OBJECTIVE: To observe the efficacy of chimeric antigen receptor T cell (CAR-T) in the treatment of children with refractory/recurrent B acute lymphocytic leukemia (B-ALL). METHODS: Thirty-two patients with r/r B-ALL were treated by CAR-T, the recurrence and death respectively were the end point events to evaluate the efficacy and safety of CAR-T. RESULTS: The median age of the patients was 7.5 (2-17.5) years old; 40 times CAR-T were received in all patients and the median number of CAR-T was 0.9×107/kg; efficacy evaluation showed that 2 cases died before the first evaluation. Thirty patients showed that 3, 6, and 9-moth RFS was (96.3±3.6)%, (81.4±8.6)% and (65.3±12.5)%, respectively, while 3, 6, and 9-month OS was all 100%, and 12, 24-month OS was (94.7±5.1)% and (76±12.8)%. BM blasts≥36% before reinfusion and ferritin peak≥2 500 ng/ml within two weeks of CAR-T cell reinfusion were associated with recurrence. Adverse reactions mainly included cytokine release syndrome (CRS) and CART-cell-related encephalopathy syndrome (CRES), CRS appeared in 26 patients within a week of CAR-T cell reinfusion. CRES reaction was detected in 12 patients. Eighteen patients received intravenous drip of tocilizumab, among them, 12 combined with glucocorticoid. CRS and CRES reactions were relieved within one week after treatment. Hormone dosage was related to the duration of remission in patients, and the cumulative dose of methylprednisolone≥8 mg/kg showed a poor prognosis. CONCLUSION CAR-T is a safe and effective treatment for r/r B-ALL, most CRS and CRES reactions are reversible. BM blasts ≥36% before reinfusion and cumulative dose of methylprednisolone ≥8 mg/kg after reinfusion both affect the therapeutic effect. Ferritin≥2 500 ng/ml within two weeks after reinfusion is related to disease recurrence and is an independent prognostic risk factor.
Adolescent ; Antigens, CD19 ; Child ; Child, Preschool ; Chronic Disease ; Ferritins ; Humans ; Immunotherapy, Adoptive ; Methylprednisolone ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy* ; Receptors, Antigen, T-Cell ; Receptors, Chimeric Antigen/metabolism* ; Recurrence ; T-Lymphocytes

Chimeric antigen receptor (CAR) T cell therapy has exhibited dramatic anti-tumor efficacy in clinical trials. In this study, we reported the transcriptome profiles of bone marrow cells in four B cell acute lymphoblastic leukemia (B-ALL) patients before and after CD19-specific CAR-T therapy. CD19-CAR-T therapy remarkably reduced the number of leukemia cells, and three patients achieved bone marrow remission (minimal residual disease negative). The efficacy of CD19-CAR-T therapy on B-ALL was positively correlated with the abundance of CAR and immune cell subpopulations, e.g., CD8 T cells and natural killer (NK) cells, in the bone marrow. Additionally, CD19-CAR-T therapy mainly influenced the expression of genes linked to cell cycle and immune response pathways, including the NK cell mediated cytotoxicity and NOD-like receptor signaling pathways. The regulatory network analyses revealed that microRNAs (e.g., miR-148a-3p and miR-375), acting as oncogenes or tumor suppressors, could regulate the crosstalk between the genes encoding transcription factors (TFs; e.g., JUN and FOS) and histones (e.g., HIST1H4A and HIST2H4A) involved in CD19-CAR-T therapy. Furthermore, many long non-coding RNAs showed a high degree of co-expression with TFs or histones (e.g., FOS and HIST1H4B) and were associated with immune processes. These transcriptome analyses provided important clues for further understanding the gene expression and related mechanisms underlying the efficacy of CAR-T immunotherapy.
Adult ; Antigens, CD19 ; metabolism ; Bone Marrow ; metabolism ; CD8-Positive T-Lymphocytes ; immunology ; Female ; Gene Expression Regulation, Leukemic ; Gene Regulatory Networks ; Humans ; Immunotherapy, Adoptive ; Male ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology ; therapy ; RNA, Long Noncoding ; genetics ; metabolism ; Receptors, Antigen, T-Cell ; Transcription Factors ; metabolism ; Transcriptome ; genetics

