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

Cytokine-activated T cells (CATs) can be easily expanded and are widely applied to cancer immunotherapy. However, the good efficacy of CATs is rarely reported in clinical applications because CATs have no or very low antigen specificity. The low-efficacy problem can be resolved using T cell antigen receptor-engineered CAT (TCR-CAT). Herein, we demonstrate that NY-ESO-1 HLA-A*02:01-specific high-affinity TCR (HAT)-transduced CATs can specifically kill cancer cells with good efficacy. With low micromolar range dissociation equilibrium constants, HAT-transduced CATs showed good specificity with no off-target killing. Furthermore, the high-affinity TCR-CATs delivered significantly better activation and cytotoxicity than the equivalent TCR-engineered T cells (TCR-Ts) in terms of interferon-γ and granzyme B production and in vitro cancer cell killing ability. TCR-CAT may be a very good alternative to the expensive TCR-T, which is considered an effective personalized cyto-immunotherapy.
Cell Line, Tumor ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; Genetic Engineering ; HLA-A2 Antigen ; metabolism ; Humans ; Immunotherapy, Adoptive ; methods ; Lymphocyte Activation ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes ; immunology

Novel biologics that redirect cytotoxic T lymphocytes (CTLs) to kill tumor cells bearing a tumor associated antigen hold great promise in the clinic. However, the ability to safely and potently target CD3 on CTL toward tumor associated antigens (TAA) expressed on tumor cells remains a challenge of both technology and biology. Herein we describe the use of a Half DVD-Ig format that can redirect CTL to kill tumor cells. Notably, Half DVD-Ig molecules that are monovalent for each specificity demonstrated reduced non-specific CTL activation and conditional CTL activation upon binding to TAA compared to intact tetravalent DVD-Ig molecules that are bivalent for each specificity, while maintaining good drug like properties and appropriate PK properties.
Animals ; Antibodies, Bispecific ; immunology ; Antibodies, Monoclonal ; immunology ; pharmacokinetics ; CD3 Complex ; metabolism ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; ErbB Receptors ; metabolism ; Female ; Humans ; Lymphocyte Activation ; immunology ; Mice, SCID ; Neoplasms ; immunology ; pathology ; Rats, Sprague-Dawley ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVEElevated natural killer lymphocyte cytotoxicity (NKc) has been linked with reproductive problems in women. Here we evaluate the potential benefit of cupping therapy (CT) in reproduction-related immune responses.

METHODSThis was a pilot clinical study. Participants were healthy female volunteers (n = 23) with elevated NKc, and received repeated CT 3 times over 5 d (inner pressure 40-50 kPa, 40 min; 12-15 cups). Lymphocyte subsets, NKc and NK lymphocyte activity (NKa) were measured in blood on day 0 (initial levels, before the first treatment) and days 3, 10 and 17 after the last CT treatment, using the K562-stimulated CD69 expression assay.

RESULTSAs a result of CT manipulations NKa was reduced on days 3 and 10, and NK percentage was reduced on day 10. NKc was most sensitive to CT treatment, resulting in their decreased counts at 3, 10 and 17 d post CT. CT treatment decreased NKc in the majority of individuals (87%), but the magnitude of the effect was variable. Out of 23 subjects 9 (39.1%) had a 2-3 fold decrease of NKc on days 3, 10 and 17; 11 (47.8%) started to show a decrease in NKc later, or more quickly returned to base levels; and only 3 (13%) subjects displayed no effect of CT on NKc. Expectedly, no changes in T-cell subsets (CD3CD4, CD3CD8, HLADR, CD158a) were observed after CT.

CONCLUSIONCT decreased NK cell numbers, their activity and cytotoxicity. Low cost, safety, non-invasive nature and ease of administration make CT a promising approach for NKc down-regulation.

