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

OBJECTIVETo investigate the inducing effect of 'modified' cytokine cocktail on the dendritic cell maturation and migration capability.

METHODSPBMNC were isolated from human peripheral blood stem cell (PBSC) by using density gradient centrifugation, the immature DC (imDC) were induced by using GM-CSF and IL-4 in vitro. Total A549 RNA was transfected into imDC by using electroporation, which was stimulated to matuation by the "gold standard" cytokine cocktail and "modified" cytokine cocktail, respectively. The expression of DC surface markers (CD11c, HLA-DR, CD80, CD83 and CD86) and chemokine receptor (CCR5, CCR7 and CXCR4) were detected by flow cytometry; the mRNA expression levels of DC chemokine receptor (CCR2, CCR5, CCR7, CXCR3 and CXCR4) and chemokine (CCL2, CCL3, CCL5, CCL19, CCL21, CXCL10 and CXCL12) were detected by RT-PCR.

RESULTSAs compared with "gold standard cytokine cocktail", the "modified" cytokine cocktail-induced DC expressed higher levels of surface markers (CD11c, HLA-DR, CD80, CD83 and CD86), chemokine receptors (CXCR4) and chemokine (CCL2, CCL3, CCL5, CCL19, CCL21, CXCL10 and CXCL12).

CONCLUSIONThe "modified" cytokine cocktail can more effectively induce the DC maturation, enhace the migratory capability of DC and more generate the immunostimulatory DC, when compared with the "gold standard" cytokine cocktail effect.

Antigens, CD ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; Chemokines ; metabolism ; Cytokines ; pharmacology ; Dendritic Cells ; cytology ; drug effects ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; Receptors, Chemokine ; metabolism

OBJECTIVETo compare the expression of C-C chemokine receptor type 5 (CCR5) on T cells between bone marrow grafts (G-BM) and peripheral blood grafts (G-PB) nobilized by recombinant human granulocyte colony-stimulating factor (rhG-CSF), and to analyze the correlation of CCR5+ T lymphocyte expression in the grafts with the occurrence of acute GVHD.

METHODSForty-six healthy donor and their recipient pairs of related allogeneic hematopoietic stem cell transplantation (allo-HSCT) were enrolled in this study. All the recipients were received the infusion of G-BM and G-PB. The relative proportion and quantity of CCR5+ T cell subset in G-BM and G-PB were detected and compared. Then the correlation of the quantity of infused CCR5+ T cells with the occurrence of acute GVHD was analyzed.

RESULTSAfter mobilization, the proportions of CD4+ CCR5+ and CD8+ CCR5+ T cells occupying T cells in G-PB were both lower than those in G-BM. However, the absolute counts in G-PB were 15-25 times more than those in the bone marrow. And the absolute counts could not predict the occurrence of acute GVHD after transplantation (P>0.05).

CONCLUSIONThe difference of CCR5+ subsets between G-PB and G-BM may partially explain that grafts from different sources have different immunologic characteristics. Besides, the quantity of CCR5+ T cells in the grafts are not related with the occurrence of acute GVHD. However, the relative proportion of CCR5+ T cell subset in the grafts may be predictive of acute GVHD.

Bone Marrow ; metabolism ; Bone Marrow Transplantation ; Graft vs Host Disease ; pathology ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; Hematopoietic Stem Cell Transplantation ; Humans ; Receptors, CCR5 ; metabolism ; T-Lymphocyte Subsets ; metabolism ; Tissue Donors

This study was aimed to investigate the expression of granulocyte colony-stimulating factor receptor IV(G-CSFR IV) in adult acute leukemia patients and its clinical significance. The bone marrow hematopoietic stem cells from healthy persons were used as controls. The real-time RT-PCR was used to determine the expression level of G-CSFR I-IV in 99 AML, 34 ALL patients and 19 healthy persons. The results showed that the relative expression level of G-CSFR IV/G-CSFR I in AML patients was obviously elevated, as compared with that in ALL patients and controls, while the relative expression level of G-CSFR IV/G-CSFR I in ALL patients showed no statistical difference from controls. The analysis of clinical features and chemotherapeutic efficacy demonstrated that the clinical remission rate in patients with high expression of G-CSFR IV/G-CSFR I was lower than that in patients with low expression. The relative expression level of G-CSFR IV/G-CSFR I was not related with risk stratification from sex, age, blast ratio, FAB typing, chromosome and fusion gene. It is concluded that the abnormal high expression of G-CSFR IV relates with poor prognosis of AML.
Case-Control Studies ; Hematopoietic Stem Cells ; metabolism ; Humans ; Leukemia, Myeloid, Acute ; metabolism ; Protein Isoforms ; metabolism ; Receptors, Granulocyte Colony-Stimulating Factor ; metabolism

