mTOR inhibition alleviates CD8+ T-cell senescence in activated phosphoinositide 3-kinase δ syndrome 2 patients

doi:10.21203/rs.3.rs-2781253/v1

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mTOR inhibition alleviates CD8+ T-cell senescence in activated phosphoinositide 3-kinase δ syndrome 2 patients

https://doi.org/10.21203/rs.3.rs-2781253/v1

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Background

We investigated the clinical and immunological features in a Chinese cohort of activated phosphatidylinositol 3-kinase δ syndrome 2 (APDS2) and assessed the efficacy of Rapamycin therapy and the underlying mechanism.

Results

The shared clinical manifestation of patients included recurrent respiratory tract infection, lymphadenopathy, persistent or recurrent splenomegaly, and hepatomegaly. Three patients carry PIK3R1 c.1425 + 1G > A mutation, and one patient has the mutation c.1425 + 2T > G. Patients have defective humoral immunity with decreased B lymphocytes, especially memory B cells, and suffered from decreased naïve T cells and elevated senescent CD8⁺ T cells. Two patients after rapamycin therapy showed improved clinical symptoms. They also have decreased CD8⁺ effector memory T cells and terminal effector memory cytotoxic T cells. TCF1 was downregulated in CD8⁺ T cells of PIK3R1 patients but upregulated after Rapamycin treatment, which was correlated with decreased senescent CD8⁺ T cells.

Conclusions

mTOR inhibitor rapamycin improved clinical symptoms in APDS2 patients and reversed CD8⁺ T cell senescence through TCF1-dependent signal pathway.

PIK3R1

APDS2

Rapamycin

mTOR

Senescence

CD8+T cell

TCF1

Phosphoinositide 3-Kinases (PI3Ks) are a family of lipid kinases that regulate various cellular responses[1, 2]. PI3Ks consist of two classes, class IA and class IB. Class IA PI3Ks are heterodimeric proteins composed of a p110α, p110β, or p110δ catalytic subunit constitutively associated with a p85α regulatory subunit[1, 3]. In lymphocytes, PI3Kδ is the dominant form of PI3K. PI3Kδ contains a catalytic subunit, PIK3CD (p110δ), and a regulatory subunit, PIK3R1 (p85α), which inhibits the default activation of p110δ [2]. Heterozygous gain-of-function (GOF) mutations in the PIK3CD gene cause unleashed PI3Kδ activation, leading to a primary immunodeficiency disease (PID) called APDS1 (Activated Phosphoinositide 3-kinase δ Syndrome 1), also known as p110δ-activating mutations causing senescent T cells, lymphadenopathy, and immunodeficiency (PASLI) disease[4]. Loss-of-function (LOF) mutations in PIK3R1 introduce premature stop codons in PIK3R1 transcripts. Mutated p85α protein is unstable and fails to control PI3Kδ hyperactivation [5]. PIK3R1 mutations also cause PID, which was named APDS2[3, 6, 7]. The clinical symptoms of APDS2 resemble those of APDS1, characterized by recurrent respiratory infections, lymphadenopathy, hepatosplenomegaly, persistent Epstein‒Barr virus (EBV)/cytomegalovirus (CMV) viremia, and growth retardation [8–10]. APDS1 and APDS2 patients have decreased serum IgG levels, increased IgM levels, B-cell lymphopenia, decreased numbers of total CD4⁺ T cells, especially naïve CD4⁺ T cells, and increased CD8⁺ effector T cells [11].

mTOR is a serine/threonine kinase that plays an essential role in cellular metabolism and cell senescence[7, 12]. The PI3K/AKT/mTOR pathway is hyperactive in APDS patients [1, 7], and an mTOR inhibitor (rapamycin) was applied to treat them. After rapamycin therapy, APDS1 patients experienced a noticeable improvement in clinical manifestations and immunophenotype. Meanwhile, rapamycin-treated APDS2 patients presented improved clinical symptoms and significantly reduced lymphoproliferation [11, 13, 14]. However, a detailed assessment of the immune cell profile of APDS2 patients posttreatment is still missing.

