the initiation of stimulation with anti-CD3/anti-CD28–coated Dynabeads, by flow cytometry, with a BD Cytometric Bead Array (CBA) Human
Th1/Th2/Th17 Cytokine Kit, following the manufacturer’s instructions.
Statistical analysis
Statistical analysis was performed using GraphPad Prism software (GraphPad).
Quantitative variables were described using mean 6 SEM. Comparisons were
evaluated using the Kruskal–Wallis test, the Student t test, or the Mann–
Whitney U test. We used two-tailed tests for all analyses; a p value , 0.05 was
indicative of statistical significance.
Results and Discussion
GSD-1b patients are characterized by lymphopenia not observed in
GSD-1a patients and healthy controls
GSD-1 is an autosomal recessive disease that is characterized
by altered glycogen/glucose homeostasis. Our eight GSD-1b
patients showed a high frequency of autoimmunity (five
were affected by autoimmune diseases), including autoimmune
thyroiditis, myasthenia gravis, inflammatory bowel disease,
and rheumatoid arthritis, which was often associated with
more disorders in the same subject (Supplemental Fig. 1B).
Broad immunophenotyping of peripheral blood of GSD-1b
subjects revealed lymphopenia that was characterized by a
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occurrence of chronic inflammatory bowel disease (7), Crohn’s
disease (8, 9), thyroid autoimmunity (10), and myasthenia
gravis (11) in GSD-1b patients. All of these autoimmune/
inflammatory manifestations are highly debilitating and impact significantly on the patient’s quality of life by affecting
the clinical outcome of the disease, as well as survival.
Immunopathogenesis of autoimmune diseases has been
associated with a hyperactivity of autoreactive T cells and the
failure of local regulatory mechanisms that are primarily
mediated by regulatory T cells (Tregs) (12). Tregs are a subset
of CD4+ T cells that express the transcription factor FOXP3
and are involved in maintaining tolerance to self-antigens and
abrogation of autoimmune diseases. It has been shown that
different cellular metabolic pathways are able to induce effector or regulatory responses, because distinct metabolic
programs are required for the commitment of effector or Treg
responses (13). In this context, although Tregs generated
in vitro in the presence of TGF-b were shown to rely mainly
on lipid oxidation (14), recent reports have also shown a
major role for glycolysis in the induction and suppressive
function of human and mouse Tregs (15, 16), given the
capacity of the glycolytic enzyme enolase-1 to control the
expression of specific FOXP3 splicing variants in human
Tregs (16). Interestingly, in human autoimmunity (i.e.,
multiple sclerosis and type 1 diabetes), an impaired engagement of glycolysis upon suboptimal TCR stimulation of
CD4+CD252 conventional T cells (Tconvs) has been observed, and this is associated with a reduced suppressive
function of Tregs (16). These data also suggest that glycolysis
plays a key role in Treg induction and function and that its
impairment leads to an unbalanced immune response,
resulting in loss of self-immune tolerance (16). Building on
the evidence that a strong relationship among glucose metabolism, FOXP3 expression, and regulatory function exists in
human immune cells, we aimed at investigating the molecular
mechanisms linking G6PT mutations and reduced glucose
utilization with loss of self-immune tolerance and autoimmunity in GSD-1b patients.
The Journal of Immunology
Lymphopenia in GSD-1b patients is associated with an impaired
engagement of glycolysis in T cells
Recent findings have shown that cell metabolism can regulate immune responses, because the engagement of specific
cell metabolic pathways profoundly affects immune cell
differentiation, fate, and function, thereby driving the fine
balance between immune tolerance and autoimmune reactions (25). Interestingly, we found that, although the T cell
proliferation did not differ significantly among control subjects and GSD-1a and GSD-1b patients upon TCR stimulation (Supplemental Fig. 1C), a specific alteration in
glycolysis was present in GSD-1b subjects only. Indeed,
ECAR and OCR, indicators of aerobic glycolysis and oxidative phosphorylation, respectively, showed that GSD-1b patients had a lower engagement of glycolysis compared with
healthy controls and GSD-1a patients, as reflected by impaired basal and maximal glycolysis and glycolytic capacity
(Fig. 1A–D); there was little impact on OCR, with the exception of a trend toward a reduction in maximal OCR,
which did not reach statistical significance (Fig. 1E–H). Impairment of glycolysis has been linked to autoimmune conditions, such as multiple sclerosis, type 1 diabetes, and
rheumatoid arthritis (16, 26). This is because engagement of
glycolysis in Tconvs generates waves of FOXP3+ inducible
Tregs in the most metabolically active fraction of proliferating
Tconvs (16). Moreover, a role for glycolysis has been further
suggested by its presence in pTregs that, when freshly isolated,
rely on glycolysis and lipid synthesis (27).
