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

BackgroundStudies of haploidentical-related donor (HRD) stem cell transplantation using a combination of peripheral blood stem cells (PBSCs) and bone marrow as the graft have reported encouraging results for patients with hematological diseases. However, few studies specifically reported transplantation of only PBSCs from HRDs among patients with relapsed or refractory acute myeloid leukemia (AML). Here, the long-term outcomes and side effects of unmanipulated HRD PBSC transplantation (HRD-PBSCT) for relapsed/refractory AML were analyzed.

MethodsWe performed a retrospective analysis of the outcomes in relapsed/refractory AML patients who underwent PBSCT from HRDs (n = 36).

ResultsThirty-one (86.1%) patients in the HRD-PBSCT group achieved platelet recovery. The cumulative incidence of acute graft-versus-host disease (aGVHD) in the HRD-PBSCT group was 40.00%, and the cumulative incidence of grades 2-4 aGVHD in this group was 13.33%. A total of 13 patients in the HRD-PBSCT group had recurrent disease at a median of 183 days after transplantation (range: 10-1700 days), reaching cumulative incidences of relapse of 50.28% at 5 years. On multivariate analysis, donor age and patient age >40 years were independent risk factors for inferior disease-free survival or overall survival (P < 0.05). The results of the present study demonstrate rapid and complete neutrophil engraftment, a low incidence of grade 2-4 aGVHD, and promising survival rates in patients after HRD-PBSCT. Thus, granulocyte colony-stimulating factor-primed PBSCs may be a reliable graft source in unmanipulated HRD-HSCT under myeloablative conditioning when no matched sibling donor is available.

ConclusionsOur results support the feasibility, effectiveness, and tolerability of PBSCs as a graft source in unmanipulated HRD transplantation under myeloablative conditioning in patients with leukemia.

Adult ; Female ; Graft Survival ; Graft vs Host Disease ; Granulocyte Colony-Stimulating Factor ; metabolism ; Humans ; Incidence ; Leukemia, Myeloid, Acute ; therapy ; Male ; Multivariate Analysis ; Peripheral Blood Stem Cell Transplantation ; adverse effects ; methods ; Retrospective Studies

BACKGROUND/AIMS: Data on dexamethasone, cytarabine, and cisplatin (DHAP) as a mobilization regimen, compared to high-dose cyclophosphamide (HDC), for up-front autologous stem cell transplantation (ASCT) in non-Hodgkin’s lymphoma (NHL) is limited. METHODS: Consecutive patients with aggressive NHL treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or rituximab-CHOP who underwent chemomobilization using HDC or DHAP plus granulocyte-colony stimulating factor (G-CSF) for up-front ASCT were enrolled from three institutions between 2004 and 2014. RESULTS: Ninety-six patients (57 men) were included. Sixty-five patients (67.7%) received HDC; and 31 (32.3%), DHAP. The total CD34+ cells mobilized were significantly higher in patients receiving DHAP (16.1 vs. 6.1 × 106/kg, p = 0.001). More patients achieved successful mobilization with DHAP (CD34+ cells ≥ 5.0 × 106/kg) compared to HDC (87.1% vs. 61.5%, respectively; p = 0.011), particularly within the first two sessions of apheresis (64.5% vs. 32.3%, respectively; p = 0.003). Mobilization failure rate (CD34+ cells < 2.0 × 106/kg) was significantly higher in patients receiving HDC (20.0% vs. 3.2%, p = 0.032). On multivariate analysis, the DHAP regimen (odds ratio, 4.12; 95% confidence interval, 1.12 to 15.17) was an independent predictor of successful mobilization. During chemomobilization, patients receiving HDC experienced more episodes of febrile neutropenia compared to patients receiving DHAP (32.3% vs. 12.9%, p = 0.043). CONCLUSIONS: The DHAP regimen was associated with a significantly higher efficacy for stem cell mobilization and lower frequency of febrile neutropenia. Therefore, DHAP plus G-CSF is an effective for mobilization in patients with aggressive NHL who were candidates for up-front ASCT.
Blood Component Removal ; Cisplatin* ; Cyclophosphamide ; Cytarabine* ; Dexamethasone* ; Doxorubicin ; Febrile Neutropenia ; Granulocyte Colony-Stimulating Factor ; Hematopoietic Stem Cell Mobilization* ; Humans ; Lymphoma ; Lymphoma, Non-Hodgkin* ; Multivariate Analysis ; Prednisone ; Stem Cell Transplantation* ; Stem Cells* ; Vincristine

