Lymphocyte Disturbance in The [Asp521Asn] ZAP70 Mutation and An Overview of All Phenotype/Genotype

Background. Activated Zeta (ζ)-chain-associated protein kinase 70 (ZAP70) phosphorylates the TCRαβ:CD3:ζ-complex to diversify the initial TCR signal. Patients with the ZAP70 mutations could present with the phenotype of immune dysregulation. Methods. We identied the rst Taiwanese boy with the ZAP70 mutation, assessed downstream signaling, investigated lymphocyte disturbance and analyzed all available phenotype/genotype for optimal treatment. Results. With the [Asp521Asn]ZAP70 mutation, the infant who had hypogammaglobulinemia, eosinophilia, isolated CD8 lymphopenia, and impaired lymphocyte proliferation experienced recurrent pneumonia, refractory diarrhea, transient hematuria and autoimmune hepatitis. Decreased CD3/CD28 downstream phosphorylation of AKT, ZAP70 and Ca2+ inux related to the Th2-skewing T follicular helper cells, expanded transitional B, increased CD21-low B cells and lower Treg cells. To speculate clinical course for effective treatment, we overviewed 45 available published patients. Except for two asymptomatic post-transplant infants, common pathogens were oral candida (n=9), PJP (n=7), CMV (n=5), varicella (n=4), parainuenza (n=3) and disseminated BCG (n=3). Their phenotypes of immune dysregulation mainly included IBD-like diarrhea (n=12), dermatitis (n=7), hepatosplenomegaly (n=3) and nephritic syndrome (n=3). Founder-effect or hot-spot mutations (32/90 alleles) involving the kinase domain clustered on intron 12 [1624-11G>A] (in 24) and exon 12[1520C>T] (in 8). Eleven died of sepsis (n=3), CMV pneumonitis (n=2), viral encephalitis, disseminated measles-vaccine infection, ARDS, HLH-like, EBV-lymphoproliferative disorder, and lymphoma each. Whatever developing opportunistic infections (p=0.2240), immune dysregulation (p=0.5268), or failure to thrive (p=0.5215), those who receiving HSCT had signicantly better prognosis (p=0.0003). Conclusions. The Asp521Asn-ZAP70 hinders TCR-CD3 downstream phosphorylation and disturbs lymphocyte subgroup “proles” leading to autoimmune/autoinlfmmation. The lymphocyte disturbance should be validated in large-scale studies. Receiving HSCT is a signicantly better survival factor, rather than mutation types, opportunistic infections, or immune dysregulation.


Introduction
Zeta (ζ)-chain-associated protein kinase 70 (ZAP70) belonging to a member of the spleen tyrosine kinase (SYK) family is a non-receptor protein tyrosine kinase mainly expressed in intracellular T cells. 1 Upon antigen recognition, T cell antigen receptors (TCR) activate lymphocyte-speci c protein tyrosine kinases (LCK) that in turn phosphorylate ZAP70 and immunoreceptor tyrosine-based activation motifs (ITAM) of the TCRαβ:CD3:ζcomplex. 2 The activated ZAP70 is recruited into phosphorylated ITAM of the TCRαβ:CD3:ζ-complex to modulate core adaptor proteins and act as a T-cell master kinase by amplifying and diversifying the initial TCR signal. 3 Different from the Zap70−/− murine model which has been shown to block both emigrant CD4 and CD8 T cells from the thymus, 4 ZAP70-de cient human CD4 T cells can maintain su cient quantitative emigration via compensation with a higher SYK expression through downstream residual TCR signaling, although this is not seen with CD8 cells. 5 Such selective CD8 lymphopenia was rst reported by Roifman et al in 1989, 6 and the harmful genetic defect of Zap70 mutations was identi ed in 1994. 7 With CD8 lymphopenia, a poor response to mitogens and increased susceptibility to opportunistic infections, such patients are diagnosed with combined immunode ciency disease (CID) and can also exhibit immune dysregulation mimicking Omenn syndrome. 8,9 Notably, some patients only present with isolated autoimmune disorders without infection and are nally found to have hypomorphic ZAP70 mutations by whole exome sequencing (WES). 10,76 Herein, we present a 2-year-old boy with recurrent wheezing, chronic diarrhea, gross hematuria and elevated liver enzymes who was referred to our PICAR (Primary Immunode ciency Care And Research) Institute where we identi ed a homozygous ZAP70 mutation for the rst time of Taiwan aboriginal ethnicity. To clarify what phenotype and adequate intervention bene cial to survival prognosis, we investigated the function of this ZAP70 substitute and survival analysis of all available patients from a PubMed search 7,9,10,21,76 Materials And Methods