BACKGROUND: Bone marrow biopsies are routinely performed for staging patients with B-cell non-Hodgkin lymphoma (NHL). In addition to histomorphological studies, ancillary tools may be needed for accurate diagnosis. We investigated the clinical utility of multiparameter flow cytometric examination of bone marrow aspirates. METHODS: A total of 248 bone marrow specimens from 232 patients diagnosed with B-cell NHL were examined. Monoclonal antibodies directed against CD19, CD20, CD10 (or CD5), and kappa and lambda immunoglobulins were used. Multi-stage sequential gating was performed to select specific cells of interest, and the results were compared with bone marrow histology. RESULTS: The concordance rate between histomorphology and flow cytometry was 91.5% (n=227). Eight cases (3.2%) were detected by flow cytometry alone and were missed by histomorphology analysis, and 6 of these 8 cases showed minimal bone marrow involvement (0.09-2.2%). The diagnosis in these cases included large cell lymphoma (n=3), mantle cell lymphoma (n=3), and mucosa-associated lymphoid tissue (MALT) lymphoma (n=2). Thirteen cases were histopathologically positive and immunophenotypically negative, and the diagnoses in these cases included diffuse large cell lymphoma (n=7), T-cell/histiocyte-rich large B-cell lymphoma (n=2), anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (n=1), follicular lymphoma (n=1), MALT lymphoma (n=1), and unclassifiable lymphoma (n=1). CONCLUSIONS: Multi-color flow cytometry can be a useful method for assessing bone marrow in staging NHL and also plays a complementary role, especially in detecting small numbers of lymphoma cells.
Antibodies, Monoclonal/immunology ; Antigens, CD19/immunology/metabolism ; Antigens, CD20/immunology/metabolism ; Bone Marrow/*pathology ; Female ; Flow Cytometry ; Humans ; Immunophenotyping ; Lymphoma, B-Cell/*pathology ; Male ; Neoplasm Staging ; Neprilysin/immunology/metabolism

OBJECTIVETo investigate the effects of rituximab (RTX) in children with steroid-dependent nephrotic syndrome.

METHODFive cases of children with steroid-dependent nephrotic syndrome seen from May 2012 to February 2013 in whom only steroid plus calcineurin inhibitor was effective and the disease recurred on reduction of dose were enrolled into this study, including 3 males and 2 females. Calcineurin inhibitors were stopped and steroids was changed to full dose. After the general condition improved, RTX was given at a dose of 375 mg/m(2), once a week for a total of three times for one course. After urine protein became negative for five days, the dose of steroid was changed to 2 mg/kg every other day, thereafter the dose was reduced by 5 mg per every 2 weeks, until discontinuation. After regular monitoring, when peripheral blood B cells were ≥ 3%, a second RTX was added.

RESULTUrine protein was negative in 2-7 days in 5 patients after the first RTX treatment. Before treatment B lymphocytes in peripheral blood was 7.8% to 13.0% and after the first course of RTX treatment decreased to 0 in the first 6 to 8 months at the beginning of recovery, while in the first 7 to 10 months to 3.3%-6.1%, after a second RTX was given, B lymphocytes were reduced to 0, but in two cases (cases 1 and 3) B lymphocytes rose again at 16 and 17 months, in the first 17 and 18 months rose to 4.16% and 4.17%, RTX was given once again respectively. B lymphocytes were reduced to 0 again. Currently the 5 patients continued to be negative for urine protein, maintaining remission for 12 to 20 months.RTX infusion had no significant side effects, and side effects of steroid and calcineurin inhibitor disappeared.

CONCLUSIONIn children with steroid-dependent and only calcineurin inhibitor effective nephritic syndrome, relapse may still occur after improvement of nephrotic syndrome, after the first RTX treatment, regular monitoring of B lymphocytes, RTX supplementary treatment in advance can help discontinuation of steroids and immunosuppressive agents and maintain remission.

