Allogeneic Spleen Research Articles

Native Spleen Preservation During Visceral Transplantation Inhibits Graft-Versus-Host-Disease Development: Clinical and Experimental Study.

We aimed to assess whether native spleen preservation during visceral transplantation (VT) affects graft-versus-host-disease (GVHD) incidence. GVHD is one of the most severe and frequently lethal hematological complications after VT procedures. Because there is no specific treatment for GVHD, it is imperative to develop a strategy to reduce donor lymphocyte engraftment and proliferation. Our study included both clinical and experimental data. A total of 108 patients were divided into 3 groups: a native spleen preservation group, a native spleen removal with no donor spleen group, and a donor spleen included (allogeneic spleen) group. We also used an allogeneic VT rat model, in which recipients were divided into 2 groups: a native spleen preservation (+SP) group and a native spleen removal (-S) group. Skin rash appearance, histopathological changes, chimerism, and spleen effects on circulating allogeneic T-cells were assessed. The patients with native spleen preservation showed a lower rate of GVHD ( P <.001) and better survival ( P <.05) than those in the other groups. Skin and histological signs of GVHD were lower in the rats in the +SP group ( P <.05). The donor T-cell frequency in the bloodstream and skin was also significantly reduced when the native spleen was preserved ( P <.01 and P <.0001, respectively). The clinical and experimental data indicate that recipient spleen preservation protects against GVHD after VT, and donor cell clearance from the bloodstream by spleen macrophages could be the underlying mechanism. Therefore, spleen preservation should be considered in VT procedures, whenever possible.

Immunomodulatory activity of biosilica water on mouse dendritic cells

Biosilica is a material extracted from the shell (cell wall) of Melosira nummuloides, a type of sea diatom, and is one of the widely distributed biominerals. Recently, some studies have revealed that biosilica has a characteristic bio-modulatory activity compared to synthetic silica. However, there has been little research on the effects and action mechanisms of biosilica in immune cells. In this study, we investigated the effect of biosilica water on mouse dendritic cells (DCs), the most potent antigen-presenting cells in immunity, and whether it may alter the function of DCs. Biosilica water decreased the metabolic activity of DCs at 20% concentration (v/v) and the production of IL-1 beta in a concentration-dependent manner. And also, more cells with fragmented nuclei were observed in the DCs treated with 20% biosilica water compared to other treatment groups. The mixed leukocyte response experiment showed the biosilica water-treated DCs significantly modulated the metabolic activity and proliferation of allogeneic spleen cells compared to control DCs. This result suggests that biosilica water may modulate the antigen-presenting capability of DCs. Taken together, this study shows the immunomodulatory activity of biosilica on DCs and may affect immune responses. Further research is needed to investigate the immunological activity of biosilica water.

AMBRA1 controls antigen-driven activation and proliferation of naive T cells.

AMBRA1 (activating molecule in Beclin1-regulated autophagy) is a member of the BECN1 (BECLIN1) protein complex, and it plays a role in autophagy, cell death, tumorigenesis and proliferation. We recently reported that on T-cell receptor (TCR) stimulation, AMBRA1 controlled both autophagy and the cell cycle with metabolic regulation. Accumulating evidence has shown that autophagy and metabolic control are pivotal for T-cell activation, clonal expansion and effector/memory cell fate decision. However, it is unknown whether AMBRA1 is involved in T-cell function under physiological conditions. We found that T cells in Ambra1-conditional knockout (cKO) mice induced an exacerbated graft versus host response when they were transplanted into allogeneic BALB/c mice. Furthermore, Ambra1-deficient T cells showed increased proliferation and cytotoxic capability toward specific antigens in response to in vivo stimulation using allogeneic spleen cells. This enhanced immune response mainly contributed to naive T-cell hyperactivity. The T-cell hyperactivity observed in this study was similar to those in some metabolic factor-deficient mice, but not those in other pro-autophagic factor-deficient mice. Under the static condition, however, naive T cells were reduced in Ambra1-cKO mice, the same as in pro-autophagic factor-deficient mice. Collectively, these results suggested that AMBRA1 was involved in regulating T cell-mediated immune responses through autophagy-dependent and -independent mechanisms.

