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

The effects of Guizhi Fuling capsule and its active ingredient combination within different concentration on SPL proliferate were observed by MTT method. The ratio of CD80/86, CD3CD25 and CD3CD69 was used to evaluate cell activation effects of Guizhi Fuling capsule and its active ingredient combination by FCM. Guizhi Fuling capsule with concentration of 400 mg · L(-1)can promote spleen lymphocyte proliferation, as well as the active ingredient combination, which showed the obvious dose-effect relationship. Compared with control group, the difference has statistical significance (P≤0.01). The result of FCM showed that Guizhi Fuling capsule and its active ingredient combination can promote CD80 and CD86 expression on spleen lymphocyte, and also can increase CD25 and CD69 ratio between spleen CD3+ cells. Compared with control group, the difference has statistical significance (P≤0.01). Thus, Guizhi Fuling capsule and its active ingredient combination may have immune-modulate effects, and the mechanism may have a close relationship with the lymphocyte activation.
Animals ; Capsules ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Immunologic Factors ; pharmacology ; Lymphocyte Activation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes ; drug effects ; immunology

OBJECTIVETo investigate the effects of intragastric administration of human interferon-α (hIFN-α)-transformed Bifidobacterium on immune functions of mice.

METHODSThe E.coli-Bifidobacterium shuttle expression vector containing hIFN-α gene was constructed and transformed into Bifidobacterium. The hIFN-α-transformed Bifidobacterium suspension (1010 /ml) was prepared after induction with 0.2% L-arabinose for hIFN-α expression and administered intragastrically in male Balb/C mice at the dose of 0.1 ml every other day for 2 weeks, with the mice receiving empty vector-transformed Bifidobacteria as the negative control and those having an equal volume of saline as the blank control. The percentages of mononuclear cell subsets in the thymus, spleen and blood were detected in the mice by flow cytometry, and the serum levels of IL-4, IL-12, IFN-γ and TNF-α were assayed using mouse cytokine FlowCytomix Kit.

RESULTSThe percentages of CD3⁺CD8⁺ and CD4⁺CD8⁺ cells in the thymus, CD3⁺CD4⁺, CD3⁺CD8⁺ and CD4⁺CD8⁺ cells in the spleen, and CD3⁺CD8⁺ cells in the blood all increased significantly in IFN group as compared with those in the negative and blank control groups (P<0.01 or 0.05). The serum level of IFN-γ also increased significantly (P<0.05) while IL-4 level remained unchanged in IFN group compared with those in the two groups.

CONCLUSIONIntragastric administration of hIFN-α-transformed Bifidobacterium promotes lymphocyte proliferation and maturation and increases the serum levels of Th1 cytokines in mice.

Animals ; Bifidobacterium ; Cell Proliferation ; Genetic Vectors ; Humans ; Interferon-alpha ; pharmacology ; Interferon-gamma ; blood ; Interleukin-12 ; blood ; Interleukin-4 ; blood ; Lymphocyte Activation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; pharmacology ; Spleen ; cytology ; Th1 Cells ; cytology ; Thymus Gland ; cytology ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo explore the in vitro anti-tumor effect and mechanism of dendritic cell (DC) tumor vaccine induced by astragalus polysacharin (APS).

METHODSPeripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood. DCs obtained from human peripheral blood were cultivated and added with culture solution for in vitro inducing them to immature DCs. On the 5th day of culture, 100 microg/mL (as the final concentration) APS was added to cells in the APS group. DCs were induced to mature in the cytokine groups by adding 20 ng/mL rhTNF-alpha (as the final concentration). Changes of morphology and phenotype of DCs were observed. Mature DCs were sensitized with tumor antigen SGC-7901 and co-cultured with allogeneic T cells. The proliferative function of T lymphocytes was detected by MTT assay. Levels of IL-12 and IFN-gamma in co-cultured supernatant were detected by ELISA. Cytotoxic lymphocytes (CTL) activated by DC were co-cultured with tumor cell SGC-7901. The specific killing capacity of CTL to target cells was detected by LDH release assay.

RESULTSThe morphological observation and phenotypic identification of APS induced DCs were in accordance with the characteristics of mature DCs. APS induced mature DCs could stimulate the proliferation of allogeneic T lymphocytes. The proliferation index of T cells increased with increased ratio of stimulator cells to effector cells (P < 0.05). Levels of IL-12 and IFN-gamma in co-culture supernatant significantly increased in a time-dependent manner (P < 0.05). CTL cells activated by sensitization of DCs could significantly kill tumor cells, and the killing effect increased along with increased effector-to-target ratio.