Ethics Statement
All human samples were obtained under protocols approved by the Institutional Review Board at Chang Gung Memorial Hospital and met the Institutional Review Board standards (IRB-201901385A3 and IRB-202001665A3) for ethical conduct of research with human subjects in accordance with the Declaration of Helsinki. All experimental protocols in this study, and the patient's parents or guardians provided informed consent. All methods were performed in accordance with the relevant guidelines and standard regulations.

Basic immunologic function assessments
To induce lymphocyte proliferation, peripheral blood mononuclear cells (PBMCs; 10 5 /well) were incubated with different concentrations of phytohemagglutinin (PHA), pokeweed (PWM), ConA, and CD3/CD28 for 3 days, or the Candida antigen and Bacillus Calmette-Guérin vaccine for 7 days, followed by incubation with [ 3 H]-thymidine overnight. 15,16 Carboxy uo rescein diacetate succinimidyl ester (CFSE) was added to stimulate PHA, and the proliferation index of mitotic cells was analyzed using FlowJo software 7.2.5 (TreeStar®). The lymphocyte subsets of T follicular helper cells, Th17 cells and Treg cells, transitional B cells and CD21-low B cells were assessed to correlate with his autoimmune phenotype.
Candidate gene approach CD8 lymphopenia and impaired lymphocyte proliferation can occur in patients with MHC class I de ciency. 17 Because of a normal MHC class I expression (Supplemental Fig. 1), the candidate ZAP70 gene responsible for CID or leaky SCID phenotype was sequenced. Total RNA was extracted from the PBMCs using TRIzol (Life Tech., Carlsbad, CA), followed by RT-PCR. The two pairs of primer sequences of the ZAP70 gene were based on human genome sequences NM_001079 and designed to cover the whole coding region: ZAP70-102F: ATT CAG AAC CGG CTC TCC AT, ZAP70-1305R: CAC GTC GAT CTG CTT CTT GC; ZAP-70 1091F: AGC CAG CAC GCA TAA CGT CC, ZAP70-2176R: CAG CTG TGT GTG GAG ACA AC. At the same time, the genomic DNA in exon 12 (forward: TGA ACA CAT GGT CAC CTG; backward: TGG TGT GTT GGA GAG CTG) was con rmed based on NT_005403.18.
Flowcytometry for candidate protein expressions and intracellular Ca2+ ux downstream CD3/CD28 signaling For phospho-ow, PBMCs were stimulated with anti-CD3 (10 μg/ml) and anti-CD28 (10 μg/mL) for an adequate period, then xed (BD Biosciences®, Cyto x), stained for surface antigens of CD4 or CD8, permeabilized (BD Biosciences®, Perm buffer III) and stained with anti-ZAP70 antibodies (eBiosciences), phospho-ZAP70 (BD Biosciences®, BD Phos ow), and phospho-AKT (BD Biosciences®, BD Phos ow). 18 l intracellular Ca2+ ; d by dTo study Ca2+ ux, CD4 T cells were further enriched using a human CD4 T cell negative selection kit (Biolegend, San Diego, CA). The cells were then washed twice with loading buffer HBSS with 25 mM HEPES (ThermoFisher, Eugene, OR). Fluo-4-AM (ThermoFisher, Eugene, OR) was loaded at a nal concentration of 5 mM and the samples were incubated for 30 min in the dark at 37°C. After washing twice with loading buffer, the cells were either untreated or stimulated with anti-CD3 (Biolegend, San Diego, CA) plus anti-CD28 (Biolegend, San Diego, CA) in the presence of rabbit anti-mouse IgG (Jackson, West Grove, PA), each at 10 mg/mL. The uorescent Ca 2+ signal was detected using ow cytometry (BD, FACSCanto II). The ow data were then analyzed using FlowJo software version 7.6.1. l intracellular Ca2+ ; d by d18, 19 Whole genome sequencing (WGS) Because bi-genetic mutations have been reported in rare disorders, WGS was performed to explore other genetic defects responsible for his autoimmune phenotype, especially those not yet reported for hepatitis. His DNA was extracted from whole blood and WGS was performed on an Illumina NoveSeq 6000 system (Illumina Inc., San Diego, CA) using a PCR-free protocol. The data were processed using CLC Genomic Workbench 11 software (Qiagen). Brie y, paired-end reads were removed from low quality bases (Q < 30, Phred scale) and aligned to a human reference genome (GRCh37/hg19). The mapping parameters were set to default except for mapping length which was set to 0.9 and mapping similarity which was also set to 0.9, and the mapped reference genome was only read once. Variants were ltered based on sequencing coverage (removed if the depth was < 10) and by comparisons with a common variant database (dbSNP version 150). [20][21][22] Clinical features and survival prognosis of all patients with ZAP70 mutations To understand the whole disease course, we searched for all reported patients with ZAP70 mutations in a Medline search. Survival curves were estimated using Kaplan-Meier analysis to compare their prognoses based on opportunistic infections (Oi), failure to thrive (Ft), immune dysregulation (Id) including autoimmune disorders, chronic or IBD-like diarrhea, skin lesions, lymphoproliferative disorders and lymphoma, and whether or not they received HSCT. The rst follow-up day was de ned as the initial presentation date of the patients with ZAP70 mutations. The last follow-up day and duration were de ned according to those reported in the studies. All analyses were performed using GraphPad Prism software, and a p value of <0.05 was de ned as being statistically signi cant.