Tolerance Induction in Neonatal Mice: Exhaustion of CD8 T Cells in Allogeneic Bone Marrow Tolerizing Inoculum Fails to Induce GVHD and Leads to Prolonged Donor Heart Graft Survival

Adult allogeneic (allo) spleen cells (SC) induce lethal aGVHD in newborn mice whereas bone marrow cells (BMC) induce non-robust transplant tolerance. To understand these differences we examined CD8 T cells from both inocula before and after injection into neonatal mice, analyzing their molecular phenotypes and trafficking/fates. Neonatal C3H (H-2k) mice were injected iv with either total adult B6 (H-2b) GFP+ SC or BMC, or purified CD8 T cells. Cell trafficking and fates were monitored by whole body/organ imaging. GVHD was defined by reduced growth/viability and tolerance by donor-type heart transplant survival. Cell phenotypes were examined by flow cytometry. Neonates injected with allo-SC developed lethal aGVHD whereas those injected with allo-BMC acquired a non-robust heart transplant tolerance. Both allo-SC and -BMC trafficked to secondary lymphoid organs, proliferated and then spread throughout the body by day 6, suggesting systemic inflammation. CD8 T cells purified from both inocula also proliferated and spread throughout the mice, indicating direct activation. Despite similar proliferation/ trafficking, purified SC CD8 T cells induced lethal aGVHD in 100% mice (n=4/4) whereas the same number of purified BMC CD8 T cells did so infrequently (n=1/4). To understand these differences flow cytometry was performed on SC and BMC CD8 T cells before and after injection (isolated from liver on day 6). CD8 T cells from BMC inocula expressed high levels of CD28, KLRG1, CTLA-4, PD-1 and CD44 unlike those from SC, indicating SC CD8 T cells to be naïve and BMC CD8 T cells to have a memory-like phenotype. At 6 days post-injection, CD8 T cells from both SC and BMC showed higher CTLA-4 and PD-1 expression than pre-injection, implying dampening of their immune responses. KLRG1 expression in BMC CD8 T cells was lower on day 6 than in the starting inoculum, which together with higher CTLA-4 and PD-1 expression suggests they were undergoing exhaustion. At day10, injected BMC CD8 T cells were observed in liver interacting with host CD8 T cells and undergoing fragmentation. In tolerizing inocula, BMC CD8 T cells start to undergo exhaustion and are attacked by the neonatal host immune system, mitigating the risk of lethal aGVHD and ultimately prolonging donor-type heart graft survival.

The kidney injury caused by the onset of acute graft-versus-host disease is associated with down-regulation of αKlotho

Acute graft-versus-host disease (aGVHD) and kidney injury are the major complications after allogeneic hematopoietic stem cell transplantation (HSCT). Although the underlying mechanisms for the development of these complications are not yet fully understood, it has been proposed that emergence of aGVHD contributes to the development of kidney injury after HSCT. We have shown previously that aGVHD targets the kidney in a biphasic manner: at the onset, inflammatory genes are up-regulated, while when aGVHD becomes established, donor lymphocytes infiltrate the kidney. Here, we characterize renal manifestations at the onset of aGVHD. Mice receiving allogeneic bone marrow and spleen cells displayed symptoms of aGVHD and elevated serum levels of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) within 4 days. There was concurrent kidney injury with the following characteristics: (1) elevated expression of the kidney injury biomarker, neutrophil gelatinase-associated lipocalin (NGAL), (2) accumulation of hetero-lysosomes in proximal tubule epithelial cells, and (3) reductions in αKlotho mRNA and protein and increased serum levels of fibroblast growth factor 23 (Fgf23), phosphate and urea. This situation resembled acute renal injury caused by bacterial lipopolysaccharide. We conclude that the onset of aGVHD is associated with kidney injury involving down-regulation of αKlotho, a sight that may inspire novel therapeutic approaches.