CONCLUSIONAPS could in vitro induce DCs to mature, promote its antigen-presenting capacity, effectively activate CTLs, and enhance anti-tumor function of the organism.

Antigen-Presenting Cells ; cytology ; drug effects ; immunology ; Cancer Vaccines ; immunology ; Cell Line ; Cell Proliferation ; drug effects ; Coculture Techniques ; Dendritic Cells ; cytology ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Interferon-gamma ; immunology ; Interleukin-12 ; immunology ; Leukocytes, Mononuclear ; cytology ; immunology ; Lymphocyte Activation ; T-Lymphocytes, Cytotoxic ; cytology ; drug effects

The adhesion and polarization of T lymphocytes involved in the adhesive interaction of lymphocyte function-associated antigen 1 (LFA-1) with its ligand intercellular adhesion molecule 1 (ICAM-1). This study was aimed to investigate the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) stimulation in vitro on the adhesion and polarization of CD4⁺ T cells of healthy human in peripheral blood. The peripheral blood mononuclear cells were collected from 12 healthy volunteers. The CD4⁺ T cells were sorted by miniMACS. The sorted CD4⁺ T cells were incubated with rhG-CSF for 24 h, then the adhesion and polarization of CD4⁺ T cells activated by stroma cell-derived factor -1α (SDF-1α) and ICAM-1 were detected by ELISA and inverted microscope. The results showed that the percentage of adhesion CD4⁺T cells in the experimental group (rhG-CSF acting on the healthy adult volunteers) (61.9 ± 5.9)% was lower than that in the control group (healthy adult volunteers without rhG-CSF stimulation) (68.3 ± 7.3)% (P < 0.05). The percentage of polarized CD4⁺T cells in the experimental group (24.3 ± 4.3)% was also lower than that in control group (47.1 ± 5.1)% (P < 0.05). It is concluded that the adhesion and polarization of CD4⁺T lymphocytes can be inhibited after rhG-CSF stimulation.
Aged ; CD4-Positive T-Lymphocytes ; drug effects ; Cell Adhesion ; drug effects ; Cell Movement ; Cell Polarity ; drug effects ; Chemokine CXCL12 ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Humans ; In Vitro Techniques ; Intercellular Adhesion Molecule-1 ; Leukocytes, Mononuclear ; Lymphocyte Activation ; Lymphocyte Function-Associated Antigen-1 ; Middle Aged ; Recombinant Proteins

OBJECTIVEAntigen-presenting cells such as monocytes and dendritic cells (DCs) stimulate T-cell proliferation and activation during adaptive immunity. This cellular interaction plays a role in the growth of atherosclerotic plaques. Tanshinone II A (TSN) had been shown to decrease the growth of atherosclerotic lesions. We therefore investigated the ability of TSN to inhibit human monocyte-derived DCs and their T-cellstimulatory capacity.

METHODSDCs derived from human monocytes cultured with recombinant human interleukin (IL)-4 and recombinant human granulocyte-macrophage colony-stimulating factor were co-cultured with TSN and lipopolysaccharide for 48 h. Phosphate-buffered saline was used as a negative control. Activation markers and the capacity of DCs for endocytosis were measured by flow cytometry, and proinflammatory cytokines were measured by enzyme-linked immunosorbent assays. DCs were co-cultured with lymphocytes to measure T-cell proliferation and IL-2 secretion by mixed lymphocyte reactions.

RESULTSTSN dose-dependently attenuated DC expression of costimulatory molecules (CD86), and decreased expression of major histocompatibility complex class II (human loukocyte antigen-DR) and adhesion molecules (CD54). Moreover, TSN reduced secretion of the proinflammatory cytokines IL-12 and IL-1 by human DCs, and restored the capacity for endocytosis. Finally, TSN-preincubated DCs showed a reduced capacity to stimulate T-cell proliferation and cytokine secretion.

CONCLUSIONSTSN inhibits DC maturation and decreases the expression of proinflammatory cytokines, while impairing their capacity to stimulate T-cell proliferation and cytokine secretion. These effects may contribute to the influence of TSN on the progression of atherosclerotic lesions.