OBJECTIVE: To identify differentially expressed genes in peripheral blood mononuclear cells between patients with continuous mild-to-moderate asthma and healthy controls using mRNA microarray in order to explore the underlying signaling pathways and clarify the roles of CD4+ T cells in the pathogenesis of asthma. METHODS: Global transcriptomic profiles of the CD4+ T cells were defined by using Agilent Sure Print G3 Human GE 8×60K microarray. Enrichment pathways were analyzed with Ingenuity Pathway Analysis (IPA) software. RESULTS: Compared with controls, 805 genes were up-regulated, 192 were down-regulated in asthma patients. Among these, the expression of 38 annotated genes have varied by 4 times or more. Expression of CD300A was inversely proportional to the absolute value of eosinophils (r=-0.89, P=0.02) as well as the proportion of eosinophils (r=-0.94, P=0.004), while CSF1R was inversely proportional to PD20 (r=-0.83, P=0.04) and AQLQ (r=-0.88, P=0.02) by correlation analysis. CONCLUSION Numerous pathophysiological pathways may be involved in the pathogenesis of asthma. Above findings have provided a basis for the delineation the pathogenesis of asthma.
Antigens, CD ; genetics ; Asthma ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; Case-Control Studies ; Eosinophils ; Gene Expression Profiling ; Humans ; Leukocytes, Mononuclear ; Oligonucleotide Array Sequence Analysis ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Receptors, Immunologic ; genetics ; Transcriptome

OBJECTIVETo observe the effects of recombinant fusion protein interleukin (IL)-18 on the expression of immune-inflammatory factors in the mice infected with Staphylococcus aureus (SA), and to investigate the mechanism of action of IL-18 in defense of SA infection in vivo.

METHODSA total of 40 specific pathogen-free female BLAB/c mice were randomly divided into four groups: control, SA infection, immunized, and intervention. A mouse model of SA infection was established by nasal inoculation with SA liquid. The immunized group and the intervention group were intranasally given IL-18 before SA modeling, and then the SA infection group and the intervention group received the nasal inoculation with SA liquid; the control group was treated with phosphate buffered saline instead. The levels of IL-4, interferon (IFN)-γ, tumor necrosis factor (TNF), granulocyte colony-stimulating factor (G-CSF), IgM in the serum and bronchoalveolar lavage fluid (BALF) of mice were measured by enzyme-linked immunosorbent assay. The expression of macrophage inflammatory protein (MIP)-1α mRNA and MIP-2β mRNA in the lung tissue of mice were determined by real-time fluorescent quantitative PCR.

RESULTSCompared with the control group, the SA infection group and the immunized group had significantly higher levels of IL-4, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue (P<0.05); the SA infection group had a significantly lower level of IFN-γ and a significantly higher level of TNF in the serum and BALF (P<0.05); the immunized group had a significantly higher level of IFN-γ in the serum and BALF (P<0.05). Compared with the SA infection group, the intervention group had significantly higher levels of IL-4, IFN-γ, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA in the lung tissue. In contrast, the intervention group showed a significantly lower level of TNF in the serum and BALF and expression of MIP-2β mRNA in the lung tissue (P<0.05). All the above indicators in the intervention group were significantly higher than those in the control group (P<0.05), except the serum level of IFN-γ.

CONCLUSIONSIn the mice infected with SA, the recombinant fusion protein IL-18 by mucosal immunity can affect inflammatory factors in the serum and BALF and the expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue to promote the anti-infective immune response and enhance the ability to clear pathogens.

Animals ; Chemokine CCL3 ; analysis ; Female ; Granulocyte Colony-Stimulating Factor ; blood ; Interferon-gamma ; blood ; Interleukin-18 ; therapeutic use ; Interleukin-4 ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; pharmacology ; therapeutic use ; Staphylococcal Infections ; drug therapy ; immunology

OBJECTIVETo observe the short- term effects of hemogram in donors after peripheral blood stem cell（PBSC）collection and donors' tolerance.

METHODSA total of 166 related allogeneic donors were selected from The First Affiliated Hospital of Medical School of Zhejiang University between January 2013 and December 2014, including 86 male and 80 female. All donors accepted granulocytecolony- stimulating factor（G-CSF）5-10 μg·kg⁻¹·d⁻¹ until collection finished and were measured by blood cells count before and after PBSC collection.