OBJECTIVETo investigate the effect of all-trans retinoid acid (ATRA) and granulocyte colony-stimulating factor (G-CSF) on the growth, apoptosis, differentiation and expression of RARα2 of myeloma cells.

METHODSMyeloma cell lines OPM2 (RARα2 positive) and U266 (RARα2 negative) were treated with ATRA in the presence or absence of G-CSF. The cells were divided into 6 groups: control groups, G-CSF groups (treated with 1000 U/ml and 2000 U/ml), ATRA groups (treated with 1.0 μmol/L ATRA) and combined groups (treated with 1000 U/mL or 2000 U/mL G-CSF plus 1.0 μmol/L ATRA). The cell viability, growth and apoptosis were examined by MTT method, inverted microscopy and Annexin-V/PI staining, respectively; RARα2 expression was detected by reverse transcription PCR; morphology change was evaluated by Wright-Giemsa staining; CD49e expression were analyzed by flow cytometry.

RESULTSThe proliferation of OPM2 cells was inhibited by ATRA treatment (P<0.05) . The growth inhibition rates in combined groups were higher than corresponding single ATRA groups (P<0.05). However, the above effects in U266 cells were not significant (P >0.05). The OPM2 cell stained by Wright-Giemsa in ATRA groups showed that the cell nucleus became smaller, chromatin condensed, number of nucleolus reduced, the volume of cytoplasm increased and the cytoplasm became dark blue. Expression rates of CD49e were low in both U266 and OPM2 cells. Expression of RARα2 in OPM2 cells of combination groups were higher than those of control group and corresponding single groups (P<0.05); and there was no significant difference between control group and G-CSF groups (P>0.05). Expression of RARα2 in U266 cells of control group and G-CSF groups was not detected; and ATRA groups and combination groups had weak expression.

CONCLUSIONATRA can induce proliferation inhibition in RARα2-expressing myeloma cells, and it may also play a certain role in promoting differentiation of RARα2 positive myeloma cells.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Humans ; Multiple Myeloma ; metabolism ; pathology ; Receptors, Retinoic Acid ; metabolism ; Retinoic Acid Receptor alpha ; Tretinoin ; pharmacology

This study was to investigate the differential regulation of CCR5 expression on T cells in healthy donors after mobilization with recombinant human granulocyte colony-stimulating factor (rhG-CSF) and analyze its correlation with acute graft-versus-host disease (aGVHD) so as to understand the possible mechanisms underlying rhG-CSF-induced immune tolerance. Sixty-eight related healthy donor and their corresponding recipient for allogeneic hematopoietic stem cell transplantation (allo-HSCT) were enrolled in this study. The expression of CCR5 on CD4(+) and CD8(+) T cells in the peripheral blood (PB) before and after mobilization were detected by using flow cytometry (FCM) respectively. According to the changes of CCR5 expression on CD4(+) and CD8(+) T cells, the Sixty-two evaluable donors were divided into the downregulated and unchanged/upregulated (non-downregulated) groups, and the incidence of grades II to IV aGVHD in two groups were compared. The results showed that the mean value of CCR5 expression on CD4(+) and CD8(+) T cells in PB was not different significantly after mobilization (P > 0.05). Apparent inconsistency was showed among different individuals. Thirty-four (50%) donors displayed downregulation of CCR5 expression, while 34 (50%) donors manifested unchanged or upregulated CCR5 expression on CD4(+) T cells. CCR5 expression on CD8(+) T cells was downregulated in 42 (61.8%), unchanged or upregulated in 26 (38.3%) donors. The cumulative incidence of grades II to IV aGVHD in the downregulated and non-downregulated groups for CD4(+) T cells were 16.1% and 41.9% (P = 0.032), and recipients with CCR5 downregulation on CD8(+) T cells showed an increased tendency of developing aGVHD (37.8% vs 16.0%, P = 0.065). In conclusion, rhG-CSF mobilization could lead to differential regulation of CCR5 expression on T cells, which might influence the migration of T cells in vivo, decrease T cell trafficking towards GVHD target organs, and thus reduce the incidence of aGVHD after transplantation.
Adolescent ; Adult ; Blood Donors ; Child ; Child, Preschool ; Female ; Gene Expression Regulation ; Graft vs Host Disease ; pathology ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Male ; Middle Aged ; Receptors, CCR5 ; metabolism ; T-Lymphocytes ; drug effects ; metabolism ; Young Adult

