BACKGROUNDLymphocyte subsets play important roles in rejection in liver transplant recipients, and the effect of splenic function on these roles remains unknown. The aim of this study was to explore the feasibility to adjust immunosuppressive agents based on splenic function status through detecting the lymphocyte subsets in liver transplantBeijing recipients.

METHODSThe lymphocyte subsets of 49 liver transplant recipients were assessed in the 309th Hospital of Chinese People's Liberation Army between June 2014 and August 2015. The patients were divided into splenectomy group (n = 9), normal splenic function group (n = 24), and hypersplenism group (n = 16). The percentages and counts of CD4+ T, CD8+ T, natural killer (NK) cell, B-cell, regulatory B-cell (Breg), and regulatory T-cell (Treg) were detected by flow cytometer. In addition, the immunosuppressive agents, histories of rejection and infection, and postoperative time of the patients were compared among the three groups.

RESULTSThere was no significant difference of clinical characteristics among the three groups. The percentage of CD19+CD24+CD38+ Breg was significantly higher in hypersplenism group than normal splenic function group and splenectomy group (3.29 ± 0.97% vs. 2.12 ± 1.08% and 1.90 ± 0.99%, P = 0.001). The same result was found in CD4+CD25+FoxP3+ Treg percentage (0.97 ± 0.39% vs. 0.54 ± 0.31% and 0.56 ± 0.28%, P = 0.001). The counts of CD8+ T-cell, CD4+ T-cell, and NK cell were significantly lower in hypersplenism group than normal splenic function group (254.25 ± 149.08 vs. 476.96 ± 225.52, P= 0.002; 301.69 ± 154.39 vs. 532.50 ± 194.42, P= 0.000; and 88.56 ± 63.15 vs. 188.33 ± 134.51, P = 0.048). Moreover, the counts of CD4+ T-cell and NK cell were significantly lower in hypersplenism group than splenectomy group (301.69 ± 154.39 vs. 491.89 ± 132.31, P= 0.033; and 88.56 ± 63.15 vs. 226.00 ± 168.85, P = 0.032).

CONCLUSIONSplenic function status might affect the immunity of liver transplant recipients, that should be considered when we make immunosuppressive protocols.

CD4-Positive T-Lymphocytes ; drug effects ; immunology ; Female ; Humans ; Hypersplenism ; immunology ; Immunosuppressive Agents ; administration & dosage ; therapeutic use ; Killer Cells, Natural ; drug effects ; immunology ; Liver Transplantation ; methods ; Lymphocyte Subsets ; drug effects ; immunology ; Male ; Middle Aged ; Retrospective Studies ; Sirolimus ; administration & dosage ; therapeutic use ; Spleen ; drug effects ; immunology ; T-Lymphocytes, Regulatory ; drug effects ; immunology

To investigate the effect of Tanreqing injection on immune activity of peripheral blood lymphocytes of patients with lung cancer. The peripheral blood lymphocytes of patients with lung cancer and healthy persons were separated by the density gradient centrifugation method for subsequent experiments, with those from healthy persons as the positive control. The effect of Tanreqing injection on stimulating the proliferation of lymphocytes with phytohemagglutinin (PHA) was determined by MTT method. The effect of Tanreqing injection on the lymphocyte secretions of IFN-γ and TNF-α and the subset ratio of lymphocytes cultured separately or with Tanreqing injection of different concentrations were examined by ELISA and flow cytometry (FCM) respectively. In addition, the LDH release assay was used to detect the cytotoxicity of cytotoxic T cells (CTL) and natural killer cells (NK). According to the findings, all of immunological indexes of lymphocytes from patients with lung cancer were weaker than that of healthy persons, but with the obvious increases in proliferation activity and IFN-γ and TNF-α secretions of lymphocytes co-cultured with Tanreqing Injection (P < 0.05). Among lymphocyte subsets co-cultured with Tanreqing Injection, CD3+, CD3+ CD4+ and CD3- CD16 + 56+ cell ratios notably increased, whereas CD4+ CD25+ Treg cell ratio obviously decreased (P < 0.05). In the meantime, Tanreqing injection can markedly promote the cytotoxicities of CTL and NK (P < 0.05). In conclusion, Tanreqing injection shows a significant effect in promoting the immune activity of lymphocytes from patients with lung cancer and their anti-tumor immunity.
Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Interferon-gamma ; genetics ; immunology ; Killer Cells, Natural ; drug effects ; immunology ; Lung Neoplasms ; drug therapy ; genetics ; immunology ; physiopathology ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Tumor Necrosis Factor-alpha ; genetics ; immunology

OBJECTIVETo investigate NK cell cytotoxicity to leukemic cell by NKG2D receptors and NKG2D ligands interaction upregulated by hydroxyurea (HU).