RESULTSAfter PBSC collection, the hemoglobin level decreased from 145（94-181）g/L to 138（93-167）g/L, and the platelet counts decreased in all donors from 231（105- 490）× 10⁹/L to 95（39- 210）× 10⁹/L. The amount of hemoglobin contamination in collection products was weak correlated with the decreased hemoglobin in peripheral blood（r=0.297, P=0.017）, and the platelet contamination was high correlated with that decreased in peripheral blood（r=0.719, P<0.001）. The decline of hemoglobin level after twice PBSC collection was of no significant difference between four groups in different ages（P≥0.05）. The decline of platelet counts was out of a significant difference（P> 0.05）. In addition, the decline of hemoglobin level after once and twice PBSC collection was of a significant difference between four groups in different body mass index（BMI）（P=0.003 and P<0.001）, especially in thinner group with obvious decrease. But the decline of platelet counts was out of a significant difference （P>0.05）.

CONCLUSIONThe hemoglobin level decreased mildly in healthy allogeneic hematopoietic stem cell donors after PBSC collection and it is better to adjust parameters every time to ensure their safety for thinner donors. However, it will increase the risk of platelet decline, which is unrelated with ages and BMI and can be tolerated.

Blood Donors ; Blood Platelets ; Female ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cells ; cytology ; Hemoglobins ; analysis ; Humans ; Male ; Platelet Count

OBJECTIVE: To explore the effect of nerve growth factor (NGF) on the  differentiation of murine bone marrow-derived dendritic cells (DCs) in vitro.
 METHODS: The bone marrow cells of femur and tibia from healthy C57B -L/6 mice were isolated and divided into 4 groups: a phosphate buffered saline (PBS) group (PBS group), a NGF group, a granulocyte monocyte colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) group (GM-CSF+IL-4 group), and a GM-CSF plus IL-4 and NGF group (n=6 in each group). The positive rate of CD11c+ and the proportion of CD8a- were compared at the 7th day among the different groups by flow cytometry. The immature DCs were acquired by classic methods with GM-CSF and IL-4. The purified DCs were obtained by magnetic bead positive selection for CD11c+ cells. The immature DCs were divided into 4 groups: a PBS group, a NGF group, a LPS group, and a NGF+LPS group (n=6 in each group), which were incubated with PBS, NGF, LPS and NGF+LPS, respectively. Cytokine levels of IL-6, IL-10 and IL-12 were detected by ELISA after 24 hours..
 RESULTS: 1) the percentage of CD11c+ DCs in the NGF group were more than that in the PBS group, and lower than that in the the GM- CSF+IL-4 group (both P<0.05). There was no difference between the GM-CSF + IL-4 group and the NGF+GM-CSF+IL-4 group (P>0.05). CD8a- DCs were dominant in these four groups; 2) NGF could further up-regulate the LPS-induced cytokine secretion from DCs, such as IL-6, IL-10, and IL-12 (all P<0.05), but NGF alone had no such effect (all P<0.05).
 CONCLUSION NGF can promote the murine bone-marrow cells differentiation into CD11c+ DCs, with CD8a-subset; NGF could enhance LPS-induced cytokine secretion from DCs (IL-6, IL-10 and IL-12).
Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Interleukin-10 ; analysis ; Interleukin-12 ; analysis ; Interleukin-4 ; pharmacology ; Interleukin-6 ; analysis ; Mice ; Mice, Inbred C57BL ; Nerve Growth Factor ; pharmacology

This study was aimed to explore the effect and feasibility of reduced conditioning intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of relapsed ETO positive acute myeloid leukemia (AML) patients. Fifteen cases of relapsed AML received the reducing conditioning intensity allo-HSCT from January 2011 to January 2013 in Beijing Military Command General Hospital. All patients were high-risk type of relapsed or refractory AML, including 10 males and 5 females, aged from 16 to 48 years old with mean age of 32.5 years. Ten cases are HLA-identical matching and other 5 cases are HLA-haploidentical.donors received granulocyte colony-stimulating factor (G-CSF) to mobilize the peripheral blood stem cell for transplantation. Conditioning regimen was fludarabine combined with busulfex, cytarabine and cyclophosphamide. The preventive donor's peripheral blood stem cell infusion were performed after 3 months of transplantation, and the toxicity, GVHD and disease-free survival were observed in patients after transplantation. The results showed that all patients achieved hematopoietic reconstitution, the average time of neutrophils ≥ 0.5 × 10⁹/L and platelets ≥ 20 × 10⁹/L were 15.5 d and 16.8 d respectively. Implantation was confirmed by the evidence of 100% donor hematopoiesis. Follow-up to June 2014, with a median follow-up duration of 27.5 months (18-54 months), GVHD occurred in 8 cases of all patients, one died of complication, the other 4 cases died of relapse and the other three patients remained in disease-free survival. The disease-free survival rate of 2-year was 66.7%,the longest disease-free survival time was up to 54 months. It is concluded that the reduced conditioning intensity allo-HSCT is the effective and safe method for relapsed AML with ETO-positive, and it may be chosen as a treatment method for relapsed ETO positive AML patients.
Adolescent ; Adult ; Allografts ; Cytarabine ; Disease-Free Survival ; Erythropoietin ; analysis ; Female ; Granulocyte Colony-Stimulating Factor ; Hematopoietic Stem Cell Transplantation ; Humans ; Leukemia, Myeloid, Acute ; therapy ; Male ; Middle Aged ; Transplantation Conditioning ; Vidarabine ; analogs & derivatives ; Young Adult