Quantitative and qualitative alterations in pTregs and impaired
induction of FOXP3 in Tconvs from GSD-1b patients
We next investigated whether alteration of T cell metabolism
in GSD-1b patients could relate to a reduced function of
Tregs and with an impairment in FOXP3 induction in
Tconvs. Specifically, we observed a lower peripheral frequency of CD4+FOXP3+ pTregs with respect to healthy
controls and GSD-1a patients that was associated with a lower
expression of FOXP3 (Fig. 2A, 2B). Ex vivo proliferation of pTregs
from GSD-1b patients was impaired, as indicated by the reduced
percentage of pTregs expressing the proliferation marker Ki67
FIGURE 1. Impaired engagement of glycolysis in T cells of GSD-1b patients. (A) Kinetic profile of ECAR in 12-h anti-CD3 (OKT3 mAb)–stimulated PBMCs
from healthy controls (n = 8), GSD-1a patients (n = 7), and GSD-1b patients (n = 6). ECAR was measured in real time under basal conditions and in response to
glucose, oligomycin, and 2-deoxy-d-glucose (2-DG). Indices of glycolytic pathway activation, calculated from the ECAR profiles of PBMCs: basal glycolysis
(before addition of glucose) (B), maximal glycolysis (after the addition of oligomycin) (C), and glycolytic capacity (calculated as the difference between oligomycin
rate and 2-DG rate) (D). (E) Kinetic profile of OCR in 12 h anti-CD3 (OKT3 mAb)–stimulated PBMCs from healthy controls (n = 8), GSD-1a patients (n = 7),
and GSD-1b patients (n = 6). OCR was measured in real time under basal conditions and in response to oligomycin, carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and Antimycin A and Rotenone (Ant-Rot). Indices of mitochondrial respiratory function, calculated from the OCR profiles of
PBMCs: Basal OCR (before addition of oligomycin) (F), maximal OCR (calculated as the difference of FCCP rate and Ant-Rot rate) (G), and ATP-linked OCR
(calculated as the difference of basal rate and oligomycin rate) (H). Data are expressed as mean 6 SEM. *p , 0.05, **p , 0.005, Kruskal–Wallis ANOVA,
followed by the Dunn post hoc test.
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reduction in total lymphocytes and CD3+, CD3+CD45RA+
naive, CD4+, CD4+CD45RA+ naive, CD4+CD28+, CD8+,
and NK cells compared with GSD-1a patients and controls
(Supplemental Table I). This trend was also maintained when
we divided all of the GSD-1b patients into two groups based
on the presence or absence of autoimmune disorders; a more
severe lymphopenia was observed only in patients affected by
at least one autoimmune disease (data not shown). These data
suggest an association between lymphopenia and skewing of
the autoimmune phenotype in GSD-1b subjects. This association is supported by studies showing a link between lymphopenia and autoimmunity (18). Indeed, in autoimmune
disorders, such as type 1 diabetes (19), celiac disease (20), and
Crohn’s disease (21), a reduced number of lymphocytes was
reported in the periphery. Lymphopenia may facilitate destructive autoimmunity through compensatory homeostatic
proliferation, which is a normal compensatory response that
leads to restoration of normal T cell count during lymphopenic conditions. However, chronic lymphopenia might lead
to the proliferation of immune populations that respond to
self-antigens, thus promoting autoimmunity (22). In this
context, it has been shown that lymphopenia and compensatory homeostatic expansion drive type 1 diabetes in NOD
mice through a mechanism supported by IL-21 (22). On the
contrary, lymphoproliferation also associates with autoimmunity, as suggested by the finding that genetic ablation of
tolerance-inducing molecules, such as CTLA-4 and PD-1, in
mice alters the main pathways controlling T cell proliferation
and Treg function (23, 24).
3
4
CUTTING EDGE: REDUCED GLYCOLYSIS AND Treg FUNCTION IN GSD-1b
(Fig. 2C, Supplemental Fig. 1D), together with a lower expression
of surface markers that are characteristic of Tregs, such as CD25
and CTLA-4 (Fig. 2C, Supplemental Fig. 1D). To determine
whether the metabolic perturbations associated with altered FOXP3
expression correlated with an impaired regulatory function, we
assessed the ability of pTregs to suppress the proliferation of autologous CD4+CD252 Tconvs in vitro. GSD-1b pTregs displayed
less suppressive function than did pTregs from GSD-1a patients
and healthy controls (Fig. 2D, left panel). Consistent with the reduced suppressive capacity of pTregs from GSD-1b patients, we
also observed a reduced capacity of these cells to inhibit cytokine
production from autologous Tconvs in coculture experiments (Fig.