METHODSLeukemic cell lines OUN-1 and primary leukemic cells were cultured for 24 hours in the presence of HU, then the NKG2D ligands expressions were analyzed by flow cytometry (FCM). Isolated NK cells from healthy individual cultured for 72 hours in presence of IL-2 were used as effect cell, and leukemic cell line OUN-1 treated with HU was used as target cell, NK cell cytotoxicity against leukemic cell line was assessed using chromium-51 release assay.

RESULTSLeukemic cell lines showed upregulation of MIC A/B (MFI: 8.9 ± 0.9 vs 23.5 ± 3.4, P = 0.01) and ULBP2 (MFI: 14.5 ± 0.6 vs 33.5 ± 4.8, P = 0.03) following incubation with HU. HU also upregulated the NKG2DLs on primary leukemia cells from patients with acute myeloid leukemia. Treatment of OUN-1 with HU significantly increased the cytotoxicity of NK cells isolated from healthy individual \[(62.0 ± 5.6)% vs (76.0 ± 5.3)%, P = 0.02\], and the enhancing effect of HU was partly blocked by anti-NKG2D Abs \[(76.0 ± 5.3)% vs (46.0 ± 4.5)%, P = 0.00\].

CONCLUSIONHU selectively upregulated NKG2D ligand expression on leukemic cell lines, and enhanced NK cell cytotoxicity against leukemic cells through NKG2D receptors and NKG2D ligands interaction.

Cell Line, Tumor ; Humans ; Hydroxyurea ; pharmacology ; Killer Cells, Natural ; drug effects ; immunology ; Leukemia ; immunology ; Ligands ; NK Cell Lectin-Like Receptor Subfamily K ; immunology

Animals ; Fatty Liver ; immunology ; pathology ; Male ; Natural Killer T-Cells ; drug effects ; Non-alcoholic Fatty Liver Disease ; Probiotics ; pharmacology ; Rats ; Rats, Wistar

OBJECTIVETo investigate the effect of blocking the inhibitory receptors KIR2DL1 and KIR2DL2/2DL3 with monoclonal antibody on cytotoxic activity of human NK cells.

METHODSHuman peripheral blood NK cells were isolated by Rosettesep NK sorting kit. The cytotoxic activity of NK cells against human leukemia NB4, K-562, Raji cells and allogeneic mature or dendritic cells (DCs) was detected before or after KIR2DL1 and KIR2DL2/2DL3 were blocked. The effect of NK cells on T lymphocyte proliferation was detected by mixed lymphocyte reaction and TGF-β1 concentration in culture supernatant was measured.

RESULTSThe cytotoxicity of NK cells to NB4 cells was augmented with increasing concentration of the antibody. Combination of both antibodies enhanced killing activity of NK cells. NK cells had strong cytotoxicity to K-562 cells, but were not enhanced by the blockade of inhibitory receptors. The cytotoxicity to Raji cells was not evidently augmented. The cytotoxicity of NK cells to mature DC was enhanced remarkably with the increase of concentration of the antibodies (2.20% ±1.10% compared with 37.59% ±5.06%, P<0.05). In mixed lymphocyte reaction, the blockade of two antibodies enhanced the inhibition effect of NK cells on T cell proliferation (77.85% ± 8.31% compared with 43.05% ± 5.95%, P<0.05) and the content of TGF-β1 in the supernatant was increased.

CONCLUSIONThe cytotoxic effects of human NK cells against target cells were significantly enhanced with the blockade of inhibitory KIR receptor; and the cytokine TGF-β1 secreted by NK cells further inhibits T cells proliferation.

Antibodies, Monoclonal ; immunology ; pharmacology ; Cell Line ; Cells, Cultured ; Cytotoxicity, Immunologic ; drug effects ; immunology ; Dendritic Cells ; immunology ; Humans ; Killer Cells, Natural ; drug effects ; immunology ; metabolism ; Lymphocyte Culture Test, Mixed ; Receptors, KIR2DL1 ; drug effects ; immunology ; Receptors, KIR2DL2 ; drug effects ; immunology ; Receptors, KIR2DL3 ; drug effects ; immunology ; T-Lymphocytes ; immunology ; Transforming Growth Factor beta1 ; metabolism

BACKGROUNDActivation in vitro of natural killer T (NKT) cells in systemic lupus erythematosus (SLE) with α-galactosylceramide (α-GalCer) and dendritic cells (DC) may affect the immunoregulatory role of NKT cells. This study was designed to compare the number of NKT cells in patients with SLE to the number in healthy volunteers and measure the cytokines secreted from these NKT cells in vitro.