Previous studies confirmed upregulated CD57 expression (a typical T-cell senescence marker), shortened telomeres, and enriched terminally differentiated CD8⁺ T cells in APDS patients, linking APDS with T-cell senescence[7, 10]. The T-cell exhaustion markers CD160, CD244, and programmed death receptor (PD-1) were also increased on CD8⁺ T cells of APDS1 patients [15, 16]. These data suggested that hyperactivated PI3Kδ results in abnormal T-cell function and phenotypes. Transcription Factor T-cell Factor 1 (TCF-1) is a direct downstream effector of the WNT signalling pathway. TCF-1 (TCF7) functions as a vital regulator of T lymphocyte thymic development and mature T-cell differentiation [17, 18]. TCF-1 is highly expressed in naïve and central memory T cells (TCM) and is critical for TCM development, maintaining long-term immunity and proliferative capacity[19]. Meanwhile, TCF-1 also participates in T-cell exhaustion and T-cell senescence[17, 20, 21]. APDS1 patients’ T cells have downregulated TCF-1 expression[22–24], but whether APDS2 patients exhibit the same phenotype is unknown.

Here, we studied four APDS2 patients and evaluated their immunological phenotype before and after rapamycin treatment. We hypothesized that in the absence of PIK3R1, overactivated PI3Kδ downregulating TCF-1 contributes to abnormal CD8⁺ T-cell function and phenotype, which can be rescued by rapamycin in APDS2 patients.

The study was approved by the Ethics Committee of the Children’s Hospital of Fudan University.

Patients and Clinical Manifestation

The APDS2 patients enrolled in the study were recruited with written informed consent from their legal guardians. Blood from healthy donors was obtained at the national children’s medical centre under approved protocols. We retrospectively summarized the clinical data of the patients.

Evaluation of Immunological Function

Nephelometry was performed to detect IgG, IgA, and IgM, and IgE was assessed by UniCAP (Pharmacia, Uppsala, Sweden). A FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) was used to analyse the lymphocyte subsets[25, 26].The whole blood was applied for immunostaining lymphocyte surface markers after red cell lysis and then analysed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). The following antibodies were used for flow cytometry as previously reported[25, 26]: anti-CD3 (UCHT1), anti-CD8 (RPA-T8), anti-CD27 (M-T271), anti-CD45RA (HI100), anti-CD4 (RPA-T4), anti-TCRαβ (T10B9.1A-31), anti-TCRγδ (B1), anti-CD19 (HIB19), anti-CD24 (ML5), anti-CD38 (HIT2), anti-IgD (IA6–2), and anti-CD57 (NK-1) (all from BD Biosciences). The human regulatory T-cell staining kit was from Invitrogen.

Whole-exome sequencing and genetic analysis

Whole-exome sequencing (WES) was performed on all four patients and their parents. Mutations in PIK3R1 were confirmed by Sanger sequencing.

Cell culture and stimulation

PBMCs (peripheral blood mononuclear cells) were enriched by Ficoll-Paque plus density gradient centrifugation and washed twice with PBS. Resuspended cells were cultured with Sigma RPMI 1640 medium containing 10% sigma FBS, 2 mM glutamine, and 100 U/ml penicillin and streptomycin. Cells were then activated with 5 µg/ml concanavalin A (ConA). For T-cell effector function, PBMCs were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and monensin was then added for 4–6 h. Cells were stained with anti-human CD3 (UCHT1), anti-CD8a (RPA-T8), anti-CD4 (RFA-T4), anti-CD69 (FN50), anti-IL-2 (MQ1-17H12), and anti-TNF-α (MAB11). To evaluate exhausted T cells, we examined cells with anti-PD-1 (MIH4), anti-Tim-3 (7D3), anti-TCF7/TCF-1 (6709S Cell Signaling Technology), and anti-Foxo1 (14262S Cell Signaling Technology) antibodies. All antibodies were purchased from BioLegend unless otherwise indicated. A total of 5x10⁴-1x10⁵ live cells were collected with a BECKMAN CytoFLEX Quanta and analysed with FlowJo_V10.

T cells from P1 were treated with 5 µg/ml ConA, 10 µmol/ml PI3K α/δ inhibitor (s1065 Selleck), or ConA and PI3K α/δ inhibitor together for 48 hours.

Western blot

PBMCs (1×10⁶ cells) from patients and healthy donors were lysed by 1X SDS‒PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat powdered milk in PBS with 0.01% Tween-20 (PBST) for 30 minutes at room temperature and then incubated with primary antibody overnight at 4 ℃. After washing three times with PBST for 10 minutes, the membrane was incubated with HRP-conjugated secondary antibody for one hour at room temperature, followed by PBST washing for another 30 minutes. Finally, the membrane was developed through chemiluminescent imaging. Primary antibodies against pAKT S473, GAPDH, Foxo1, and TCF-1 were all purchased from Cell Signalling Technology.