BACKGROUND: Peripheral blood stem cell (PBSC) transplantation is a curative treatment in various hematologic malignancies and some solid cancers. Effective mobilization and collection of PBSC is essential for successful PBSC transplantation. The aim of this study was to investigate the useful factors for predicting PBSC collection using multivariate analysis. METHODS: We retrospectively reviewed the medical records of 170 allogeneic and 389 autologous donors at Chonnam National University Hwasun Hospital between 2005 and 2012. Donor groups were divided into three groups (failure group, suboptimal group, and optimal group) according to the total CD34+ yield. Donors were compared regarding age, sex, body weight, disease, complete blood count, hematopoietic progenitor cell (HPC) parameter of automated cell counter, process volume, number of leukapheresis procedures, prior mobilization history, type of vascular access and instrument. RESULTS: In allogeneic PBSC collections (n=170), the collection failure group showed lower baseline (premobilization) white blood cell (WBC) (P=0.004) and HPC (P<0.001) than the optimal group. In autologous PBSC collections (n=389), the collection failure group showed lower baseline HPC and more frequent prior mobilization history (P<0.001) than the suboptimal and optimal group. In multivariate analysis, older age, lower number of leukapheresis procedures, and prior mobilization history were risk factors associated with mobilization failure. CONCLUSION: Our data suggest that baseline WBC and HPC would be useful for predicting poor mobilizer in allogeneic PBSC collection, whereas baseline HPC would be useful in autologous PBSC collection. Conventional chemotherapy and G-CSF based remobilization would not be helpful to proven poor mobilizer in previous mobilization.
Blood Cell Count ; Body Weight ; Cell Count ; Drug Therapy ; Granulocyte Colony-Stimulating Factor ; Hematologic Neoplasms ; Hematopoietic Stem Cell Mobilization ; Hematopoietic Stem Cells ; Humans ; Jeollanam-do ; Leukapheresis ; Leukocytes ; Medical Records ; Multivariate Analysis ; Retrospective Studies ; Risk Factors ; Stem Cells* ; Tissue Donors ; Transplantation

Severe congenital neutropenia (SCN) is a heterogeneous group of disorders with a defect in granulopoiesis causing marked neutropenia and severe bacterial infections. A 17-month-old girl (patient 1) was admitted due to cervical lymphadenitis caused by methicillin-resistant Staphylococcus aureus, with neutropenia. She had Pseudomonas aeruginosa sepsis and peritonitis with perforated appendicitis at 8-month of age. Her sister, a 37-month-old girl (patient 2), had recurrent stomatitis with profound neutropenia, and her mother, a 32-yr-old woman (patient 3), had had recurrent stomatitis until her early 20s with neutropenia. We found an ELANE gene mutation (c.597+1G > A) from them in direct DNA sequencing analysis. Patients 1 and 2 did not respond to granulocyte colony stimulating factor and patient 1 was treated with prolonged antibiotics and excision. We demonstrated inherited SCN cases showing different severity even with the same mutation of the ELANE gene in a family.
Adult ; Child, Preschool ; DNA Mutational Analysis ; Female ; Granulocyte Colony-Stimulating Factor/therapeutic use ; Humans ; Infant ; Leukocyte Elastase/*genetics ; Methicillin-Resistant Staphylococcus aureus/isolation & purification ; Mutation/genetics ; Neutropenia/*congenital/diagnosis/drug therapy/genetics ; Pedigree ; *Phenotype ; Polymorphism, Single Nucleotide ; Recurrence ; Staphylococcal Infections/diagnosis/microbiology ; Stomatitis/diagnosis ; Tomography, X-Ray Computed