Anti-Inflammatory Agents ; administration & dosage ; therapeutic use ; Antibodies, Monoclonal, Murine-Derived ; administration & dosage ; therapeutic use ; Antigens, CD19 ; metabolism ; B-Lymphocytes ; drug effects ; metabolism ; Calcineurin Inhibitors ; administration & dosage ; therapeutic use ; Child ; Child, Preschool ; Drug Administration Schedule ; Female ; Follow-Up Studies ; Humans ; Immunosuppressive Agents ; administration & dosage ; therapeutic use ; Lymphocyte Count ; Male ; Nephrotic Syndrome ; drug therapy ; metabolism ; Proteinuria ; urine ; Recurrence ; Remission Induction ; Rituximab ; Treatment Outcome

PURPOSE: The function of regulatory B lymphocytes is known to be abnormal in inflammatory diseases. However, a recent study indicates that IL-10+ B cells seem to be expanded in rheumatoid arthritis (RA). Therefore, the state of IL-10+ B cells in the peripheral blood from RA patients and healthy controls were investigated. MATERIALS AND METHODS: CD19+ cells in peripheral blood mononuclear cells were purified from blood samples of RA patients and age and gender-matched healthy controls, and stimulated with CD40 ligand and CpG for 48 hours. Then, intracellular IL-10 in CD19+ cells was analyzed using flow cytometry. RESULTS: There was no significant difference in the proportion of IL-10+ B cells between 10 RA patients and 10 healthy controls (RA, 0.300+/-0.07 vs. healthy control 0.459+/-0.07, p=0.114). The proportion of induced IL-10+ B cells to total B cells in RA patients was significantly higher than those in controls (RA, 4.44+/-3.44% vs. healthy control 2.44+/-1.64%, p=0.033). However, the proportion of IL-10+ B cells to total B cells correlated negatively with disease activity in RA patients (r=-0.398, p=0.040). Erythrocyte sedimentation rate or C-reactive protein or medication was not associated with the proportion of IL-10+ B cells. CONCLUSION: The proportion of induced IL-10+ B cell increased in RA patients compared to healthy control, however, negatively correlated with disease activity in RA.
Adult ; Aged ; Antigens, CD19/metabolism ; Arthritis, Rheumatoid/blood/*immunology/pathology ; B-Lymphocytes, Regulatory/metabolism/*physiology ; Biological Markers/blood ; Female ; Humans ; Interleukin-10/metabolism ; Male ; Middle Aged ; Severity of Illness Index

Immunophenotype is critical for diagnosing common B-cell acute lymphoblastic leukemia (common ALL) and detecting minimal residual disease. We developed a protocol to explore the immunophenotypic profiles of common ALL based on the expression levels of the antigens associated with B lymphoid development, including IL-7Rα (CD127), cytoplasmic CD79a (cCD79a), CD19, VpreB (CD179a), and sIgM, which are successive and essential for progression of B cells along their developmental pathway. Analysis of the immunophenotypes of 48 common ALL cases showed that the immunophenotypic patterns were highly heterogeneous, with the leukemic cell population differing from case to case. Through the comprehensive analysis of immunophenotypic patterns, the profiles of patient-specific composite leukemia cell populations could provide detailed information helpful for the diagnosis, therapeutic monitoring, and individualized therapies for common ALL.
Adult ; Antigens, CD19 ; metabolism ; B-Lymphocytes ; immunology ; metabolism ; CD79 Antigens ; metabolism ; Female ; Humans ; Immunoglobulin Light Chains, Surrogate ; metabolism ; Immunophenotyping ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; immunology ; pathology ; Receptors, Interleukin-7 ; metabolism

OBJECTIVETo investigate the change of lymphocyte subsets before and after chemotherapy in colorectal carcinoma patients.

METHODSTwenty-one peripheral blood lymphocyte subsets from 62 colorectal carcinoma patients before and after FOLFOX4(including oxaliplatin, 5-fluorouracil and leucovorin) , FOLFRI(including irinotecan, 5-fluorouracil and leucovorin) , or XELOX(including oxaliplatin and capecitabine) regimen chemotherapy were examined by flow cytometry.The differences of these lymphocyte subsets were analyzed.