Case demonstration
This 1-year 4-month-old boy was born to aboriginal parents via cesarean section without any adverse events. He was hospitalized for pneumococcus pneumonia three times from the age of 6 months, and experienced frequent coughing and wheezing thereafter. Respiratory syncytial virus, enterovirus and adenovirus infections caused recurrent wheezing. Neither infective cultures nor reverse transcription polymerase chain reaction ampli cation (RT-PCR) in bronchial lavage revealed any evidence of pneumocystis jirovecii pneumonia (PJP), aspergillosis, fungus and mycobacterium. Because of his failure to thrive since 2 months of age, he only received hepatitis B, diphtheria-pertussis-tetanus and Hib vaccines and avoided any live attenuated vaccines in spite of his normal TREC value (252 copies) on his neonatal Guthrie card. When he was 13 months old, Pseudomonas aeruginosa caused severe colitis, sepsis and hypovolemic shock that met the diagnosis criteria of Shanghai fever. 11 Ceftazidime, amikin, cipro oxacin and hydration rescued his life-threatening infection. A hematologic evaluation showed mild neutropenia (1034/mm 3 ), normal lymphocyte count (3290/mm 3 ), eosinophilia (12%), anemia (Hb 7.6 g/dL) and thrombocytosis (platelets 760 k/UL). Hypogammaglobulinemia (IgG 74.8 mg/dL, IgM 24.6 mg/dL, but higher IgE 1430 IU/mL), a profound decrease in CD8 lymphocytes to 5% (CD4 46%, NK 16%, and CD19 24%) and obviously impaired lymphocyte proliferation (Supplemental Table 1) were compatible with combined B and T cell immunode ciency. Regular immunoglobulin infusions (0.8g/kg per month) and prophylactics for bronchiectasis (25 mg/kg/day Augmentin), PJP (5mg/kg/day Trimethoprim-Sulfamethoxazole) and fungal infections (5 mg/kg/day uconazole) were given until successful HSCT engraftment.
During the waiting period, his ALT and AST levels increased to over 3000, but levels of ALP, γ-GT, and bilirubin remained normal. Any kinds of antibiotics with the potential for hepatic toxicity were discontinued, and RT-PCR for hepatitis viral load excluded EBV, CMV, HSV, hepatitis A, B, C and E. A liver biopsy did not favor medication-related or viral hepatitis. Liver enzyme levels often returned to normal after monthly immunoglobulin infusion. Mildly elevated anti-smooth muscle antibodies (1:40) supported autoimmune hepatitis, but negative for anti-nuclear (ANA) and anti-liver-kidney-microsomal (LKM) antibodies. Gross hematuria once occurred, but urine cultures and RT-PCR ampli cation for adenovirus and BK virus were negative. One month later, he had chronic diarrhea and acute exacerbation leading to abdominal distension, ileus, and moderate ascites. Under the impression of sepsis from colonic pathogens (especially previous pseudomonas aeruginosa), hypovolemic shock and severe hematochezia ( Fig. 1A) with an Hb level down to 4 g/dL, aggressive antibiotic treatment was given over 2 weeks with a continuous blood transfusion for 7 days. Abdominal CT, angiography and Meckel's scan did not reveal any signi cant ndings for bleeders. Colonoscopy showed small white nodules of 5 x 5 mm in size protruding into the intestinal lumen (Fig. 1B). Mucosa nodular lymphocyte aggregation and submucosa oozing-like edema have been reported to be a possible prodrome of in ammatory bowel diseases ( Fig. 1C and 1D) that resemble intestinal lymphoproliferation in patients with activated phosphoinositide 3-kinase δ syndrome (APDS) 12 and common variable immunode ciency (CVID) 13,14 With regular immunoglobulin infusion and adequate prophylactics, n the near future, ing donor is ready to hepatitis, hematuria and chronic . kinds of antibiotics low-dose prednisolone (0.5mg/kg/day) was added for his phenotype of immune dysregulation including hepatitis, hematuria and chronic diarrhea. Fortunately, he received an HSCT from an HLA-matching sibling donor when he was 1 year and 11 months of age. At present, he was 3 years 6 months and free of IVIG infusion and GvHD medication.