Comparative Behaviour of Allogeneic Spleen versus Bone Marrow Cells in Non-Conditioned Neonatal Mice: Insights into Robust Heart Transplant Tolerance

Purpose Understanding the in vivo/in situ behaviour of fully allogeneic spleen (SC) vs bone marrow cells (BMC) in neonatal mice is important for optimizing appropriate synergistic interactions between the two to induce robust transplant tolerance. Allo-SC and -BMC were therefore injected either as a mixture or separately into neonates; their trafficking, fates, and effects on GVHD, HVGD and tolerance compared. Methods C3H (H-2k) neonates were injected with adult B6 (H2-2b) GFP+ SC or BMC. Cell trafficking, interactions and fates were monitored by whole body/organ imaging and high-resolution microscopy. GVHD was defined by reduced mouse growth/viability, HVGD by donor cell fragmentation and tolerance by donor-type heart transplantation. Results Allo-SC inoculum contained more immune effector cells than allo-BMC as determined by flow cytometry (CD8 T, NK, CD4 T cells). Neonates injected with allo-SC developed aGVHD with diarrhea, severe reduction in growth and death by 14 days post-injection (n=7/7) whereas allo-BMC-injected mice developed aGVHD infrequently (n=1/8). Mice injected with the SC/BMC mixture showed intermediate disease progression (n=6/7). Whole body/organ imaging showed allo-SC and -BMC each trafficked to secondary lymphoid organs, became activated, proliferated and spread throughout the mice suggesting systemic inflammation. GFP signal was stronger in mice injected with allo-SC than -BMC. Proliferating allo-SC and -BMC both contained T cells, including CD8 T cells. GFP signal intensities in syngeneic-SC and -BMC-injected mice were low at day 6 and confined to lymphoid organs indicating proliferation due to lymphopenia was limited. Surviving allo-BMC-injected mice transplanted as adults (n=6) with donor-type hearts showed moderate (2-3) to low (≤1) allograft beat scores at 100 days post-transplant. A 100-day allograft (score=1) showed cellular infiltration consisting of macrophages and T cells including CD8 T cells. Conclusion Both injected allo-SC and -BMC trafficked to secondary lymphoid organs, proliferated and spread throughout neonatal mice; allo-SC consistently caused aGVHD whereas allo-BMC induced non-robust heart transplant tolerance. These promising results suggest that robust tolerance induction will require different optimization procedures for allo-SC and -BMC. Understanding the in vivo/in situ behaviour of fully allogeneic spleen (SC) vs bone marrow cells (BMC) in neonatal mice is important for optimizing appropriate synergistic interactions between the two to induce robust transplant tolerance. Allo-SC and -BMC were therefore injected either as a mixture or separately into neonates; their trafficking, fates, and effects on GVHD, HVGD and tolerance compared. C3H (H-2k) neonates were injected with adult B6 (H2-2b) GFP+ SC or BMC. Cell trafficking, interactions and fates were monitored by whole body/organ imaging and high-resolution microscopy. GVHD was defined by reduced mouse growth/viability, HVGD by donor cell fragmentation and tolerance by donor-type heart transplantation. Allo-SC inoculum contained more immune effector cells than allo-BMC as determined by flow cytometry (CD8 T, NK, CD4 T cells). Neonates injected with allo-SC developed aGVHD with diarrhea, severe reduction in growth and death by 14 days post-injection (n=7/7) whereas allo-BMC-injected mice developed aGVHD infrequently (n=1/8). Mice injected with the SC/BMC mixture showed intermediate disease progression (n=6/7). Whole body/organ imaging showed allo-SC and -BMC each trafficked to secondary lymphoid organs, became activated, proliferated and spread throughout the mice suggesting systemic inflammation. GFP signal was stronger in mice injected with allo-SC than -BMC. Proliferating allo-SC and -BMC both contained T cells, including CD8 T cells. GFP signal intensities in syngeneic-SC and -BMC-injected mice were low at day 6 and confined to lymphoid organs indicating proliferation due to lymphopenia was limited. Surviving allo-BMC-injected mice transplanted as adults (n=6) with donor-type hearts showed moderate (2-3) to low (≤1) allograft beat scores at 100 days post-transplant. A 100-day allograft (score=1) showed cellular infiltration consisting of macrophages and T cells including CD8 T cells. Both injected allo-SC and -BMC trafficked to secondary lymphoid organs, proliferated and spread throughout neonatal mice; allo-SC consistently caused aGVHD whereas allo-BMC induced non-robust heart transplant tolerance. These promising results suggest that robust tolerance induction will require different optimization procedures for allo-SC and -BMC.