RESULTSAfter chemotherapy, the percentages of CD3(+), CD3(+)CD8(+), CD29(+), CD4(+)CD29(+), and CD4(+)CD25(+) cells in peripheral blood of colorectal carcinoma patients increased significantly, while the percentages of CD19(+) and human leukocyte antigen(locus) DR(HLA-DR) (+) cells decreased significantly(P<0.05) .The results of subgroup analysis showed that the patients' CD3(+)CD8(+) and CD4(+)CD25(+) cells increased significantly, CD19(+) and HLA-DR(+) cells decreased significantly after FOLFOX4 regimen chemotherapy(P<0.05) ;CD3(+)CD8(+) cells increased significantly and CD19(+) cells decreased significantly after XELOX regimen chemotherapy(P<0.05) ;while after FOLFRI regimen chemotherapy, there were no significant changes in all 21 lymphocyte subsets(P>0.05) . CD3(+), CD3(+)CD8(+), memory T lymphoctye(45RO(+)) , and CD4(+)CD45RO(+) cells increased significantly(P<0.05) in patients who received no more than 4 cycles of chemotherapy. However, in patients that received 5 to 8 cycles and more than 9 cycles chemotherapy, we only found significant decrease of HLADR(+) cells and significant increase of CD29(+) cells, respectively(P<0.05) .

CONCLUSIONSThe humoral immunity is attenuated after chemotherapy in colorectal carcinoma patients. FOLFOX4 may suppress the cellular immunity.Chemotherapy that is less than 4 cycles will strengthens the cellular immunity by modulating body immunity arrangement;however, along with the increase of chemotherapy cycles, the cellular immunity gradually declines in these patients.

Adult ; Aged ; Aged, 80 and over ; Antigens, CD19 ; metabolism ; Antineoplastic Combined Chemotherapy Protocols ; administration & dosage ; Camptothecin ; administration & dosage ; analogs & derivatives ; Colorectal Neoplasms ; drug therapy ; immunology ; Deoxycytidine ; administration & dosage ; analogs & derivatives ; Female ; Fluorouracil ; administration & dosage ; analogs & derivatives ; Humans ; Leucovorin ; administration & dosage ; Lymphocyte Subsets ; pathology ; Male ; Middle Aged ; Organoplatinum Compounds ; administration & dosage

OBJECTIVETo study the immunophenotype and chromosome karyotype characteristics of leukemic stem cells (LSC) in Uyghur leukemia pediatric patients.

METHODSThe morphological features of LSC in culture in vitro was observed by flow cytometry. The immunophenotype was assessed by detective flow cytometry. The chromosome karyotype was analyzed by R-banding technique.

RESULTSThe LSC showed suspended floating colonies growing in the culture medium, and grew well and proliferated constantly in culture over 8 months. Among the 13 children with AML, there were 10 CD34(+)CD38(-)CD123(+) and CD33(+) cases, 10 CD44(+) cases, 10 CD96(+) cases, and 5 CD90(+) cases. Among the 13 children with B-ALL, there were 6 CD34(+)CD20(-)CD19(+) cases, 7 CD9(+) cases, and 5 CD123(+) cases. Among the 9 children with acute T lymphoblastic leukemia (T-ALL), there were 5 CD34(+)CD7(-) and CD90(+) cases, and 4 CD123(+) cases. Among the 13 cases of AML, 5 cases showed chromosome translocation t(15;17), one case chromosome translocation t(8;21), and 7 cases showed no chromosome karyotype abnormality. Among the 22 ALL cases, there were chromosome translocation t(12;21) in 1 case, t(9;22) in 3 case, hyperdiploid in 2 cases, and 16 cases without karyotype abnormalities. Twenty-nine children received induction remission therapy. Among them, 12 died, including 9 CD96(-)positive cases and 3 CD96(-)negative cases, with a statistically significant difference (P < 0.05).

CONCLUSIONSThe LSC of Uyghur leukemia pediatric patients in Xinjiang express CD9 and CD19 in ALL, and express CD123 and CD90 simultaneously in ALL and AML. The expression of CD96 is one of factors of poor prognosis.

Adolescent ; Antigens, CD ; metabolism ; Antigens, CD19 ; metabolism ; Child ; China ; ethnology ; Diploidy ; Humans ; Immunophenotyping ; Interleukin-3 Receptor alpha Subunit ; metabolism ; Karyotyping ; Leukemia, Myeloid, Acute ; drug therapy ; genetics ; immunology ; pathology ; Neoplastic Stem Cells ; immunology ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; genetics ; immunology ; pathology ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; genetics ; immunology ; pathology ; Remission Induction ; Tetraspanin-29 ; metabolism ; Thy-1 Antigens ; metabolism ; Translocation, Genetic