Immune functional assessment
Obvious decreases in mitogen and antigen proliferation were shown by [ 3 H]-thymidine incorporation (Supplemental Table 1). Compared with the parallel control, PHA stimulation and lymphocyte proliferation of less 10% of normal was de ned as being "absent" function and was considered to be an indication for HSCT. Such profound impairment of lymphocyte proliferation was also demonstrated by a CFSE evaluation of the proliferation index ( Fig. 2A).

Genetic analysis
Using a candidate gene approach, Sanger sequencing revealed that the 1561st nucleotide G was homozygously replaced by A in exon 12 leading to a missense mutation of Asp 521 Asn in the ZAP70 gene (Fig. 3). We further assessed the effect of 521Asn-ZAP70 on CD3/CD28 downstream signaling, and also utilized WGS to investigate whether a gene other than ZAP70, or even bi-genetic mutations 23 were related to his complex phenotype including uctuating higher levels of liver enzymes, gross hematuria and hematochezia. However, only the same mutation was consistently identi ed instead of revealing any other responsible novel gene (Supplemental Table 2). His parents and older sibling were all carriers.

ZAP70 expression and intracellular Ca2+ dynamic in ux downstream the CD3CD28 signaling
In an in-depth study of the mutant 521Asn-ZAP70 function, we investigated the expressions of phosphorylated translation factors of ZAP and AKT in downstream signaling. Compared to that in the control, the intracellular expression of 521Asn-ZAP70 obviously decreased in CD4 cells and CD8 cells without/with CD3CD28 stimulation for 10 min (Fig. 4). In parallel, the downstream signal expressions of phosphorylated-ZAP70 and -AKT were also decreased (Fig. 4). Thus, such alterations attenuated its binding capacity and signal pathway. Asp521-ZAP70 is an acidic residue which forms hydrogen bonds with H459 and R460 and stabilizes an activation loop, a crucial component of kinase function. 24,25 Changes in Asp521Asn can cause a conformational change in the activation loop and impact signaling cascades, compatible with the SIFT predicted scoring system (Score 0 meaning damage, http://sift.jcvi.org/) and PolyPhen2 (Score 1 also meaning damage, http://genetics.bwh.harvard.edu/pph2/index.shtml).
We then conducted calcium in ux assays to evaluate the downstream function of ZAP70 in T cell activation. As shown in Figure 5A, calcium in ux in the patient's T cells was nearly undetectable when stimulated. Compared to the patient (Fig. 5A), the results of calcium in ux in the carrier was evident (Fig. 5B), even though the in ux of maximum calcium concentration was not as high as in the normal control (Fig. 5C). To further compare the differences between these three subjects, kinetic plots of each calcium in ux were superimposed to reveal the functional de cit (Fig. 5D). After stimulation, the response time and ascending slope of the calcium in ux in the carrier were nearly the same as those in the control. However, the carrier did not reach the same level of calcium concentration as the control, and the descending slope was also signi cantly faster than that of the control.
Infections were the most common presentations of combined T and B immunode ciency. The identi ed pathogens over two eventful episodes included candida (9 episodes), PJP (n=7), CMV (n=5), varicella (n=4), parain uenza (n=3) and BCG vaccine (n=3) that was re ected by their CD8 lymphopenia as they were mainly unable to resist viral pathogens rather than bacteria (Table 1). To compare survival based on their phenotypes, we de ned the phenotype of immune dysregulation encompassing autoimmune disorders as the patients who developed in ammatory bowel disease (IBD)-like chronic diarrhea, eczematous dermatitis, hepatosplenomegaly, lymphoadenopathy, hemolytic anemia, nephritic syndrome or brain infarction and lymphoma ( Table 2).
In complex-phenotype comparisons (in Table 3 and Supplemental Table 4), we classi ed the major phenotypes of these patients as "opportunistic infection (Oi)", "immune dysregulation encompassing autoimmune disorders (Id)" and "failure to thrive (Ft)". The onset age and published age of the patients with the Oi-Id-Ft phenotype were signi cantly older than those with the Oi-Id phenotype (p=0.0399; p= 0.0143) because failure to thrive (Ft) often appeared after 3 months old, but not signi cantly in each-other mutual comparisons ( Table 3). The oldest patient was 33 years old at time of writing and presented with the Oi-Id-Ft phenotype. 46 In the strict sense, no relationship between genotype and phenotype existed as the recent review. 77 For Kaplan-Meier survival analysis, those receiving HSCT showed an obviously and signi cantly higher survival (p=0.0003) than without, but not in the other comparisons of opportunistic infections (p=0.2240), immune dysregulation (p=0.5268), and failure to thrive (p=0.5215). (Fig. 6).

