The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 helper T (T) cells and CD8 cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T1 and T2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent T1 or T2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T1/T2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T1/T2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.
Antigens ; metabolism ; CTLA-4 Antigen ; metabolism ; Cell Differentiation ; drug effects ; Cell Line ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Lymphocyte Activation ; drug effects ; immunology ; Lymphocyte Subsets ; drug effects ; metabolism ; Phenotype ; Proteomics ; Receptors, Chimeric Antigen ; metabolism ; Single-Cell Analysis ; methods ; T-Lymphocytes, Regulatory ; drug effects ; metabolism ; Th1 Cells ; cytology ; drug effects ; Th2 Cells ; cytology ; drug effects ; Transcription, Genetic ; drug effects ; Up-Regulation ; drug effects

Asthma is a chronic disease of airway inflammation due to excessive T helper cell type 2 (Th2) response. Present treatment based on inhalation of synthetic glucocorticoids can only control Th2-driven chronic eosinophilic inflammation, but cannot change the immune tolerance of the body to external allergens. Regulatory T cells (Tregs) are the main negative regulatory cells of the immune response. Tregs play a great role in regulating allergic, autoimmune, graft-versus-host responses, and other immune responses. In this review, we will discuss the classification and biological characteristics, the established immunomodulatory mechanisms, and the characteristics of induced differentiation of Tregs. We will also discuss the progress of Tregs in the field of asthma. We believe that further studies on the regulatory mechanisms of Tregs will provide better treatments and control strategies for asthma.
Antigens, CD/analysis* ; Apyrase/analysis* ; Asthma/immunology* ; Cell Differentiation ; Cytokines/metabolism* ; Humans ; Lymphocyte Transfusion ; T-Lymphocytes, Regulatory/immunology*

OBJECTIVETo study the clinical significance of CD163 in the diagnosis and the evaluation of severity and prognosis of childhood hemophagocytic lymphohistiocytosis (HLH).

METHODSNinety-four children were classified into Epstein-Barr virus (EBV)-positive (n=55) and EBV-negative groups (n=39; control group). The EBV-positive group was subgrouped into infectious mononucleosis (IM; n=47) and HLH (n=8). Serum levels of soluble CD163 were measured using ELISA. Expression of CD163 on mononuclear cells was detected by flow cytometry.

RESULTSThe serum levels of soluble CD163 were>10 000 ng/mL in all eight HLH patients (>30 000 ng/mL in 3 cases). The mean serum levels of soluble CD163 in the HLH group were significantly higher than in the control and IM groups (P<0.05). The serum levels of soluble CD163 in EBV-positive children were positively correlated with EBV-DNA copies and serum levels of ferritin and LDH, but were negatively correlated with white blood cell count, neutrophil count, hemoglobin and platelet count. The follow-up after treatment for three HLH patients showed that serum levels of soluble CD163 were significantly reduced, but the soluble CD163 levels rebounded in one patient who was complicated by fungal pneumonia infection.

CONCLUSIONSThe levels of serum soluble CD163 may be related to the severity in children with HLH. The EBV-positive children with soluble CD163 levels >10 000 ng/mL should be considered the possibility of HLH.

Adolescent ; Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Child ; Child, Preschool ; Epstein-Barr Virus Infections ; immunology ; Female ; Humans ; Infant ; Male ; Receptors, Cell Surface ; analysis

OBJECTIVETo explore the clinicopathologic features, immunophenotype, differential diagnosis and gene mutation status of the Erdheim-Chester disease (ECD).

METHODSClinical and pathologic findings of 3 ECD cases were examined by gross, microscopic, immunohistochemical methods and BRAF V600E mutation. Related literatures were reviewed.

RESULTSTwo male patients and one female patient presented clinically with multiple skin nodules, bone pain and bony lesions by imaging study. Microscopically, the lesions were composed of spindle-shaped fibroblasts, foamy histiocytes and scattered Touton-type giant cells embedded in reactive fibrous tissue. Lymphocytes, plasma cells, and multinucleated giant cells were also found. Immunohistochemically, all histiocytes were positive for CD68, none of which expressed CD1a, although 2 cases focally expressed weak S-100 stain. In 2 cases,BRAF V600E mutation was detected.

