Indoleamine 2,3-dioxygenase and inducible nitric oxide synthase mediate immune tolerance induced by CTLA4Ig and anti-CD154 hematopoietic stem cell transplantation in a sensitized mouse model.

Cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4Ig) and anti-cluster of differentiation 154 (anti-CD154) are able to block B7/CD28 and CD40/CD154 co-stimulatory signals in T cells. Additionally, they promote hematopoietic stem cell transplantation (HSCT) in sensitized recipients and are able to induce immune tolerance and complete hematopoietic reconstitution. Indoleamine 2, 3-dioxygenase (IDO) and nitric oxide (NO) have been implicated in T cell immune tolerance. The aim of the present report was to study the in vivo tolerogenic mechanisms by which CTLA4Ig and anti-CD154 induce transplantation survival in mice receiving HSCT. BALB/c mice were sensitized via splenocyte transfusion and pretreated with CTLA4Ig plus anti-CD154 on day-7. IDO and inducible nitric oxide synthase (iNOS) inhibitors were applied on days-7 to 0 and the mice were divided into 4 groups (n=10) and injected with IDO every other day. The mice were sacrificed on day 0, and splenocytes were separated to identify CD11c+ antigen-presenting cells, which were subsequently assessed for IDO expression and activity. The concentration of NO was tested using a nitrate reductase kit. Following the acceptance of allogeneic HSCT, mice were tested for homing and engraftment, as well as survival rate. Application of the IDO inhibitor increased the concentration of NO, whereas a decrease in NO resulted in increased IDO activity. Immune tolerance was abrogated in the presence of both IDO and iNOS inhibitors, whereas this effect was not observed with either compound alone. CTLA4Ig and anti-CD154 may induce immune tolerance by affecting the activity of IDO and iNOS. This tolerance was abrogated in the presence of both IDO and iNOS inhibitors. A cross-regulatory pathway was observed between the IDO and NO pathways, in which the inhibition of IDO stimulated the iNOS pathway and vice versa.

[Influence of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice].

This study was aimed to explore the effects of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice', and provide the evidences of acquired immune tolerance for allogeneic bone marrow transplantation. The mice sensitized on 7 day before transplant were divided into 4 groups: (1)CTLA4Ig+ anti-CD154 isotype control IgG; (2)anti-CD154 +CTLA4Ig isotype control IgG; (3)CTLA4Ig and anti-CD154; (4)isotype control IgG of CTLA4Ig and anti-CD154. CTLA4Ig and anti-CD154 used in normal BALB/c mice as isotype control IgG. Each mouse in all groups received CTLA4Ig and anti-CD154 (or corresponding isotype control IgG) 500 µg respectively, and was injected via tail vein on 7 day before transplant. There were 5 mice in each group. The mice were sacrificed on day 0, then the number of CD19(+)CD69(+)B cells, CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- T cells were measured by flow cytometry. Changes of cytokines and sensitized antibody were tested by ELISA or flow cytometry. The results showed that the numbers of CD19(+)CD69(+)B cells were significantly increased in comparison with the normal group (P < 0.01) , whereas the numbers of cells were significantly decreased when blocking B7/CD28 or /and CD40/CD154 co-stimulatory signals (P < 0.01) . Blocking these 2 signals together displayed a synergistic effect (P < 0.01) . The central memory and effector T cells were defined as CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- respectively, those increased significantly after sensitized in comparison with those in normal group, whereas their numbers decreased when blocking B7/CD28 or/and CD40/CD154 co-stimulatory signals. Blocking these two signals together, displayed a synergistic effect (P < 0.01). Cytokines, IgG and IgM in all groups were not significantly different. Sensitizing antibody test showed that the fluorescence intensity of sensitized group significantly increased as compared with normal group, whereas fluorescence intensity of CTLA4Ig or/and anti-CD154 treated groups significantly decreased as compared with sensitized group (P < 0.01) . It is concluded that blocking the B7/CD28 or/and CD40/CD154 co-stimulatory signal can inhibit the cellular and humoral immune function, whereas blocking these two signals together displays a synergistic effect.

[Effects of CD4(+)CD25(+) regulatory T cells on allogeneic hematopoietic stem cell transplantation in sensitized mice].

The aim of this study was to investigate the effects of CD4(+)CD25(+) regulatory T cells (Treg) on allogeneic hematopoietic stem cell transplantation (HSCT) in sensitized mice so as to provide experimental evidence for clinical treatment of allogeneic HSCT rejection in sensitized recipients. The BALB/c mice were divided into 5 groups: group A - mice were sensitized with injection of splenocytes; group B - mice were sensitized with splenocytes and treated with >5×10(5) Treg on day 7 before transplantation; group C - mice were sensitized with splenocytes and treated with 5×10(5) Treg on day 13 and 7 before transplantation; group D - mice were not sensitized, but treated with equal volume of PBS as control; group E - blank control. Each group had 15 mice. On day 0 of transplantation, mice in each group were irradiated lethally with 8 Gy by linear accelerator, and the bone marrow cells of C57BL/6 labeled by fluorescence staining were intravenously injected via the tail vein. The fluorescent cells in peripheral blood and organ tissue were detected by flow cytometry on different time points for homing assessment. Survival rates and hematopoietic reconstitution were also recorded and monitored. The results showed that on 12 and 24 hours after transplantation, as compared with the sensitized group, the number of fluorescence homing cells in different tissue of the applied Treg groups increased significantly and the differences were statistically significant (P < 0.05). The mice in sensitized group and blank control group all died on the 6-13 day, whereas the median survival time of mice in applied Treg once and twice were 15 days and 16 days respectively. Comparing with sensitized group, the difference was statistically significant (P < 0.001), but there was no significant difference between these two groups applied regulatory T cell (P > 0.05). It is concluded that applying Treg can induce immune tolerance of sensitized recipient to allogeneic HSCT and inhibit immune destruction and prolong the survival time, but can not induce full immune tolerance and at last sensitized mice died of rejection of hematopoietic stem cells.

[Effects of blocking co-stimulatory signals on immunotolerance rejection of sensitized recipient after hematopoietic stem cell transplantation].

This research was aimed to explore the effects of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on engraftment of hematopoietic stem cell in the sensitized recipient so as to provide the experimental evidence for the treatment of sensitized recipient's immune rejection after clinical allogeneic hematopoietic stem cell transplantation (HSCT). The BALB/c mice were divided into 4 groups: (1)mice sensitized on 7 day before transplant; (2)mice were sensitized on 7 day before transplant, and injected CTLA4Ig+anti-CD154 applied; (3)normal mice injected by corresponding isotype control IgG of CTLA4Ig and anti-CD154; (4)normal blank control mice. Each group had 15 mice. On day 0, mice of each group were irradiated lethally 8 Gy by linear accelerator, and the bone marrow cells of C57BL/6 labeled by fluorescence staining were injected via the tail vein. The fluorescent cell level in peripheral blood and organ tissue at different time points were detected by flow cytometry (FCM) for homing assessment. Survival rates and hematopoietic reconstitution were also monitored and recorded. The results showed that application of CTLA4Ig anti-CD154 could promote implantation of allogeneic HSC in sensitized recipients, induce the immune tolerance, prolong their survival time and accelerate the hematopoietic reconstitution within 28 days, compared with the sensitized group. It is concluded that applying CTLA4Ig and anti-CD154 can enhance the engraftment of HSCT and induce immune tolerance in the sensitized recipient after allogeneic HSCT and accelerate the hematopoietic reconstitution.

20S-Ginsenoside Rh2 induces apoptosis in human Leukaemia Reh cells through mitochondrial signaling pathways.

20(S)-Ginsenoside Rh2 (GRh2) and ginsenoside Rg3 (GRg3) are members of the protopanaxadiol family and have been investigated for possible chemopreventive activity. This study explored the biological and apoptotic mechanisms induced by 20(S)-GRh2 in human acute leukaemia line-Reh cells. Reh cells were treated with different concentration of 20(S)-GRh2 in vitro. Cell viability was determined by Cell Counting Kit-8 and Annexin V/7-AAD assays. Mitochondrial membrane potential (MMP) was examined through JC-1 staining. Activation of caspases associated with the mitochondria-mediated apoptosis pathway was determined by Western blot. We observed that survival of Reh cells decreased after exposure to 20(S)-GRh2 in a concentration-dependent manner. Moreover, 20(S)-GRh2 can induce mitochondria depolarization of Reh cells as evident in the shift in JC-1 fluorescence from red to green. In addition, 20(S)-GRh2 induced the release of mitochondrial cytochrome c and activation of caspase-9 and caspase-3 in Reh cells. These results indicate that 20(S)-GRh2 could induce apoptosis through the mitochondrial pathway, demonstrating its potential as a chemotherapeutic agent for leukaemia therapy.

[Experimental study on rejection of allogeneic donor bone marrow cells in sensitized recipients].

OBJECTIVE: To establish a murine model of sensitization, and investigate the effect and mechanism of sensitization on allogeneic donor bone marrow cells (BMCs). METHODS: Sensitized BALB/c mice were established by transfusions of allogeneic splenocytes. The donor reactive antibodies were detected by binding and complement-dependent cytotoxicity assays. After irradiation, 1 × 10(7) BMCs of C57BL/6 donor mice were injected into non-sensitized or sensitized BALB/c recipient mice. The distribution pattern of donor BMCs in peripheral blood, spleen and bone marrow of recipient mice were analyzed at different time points (2 h, 12 h and 48 h) post transplantation. Hematopoietic recovery post transplantation was assessed, and survival was monitored. Moreover, sera and splenocytes derived from non-sensitized or sensitized recipients were incubated with allogeneic BMCs in vitro, and the cytotoxic indexes were calculated in the immune experiments. RESULTS: The binding and complement-dependent cytotoxicity assays showed that a high level of donor reactive antibodies was presented in sensitized sera. Compared with the non-sensitized recipients, the homing assay showed significantly decreased distributions of allogeneic donor BMCs in peripheral blood, spleen and femur of sensitized recipients. Non-sensitized recipients survived long term after irradiation, while all the sensitized recipients died within 12-15 days. Fourteen days post transplantation, the white blood cells and BMCs of non-sensitized recipients were (3240 ± 300) × 10(6)/L and (396 ± 27) × 10(6)/femur, respectively; while the white blood cells and BMCs of sensitized recipients were (320 ± 80) × 10(6)/L and (6 ± 2) × 10(6)/femur, respectively; the differences were statistically significant between this two groups (P < 0.05). Seven days post transplantation, the percentage of donor cells in bone marrow of non-sensitized and sensitized recipients was (48.07 ± 4.70)% and (0.77 ± 0.11)%, respectively, and the differences were statistically significant (P < 0.05). Furthermore, the white blood cells and BMCs following transplantation decreased along with time in sensitized recipients. The immune experiments of complement-dependent cytotoxicity reaction, cytotoxic T lymphocytes reaction and antibody-dependent cellular cytotoxicity showed the cytotoxic indexes were higher in sensitized group than the non-sensitized group. CONCLUSION: A sensitized model was established by transfusions of allogeneic spleen cells. Allogeneic donor BMCs were rejected in sensitized recipients, and its mechanism might be through immune impairment pathways.