Discussion
Patients with ZAP70 mutations have hypogammaglobulinemia, CD8 lymphopenia and impaired lymphocyte proliferation, therefore suffered from recurrent infections. As well as approximate 60% (26/43) developed the phenotype of immune dysregulation, our patient with the [Asp521Asn] ZAP70 mutation experienced chronic diarrhea, non-virus autoimmune hepatitis (with positive anti-smooth antibodies), hematuria and eosinophilia with a relatively high level of IgE, mimicking Omenn syndrome. Omenn syndrome is a T-B-NK+ SCID subgroup rst identi ed by hypomorphic RAG mutations interrupting T and B cell receptor recombination and mainly orchestrating oligoclonal T cells to in ltrate multiple organs of intestine, liver and kidney. 53 Based on Freiburg 13 and EUROclass 14 classi cation, markedly increased proportion of CD21-low B cells (more than 10%), reduced switched memory B cells (less than 2%), and expanded transitional B cells (IgM++CD38++ more than 9%) trend to develop autoimmune cytopenia, lymphadenopathy, splenomegaly, and granulomatous disease. 54 Furthermore, an imbalance between Treg and Th17cells (the ratio of Th17/Treg) is a feature of autoimmunity in systemic lupus erythematosus, 57 vasculitis (such as Kawasaki disease) 58 and exacerbated asthma. [59][60][61] Taken together, expanded transitional B cells, augmented CD21-low B cells, Tfh shift to Th2 deviation and higher Th17/Treg ratio due to lower Treg cell contribute to immune dysregulation of autoimmunity/autoin ammation as well as reduced switched memory B cells and T EM cells 55,56 that also increase susceptibility to infections.
Immune imbalance could affect peripheral and central tolerance, trigger autoimmune/autoin ammation process and possibly trend to malignancy transformation. 8,62 Thus, in a broad sense, primary immune "dysfunction or dysregulation" (PID) is a more appropriate term than "de ciency" since that the four aspects of autoimmunity, autoin ammation, malignancy and refractory allergy have diversi ed the PID phenotype. 63 Patients with mutations of the DCLRE1C, IL7RA, RMRP, ADA, LIG4, IL2RG, and AK2 genes and those responsible for DiGeorge syndrome are observed to resemble Omenn syndrome and listed in the 2014 PID classi cation. 64,65 Such "Omenn" syndrome combining T cell de ciency and immune dysregulation has been omitted in the 2017 classi cation because advanced WGS/WES 66 reveal novel mutations of the LRBA, 67 CTLA4, 68,69 , PPIK3CD 70 and PIK3R1 71,72 genes in previously termed "Omenn" syndrome. Nevertheless, the ZAP70 gene could be still a potential candidate gene for Omenn-like syndrome after validation by a large cohort study.
Distinct from the recent review summarized by Shari nedjad et al, 77 our study emphasized more on optimal treatment. In practice, all of the patients with ZAP70 mutations received regular immunoglobulin infusion and prophylactics for opportunistic infections. 73 Immunosuppressants such as prednisolone and thalidomide were not uncommon for patients with IBD-like chronic diarrhea and recurrent intestinal stenosis. 48 In an extreme case, nephrectomy was performed in one Turkish male patient who suffered from congenital nephritic syndrome, persistent hypertension and silent brain infarction. 47 Receiving mini-or myeloablative conditioning for partialmatched donors or non-conditioning for sibling-matched donors, 19 patients were still alive despite of receiving 26 HSCTs due to graft failure in some. The donors included 15 from bone marrow, 8 from peripheral stem cells, and 3 from cord blood. Like our toddler boy, three receiving sibling-matched donors without conditioning are still well 20 years post-transplant. 