Most of the previous studies on immune dysregulation in end-stage renal disease (ESRD) have focused on T cell immunity. We investigated B cell subpopulations in ESRD patients and the effect of hemodialysis (HD) on B cell-associated immune profiles in these patients. Forty-four ESRD [maintenance HD patients (n = 27) and pre-dialysis patients (n = 17)] and 27 healthy volunteers were included in this study. We determined the percentage of B cell subtypes, such as mature and immature B cells, memory B cells, and interleukin (IL)-10+ cells, as well as B cell-producing cytokines (IL-10, IL-4 and IL-21) by florescent activated cell sorting (FACS). B cell-associated gene expression was examined using real-time PCR and B cell producing cytokines (IL-10, IL-4 and IL-21) were determined using an enzyme-linked immunosorbent assay (ELISA). The percentage of total B cells and mature B cells did not differ significantly among the three groups. The percentages of memory B cells were significantly higher in the pre-dialysis group than in the HD group (P < 0.01), but the percentage of immature B cells was significantly lower in the pre-dialysis group than in the other groups. The percentages of IL-10-expressing cells that were CD19+ or immature B cells did not differ significantly (P > 0.05) between the two subgroups within the ESRD group, but the serum IL-10 concentration was significantly lower in the pre-dialysis group (P < 0.01). The results of this study demonstrate significantly altered B cell-associated immunity. Specifically, an imbalance of immature and memory B cells in ESRD patients was observed, with this finding predominating in pre-dialysis patients.
Adaptor Proteins, Signal Transducing/genetics ; Adult ; Antigens, CD19/metabolism ; B-Lymphocyte Subsets/immunology/metabolism ; B-Lymphocytes/*immunology/metabolism ; Cytokines/biosynthesis ; Female ; Humans ; Immunophenotyping ; Interleukin-10/metabolism ; Kidney Failure, Chronic/*immunology/metabolism ; Leukocytes, Mononuclear/metabolism ; Male ; Middle Aged ; Proto-Oncogene Proteins/genetics ; T-Lymphocytes, Regulatory/immunology/metabolism

BACKGROUND: We developed a single-color multitarget flow cytometry (SM-FC) assay, a single-tube assay with graded mean fluorescence intensities (MFIs). We evaluated the repeatability of SM-FC, and its correlation with multicolor flow cytometry (MFC), to assess its application as a routine FC assay. METHODS: We selected CD19, CD3, CD4, and CD8 as antigen targets to analyze a lymphocyte subset. MFIs were graded by adjusting monoclonal antibody (mAb) volumes to detect several cell populations. Dimly labeled mAb was prepared by decreasing mAb volume and the optimum diluted volume was determined by serial dilution. SM-FC repeatability was analyzed 10 times in 2 normal controls. The correlation between SM-FC and MFC was evaluated in 20 normal and 23 patient samples. RESULTS: CV values (0.8-5.0% and 1.3-4.1% in samples 1 and 2, respectively) acquired by SM-FC with CD3-fluorescein alpha-isothyocyanate (FITC)dim+CD4-FITCbright and with CD19-FITCdim+CD3-FITCbright showed good repeatability, comparable to that acquired by MFC (1.6-3.7% and 1.0-4.8% in samples 1 and 2, respectively). Excellent correlation was observed between the 2 methods in the 20 normal samples (B cells, T cells, non-Thelper cells, and Thelper cells; r2=0.87, 0.97, 0.97, and 0.98, respectively; P<0.05). There were also linear relationships between SM-FC with CD19-FITCdim+CD3-FITCbright and CD8-PEdim+CD4-PEbright, and MFC, in the 23 patient samples (B cells, T cells, Tcytotoxic cells, and Thelper cells; r2> or =0.98, 0.99, 0.99, and 0.99, respectively; P<0.05). CONCLUSIONS: The multicolor, single-tube SM-FC technique is a potential alternative tool for identifying a lymphocyte subset.
Antibodies, Monoclonal/chemistry/*immunology ; Antigens, CD19/chemistry/metabolism ; Antigens, CD3/chemistry/metabolism ; Antigens, CD4/chemistry/metabolism ; Antigens, CD8/chemistry/metabolism ; B-Lymphocyte Subsets/immunology/metabolism ; Color ; Flow Cytometry/*methods ; Fluorescein-5-isothiocyanate/*chemistry ; Humans ; T-Lymphocyte Subsets/immunology/metabolism