2D, right panel, Supplemental Fig. 1E). To rule out that the defect
in suppressive capacity observed in GSD-1b was secondary to impaired Tconv proliferation, we performed criss-cross experiments in
which we measured the suppressive function of glycogen storage
disease patients’ pTregs against heterologous Tconvs from healthy
controls. Again, we found a lower capacity of pTregs from GSD-1b
patients to suppress proliferation and cytokine production of
Tconvs from healthy controls (Fig. 2E, Supplemental Fig. 1F).
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FIGURE 2. Reduced pTreg function and impaired capacity to induce FOXP3 in Tconvs from patients with GSD-1b. (A) Representative flow cytometry
plots of FOXP3 expression in freshly isolated PBMCs from healthy controls and GSD-1a and GSD-1b patients. Numbers in plots indicate the percentage of
FOXP3+ cells (gated on CD4+ cells) and mean fluorescence intensity (MFI) (in parentheses). (B) Percentage (left panel) and MFI (right panel) of FOXP3
expression (gated on CD4+ cells) in freshly isolated PBMCs from healthy controls and GSD-1a and GSD-1b patients. Data are from seven healthy controls,
five GSD-1a patients, and six GSD-1b patients in technical quadruplicates. (C) Percentage of Ki67+ cells (upper panel) and MFI of CD25 and CTLA4 (lower
panels) expression in freshly isolated PBMCs (gated on CD4+FOXP3+ cells) from healthy controls and GSD-1a and GSD-1b patients. Data are from at least
seven healthy controls, four GSD-1a patients, and six GSD-1b patients. (D) Percentage of proliferation (left panel) and IL-2 production (right panel) by
Tconvs from healthy controls and GSD-1a and GSD-1b patients cultured in vitro, alone (1:0) or in the presence of autologous freshly isolated pTregs at
various ratios (Tconvs/pTregs, 1:0.25 to 1:1). Data are from three independent experiments with technical duplicates from four healthy controls, two GSD1a patients, and three GSD-1b patients (left panel) or from one healthy control, one GSD-1a patient, and one GSD-1b patient in technical duplicates and are
representative of two healthy controls, two GSD-1a patients, and two GSD-1b patients (right panel). The asterisks indicate significant differences between
healthy controls and GSD-1b patients. (E) Criss-cross experiment showing percentage of proliferation (left) and IL-2 production (right) of heterologous
Tconvs from healthy controls cultured in vitro alone (1:0) or in the presence of freshly isolated pTregs from healthy controls, GSD-1a or GSD-1b patients, at
various ratios (Tconvs/pTregs, 1:0.25 to 1:1). Data are from three healthy controls, three GSD-1a patients, and three GSD-1b patients in technical duplicates
(left) and from four healthy controls, three GSD-1a patients, and three GSD-1b patients in technical triplicates. The asterisks indicate significant differences
between healthy controls and GSD-1b patients. (F) Representative immunoblot analysis of the 44- and 47-kDa forms of total FOXP3 (probed with mAb
PCH101 against a common epitope of the N terminus) normalized to total Erk1/2 in freshly isolated pTregs purified from healthy control and GSD-1a and
GSD-1b patients (left panel). Densitometry of 44–47-kDa forms of FOXP3 normalized against total Erk1/2. The average value of densitometry was obtained
from the scan of at least five films with different exposures (see Materials and Methods for details) (right panel). (G) Representative immunoblot analysis of
total FOXP3 and FOXP3 containing exon 2 (FOXP3-E2) splicing variants (probed with mAb PCH101 against a common epitope of the N terminus or with
mAb 150D/E4 against an epitope encoded by exon 2, respectively) and total Erk1/2 in Tconvs, stimulated for 24 and 36 h in vitro with anti-CD3/antiCD28–coated Dynabeads (0.1 beads per cell), purified from healthy control and GSD-1a and GSD-1b patients. (H and I) Densitometry of the 44–47 kDa
forms of FOXP3 and of splicing variants containing FOXP3-E2, normalized against total Erk1/2. The average value of densitometry was obtained from the
scan of at least three films with different exposures (see Materials and Methods for details). Data are from three independent experiments with four healthy
controls, three GSD-1a patients, and three GSD-1b patients (H) and from two independent experiments with three healthy controls, two GSD-1a patients,
and two GSD-1b patients (I), all in technical triplicates. All data are expressed as mean 6 SEM. *p , 0.05, **p , 0.005, ***p , 0.0005, two-tailed Student
t test (B and H), two-tailed Mann–Whitney U test (C–F and I).