28 Unexpectedly, one Turkish male infant developed cholangitic hepatitis and portal brosis, possibly ascribed to persistent hepatitis and/or an adverse event of myeloablative conditioning (melphalan 140 mg/m2 and udarabine 160 mg/m2), which was resolved by a liver transplantation 2 years post-transplant. 43 Notably, one male 33-year-old patient with a homozygous c1272 C>T mutation had a minor product of the wild form with a silent mutation p.G355G, but more truncation products from creating a donor splice (within exon 10) p.G355G fs10X losing the kinase domain. An antisense morpholino oligonucleotide (AMO) was designed to recognize this segment (20 nucleotides) and to prevent the aberrant splicing. 46 In vitro, AMO treatment restored CD3/CD28 signaling by predominantly deviating to wild-type transcription. AMO driving to intact transcription has been utilized to treat inborn errors of muscle and metabolism. 74 Newborn TREC screening using the Gathrie card may prompt diagnosis of patients with ZAP70 mutations. A Canadian Mennonite patient with the splicing mutation IVS 12 (-11) G>A had an obviously decreased TREC value when he clinically deteriorated at around 8 months of age. 51 Because the TREC value correlates to the number of naïve CD4CD45RA T cells, 75 the normal number of CD3+ and CD4+CD45RA+ lymphocytes could cover the CD8+ lymphopenia and showed a normal TREC value in the newborn Guthrie card as our patient.
In contrast to the Iranian boy with the same missense mutation who suffered form autoimmune hemolytic anemia and immune thrombocytopenia anemia (in supplemental Table 3), 52  signaling to open an alternative therapeutic avenue. Second, the patients receiving HSCT had signi cantly better survivor despite the rst graft failure and unrecognized in newborn TREC screening. Multidisciplinary care team with a certain level of successful HSCT should save all patients if suitable donors available. Third, bi-genetic defects have been identi ed in a blended syndrome with ZAP70 and RNF168 mutations. 23 The genetic candidate one-by-one approach for complex phenotypes may not reveal the second genetic defect. More advanced sequencing techniques and analysis software may explore signi cant genetic variance that is not recognized at present.
In conclusion, the [Asp521Asn] ZAP70 missense mutation decreased downstream TCR-CD3 phosphorylation of Akt, ZAP70 and Ca2+ in ux. The correspondent cellular phenotype of immune imbalance included Th2-skewing T follicular helper cells, lower Trg cell (higher Th17/Treg ratio), increased transitional B and CD21-low B cells. Those who presented with CD8-lymphopenia related CID phenotypes caused by the ZAP70 mutations and received suitable HSCT had signi cantly higher survival even though they suffered from graft failure and severe infection. Two patients diagnosed prenatally were asymptomatic and then received stem cell transplantation.    23.2% vs. 42.0%) (F). CM means T central memory cells and EMRA means terminally differentiated effector memory cells re-expressing CD45RA, which is a marker usually found on naive T cells.

Figure 3
In exon 12 of the ZAP70 gene, the 1561th nucleotide G was replaced by A, causing amino acid Asp 521 to be substituted by Asn. His parents and sibling were all carriers.

Figure 4
Under TCR activation stimulated by CD3CD28 for 10, 30 and 60 mins, the most obvious augmentation of downstream phosphorylation was at 10 mins. Therefore, we decided to detect phosphorylated AKT and ZAP70 at 10 mins. Compared to that in the healthy control who had augmentation after CD3CD28 stimulation, the patient was observed to have obviously decreased ZAP70 expression, downstream phosphorylation of ZAP70 and AKT (all <3%) in CD4 and CD8 gating. Black lines meant ZAP70, phospho-ZAP70 and phosphor-AKT antibodies by FlowJo histogram. Gray-ground area meant respective isotypes. Duplicated experiments were done before steroids intervention.