Quantitative PCR

Total RNA was extracted from PBMCs using TRIzol reagent (Thermo Fisher Scientific). Then, cDNA was synthesized using Transcript RT Master Mix (Takara). Quantitative PCR was then performed using Takara SYBR Fast qPCR Mix (Takara). The primer sequences are as follows:

TCF7: Forward: 5'-CGGTCATAATGGGTGAGAGTCT-3',

Reverse: 5’-ACCAGAACCTAGCATCAAGGA-3’;

FOXO1: Forward: 5'-TGATAACTGGAGTACATTTCGCC-3',

Reverse: 5'-CGGTCATAATGGGTGAGAGTCT-3'.

β-actin was used as an internal reference. Fold changes in patients vs. healthy controls were calculated by the 2^-ΔΔCT method, and the data were analysed using t tests.

Statistical Analysis

The data were analysed using Student’s t test or one-way analysis of variance (ANOVA) and Duncan’s test. P values < 0.05 were considered significant.

Clinical Characteristics of PIK3R1 (APDS2) patients

Our clinical studies identified 4 Chinese APDS2 patients from 4 unrelated families. The clinical features of the 4 subjects are shown in Table 1. All patients experienced recurrent respiratory tract infections and lymphadenopathy, and lymph node biopsy revealed a high proliferation of lymphocytes. Lymphadenopathy was especially observed in cervical, mediastinal, intrathoracic, coeliac, and inguinal lymph nodes. Three subjects progressed to pneumonia, and growth retardation was found in two patients.

Furthermore, septic arthritis was diagnosed in three patients with a single knee joint. Three patients had persistent or recurrent splenomegaly or hepatomegaly. Persistent or recurrent EBV viraemia was detected in two patients. In addition, one patient exhibited cardiac anomalies, demonstrating tetralogy of Fallot.

Immunological profile of four PIK3R1 (APDS2) patients

We summarized the immune cells and immunoglobulin profiles of all four subjects in Table 2. Generally, they all had decreased IgG and IgA levels at the time of diagnosis. Reduced IgM levels were observed in two patients, while the other patients presented with higher IgM levels. All patients had normal IgE.

All patients showed decreased B lymphocytes, especially memory B cells. In contrast, they had an increased proportion of transitional B lymphocytes, which indicated an impairment of B-cell maturation. Most of our patients had a decreased number of naïve CD4⁺ T lymphocytes and CD8⁺ T lymphocytes but increased central memory CD4⁺ T cells and cytotoxic CD8⁺ T cells. Other CD4⁺ and CD8⁺ subsets were not consistently changed in all four patients. Given that PIK3R1 patients are characterized by inflammatory arthritis, elevated erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP), we evaluated the regulatory T cells (Tregs) of two patients (P1, P2). The percentage of Treg cells among total CD4⁺ T cells was 1.9% in P1 and 4% in P2, which was lower than that of age-matched healthy controls (Supplementary Fig. 1).

Genetic analysis of PIK3R1 (APDS2)

Genetic variations were analysed and confirmed by WES. In addition to PIK3R1 gene mutations, there were no other pathogenic gene mutations. All of these mutations were de novo (Fig. 1A). Three of 4 patients, P1, P2, and P4, carried the previously described mutation c.1425 + 1G > A on the PIK3R1 gene [3, 7], and patient P3 had the mutation c.1425 + 2T > G [11], resulting in skipping of exon 11 (Fig. 1B).

T-cell activation and the expression of T-cell exhaustion markers are not changed in APDS2 patients

Previous studies confirmed that PIK3CD patients exhibit upregulation of the immunosuppressive molecule PD-1 and reduced proliferative capacity of CD8⁺ T cells, which indicates an exhaustion phenotype[15]. We investigated whether PIK3R1 patients have similar characteristics.