CONCLUSIONSECD is a rare disease of xanthogranulomatous histiocytosis.Its diagnosis relies on pathological and immunohistochemical findings, but correlation with clinical information, especially radiographic findings should be performed.No effective treatment of the disease is currently available.

Antigens, CD ; analysis ; Antigens, CD1 ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Diagnosis, Differential ; Erdheim-Chester Disease ; genetics ; immunology ; pathology ; Female ; Humans ; Male ; Mutation ; S100 Proteins ; analysis ; Treatment Outcome

Toxoplasma gondii is an intracellular protozoan parasite that causes a Th1 cellular immunity. Our previous study showed that T. gondii lysate antigen (TLA) treatment in S180 tumor-bearing mice resulted in tumor reduction by suppressing CD31 expression, a marker of angiogenesis. In the present study, to investigate tumor suppressive effect of TLA under the absence of T lymphocytes, athymic nude mice were compared with euthymic mice in the anti-tumorigenic effect triggered by TLA in CT26 tumors. According to the results, intratumorally injected TLA reduced tumor growth and TIMP-1 level, a metastatic marker, in both euthymic and athymic mice. TLA treatment led to a sharp increase in IL-12 expression in serum cytokine profiling of athymic mice, and increased MyD88 signals in macrophages derived from the bone marrow, implying the activation of innate immunity. The selective induction of IL-12 by TLA treatment had an anti-tumorigenic effect.
Animals ; Antigens, Protozoan/*immunology ; Disease Models, Animal ; Immunity, Innate ; Immunotherapy/*methods ; Interleukin-12/*blood ; Macrophages/immunology ; Mice, Inbred BALB C ; Mice, Nude ; Myeloid Differentiation Factor 88/analysis ; Neoplasms/pathology/*therapy ; Toxoplasma/*immunology ; Treatment Outcome

OBJECTIVETo investigate the changes of serum soluble CD 163 (sCD 163) level, to assess the severity of critical illness and to evaluate the immune status of sepsis or severe sepsis in children.

METHODA prospective study was conducted. The sCD 163 was determined in 50 cases with sepsis or severe sepsis in pediatric intensive care unit (PICU) and 23 cases of age- and gender-matched healthy children were enrolled as control during the period from April 2010 to March 2011. Double-antibody sandwich ELISA was used for sCD 163 measurement. The relationship with sCD 163 level and disease severity score (pediatric critical illness score, PCIS; and pediatric risk of mortality III, PRISM III), lymphocyte subsets, C-reactive protein (CRP), tumor necrosis factor α (TNFα) were analyzed.

RESULTThe sCD 163 in sepsis/severe sepsis groups (171.04 ± 177.85) mg/L was significantly higher than that in control group (44.19 ± 86.48) mg/L (P < 0.01).sCD 163 in sepsis group [(105.32 ± 145.87) mg/L] was significantly lower than that of severe sepsis group [(233.32 ± 171.78) mg/L] (P < 0.05). sCD 163 level was significantly higher in lower PCIS score patients. (P < 0.01). The sCD 163 levels was higher in PRISM III ≥ 10 than the PRISM III < 10 group. The sCD 163 levels were higher in death group than the survival group. The sCD 163 was negatively correlated with CD4 +, CD4 +/CD8 + (R = -0.820, P < 0.05; R = -0.839, P < 0.01).

CONCLUSIONDetection of sCD 163 was helpful in predicting the severity of sepsis and severe sepsis, and sCD 163 may reflect the immune status of critically ill children with sepsis.