Heart Transplant Tolerance Induction in Mice: Understanding the Complex Interplay Between Tolerizing Cells and Host Immune System by Systematic Depletion of Effector Cells

To induce robust heart transplant tolerance in neonates with fully allogeneic spleen and bone marrow cells (SC/BMC) it is crucial to define the complex interplay amongst the different cells of the neonatal host and adult-derived tolerizing inoculum. We used specific and systematic depletion of donor and host effector cells, studying the effects of each depletion on immune outcome.

IJU this issue

In this issue of International Journal of Urology, six Review Articles with three Editorial Comments, four Original Articles and two Urological Notes were published. Husmann (Rochester, USA) reviewed 385 patients that have experienced an enteric bladder augmentation as a result of spina bifida or bladder exstrophy, using either the ileum or colon. Based on the finding of increasing complications with body mass index, the author concluded long-term follow-up evaluations and the nutritional management of patients undergoing an enteric bladder augmentation are necessary to prevent the long-term sequela of this procedure. As the reviewer notes in the Editorial Comment, urologists should bear in mind that complicated surgeries are not a goal, but the start of a new daily care routine of their bladder. Takeuchi et al. (Fukuoka, Japan) reviewed the mechanism of immunological tolerance induced by cyclophosphamide (CY) and other immune factors in kidney transplantation. The authors established their own CY-induced experimental chimera mouse model consisting of intravenous injection of allogeneic bone marrow and spleen cells, followed by intraperitoneal injection of CY. In this model, they showed destruction of allo-reactive T cells by CY. Interestingly, they modified their tolerance system for the experimental treatment of malignancy. Inamoto et al. (Osaka, Japan) compared the oncological and quality of life results of bladder preservation therapy including trimodal therapy with that of radical cystectomy, and concluded that trimodal therapy has emerged as a potential alternative treatment option for muscle-invasive bladder cancer. As muscle-invasive bladder cancer is a systemic disease, trimodal therapy might become an attractive alternative when considering the combination or adjuvant setting of immuno-oncology drugs because of the abscopal effect as a result of radiation therapy. Several interesting Original Articles have been published. Nishioka et al. (Osaka, Japan) reported the utility and safety of high-dose mizoribine combination therapy using cyclosporine and tacrolimus as calcineurin inhibitors in patients undergoing kidney transplant from a Japanese multicenter study that enrolled 156 kidney transplant patients between 2009 and 2013. The authors concluded that two calcineurin inhibitors yielded equivalent efficacy, and high-dose (6 mg/kg at the start) mizoribine in combination with calcineurin inhibitors basiliximab, and corticosteroids can provide effective immunosuppression. This study is worthwhile, not only for showing the utility and safety of this regimen, but also for showing the commitment of Japanese urologists to kidney transplantation worldwide. Urakami et al. (Tokyo, Japan) studied DNA mismatch repair proteins for 143 patients with upper urinary tract urothelial cancer, and found seven (5%) had loss of mismatch repair proteins, so-called microsatellite instability (MSI). Malignant tumors show an inherent genetic instability that can be classified as MSI or chromosomal instability. Although the frequency of MSI was reportedly higher in upper urinary tract urothelial cancer than bladder cancer, the figure and clinical characteristics were almost identical to bladder cancer (9/100; 9%, all with a low grade without tumor progression; Yamamoto T. Clin Cancer Res 2006; 12: 2752). Study of the genetic status of MSI will be more important because of the higher mutational burden than chromosomal instability with a good response to immuno-oncology drugs. Unfortunately, other interesting papers could not be published in this issue due to limited space. None declared.