METHODSThree sets of culture conditions using (i) α-GalCer, (ii) DC, or (iii) both α-GalCer and DC (α-GalCer+DC) were adopted to expand NKT cells from peripheral blood mononuclear cells (PBMC) of patients with SLE and healthy volunteers. Flow cytometry was used to assess the levels of interleukin (IL)-4, IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α produced by the Vα24(+)Vβ11(+) NKT cells.

RESULTSAfter 14 days in culture, the total cell count and percentage of Vα24(+)Vβ11(+) NKT cells were increased under all conditions but were highest in the α-GalCer+DC group. The level of IL-4 and IL-10 secreted by Vα24(+)Vβ11(+) NKT cells from patients with active SLE was found to be higher than that of inactive patients and the control group (P < 0.05), while the levels of IFN-γ and TNF-α were lower than those found in the inactive and control groups (P < 0.05).

CONCLUSIONSVα24(+)Vβ11(+) NKT cells showed the greatest expansion in vitro with α-GalCer and DC. Th2-type cytokines from Vα24(+)Vβ11(+) NKT cells are the predominant type in patients with SLE, while Th1 cytokines predominate in the control group. This evolution of NKT cell function during the progression of the disease may have important implications in understanding the mechanism of SLE and for the development of possible therapies using NKT cell agonists.

Adolescent ; Adult ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cytokines ; metabolism ; Dendritic Cells ; metabolism ; Female ; Flow Cytometry ; Galactosylceramides ; pharmacology ; Humans ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-4 ; metabolism ; Lupus Erythematosus, Systemic ; immunology ; metabolism ; Male ; Middle Aged ; Natural Killer T-Cells ; cytology ; drug effects ; metabolism ; Receptors, Antigen, T-Cell ; metabolism ; Receptors, Antigen, T-Cell, alpha-beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Young Adult

The aim of this study was to explore the cytotoxicity of fresh cord blood(CB) NK cells and the influence of IL-12 and IL-15 on activity of the NK cells killing K562 and Jurkat cells lines. The NK cells were isolated from cord blood by depleting CD3(+) cells and then enriching CD56(+) cells using sorting with immunomagnetic beads. The experiment was divided into 3 groups: group A (fresh CB-NK cells without cytokines), group B (CB-NK cells cultured by IL-2) and group C (CB-NK cells cultured by IL-2 and IL-15). The purity of NK cells was determined by flow cytometry; the cytotoxity of fresh and different cytokine-treated CB-NK cells on K562 and Jurkat cell lines was detected by LDH release test. The results showed that the purity of NK cells before and after sorting was 14.88 ± 9.2% and 92.39 ± 0.8% respectively. After culture for 3 days, NK-forming colony amounts in group B and group C were 148.60 ± 13.0 and 831.80 ± 23.0 respectively, the comparison between group B and group C showed the significant difference (p < 0.05). The cytotoxicities of NK cells in group A, B and C on K562 and Jurkat cell lines were 27.76 ± 8.8%, 61.90 ± 9.1% and 87.62 ± 3.7%; 29.32 ± 2.5%, 69.43 ± 4.4% and 92.95 ± 3.2% respectively, the difference was significant (p < 0.05). It is concluded that the fresh isolated CB-NK cells show low cytotoxic activity. After stimulated with IL-2 or IL-2 plus IL15, cytotoxicity of CB-NK cells increases obviously, the effect of IL-2 plus IL-15 is much better than IL-2 alone for promoting the growth and enhancing the cytotoxicity of CB-NK cells.
Cytotoxicity, Immunologic ; drug effects ; immunology ; Fetal Blood ; drug effects ; immunology ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Jurkat Cells ; K562 Cells ; Killer Cells, Natural ; drug effects ; immunology

OBJECTIVETo explore the effect of Yinqiao detoxifcation oral liquid on activity of natural killer cells (NK) and serum content of TNF-alpha, TGF-beta1 of BALB/C nude mouse infected by influenza virus.

METHODTo establish infected mice model by FM1 followed by intragastric administration of Yinqiao detoxifcation oral liquid for treatment. LDH method was used to observe NK cells. ELISA method was used to determine the levels of TNF-alpha, TGF-beta1, in serum on 1st, 3rd, 5th, 7th days after infection.