To explore the reasonable procedures and strategies of diagnosis and treatment of congenital neutropenia (CN), clinical data and laboratory examination results of a boy suspected of CN were collected; gene ELA2, GFI1, HAX1, and WASp of whom were sequenced, granulocyte colony-stimulating factor receptor (G-CSFR) expression on neutrophil was analyzed, and cytoplasmic domain of G-CSFR was sequenced. The results showed that the diagnosis of non-syndromic variants of CN (NSVCN) was made on this patient according to the criteria; sequencing results revealed no mutation occurred in ELA2, GFI1, HAX1 and WASp; a normal expression level of G-CSFR on neutrophil from this patient was detected and no truncated mutation was found in the intracellular domain of G-CSFR. It is concluded that reasonable procedure of diagnosis and treatment of CN is established, and a sporadic NSVCN with no recognized pathogenic mutation is confirmed in this patient.
Child ; DNA Mutational Analysis ; Humans ; Male ; Neutropenia ; congenital ; diagnosis ; genetics ; therapy ; Receptors, Granulocyte Colony-Stimulating Factor ; metabolism

Granulocyte-colony stimulating factor (G-CSF) is a naturally occurring glycoprotein that stimulates the proliferation and maturation of precursor cells in the bone marrow into fully differentiated neutrophils. Several reports of G-CSF-producing malignant tumors have been published, but scarcely any in the hepatobiliary system, such as in hepatocellular carcinoma (HCC). Here, we encountered a 69-yr-old man with a hepatic tumor who had received right hepatic resection. He showed leukocytosis of 25,450/microL along with elevated serum G-CSF. Histological examination of surgical samples demonstrated immunohistochemical staining for G-CSF, but not for G-CSF receptor. The patient survived without recurrence for four years, but ultimately passed away with multiple bone metastases. In light of the above, clinicians may consider G-CSF-producing HCC when encountering patients with leukocytosis and a hepatic tumor. More cases are needed to clarify the clinical picture of G-CSF-producing HCC.
Aged ; Bone Neoplasms/secondary ; Carcinoma, Hepatocellular/*metabolism/pathology ; Fatal Outcome ; Granulocyte Colony-Stimulating Factor/*metabolism ; Humans ; Liver Neoplasms/*metabolism/pathology ; Male ; Receptors, Granulocyte Colony-Stimulating Factor/metabolism

This study was purposed to investigate the effect of gemcitabine (GEM) on granulocyte colony-stimulating factor receptor (G-CSFR) and bcr/abl mRNA in patients with chronic myeloid leukemia (CML). 23 cases of CML in chronic phase, 8 cases of CML in blastic phase and 10 cases of non-hematologic diseases with normal bone marrow were enrolled in this study. The bone marrow from all these cases was collected and divided into 2 group: GEM group (bone marrow cells of CML patients and normal bone marrow cells were cultured with 10 µg/ml GEM for 48 hours) and control group (above-mentioned bone marrow cells were cultured without GEM for 48 hours). The expression of G-CSFR was detected by flow cytometry, the expression of bcr/abl mRNA was assayed by RT-PCR. The results showed that the G-CSFR expression rates of bone marrow in CML chronic phase and blastic phase as well as normal bone marrow in GEM group were (50.72±8.57)%, (36.32±4.25)% and (59.42±7.62)% respectively, while the G-CSFR expression rates of above-mentioned bone marrow in control group were (45.42±6.52)%, (30.58±5.68)% and (58.56±5.54)% respectively. The comparison of G-CSFR expression rates between bone marrow of CML and normal bone marrow, between bone marrow of chronic phase and blastic phase and between bone marrow of GEM group and control group all demonstrated significant difference (p<0.05). The RT-PCR assay showed that the expressions of bcr/abl mRNA in CML chronic and blastic phases of GEM group were (0.59±0.15)% and (0.60±0.13)% respectively, while above-mentioned indicators of control group were (0.60±0.10)% and (0.63±0.11)%; there was no significant difference on expression of bcr/abl mRNA between GEM and control groups (p>0.05). The negative correlation of G-CSFR expression rate with bcr/abl mRNA expression level was observed in GEM and control groups as well as in CML chronic phase and blastic phase of GEM group (r=-0.747, p<0.01; r=-0.803, p<0.01 respectively). It is concluded that the GEM can in vitro enhance the expression rate of bone marrow G-CSFR in CML patients at chronic or blastic phases, but no significant effect on expression of bcr/abl mRNA. The negative correlation of G-CSFR expression rate with bcr/abl mRNA expression level exists in CML patients at chronic or blastic phases.
Adolescent ; Adult ; Bone Marrow ; metabolism ; pathology ; Case-Control Studies ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; pathology ; Middle Aged ; RNA, Messenger ; genetics ; Receptors, Granulocyte Colony-Stimulating Factor ; genetics ; metabolism ; Tumor Cells, Cultured ; Young Adult