We examined T-cell activation marker CD69 and interleukin (IL)-2 secretion after mitogen ConA stimulation and found slightly reduced CD69 in P1 and P2 compared with healthy controls (Fig. 2A). There was no difference in the effector cytokine IL-2 between P1 and healthy control. Moreover, P1 cells exhibited even higher TNF-α secretion than control T cells (Fig. 2B). In addition, we evaluated the T-cell exhaustion marker PD-1, T-cell immunoglobulin domain and mucin domain-3 (Tim-3), and TCF-1 in PIK3R1 patients (P1, P2). Nevertheless, PD-1 and Tim-3 expression were equal between APDS2 patients and healthy controls in both CD4⁺ T (Fig. 2C) and CD8⁺ T cells (Fig. 2D).

LOF mutation in PIK3R1 downregulated TCF-1 expression through a PI3K-AKT-Foxo1 signalling axis

Hyperactivated PI3K enhances downstream AKT phosphorylation, which activates mTOR and Foxo1/3 to promote cell growth, proliferation, and survival [1, 3, 7, 27]. We detected the phosphorylation of AKT (pAKT S473) and total Foxo1 in PBMCs from P1 and healthy control. The AKT phosphorylation (pAKT S473) was increased (Fig. 3A) and total Foxo1 expression was decreased in P1 compared with healthy control (Fig. 3B). Foxo1 is the upstream regulator of TCF-1 [12]. We found normal TCF-1 expression in CD4⁺ T cells (Fig. 3C) but reduced TCF-1 in CD8⁺ T cells from PIK3R1 patients (Fig. 3D). Consistent with the protein expression level, TCF-1 transcription in CD8⁺ T cells of P1 and P2 was lower than that in the healthy controls (Fig. 3E). Therefore, we speculated that the aberrant PI3K-AKT-Foxo1 pathway may deregulate TCF-1 expression in CD8⁺ T cells.

The expression of the T-cell senescence marker CD57 was increased on CD8⁺ T lymphocytes but not on CD4⁺ T lymphocytes in PIK3R1 patients (P1, P2, P3) compared with healthy controls (Fig. 3F), which suggested that PIK3R1 mutations resulted in CD8⁺ T-cell senescence specifically. Furthermore, CD57 expression in T cells negatively correlated with TCF-1 expression. In healthy controls (Fig. 3G), P1 and P2 (Fig. 3H), the expression of CD57 in TCF-1^low CD8⁺ T cells was higher than that in TCF-1^hi CD8⁺ T cells. This finding indicates that CD57 expression is tightly correlated with TCF-1 expression. Further assessment of TCF-1 expression in CD8⁺ T-cell subsets of healthy controls revealed that TCF-1 expression was higher in naïve CD8⁺ and CD8⁺ TCM cells but lower in CD8⁺ effector memory T cells (TEM) and terminal effector memory cytotoxic T cells (TEMRA) (Fig. 3I). There was no significant difference in TCF-1 expression in CD8 + T-cell subsets between patients (P1 and P2) and healthy controls (Fig. 3J). Contrary to TCF-1, CD57 expression was lower in naïve CD8 and CD8⁺ TCM cells but higher in CD8⁺ TEM and CD8⁺ TEMRA cells of healthy controls (Fig. 3K). CD57 expression in CD8⁺ T-cell subsets exhibited no difference between P1, P2 and healthy controls (Fig. 3L).

Efficacy of Treatment

To prevent recurrent infections, the APDS2 patients received anti-infection prophylaxis and immunoglobulin replacement therapy due to hypogammaglobulinemia and decreased B lymphocytes. Two patients (P1 and P2) received mTOR inhibitor rapamycin treatment at 1 mg/m² once daily. Both patients exhibited benefits. After rapamycin treatment, the patients presented with a reduced sinopulmonary infection frequency and decreased cervical lymph node size. The infection frequency in P1 was reduced from 2–3 times a month to once in 3–4 months, and for P2, it was reduced from once every six months to once a year. The total B-cell counts were not increased in treated PIK3R1 patients (P1, P2) (Fig. 4A), and the cell counts of whole T cells (Fig. 4B) and the inversion of the CD4/CD8 T-cell ratio (Fig. 4C) were not improved. Naïve T cells were not significantly increased in P2 but were slightly increased in P1 after treatment (Fig. 4D, Fig. 4E). Moreover, the proportion of CD8⁺ TCM cells increased at both P1 and P2 (Fig. 4F), and CD8⁺ TEM (Fig. 4G) and CD8 + TEMRA (Fig. 4H) decreased at P1, especially at 15 months. P2 exhibited decreased CD8⁺ TCM within four months of rapamycin treatment. Meanwhile, the proportion of senescent CD8⁺ CD57⁺ T cells was reduced in both treated patients (Fig. 4I). Consistent with the clinical and immunological improvements, the expression of TCF-1 in patients’ cells increased after rapamycin treatment (Fig. 4J). Furthermore, we found reduced phosphorylation of AKT (pAKT S473) (Fig. 4K) after rapamycin treatment at P1. We observed the same effects when cells were treated with a PI3K α/δ inhibitor. Increased Foxo1 and TCF-1 expression and decreased phosphorylation of AKT (pAKT S473) were observed after PI3K α/δ inhibitor treatment in T cells at P1 (Fig. 4L).