RESULTComparing to the normal group, the NK activity of the model group was significantly increased on 1 dpi (day post infection), and significantly decreased on 3, 5, 7 dpi. The NK activity of three dosage groups (5, 10, 20 g x kg(-1)) of Yinqiao detoxifcation oral liquid were respectively higher than that of the model on 3, 5, 7 dpi, especially with high dose (P < 0.01). The serum level of TNF-alpha and TGF-beta1 of model group is higher than that of normal group on 1, 3, 5, 7 d. Compared with model group, the serum level of Yinqiao detoxifcation oral liquid groups (5, 10, 20 g x kg(-1)) were decreased in different degree on every time point, especially the serum level of the higher dose of Yinqiao detoxifcation oral liquid decreased on 3 dpi (P < 0.05), Yinqiao detoxifcation oral liquid inhibit the serum level of TGF-beta1 in a dose-dependent manner.

CONCLUSIONYinqiao detoxifcation oral liquid could enhance the activity of NK cell and decrease the serum level of TNF-alpha and TGF-beta1 of the mice infected by influenza virus.

Administration, Oral ; Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Influenza A virus ; immunology ; physiology ; Influenza, Human ; drug therapy ; immunology ; virology ; Killer Cells, Natural ; drug effects ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Random Allocation ; Transforming Growth Factor beta1 ; immunology ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVETo explore the dynamic effects of Yinqiao detoxifcation oral liquid (YQD) on NK cell lysis in spleen and serum concentration of IFN-gamma in SCID mouse infected by influenza A virus FM1.

METHODThe mice were divided into six different groups randomly. Except normal control, other mice were intranasally instilled with 15 TCID of virus. Three dosage groups of YQD (5.0, 10.0, 20.0 g x kg(-1)) were respectively fed with YQD. Positive control group was administrated orally with 0.07 g x kg(-1) of ribavirin. Normal control and model group were fed with physiological saline. After 1, 3, 5 and 7 days' infection, spleens and serum were collected. Then the NK cell lysis was detected by LDH release kit and the concentration of IFN-gamma was examined by ELISA assay.

RESULTContents of IFN-gamma reached to peak value on the 3th day and until the 5th day. Later, the level of IFN-gamma returned to normal level. The variation tendency of activity of NK cell in spleen was according with that of IFN-gamma. But it reached the maximum value until the 5th day. The activity of NK cell lysis in three groups of YQD was well above that i n model group. In addition, therapeutic action of both 10.0 g x kg(-1) and 20.0 g x kg(-1) of YQD treatment groups was better than that of 5.0 g x kg(-1).

CONCLUSIONThe data showed that serum concentration of IFN-gamma and NK cell lysis were improved by YQD at different time, which was demonstrated YQD could perform well in immune system.

Animals ; Cell Survival ; drug effects ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Humans ; Influenza, Human ; blood ; drug therapy ; immunology ; physiopathology ; Influenzavirus A ; physiology ; Interferon-gamma ; blood ; Killer Cells, Natural ; drug effects ; physiology ; Mice ; Mice, SCID ; Random Allocation ; Spleen ; immunology ; physiopathology

Immune reconstitution is crucially relevant for patients receiving hematopoietic stem cell transplantation (HSCT). This study was purposed to investigate the ability of α-GalCer (α-galactosylceramide), a well-known activator of natural killer T cells (NK-T), to enhance immune and hematological reconstitution. Lethally irradiated BALB/c mice were transplanted with allogeneic C57BL/6 bone marrow cells and splenocytes. α-GalCer was administered immediately after HSCT. After transplantation, the weight, activity, hairs, diarrhea and survival time of mice were observed daily; the blood routine test was performed once weekly; the donor chimeras, amount of mononuclear cells in spleen (MNC) and relative levels of CD3(+), CD4(+), CD8(+), B220(+), CD11c(+), CD40(+), CD86(+) and CD80(+) cells were detected by FACS on day 2, 7, 14, 27, 70 after transplantation. The results indicated that the MNC counts and relative levels of CD3(+) and CD4(+) in group treated with α-GalCer on day 2 after transplantation were higher than those in control group; at the same time, the detected donor chimeras were complete recipient type chimeras, then gradually transformed into donor type, on day 7 - 14 donor chimeras in α-GalCer group were enhanced significantly as compared with control group, on day 27 the chimeras in two groups were complete donor type chimeras thereafter to day 70, the MNC count and relative levels of CD3(+), CD4(+), CD8(+), B220(+), CD40(+), CD86(+) cells in α-GalCer group were obviously higher than those in control group, at the same time, the hematopoietic reconstitution in α-GalCer group was accelerated as compared with control group. It is concluded that the α-GalCer administration after allogeneic bone marrow transplantations accelerates immune and hematological reconstitution.
Animals ; Bone Marrow Transplantation ; immunology ; methods ; Chimera ; Female ; Galactosylceramides ; pharmacology ; Leukocyte Count ; Lymphocyte Activation ; drug effects ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Natural Killer T-Cells ; drug effects ; immunology ; Postoperative Period