Adolescent ; Adult ; Complementary Therapies ; Cytotoxicity, Immunologic ; Female ; Humans ; Killer Cells, Natural ; immunology ; Lymphocyte Count ; Pilot Projects

PURPOSE: To evaluate tumor-specific immunity and define the mechanisms involved in the cryoimmunologic response, we compared the tumor control efficacy and immunologic responses of cryoablation with those of surgical excision in a tumor rechallenge model. MATERIALS AND METHODS: Sixty BALB/c mice with RENCA tumors that were generated in the left flank area underwent cryoablation or radical excision. The mice successfully treated were rechallenged with RENCA or an undifferentiated colon carcinoma cell line, CT26, in the contralateral right flank area. The recurrence rate after tumor rechallenge in each group was then observed. To assess the immunologic response of each treatment modality, fluorescent-activated cell sorting (FACS) analysis and a cytotoxicity assay using 51Cr release were performed. RESULTS: After reinoculation of the RENCA cells, the rate of tumor growth was significantly higher in the surgical excision group than in the cryoablation group (94.4% vs. 11.1%, p=0.001). In the cryoablation group, the tumor growth rate was significantly increased after rechallenge of CT26 cells compared with RENCA (94.1% vs. 11.1%, p=0.001). The cryoablation group showed an elevated CD3, CD4, CD8 T, and natural killer cell count in the FACS analysis and also showed significantly increased cytotoxicity in the 51Cr release assay compared with the excision group. CONCLUSIONS: These results showed that cryoablation, compared to surgical resection, was more effective in preventing tumor growth after rechallenge with RENCA cells and that this response was tumor-specific, because the CT26 cells did not have the same effect.
Animals ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Carcinoma, Renal Cell/*immunology/pathology/*surgery ; Cell Death ; Cryosurgery/*methods ; Cytotoxicity, Immunologic ; Disease Models, Animal ; Kidney Neoplasms/*immunology/pathology/*surgery ; Lymphocyte Count ; Lymphocytes, Tumor-Infiltrating/immunology ; Mice, Inbred BALB C ; Neoplasm Recurrence, Local/immunology ; Neoplasm Transplantation

Achyranthes bidentata polysaccharides (ABPS) was extracted from the root of A. bidentata. Dendritic cells (DC), which were stimulated with ABPS and/or tumor antigen SW480, were co-cultured with cytokine induced killer cells (CIK) to test the cytotoxic effect on colon cancer cell line SW480. Peripheral blood mononuclear cells (PBMNCs) which were separated from human peripheral blood were cultured to DC and CIK separately. (1) DC were divided into four groups: pure DC served as control group; ABPS (50 mg x L(-1)) stimulated DC served as experimental group; SW480 tumor antigen stimulated DC served as the second experimental group; ABPS (50 mg x L(-1)) and SW480 tumor antigen co-stimulated DC served as the third experimental group. Flow cytometry was used to detect the difference of the positive rate of molecules in the cell surface of DC, include CD80, CD86, CD1c, CD40, HLA-DR (6 samples for each group). (2) The four DC groups were mixed with CIK at the ratio 1:5 and acted as effect cells (DC + CIK groups), and the colon cancer cell line SW480 acted as target cells. The effect cells and the target cells were mixed together at the ratio 30: 1, 20:1 and 10:1 separately, and the CCK-8 kit was used to test the cytotoxic effect on colon cancer cell line SW480. (3) At the mixing ratio 30:1 of effect cells and target cells, ELISA was used to test the level of cytokines secretion, including IL-2, IL-12p70, IL-17 and TNF-alpha, in the liquid supernatant of every test group (3 duplication per sample). The results showed as following: (1) The positive rates of CD80, CD11c, HLA-DR, in the cell surface of DC which was co-stimulated by ABPS (50 mg x L(-1)) and SW480 tumor antigen, were obviously higher than the other DC groups (P < 0.05), and the positive rates of CD86, CD40 were obviously higher than the pure DC group (P < 0.05), and there was no remarkable difference with the other two DC groups. (2) At the mixing ratio 30:1, 20:1 and 10:1 of the effect cells and the target cells, the cytotoxic effect of ABPS stimulated DC + CIK group and SW480 tumor antigen stimulated DC + CIK group was obviously higher than DC + CIK group (P < 0.05), the cytotoxic effect of ABPS and SW480 tumor antigen co-stimulated DC + CIK group was obviously higher than all the other groups. (3) At the mixing ratio 30:1 of the effect cells and the target cells, the secretion levels of IL-12p70 and TNF-alpha in the liquid supernatant of the ABPS and SW480 tumor antigen co-stimulated DC + CIK group were obviously higher than all the other groups (P < 0.05), the secretion levels of IL-2 and IL-17 in the liquid supernatant of every test group have no remarkable difference. The cytotoxic effect of ABPS stimulated DC + CIK on SW480 was obviously increased. The cytotoxic effect of ABPS and SW480 tumor antigen co-stimulated DC + CIK group was obviously higher than all the other.
Achyranthes ; chemistry ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Colonic Neoplasms ; drug therapy ; immunology ; physiopathology ; Cytokine-Induced Killer Cells ; drug effects ; immunology ; Cytotoxicity, Immunologic ; drug effects ; Dendritic Cells ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Interferon-gamma ; immunology ; Interleukin-2 ; immunology ; Polysaccharides ; pharmacology