PIK3R1 and PIK3CD patients exhibited similar clinical symptoms and immunological characteristics, especially abnormal CD8⁺ T-cell differentiation, such as T-cell exhaustion and senescence. T-cell exhaustion represents impaired T-cell effector function, including retarded effector cytokine secretion and increased chemokine expression [28]. Although exhaustion markers, such as PD-1 and Tim-3, on CD8⁺ T cells were upregulated in APDS1 patients [15, 16], we found no significant difference between 2 APDS2 patients and healthy controls. CD8⁺ T-cell activation and effector cytokine secretion in APDS2 patients were also not significantly impaired. CD57 is a typical human senescent T-cell marker, and previous studies confirmed upregulated CD57 expression in APDS1 and APDS2 patients [17]. We confirmed the increased CD57⁺ senescent CD8⁺ T cells, but not CD4⁺ T cells, in our APDS2 patients.

The critical transcription factor TCF-1 has been widely verified to participate in T-cell exhaustion and senescence [17, 19, 28–31]. TCF-1 expression in our cohort was negatively correlated with CD57 expression in CD8⁺ T cells but was not identified in the CD4⁺ T cells of our APDS2 patients. These data suggested that PIK3R1 mutations exacerbate CD8⁺ T-cell senescence by suppressing TCF-1 transcription and indicated that mutated PIK3R1 may have a different regulatory function than GOF PIK3CD mutants in CD4⁺ T cells.

PI3Ks initiate phosphorylation-based signalling cascades through PDK1 and AKT. AKT then phosphorylates downstream mTOR complex 1 (mTORC1), Foxo1/3, and GSK3 to regulate cellular functions [7]. Activated AKT can phosphorylate Foxo1, which excludes Foxo1 from the nucleus and disables Foxo1 target gene expression[1]. Meanwhile, directly activated mTOR complex 2 (mTORC2) can also enhance this inhibitory effect of AKT on Foxo1 by promoting AKT S473 phosphorylation[2, 3, 32]. Foxo1 was reported as the upstream regulator of TCF-1 in other studies[12]. We also observed increased Foxo1 and TCF-1 expression and decreased AKT (pAKT S473) phosphorylation with PI3K α/δ inhibitor treatment in T cells at P1. The results were consistent with the PI3K-AKT/mTOR-Foxo1 signal transduction axis. The overactivated PI3K-AKT/mTOR-Foxo1 signalling pathway in APDS2 patients results in the downregulation of TCF-1 and may correlate with subsequent T-cell senescence (Supplementary Fig. 2). TCF-1 is typically an effector target of the WNT/β-catenin pathway. Although a previous study confirmed that enforced expression of the TCF-1 long isoform and stabilized β-catenin increased the generation of memory CD8⁺ T cells (17), the conclusion remained elusive [33, 34]. APDS2 patients showed increased memory T cells and reduced TCF-1 expression. Furthermore, WNT/β-catenin functions require further investigation.

The ideal way to alleviate patients’ symptoms is to inhibit the hyperactivated PI3Kδ in PIK3R1 mutation patients. PI3K inhibitors have not been approved for clinical use in China. The mTOR inhibitor rapamycin was used as a substitute treatment. Rapamycin has been widely applied to APDS1 patients with effective results[10] and APDS2 patients [13], while a detailed immunological evaluation of APDS2 patients posttreatment is also missing. Here, we investigated the T-cell profiles of APDS2 patients after rapamycin treatment. In addition to the similar improvements shown in APDS1 patients, we noticed that rapamycin specifically increased TCF-1 expression and alleviated senescence in CD8⁺ T cells of APDS2 patients.

