Study On The Characteristics Of Peripheral Blood Immune Map And Its Clinical Correlation In Children With Autism Spectrum Disorder

ObjectiveTo explore the characteristics of the peripheral blood immune map of children with autistic spectrum disorder（ASD）and their clinical associations.MethodsThe newly diagnosed children with ASD who visited the Children’s Autism Intervention Center of Guangzhou Women and Children Medical Center from 2021 to 2022 were prospectively selected as the ASD group,meeting the following inclusion criteria:Diagnostic and Statistical Manual of Mental Disorders-fifth edition（DSM-5）,Autism Diagnostic Interview Revised（ADI-R）,Autism Diagnostic Observation Schedule（ADOS）,and age between 1 and 6 years old.Typically developing children（TD）matched with the age and sex in the ASD group were included as the control group.Flow cytometry was used to determine the levels of various immune cells in peripheral blood of all enrolled children,and routine blood tests were also completed.The differences of blood routine,immune cell level and correlation among immune cells were compared between ASD group and control group.In addition,the children in the ASD group completed a rating of relevant cognitive function within one week after blood drawing:Developmental Quotient（DQ）[Gesell Developmental Diagnosis Scale（GDDS）]or Intelligence Quotient（IQ）[Wechsler Preschool and Primary Intelligence Scale IV（WPPSI-IV）/Wechsler Childhood Intelligence Scale IV（WISC-IV）]and severity of autism[Childhood Autism Rating Scale（CARS）];In addition,children in ASD group were divided into high function group（≧70 scores）and low function group（<70 scores）according to the score of DQ/IQ,and the differences of immune cells between the high function group and the low function group were compared.ADI-R and ADOS were also used to quantify the severity of key symptoms in children with ASD respectively,including:abnormal social interaction,abnormal communication,stereotypical behavior,impaired play ability,and signs of developmental abnormalities at or before 36 months.The correlation between immune cells and cognitive function,severity of autism（CARS）and severity of key symptoms（ADI-R and ADOS）was analyzed in ASD group.Results44 children with ASD and 27 children in the control group were enrolled in the study.（1）Blood routine test showed that the absolute lymphocyte count（p=0.049）and monocyte count（p<0.001）of children in the ASD group were higher than those in the control group.（2）Flow cytometry was used to measure 34 kinds of immune cells.Among the CD4+T cell subsets in children with ASD,the percentages of naive（p<0.001）T cells,central memory T cells（CM）（p<0.001）,and effector memory T cells 1（EM1）（p<0.001）were all significantly lower than those in the control group.Effector memory T cells 2（p=0.009）and terminal differentiation effector memory T cells（EMRA）were significantly increased（p=0.019）,and the decrease of CD4+EM1 cells was associated with the increase of stereotyped behavior in children with ASD（r=-0.327,p<0.05）.Increased CD4+EM2 cells were associated with increased communication disorders in children with ASD（r=0.372,p<0.05）.In the CD8+T cell subsets of children with ASD,the percentages of naive（p<0.001）T cells,central memory T cells（CM）（p<0.001）,and effector memory T cells 1（EM1）（p<0.001）were all significantly lower than those in the control group.Effector memory T cells 3（p=0.029）and terminal differentiation effector memory T cells（EMRA）were significantly increased（p=0.003）,and the increase of CD8+EM1 cells was associated with the decrease of stereotyped behavior in ASD group（r=-0.322,p<0.05）.The percentage of regulatory T cells（p<0.001）and the absolute count（p=0.003）were significantly lower than those in the control group.The absolute B-cell count in children with ASD was significantly higher than that in the control group（p=0.002）,and in ASD children,increased B cells were associated with reduced communication impairments（r=-0.361,p<0.05）.The percentage of plasmablast（PB）cells（p=0.003）and the absolute count of PB cells（p=0.018）were significantly lower than those in the control group,and the percentage of PB cells（p=0.027）and the absolute count of PB cells（p=0.0264）were significantly lower in children with ASD in the low function group.The percentage of natural killer（NK）cells（p=0.003）and absolute count（p<0.001）were significantly higher in ASD group than