OBJECTIVETo investigate the cytotoxic effect of γδ T cells from osteosarcoma patients against interferon-γ (IFN-γ)-treated osteosarcoma cells in vitro.

METHODSHuman γδ T cells were amplified by zoledronate from peripheral blood cells of osteosarcoma patients. The expression of Fas on the osteosarcoma cells were measured by flow cytometry and quantitative real-time PCR analysis before and after IFN-γ treatment. The cytotoxicity of γδ T cells against osteosarcoma cells was evaluated with LDH assay.

RESULTSIFN-γ significantly enhanced the susceptibility of the osteosarcoma cell lines HOS and U2OS to the cytotoxicity of γDelta; T cells from osteosarcoma patients (P<0.01). IFN-γ obviously up-regulated the expression of Fas in HOS and U2OS cells (P<0.01). Anti-FasL mAb failed to inhibit the cytotoxicity of γδ T cells in untreated osteosarcoma targets (P>0.05), but significantly impaired γδ T cell cytotoxicity in IFN-γ pre-treated osteosarcoma targets (P<0.01).

CONCLUSIONIFN-γ can enhance the cytotoxic effect of human γδ T cells from osteosarcoma patients against osteosarcoma cells in vitro.

Bone Neoplasms ; metabolism ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Humans ; Interferon-gamma ; pharmacology ; Osteosarcoma ; immunology ; metabolism ; Receptors, Antigen, T-Cell, gamma-delta ; immunology ; T-Lymphocytes, Cytotoxic ; cytology ; drug effects ; immunology ; fas Receptor ; metabolism

OBJECTIVETo observe the effect of mesenchymal stem cells derived from umbilical cord (UC-MSCs) on natural killer (NK) cells-mediated cytotoxicity against dendritic cells (DCs) and explore the mechanism.

METHODSMSCs were isolated from human umbilical cord by collagen digestion and cultured in vitro. NK cells were separated from healthy human peripheral blood by magnetic bead sorting. Mononuclear cells from healthy human peripheral blood were cultured in the presence of granulocyte and macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to obtain the immature DCs. The DCs were then co-cultured with UC-MSCs in the presence of tumor necrosis factor α (TNFα) for 2 days, and the expressions of CD11c and CD86 on DCs and IL-12 level in the culture medium was detected using flow cytometry and ELISA, respectively. The cytotoxicity of NK cells against DCs was analyzed by LDH-releasing assay, and the expressions of ligands for killer activator receptor (MICA/B and ULBP1-3) on the DCs were detected with flow cytometry.

RESULTSCompared with the cytokine-induced DCs, the DCs induced by co-culture with UC-MSCs showed an identical CD11c expression but lowered CD86 expression and IL-12 secretion. The natural killer cells produced a stronger cytotoxicity against UC-MSCs-induced DCs than against cytokine-induced DCs. The UC-MSCs-induced DCs also showed increased expressions of MICA and MICB on the surface.