Adolescent ; Antigens, CD ; blood ; Antigens, Differentiation, Myelomonocytic ; blood ; Biomarkers ; blood ; C-Reactive Protein ; analysis ; Case-Control Studies ; Child ; Child, Preschool ; Critical Illness ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Infant ; Intensive Care Units, Pediatric ; Lymphocyte Subsets ; immunology ; Male ; Prognosis ; Prospective Studies ; Receptors, Cell Surface ; blood ; Sepsis ; blood ; immunology ; mortality ; Severity of Illness Index ; Tumor Necrosis Factor-alpha ; blood

Many immune down-regulatory molecules have been isolated from parasites, including cystatin (cystain protease inhibitor). In a previous study, we isolated and characterized Type I cystatin (CsStefin-1) of the liver fluke, Clonorchis sinensis. To investigate whether the CsStefin-1 might be a new host immune modulator, we induced intestinal inflammation in mice by dextran sodium sulfate (DSS) and treated them with recombinant CsStefin-1 (rCsStefin-1). The disease activity index (DAI) increased in DSS only-treated mice. In contrast, the DAI value was significantly reduced in rCsStefin-1-treated mice than DSS only-treated mice. In addition, the colon length of DSS only-treated mice was shorter than that of rCsStefin-1 treated mice. The secretion levels of IFN-gamma and TNF-alpha in the spleen and mesenteric lymph nodes (MLNs) were significantly increased by DSS treatment, but the level of TNF-alpha in MLNs was significantly decreased by rCsStefin-1 treatment. IL-10 production in both spleen and MLNs was significantly increased, and IL-10+F4/80+ macrophage cells were significantly increased in the spleen and MLNs of rCsStefin-1 treated mice after DSS treatment. In conclusion, rCsStefin-1 could reduce the intestinal inflammation occurring after DSS treatment, these effects might be related with recruitment of IL-10 secreting macrophages.
Animals ; Antigens, Differentiation/analysis ; Clonorchis sinensis/*enzymology ; Colon/pathology ; Cystatins/*metabolism ; Cytokines/secretion ; Dextran Sulfate/toxicity ; Female ; Helminth Proteins/*metabolism ; Immunologic Factors/*metabolism ; Inflammation/chemically induced/*pathology ; Interleukin-10/analysis ; Intestines/*drug effects/pathology ; Lymph Nodes/immunology ; Macrophages/chemistry/*immunology ; Mice ; Mice, Inbred C57BL ; Severity of Illness Index ; Spleen/immunology

OBJECTIVETo study the effect of activated microglia grafting on rats' hind limb motor function recovery after spinal cord injury.

METHODSMicroglia were separated from primary culture and subcultured for 3 generations. Lipopolysaccharide was added to the culture medium with the terminal concentration of 10 microl/L for microglia activation 3 days before transplantation. Totally 80 adult Wistar rats were divided into transplantation group and control group, with 40 rats in each group. Spinal cord injury model of rats was set by hitting onto the spinal cord using a modified Allen impactor. With a 5 microl micro-syringe, the activated microglia suspension was injected into the injured area 7 days after the first operation. Basso, Beattie and Bresnahan (BBB) scoring for hind limb motor function was taken on the 1st, 7th, 14th, 21st, and 28th day after microglia transplantation, and 8 rats were sacrificed at each time point mentioned above, respectively. Frozen sections of the spinal cord were made for haematoxylin-eosin (HE) and Naoumenko-Feigin stainings. SPSS 11.0 software was used for statistical analysis.

RESULTSBBB scores for hind limb motor function on the 14th, 21st, and 28th day were significantly higher compared with the control group. Most liquefaction necrosis areas disappeared and only a few multicystic cavities surrounded by aggregated microglia remained in the transplantation group. Naoumenko-Feigin staining for microglia showed that the transplantation group had significantly more positive cells (P < 0.05).

CONCLUSIONSGrafting of activated microglia into the injured spinal cord can significantly promote the hind limb motor function recovery in rats with spinal cord injury and reduce the size of liquefaction necrosis area. The extent of lower limb motor function improvement has a positive correlation with the number of aggregated microglia.

Animals ; Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Cells, Cultured ; Flow Cytometry ; Fluorescent Antibody Technique, Indirect ; Hindlimb ; physiopathology ; Immunohistochemistry ; Microglia ; transplantation ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Spinal Cord Injuries ; immunology ; physiopathology ; therapy

OBJECTIVETo investigate clinicopathologic features, immunophenotypes and differential diagnoses of follicular dendritic cell sarcoma/tumor (FDCS).