Investigation for the effect of donor endothelium bcl-xL gene transfection on transplant immunoreaction in rat aorta transplantation

Objective To investigate the influence of bcl-xL overexpression in donor endothelia on recipient immunoreaction after rat aorta transplantation. Methods Enhanced green fluorescent protein (EGFP)-marked lentivirus express vector with Tie2 promoter of anti-apoptotic gene bcl-xL was constructed and transfected into donor SD rat by tail vein injection. The isogeneic and allogeneic aorta transplantation was performed between SD and SD rats as well as SD and Wistar rats. After one week of transplantation, the overexpression of bcl-xL gene in transplant aorta was detected by fluorescence microscope and real-time quantitative polymerase chain reaction (Real-time PCR). The serum from recipients was collected and transplant aortic endothelial cells, spleen cells as well as cytotoxic T lymphocytes (CTL) from donor and recipient were separated and cultured. The mixed lymphocyte reaction, CTL activity and complement-dependent antibody-mediated cytotoxicity were then performed to assess the influence of bcl-xL overexpression in donor endothelia on recipient immunoreaction after rat aorta transplantation. Results Bcl-xL gene was significantly overexpressed (8.5±1.1 vs. 1.0±0.2, t=17.050, P=0.002) in transplant aorta after transfection of lentivirus express vector with Tie2 promoter. The proliferation rate of recipient spleen cells stimulated by allogeneic donor spleen cells was significantly increased in contrast to stimulated by isogeneic donor spleen cells (14.2±1.8 vs. 1.0±0.1, t=23.599, P=0.001; 14.7±2.2 vs. 0.8±0.1, t=25.037, P=0.001). The survival rate of transplant aortic endothelial cells incubated by allogeneic recipient CTL and serum was significantly reduced in contrast to incubated by isogeneic recipient CTL and serum (14.5% vs. 92.7%, χ2=244.558, P=0.000; 8.9% vs. 95.8%, χ2=290.494, P=0.000; 15.1% vs. 96.7%, χ2=269.251, P=0.000; 10.1% vs. 94.7%, χ2=286.190, P=0.000). However, there were no significant differences of the proliferation rate (1.0±0.1 vs. 0.8±0.1, t=2.108, P=0.068; 14.2±1.8 vs. 14.7±2.2, t=-1.012, P=0.341) and survival rate (92.7% vs. 96.7%, χ2=3.078, P=0.079; 95.8% vs. 94.7%, χ2=0.497, P=0.481; 14.5% vs. 8.9%, χ2=2.917, P=0.088; 15.1% vs. 10.1%, χ2=2.286, P=0.131; and 96.6% vs. 97.3%, χ2=0.344, P=0.558; 91.5% vs. 92.1%, χ2=0.033, P=0.856; 19.8% vs. 15.4%, χ2=0.387, P=0.239; 10.2% vs. 13.8%, χ2=1.515, P=0.218) between transfected groups and nontransfected groups in both isogeneic and allogeneic transplantation. Conclusion No significant effect of bcl-xL overexpression in donor endothelia on recipient immunoreaction after rat aorta transplantation. Key words: Bcl-xL; Aorta; Transplantation; Immune

Evaluation of the toxicity of iron-ion irradiation in murine bone marrow dendritic cells via increasing the expression of indoleamine 2,3-dioxygenase 1.

High linear energy transfer radiation is known to deposit higher energy in tissues and cause greater toxicity compared to low-LET irradiation. Local immunosuppression is frequently observed after irradiation (IR). Dendritic cells (DCs) play important roles in the initiation and maintenance of the immune response. The dysfunction of DCs contributes to tumor evasion and growth. However, molecular mechanisms underlying the establishment of immune tolerance induced by heavy ion IR through this DC population are poorly understood. Therefore, here we report our findings on the dysfunction of bone marrow-derived dendritic cells (BMDCs) induced by 1 Gy iron ion radiation and promotions of expressions of JNK1/2/3, indoleamine 2,3-dioxygenase 1 (IDO1), p-ERK1/2 and p38/MAPK; and decrease of IDO2, MHC class II, CD40, CD80 expressions and IFN-γ and TNF-α secretion after total-body IR in mice. JNK+IDO1+ BMDCs showed up-expression of p-ERK1/2 and p-p38/MAPK, reduced expression of MHC class II and CD80, and were not able to effectively stimulate allogeneic spleen T cells. The inhibition of IDO1 expressions could partly restore the function of BMDCs. In all, our study shows that elevated JNK and IDO1 expression induced by Fe ion IR could result in dysfunction of BMDCs via p-p38/MAPK and p-ERK1/2 signal pathway, and it may represent a new mechanism in radiation-induced immune tolerance.