CONCLUSIONUC-MSCs can enhance NK cells-mediated cytotoxicity against DCs possibly by inhibiting DC maturation and up-regulating the ligands for killer activator receptor on the surface of the DCs.

Cells, Cultured ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; Humans ; Killer Cells, Natural ; cytology ; Mesenchymal Stromal Cells ; cytology ; Umbilical Cord ; cytology

Chuankezhi (CKZ), a new Chinese medicine, plays an important role in immunoregulation. Cytokine-induced killer (CIK) cells have been commonly used for immunotherapy in recent years. In this study, we aimed to investigate the immunoregulatory effect of CKZ on CIK cells. Peripheral blood monocytes were isolated from healthy donors, and CIK cells were generated by culturing monocytes with interferon-gamma (IFN-γ) and interleukin 2. Different concentrations of CKZ were added on day 2. After incubation for 14 days in culture, the antitumor effects of CIK cells were measured by cytotoxicity assay. Flow cytometry was used to explore the effect of CKZ on CIK cell immunophenotype, intracellular cytokine production, and apoptosis. The effect of CKZ on the antitumor activity of CIK cells in nude mice was also investigated. CKZ increased the percentage of CD3+CD56+ CIK cells but did not significantly change the percentage of CD4+, CD8+, or CD4+CD25+ CIK cells. CKZ-conditioned CIK cells showed a greater ability to kill tumor cells, as well as a higher frequency of IFN-γ and TNF-α production, compared with the CIK cells in the control group. CKZ also suppressed the apoptosis of CIK cells in vitro. Furthermore, CKZ combined with CIK cells had a stronger suppressive effect on tumor growth in vivo than the CIK, CKZ, or normal saline control groups. Our results indicate that CKZ enhances the antitumor activity of CIK cells and is a potential medicine for tumor immunotherapy.
Animals ; Apoptosis ; drug effects ; CD3 Complex ; metabolism ; CD56 Antigen ; metabolism ; Cell Line, Tumor ; drug effects ; Cytokine-Induced Killer Cells ; cytology ; drug effects ; immunology ; Cytotoxicity, Immunologic ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Epimedium ; chemistry ; Female ; Humans ; Interferon-gamma ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Morinda ; chemistry ; Neoplasm Transplantation ; Plants, Medicinal ; chemistry ; Tumor Burden ; Tumor Necrosis Factor-alpha ; metabolism

This study was aimed to investigate the cytotoxic effect of the Naja Naja Actra Venom Component (NNAVC) combined with activated immune cells on human acute myeloblastic leukemia line KG1a cells. The cytotoxic effects of NNAVC at different concentrations on KG1a cells were measured by CCK-8 method. LDH releasing assay was used to detect the cytotoxic effects of activated immune cells, NNAVC combined with activated immune cells on KG1a cells and the sensitivity of KG1a treated with NNAVC to activated immune cells. The results showed that the inhibitory rate of NNAVC on KG1a cells increased with the concentration enhancement, the cytotoxicity of activated immune cells at the different effector to target (E:T) ratios(6.25:1, 12.5:1, 25:1) on KG1a cells were 12.30%, 24.85% and 52.26%. The cytotoxicity of NNAVC combined with activated immune cells at the different E:T cell ratios (10:1, 20: 1) on KG1a cells were 56.21% and 85.59%, which were higher than that of NNAVC or activated immune cells alone. The cytotoxicity of activated immune cells at the E: T cell ratio of 10:1 on KG1a cells treated with NNAVC at different concentrations were 25.65%, 31.33%, 28.63% and 16.78%, respectively, and that at the E:T cell ratio of 20: 1 were 40.62%, 44.70%, 44.62% and 40.72%. It is concluded that:both of NNAVC and activated immune cells have lethal effect on KG1a cells, and the combination of NNAVC and activated immune cells can strengthen their effect on KG1a.
Animals ; Cell Line, Tumor ; drug effects ; Cytotoxicity, Immunologic ; Elapidae ; Humans ; Immunocompetence ; Leukemia, Myeloid, Acute ; immunology ; pathology ; Venoms ; pharmacology