Antigen-Presenting Cells ; drug effects ; Atherosclerosis ; immunology ; pathology ; B7-2 Antigen ; metabolism ; Cell Membrane ; drug effects ; metabolism ; Cytokines ; secretion ; Dendritic Cells ; drug effects ; immunology ; secretion ; Diterpenes, Abietane ; pharmacology ; Endocytosis ; drug effects ; Flow Cytometry ; Humans ; Immunity, Cellular ; drug effects ; Inflammation Mediators ; metabolism ; Lymphocyte Activation ; drug effects

OBJECTIVETo observe if VIR576, an 20-mer peptide derived from the C-proximal subfragment of a1-antitrypsin (a1-AT) which inhibits human immunodeficiency virus type 1 (HIV-1) entry into the target cells by interacting with fusion peptide (FP), can also directly inhibit CD4(+) T cell activation in vitro.

METHODSSplenocytes isolated from DO11.10 OVA Tg mice were stimulated with ovalbumin or concanavalin A to test the effects of VIR576 on antigen-specific or non-antigen-specific T cell activation. Both primary CD4(+)CD25(-) T cells from DO11.10 mice and CD4(+) T cell line A2b were activated with specific antigens to evaluate the effects of VIR576.

RESULTSVIR576 inhibited antigen-specific splenocyte activation but had no significant effect on non-antigen-specific T-cell activation, which bypassed the crosstalk between the CD3-signaling complex and TCR. We furthermore observed that VIR576 could also down-regulate antigen-specific CD4(+) T-cell activation.

CONCLUSIONSGiven the high susceptibility of activated CD4(+) T cells in the mucosa to HIV-1 infection, the inhibitory effects of VIR576 on both HIV entry into the target cells and CD4(+) T-cell activation suggest the potential of VIR576 as a microbicide for prevention of sexual transmission of HIV.

Animals ; CD3 Complex ; CD4-Positive T-Lymphocytes ; drug effects ; HIV Fusion Inhibitors ; pharmacology ; HIV-1 ; Lymphocyte Activation ; drug effects ; Mice ; Mice, Transgenic ; Ovalbumin ; Peptide Fragments ; pharmacology ; alpha 1-Antitrypsin ; pharmacology

Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; CD3 Complex ; metabolism ; Cells, Cultured ; Humans ; Kinetics ; Lectins, C-Type ; metabolism ; Lymphocyte Activation ; T-Lymphocyte Subsets ; drug effects ; immunology ; metabolism

Immunogenicity for medical devices of animal origin is the key and difficult point during immune safety evaluation for these devices. This paper firstly investigated the effect of collagen sponge of animal origin on mouse splenic lymphocyte transformation and proliferation, and then analyzed the influence factors on the MTT method and CFSE method. The results showed that collagen sponge extract cannot significantly induce transformation and proliferation of mouse splenic lymphocyte in vitro.
Animals ; Cells, Cultured ; Collagen ; pharmacology ; Lymphocyte Activation ; drug effects ; Mice ; Porifera ; chemistry ; Spleen ; cytology

This study investigated the effect of arctigenin (Arc) on the cell activation, cytokines expression, proliferation, and cell-cycle distribution of mouse T lymphocytes. Mouse lymphocytes were prepared from lymph node and treated with Phorbol-12-myristate-13-acetate (PMA)/Ionimycin (Ion) and/or Arc. CD69, CD25, cytokines, proliferation and cell cycle were assayed by flow cytometry. The results showed that, at concentrations of less than 1.00 micromol x L(-1), Arc expressed non-obvious cell damage to cultured lymphocytes, however, it could significantly down-regulate the expression of CD69 and CD25, as well as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-6 and IL-10 on PMA/Ion stimulated lymphocytes. At the same time, Arc could also inhibit the proliferation of PMA/Ion-activated lymphocytes and exhibited lymphocyte G 0/G1 phase cycle arrest. These results suggest that Arc possesses significant anti-inflammatory effects that may be mediated through the regulation of cell activation, cytokines expression and cell proliferation.
Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; Arctium ; chemistry ; Cell Cycle Checkpoints ; drug effects ; Cell Proliferation ; drug effects ; Cytokines ; metabolism ; Female ; Furans ; isolation & purification ; pharmacology ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; metabolism ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Interleukin-4 ; metabolism ; Interleukin-6 ; metabolism ; Ionomycin ; pharmacology ; Lectins, C-Type ; metabolism ; Lignans ; isolation & purification ; pharmacology ; Lymphocyte Activation ; drug effects ; Mice ; Mice, Inbred BALB C ; Plants, Medicinal ; chemistry ; T-Lymphocytes ; cytology ; drug effects ; immunology ; Tetradecanoylphorbol Acetate ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism