Third-party regulatory T cells prevent murine acute graft-versus-host disease

BACKGROUND/AIMS: Adoptive therapy with regulatory T (Treg) cells to prevent graft-versus-host disease (GVHD) would benefit from a strategy to improve homing to the sites of inflammation following hematopoietic stem cell transplantation (HSCT). Although donor-derived Treg cells have mainly been used in these models, third-party-derived Treg cells are a promising alternative for cell-based immunotherapy, as they can be screened for pathogens and cell activity, and banked for GVHD prevention. In this study, we explored major histocompatibility complex (MHC) disparities between Treg cells and conventional T cells in HSCT to evaluate the impact of these different cell populations on the prevention of acute GVHD, as well as survival after allogeneic transplantation. METHODS: To induce acute GVHD, lethally irradiated BALB/c (H-2d) mice were transplanted with 5 × 10⁵ T cell-depleted bone marrow cells and 5 × 10⁵ CD4+CD25– splenic T cells from C57BL/6 (H-2b) mice. Recipients were injected with 5 × 10⁵ cultured donor-, host-, or third-party-derived CD4+CD25+CD62L+ Treg cells (bone marrow transplantation + day 1). RESULTS: Systemic infusion of three groups of Treg cell improved clinicopathological manifestations and survival in an acute GVHD model. Although donor-derived Treg cells were immunologically the most effective, the third-party-derived Treg cell therapy group displayed equal regulation of expansion of CD4+CD25+- Foxp3+ Treg cells and suppressive CD4+IL-17+ T-helper (Th17) cells in ex vivo assays compared with the donor- and host-derived groups. CONCLUSIONS: Our findings demonstrate that the use of third-party Treg cells is a viable alternative to donor-derived Treg cellular therapy in clinical settings, in which human leukocyte antigen-matched donors are not always readily available.

The Role of Protein Kinase C in Acute Lung Injury Induced by Endotoxin / 결핵

BACKGROUND: The signal pathways and their precise roles for acute respiratory distress syndrome caused by endotoxin (ETX) has not been established. Since there has been several in vitro experiments suggesting that activation of protein kinase C (PKC) pathway may be responsible for endotoxin-induced inflammatory reaction, we performed in vivo experiments in the rats with the hypothesis that PKC-inhibition can effectively prevent endotoxin-induced acute lung injury. METHODS: We studied the role of PKC in ETX-induced ALl using PKC inhibitor (staurosporine, 5Th) in the rat. Specific pathogen free male Sprague-Dawley weighted from 165 to 270g were used for the study. Animals were divided into the normal control (NC)-, vehicle control (VC)-, ETX-, PMA (phorbolmyristateacetate)-, STP+PMA-, and STP+ETX-group. PMA (50mg/kg) or ETX (7mg/kg) was instilled through polyethylen catheter after aseptic tracheostomy with and without STP (0.2mg/kg) pretreatment. STP was injected via tail vein 30mm before intratracheal injection (IT) of PMA or ETX. Bronchoalveolar lavage (BAL) was done 3- or 6-hrs after IT of PMA or FTX respectively, to measure protein concentration, total and differential cell counts. RESULTS The results were as follows. The protein concentrations in BALF in the PMA- and ETX-group were very higher than that of VC-group (p<0.001). When animals were pretreated with STP, the %reduction of the protein concentration in BALF was 64.8 8.5 and 30.4 2.5% in the STP+PMA- and STP+ETX-group, respectively (p=0.028). There was no difference in the total cell counts between the PMA-and VC-group (p = 0.26). However the ETX-group showed markedly increased total cell counts as compared to the VC- (p=0.003) and PMA group (p=0.0027), respectively. The total cell counts in BALF were not changed after pretreatment with STP compared to the PMA- (p=0.22) and ETX-group (p=0.46). The percentage of PMN, but not alveolar macrophage, was significantly elevated in the PMA-, and ETX-group. Especially in the ETX-group, the percentage of PMN was 17 times higher than that of PMA (p<0.001). The differential cell counts was not different between the PMA and STP+PMA. On the contrary the STP+ETX-group showed decreased percentage of PMN (p = 0.016). There was no significant relationship between the protein concentration and the total or differential cell counts in each group. CONCLUSION: Pretreatment with PKC-inhibitor (staurosporine) partially but significantly inhibited ETX-in-duced ALI.

An Appreciation of Functional Role of Macrophage in the Acute Lung Injury in the Neutropenic Rat / 결핵

BACKGROUND: It has long been suggested that neutrophils and their products are implicated as the central mediators of the acute lung injuries. Contrary to the dominant role of neutrophils in ARDS, many cases of ARDS has occurred in the setting of severe neutropenia without pufrnonary neutrophil infiltration. Therefore it is certain that effector cell(s) other than neutrophil play an important role in the pathogenesis of ARDS. This experiment was performed to define the mechanism of ARDS in the setting of neutiopenia, 1) by comparing the severity of endotoxin-induced lung injury, 2) by measurement of hydrogen peroxide production and cytokine concentration in the bronchoalveolar lavage cells and fluids obtained from different rats with and without cyclophosphamide-pretreatment. METHOD: The male Sprague-Dawleys were divided into the normal control (NC)-, endotoxin (ETX)-, and cyclophosphamide (CPA)-group in which neutropenia was induced by injecting cyclophosphamide intraperitoneally. Acute lung injury was evoked by injecting lipopolysaccharide (LPS) into a tail vein. The bronchoalveolar lavage (BAL) was performed at 3 and 6 hour after administration of LPS to measure the change of cell counts and concentrations of protein and cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Hydrogen peroxide (HPO) production from BAL cel]s was measured at 6 hour after LPS administration by phenol red microassay with and without zymosan stimulation. RESULTS: The results were as follows. A change of leukocyte counts in the peripheral blood after treatment with CPA More than 95% of total leukocytes and neutrophils were reduced after CPA administration, resulting in severe neutropenia. A change of BAL cells In the ETX-group, the number of total cells (p<0.01) and of macrophage and neutrophll (p<0.05) were increased at 3 and 6 hour after LPS administration compared to those of NC- group. In the CPA-group, the number of total leukocyte and macrophage were not changed after LPS administration, but neutrophil counts were significantly reduced and jt took part in less than 0.1% of total BAL cells (p<0.01 vs NC-group). BAL cells in this group were almost all macrophages (99.7%). A change of protein concentration in the BALF In the ETX-group, protein concentration was increased at 3 hour and was more increased at 6 hour after LPS administration (p<0.05 and <0.01 vs NC-group, respectively). In the CPA-group, it was also significantly elevated at 3 hour after LPS administration (p<0.05 vs NC-group) , but the value was statistically not different from that of ETh-group. The value measured at 6 hour after LPS administration in the CPA-group became lower than that of ETX-group (p<0.05), but showed still a higher value compared to that of NC-group (p<0.05). A change of cytokine concentration in the BALF TNF-alpha and IL-6 were elevated in the ETX- and CPA-group compared to those of NC-group at both time intervals. There was no statistical difference in the values of both cytokines between the ETX- and CPA-groups. Measurement of hydrogen peroxide production from BAL cells There was no intergroup difference of HPO production from resting cells. HPO production after incubation with opsonized zymosan was significantly elevated in all groups. The percent increment of HPO production was highest in the ETX-group (89.0%, p<0.0008 vs NC-group ), and was 42.85 in the CPA-group (p = 0.003 vs NC-group ). Conclusion Acute lung injury in the setting of neutropenia might be caused by functional activation of resident alveola r macrophages.