Rapamycin inhibits mTORC1 and mTORC2 activation and controls CD8⁺ memory T-cell differentiation in a Foxo1-dependent manner. In addition, prolonged rapamycin treatment could inhibit mTORC2 assembly and AKT/PKB signalling [12, 35, 36]. In rapamycin-treated APDS2 patients, the proportions of naïve T cells or CD8⁺ TCM cells were increased, and the CD8⁺ TEM and/or TEMRA were reduced. Decreased AKT473 phosphorylation and increased TCF-1 were also observed. These data indicate that rapamycin alleviated the exacerbated PI3K-pAKT/mTOR-Foxo1 pathway in the presence of PIK3R1 mutations, corrected T-cell differentiation in PIK3R1 patients, and maintained immune responses against recurrent chronic infections. Meanwhile, we observed similar effects on T cells in APDS2 patients treated with a PI3K α/δ inhibitor in vitro compared with rapamycin treatment.

Our study also demonstrated that mTOR inhibition could specifically alleviate CD8⁺ T-cell senescence in APDS2 patients. Furthermore, we found that the expression of the T-cell senescence marker CD57 was lower in naïve CD8⁺ and CD8⁺ TCM cells but higher in CD8⁺ TEM and CD8⁺ TEMRA cells. Meanwhile, rapamycin therapy could increase naïve T cells or CD8⁺ TCM cells and reduce CD8⁺ TEM or TEMRA in APDS2 patients. We speculate that rapamycin alleviates CD8⁺ T-cell senescence by decreasing the TEM and TEMRA proportions. TCF-1 is verified to participate in T-cell senescence. We also found that TCF-1 expression was closely correlated with CD57 expression before and after rapamycin therapy. These results indicate that TCF-1 may mediate the process of rapamycin assuaging CD8⁺ T-cell senescence. Previous reports confirmed that rapamycin treatment could rectify CD4⁺ and CD8⁺ T-cell senescence in APDS1 patients [10]. Whether TCF-1 was upregulated in CD4⁺ T cells of APDS1 patients treated with rapamycin is unclear. The underlying mechanism of different CD4⁺ T-cell senescence patterns between APDS1 and APDS2 patients requires further investigation.

Only two APDS2 patients receiving rapamycin therapy were recruited in our study, but we applied multiple in vitro and ex vivo investigations to confirm rapamycin treatment efficacy in APDS2 patients. APDS2 patients benefited from rapamycin therapy with a partially rescued immune system, which supports its clinical application in APDS2 patients. We are following the patients currently undergoing treatment and recruiting more APDS2 patients to explore the uncovered molecular mechanisms.

Ethics approval and consent to participate

The study was approved by the Ethics Committee of the Children’s Hospital of Fudan University. The patients and their parents provided written informed consent for enrollment in this study. Trial registration ChiCTR2100044149. All methods were carried out in accordance with ethics guidelines and regulations of Ethics Committee of the Children’s Hospital of Fudan University.

Consent for publication

Consent for Publication-Not Applicable. Data and information are available and there is no dispute of interest.

Conflicts of Interest

The authors declare that they have no conflict of Interest.

Funding

This study was supported by the National Key Research and Development Program of China (2021YFC2701802), Leader of Health Science in Shanghai (2022XD024), Peak Climbing Program of Children’s Hospital of Fudan University (EK112520180202) and the National Natural Science Foundation of China (82071868).

Authors’ Contributions

L.H, designed and performed the research. L.L and Q.Z, H.B, W.W and B.S analyzed data. W.Y, X.H, Y.Z, H.Y and J.H provided clinical data and advice. L.H and L.L wrote the paper. X.W, Y.W, W.L and J.S provided advice on the imaging and paper. J.S finally approve the version to be submitted.

Acknowledgments

Many thanks to the patients and their parents.

Data Availability

All data generated or analyzed during this study are included in this article and its supplementary files. The data used or analyzed in this study are available from the corresponding author on reasonable request

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Table 1 to 2 are available in the Supplementary Files section.

No competing interests reported.

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mTOR inhibition alleviates CD8+ T-cell senescence in activated phosphoinositide 3-kinase δ syndrome 2 patients

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Abstract

Figures

Introduction

Materials And Methods

Patients and Clinical Manifestation

Evaluation of Immunological Function

Whole-exome sequencing and genetic analysis

Cell culture and stimulation

Western blot

Quantitative PCR

Statistical Analysis

Results

T-cell activation and the expression of T-cell exhaustion markers are not changed in APDS2 patients

Efficacy of Treatment

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

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