in control group.The percentage of CD56^dimNK cells（p=0.009）and absolute count（p<0.001）were also significantly higher in ASD group.Increased number of CD56^dimNK cells was significantly associated with decreased severity of autism（r=-0.420,p<0.05）and decreased abnormal social interaction in ASD group（r=-0.385,p<0.05）.The increased number of CD56^brightNK cells in ASD group was significantly associated with increased severity of autism（r=0.387,p<0.05）,increased stereotyped behavior（r=0.447,p<0.01）,decreased cognitive ability（r=-0.334,p<0.05）and increased signs of developmental abnormalities at or before 36 months（r=0.333,p<0.05）.The percentage of natural killer T（NKT）cells in the ASD group was significantly lower than that in the control group（p=0.011）,and the decrease of NKT cells was significantly associated with increased signs of developmental abnormalities at or before 36 months of age（r=-0.346,p<0.05）.There were no significant differences in the percentage and absolute count of intrinsic lymphoid cells（ILCs）between the ASD and the control group,but in the ASD group,increased intrinsic lymphoid cells were significantly associated with reduced stereotyped behavior（r=-0.381,p<0.05）and reduced communication impairments（r=-0.355,p<0.05）respectively.The absolute count of classical monocytes（p<0.001）and non-classical monocytes（p=0.003）in ASD group was significantly higher than that in control group,and the increased level of classical monocytes was associated with increased severity of autism（r=0.332,p<0.05）,abnormal social interaction（r=0.384,p<0.05）and decreased cognitive ability（r=-0.381,p<0.05）,while increased number of non-classical monocytes was significantly associated with increased cognitive ability（r=0.358,p<0.05）.There was no significant difference in the percentage of dendritic cells between the ASD children and the control group,but in the ASD children,an increased number of dendritic cells was significantly associated with fewer signs of developmental abnormalities at or before 36months（r=-0.477,p<0.01）.The percentage of neutrophil in children with ASD was significantly higher than that in the control group（p=0.006）,and the increased neutrophil ratio was associated with increased play impairment in children with ASD（r=0.329,p<0.05）.The percentages of myeloid-derived suppressor cells（MDSCs）（p<0.001）and their subsets of polymorphonuclear myeloid-derived suppressor cells（PMN-MDSCs）（p=0.004）were significantly increased in ASD group,while there was no significant difference between mononuclear myeloid-derived suppressor cells（M-MDSC）and control group.However,in ASD group,increased M-MDSCs cell levels were significantly associated with increased social interaction impairment in children with ASD（r=0.358,p<0.05）.ConclusionsThe children in the ASD group had an overall imbalance of immune cells,characterized by a decreased percentage of lymphocytes and an increased percentage of myeloid cells.In the CD4+T and CD8+T cell subsets of ASD children,the number of cells in their early state（na?ve-CM-EM1cells）decreased,while the number of advanced stage cells（EM2-EM3-EMRA cells）increased,indicating that young ASD children were inclined to the late differentiated and mature T lymphocyte subsets.The number of Treg cells decreased,suggesting that the immune tolerance of children with ASD may be damaged.The absolute count of B cells increased,while the level of PB cells decreased.Moreover,B cells were associated with the communication ability of the children in the ASD group,while PB cells were associated with the cognitive impairment of the children in the ASD group,suggesting the humoral immunity of ASD children was abnormal,which was related to the symptoms of ASD children.The level of NK cells increased,and the effects of different NK cell subsets on children with ASD varied:CD56^dimNK cells may help improve symptoms in children with ASD,while CD56^brightNK cells may aggravate symptoms.NKT cells and dendritic cells may be related to the signs of developmental abnormalities at or before 36 months of age in ASD children.Innate lymphoid cells in children with ASD may help alleviate stereotyped behavior and communication disorders.Elevated levels of neutrophils may affect play ability in children with ASD.The levels of MDSCs and their subgroup PMN-MDSCs were elevated in children with ASDs,while M-MDSC may affect social interaction in children with ASD.