The Journal of Immunology
altered, a profound defect in Tconvs and Tregs is also present.
Ultimately, this model could also be instrumental in the study
of how immunometabolism regulates Tconv and Treg fate and
function in humans.
Acknowledgments
We thank Salvatore De Simone from the MoFlo sorting facility for the isolation of cells and Teresa Micillo for technical support with Western blotting
analyses.
Disclosures
The authors have no financial conflicts of interest.
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Approximately eight splicing forms of FOXP3 have been
described in humans; however, their function is not fully
characterized. It was reported that glycolysis has a crucial role
in modulating the expression of specific splicing forms of
FOXP3 containing exon 2 (FOXP3-E2) that were indispensable for the suppressive function of Tregs (16). We
measured the different splicing forms of FOXP3 protein using
two specific mAbs: PCH101, which recognizes all FOXP3
slicing variants, and 150D/E4, which is specific for the variant
encoded by FOXP3-E2. We confirmed, by Western blotting,
that freshly isolated pTregs from GSD-1b patients had lower
expression of the 44- and 47-kDa forms of FOXP3 compared
with healthy subjects and GSD-1a patients (Fig. 2F,
Supplemental Fig. 1G). In addition, flow cytometry analysis
revealed that freshly isolated pTregs from GSD-1b patients
expressed less FOXP3-E2 (Supplemental Fig. 1H).
To measure the capacity of Tconvs to induce FOXP3 gene
expression, we analyzed the kinetics (24236 h) of expression
of FOXP3 in Tconvs upon suboptimal TCR stimulation.
Strikingly, immunoblot analyses with mAb PCH101 (FOXP3
all) revealed a delay of Tconvs from GSD-1b patients in the
induction of the 44- and 47-kDa FOXP3 forms at 24 h
compared with healthy controls and GSD-1a patients
(Fig. 2G, 2H, Supplemental Fig. 1I). Also, we probed, in
parallel, the same filter with FOXP3-E2–specific mAb
(150D/E4) and confirmed that the most affected FOXP3
splicing variant in GSD-1b patients was FOXP3-E2 (Fig. 2G,
2I). On the contrary, Tconvs from GSD-1a patients showed
higher amounts of the 44- and 47- kDa forms of FOXP3 and
of FOXP3-E2 (Fig. 2G–I, Supplemental Fig. 1I). These data
could be related, in part, to the altered systemic metabolic
environment that characterizes GSD-1a patients, who display
increased serum levels of lactic acid and uric acid, which is not
associated with the alteration in glucose metabolism in immune cells as occurs in GSD-1b patients, because the expression of mutated enzyme is limited to liver, kidney, and
intestine (1).
In conclusion, our data suggest that the deficit in G6PT
expression affects engagement of glycolysis in T cells and is
associated with impaired pTreg function and with the reduced capacity of Tconvs to express specific FOXP3 splicing
variants containing exon 2. G6PT is involved in the transport
of cytoplasmic G6P into the lumen of the ER and in the
translocation of inorganic phosphate in the opposite direction. It forms, together with G6Pase, the complex responsible
for glucose production through glycogenolysis and gluconeogenesis, playing a central role in homeostatic regulation of
blood glucose levels. A defect in G6PT leads to a reduced
capacity to mobilize glucose, and the primary consequence is
a reduced engagement of glycolysis upon T cell activation
and peripheral generation and function of Tregs; this process
is also associated with impaired FOXP3 expression by
Tconvs during low TCR activation. These data could support and shed light on the metabolic determinants that lead
to an increased frequency of autoimmune disorders in GSD1b subjects. Unfortunately, a major limitation of the study is
the relatively small number of GSD-1 subjects because of the
extreme rarity of the disease (our is one of the biggest cohort of
GSD-1 subjects in Italy). Nonetheless, to the best of our
knowledge, this is the first report showing that, in a monogenic
orphan disease in which glucose utilization and metabolism are
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CUTTING EDGE: REDUCED GLYCOLYSIS AND Treg FUNCTION IN GSD-1b
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metabolic requirements. [Published erratum appears in 2016 Immunity 44: 712.]
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