METHODSEight cases of FDCS were studied using histological and immunohistochemical examinations and EBER in situ hybridization, with a review of the related literatures.

RESULTSThere were 5 male and 3 female patients with a median age of 50 years. The sites of involvement included lymph node (4 cases), tonsil, nasopharynx, liver, and spleen (1 case each, respectively). The predominant microscopic features histologically included storiform, fascicular, diffuse, whorled and nodular in patterns. The neoplastic cells, dispersed by the infiltrated small lymphocytes, were characterized by abundant eosinophilic or fine granular cytoplasm with indistinct cell borders, and syncytial in appearance. The nuclei of the tumors were ovoid, round to spindled in shape with vesicular or stippled chromatin and small distinct nucleoli. Mitotic figures varied among cases. Pseudovascular spaces and perivascular cuffing were observed in some cases. One case of FDCS involving lesion in liver showed a background of abundant lymphocytes mixing with dispersed spindle or ovoid neoplastic cells having delicate chromatin, mild nuclear atypia, irregular/vesicular nuclei and distinct nucleoli. The neoplastic cells were positive for CD21, CD35, clusterin, and weakly positive for CD68, EMA, S-100 and EGFR. Ki-67 stain showed a variable expression among cases. EBER was positive in 2 cases.

CONCLUSIONSFDCS is a rare malignant tumor with a tendency to relapse and metastasis. Combined morphological and immunophenotypical analysis is necessary to reach a correct diagnosis.

Adult ; Aged ; Antigens, CD ; analysis ; immunology ; Antigens, Differentiation, Myelomonocytic ; analysis ; immunology ; Dendritic Cell Sarcoma, Follicular ; metabolism ; pathology ; Dendritic Cells ; pathology ; Female ; Giant Cells ; Humans ; Immunophenotyping ; methods ; Male ; Middle Aged ; Receptors, Complement 3d ; analysis ; immunology ; Treatment Outcome

OBJECTIVETo study the clinical and pathologic characteristics of poorly differentiated cutaneous angiosarcoma of scalp.

METHODSEight cases of poorly differentiated cutaneous angiosarcoma of scalp were enrolled into this study. The clinical manifestations and histopathologic features were analyzed. Immunohistochemical study for CD31, CD34, factor VIII-related antigen, vimentin, AE1/AE3, CAM5. 2, epithelial membrane antigen and carcinoembryonic antigen was performed.

RESULTSThe mean age of the patients was 69 years. The male-to-female ratio was 5 : 3. The tumor manifested clinically as bruise-like lesion in early phase, indurated erythematous plaque accompanied by nodules, ulcerations and bleeding in advanced phase. Histologically, the tumor was composed of solid sheets of undifferentiated spindle cells which were not easily recognizable as vascular in origin. Nuclear atypia was always present. The tumor cells in all of the 8 cases strongly expressed CD31, factor VIII-related antigen and vimentin. Weak expression of CD34, AE1/AE3 and CAMS. 2 was noted in 2, 4 and 4 cases, respectively. The staining for epithelial membrane antigen, carcinoembryonic antigen and S-100 was negative. Conclusions Angiosarcoma needs to be excluded by histologic examination whenever bruise-like and erythematous lesions occurring on scalp skin of elderly patients. The endothelial origin of the tumor cells can be confirmed with immunostaining for CD31, CD34 and factor VIII-related antigen.

Aged ; Aged, 80 and over ; Antigens, CD34 ; immunology ; Biomarkers, Tumor ; analysis ; Cell Adhesion Molecules ; Cell Differentiation ; Endothelium ; metabolism ; Female ; Hemangiosarcoma ; immunology ; Humans ; Male ; Middle Aged ; Platelet Endothelial Cell Adhesion Molecule-1 ; immunology ; Scalp ; pathology ; Skin Neoplasms ; immunology ; metabolism ; pathology ; Vimentin ; analysis