BACKGROUNDRecent studies have shown that interleukin-3 receptor alpha (CD123) is highly expressed on leukemia stem cells of patients with acute myeloid leukemia, and is correlated with tumor load and poor prognosis. The expression of CD123 may also be high in patients with myelodysplastic syndrome (MDS). In this study, the expression and clinical significance of CD123 and granulocyte colony stimulating factor (G-CSF) receptor (CD114) on the bone marrow cells of patients with MDS were investigated to explore the molecular marker of the malignant clone of MDS.

METHODSForty-two patients with MDS, who were diagnosed in the Hematological Department of General Hospital of Tianjin Medical University from 2008 to 2009, and twelve normal controls were enrolled in this study. Fluorescence activiated cell sorter (FACS) was used to measure the expression of CD123 on CD34(+)CD38(-) cells and CD114 on CD34(+) cells of the bone marrow of these patients and controls and the clinical significance was analyzed. The expression of CD114 on CD123(+)CD34(+)CD38(-) cells was further measured to explore the molecular marker of the malignant clone in MDS.

RESULTSMDS patients displayed significantly higher proportion of CD34(+)CD38(-)/CD34(+) ((14.03 +/- 5.27)%) than normal controls ((7.70 +/- 4.36)%, P < 0.05). The expression rate of CD123(+)CD34(+)CD38(-)/CD34(+)CD38(-) was significantly higher in MDS patients ((48.39 +/- 28.15)%) than that in normal controls ((8.75 +/- 11.71)%, P < 0.01). The expression level of CD123 was significantly correlated with the proportion of bone marrow blasts (r = 0.457, P < 0.05). The expression rate of CD114(+)CD34(+)/CD34(+) was lower in MDS patients ((33.05 +/- 21.71)%) than that in normal controls ((38.99 +/- 19.07)%) but was not statistically significant (P > 0.05). The expression of CD114 on CD123(+)CD34(+)CD38(-) cells ((34.82 +/- 29.58)%) was significantly lower than that on CD123(-)CD34(+)CD38(-) cells ((53.48 +/- 27.41)%) of MDS patients (P < 0.05).

CONCLUSIONSMDS patients displayed higher proportion of CD34(+)CD38(-)/CD34(+) than normal controls. CD123 was highly expressed in the bone marrow of the patients with MDS, significantly correlated with the proportion of bone marrow blasts, and thus might be the marker of MDS malignant clone. CD123(+)CD34(+)CD38(-) cells exhibited lower expression of G-CSF receptors, which might partly explain why MDS clone responds worse to G-CSF in vitro and in vivo.

ADP-ribosyl Cyclase 1 ; metabolism ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD34 ; metabolism ; Bone Marrow Cells ; metabolism ; Cells, Cultured ; Female ; Humans ; Interleukin-3 Receptor alpha Subunit ; metabolism ; Male ; Middle Aged ; Myelodysplastic Syndromes ; metabolism ; Receptors, Granulocyte Colony-Stimulating Factor ; metabolism ; Young Adult