Nonfluorodeoxyglucose-Avid Persistent Splenomegaly at Time of Transplantation Delays Neutrophil and Platelets Engraftment without Affecting Survival in Patients with Lymphomas Undergoing Allogeneic Hematopoietic Cell Transplantation

It is unclear if persistent splenomegaly in the presence of a negative positron emission tomography (PET) scan before allogeneic hematopoietic cell transplantation (HCT) influences post-transplantation outcomes in patients with lymphoma. We retrospectively reviewed records of 152 patients who underwent allogeneic HCT for various lymphomas. Centralized review of pretransplantation computed tomography (CT) and PET images was performed. Spleen volume (SV) was measured using the freehand volume segmentation tool in AW Workstation software (General Electric, Waukesha, WI). Splenic index (SI) was calculated as a product of width, thickness, and length of the spleen. Normal SV was defined as SV < 314.5 cm3 and normal SI was defined as SI ≤ 480 cm3, as described in the literature. Among the study population, 42.8% received an allogeneic HCT from an HLA-matched related donor, 36.2% from a matched unrelated donor, 12.5% from a mismatched unrelated donor, and 8.6% received a double umbilical cord blood transplantation. Most (61.8%) received myeloablative conditioning. Median age at transplantation was 52 (range, 21 to 68) years. Pre–allogeneic HCT spleen CT and PET images were available on 88% and 70.3% patients, respectively. SV ranged from 90 cm3 to 4684 cm3 with a median of 290.5 cm3 and a mean of 400.3 cm3. SI calculation showed a range from 50.3 cm3 to 8276.4 cm3 with a median of 582.1 cm3 and a mean of 771.2 cm3. The majority of patients (83.1%) had PET-negative spleen before allogeneic transplantation. Engraftment was delayed in PET-negative patients with persistent splenomegaly, with median days to neutrophil engraftment of 17 versus 16 (P = .03) and median days to platelet engraftment of 16 versus 14 (P = .04) when using SV. However, persistent splenomegaly did not appear to impact progression-free survival (P = .11) or overall survival (P = .37). Splenomegaly in the setting of a PET-negative study before allogeneic HCT delays neutrophil and platelet engraftment but does not appear to affect survival. Future studies using registry data or larger multicenter studies would be required to evaluate the impact of splenomegaly and its fluorodeoxyglucose avidity on allogeneic HCT outcomes in specific subtypes of lymphomas.

High-dose immunosuppressant alters the immunological status of New Zealand white rabbits following skin transplantation.

The aim of this study was to investigate the effect of an immunosuppressant on the immunological status of New Zealand white rabbits after skin grafting, and to evaluate a method for monitoring the immunological status of subjects with skin transplants. The rabbits were randomly divided into allograft rejection, autograft tolerance, nontransplant, allograft low-dose immunosuppressant and allograft high-dose immunosuppressant groups. The rabbits in the low- and high-dose immunosuppressant groups were treated with cyclosporine A intravenously 8 h prior to skin transplantation and once daily following transplantation at doses of 2 and 25 mg/kg, respectively. At 12 days after skin transplantation, the spleens of donor (female) rabbits and recipient (male) rabbits were harvested for the preparation of single-cell suspensions. The splenocytes from recipient and donor rabbits were labeled with 0.3 or 6 µM carboxy fluorescein diacetate succinimidyl ester, respectively, and a mixed cell suspension was prepared. The final preparation was intravenously injected into recipient New Zealand white rabbits. The ratio of the two fluorescently labeled cell populations in the peripheral blood was measured using flow cytometry at 1, 2, 4 and 8 h after the injection, and the cell death rate was calculated. Histological analysis was also performed on samples collected at the time of splenectomy. The cell death rates of the allograft rejection and low-dose immunosuppressant groups reached their highest levels 8 h after the injection of spleen cell suspension. Allogeneic spleen cells from donor male rabbits were almost completely removed within 8 h of injection. The cell death rate increased slowly in the nontransplant, autograft and high-dose immunosuppressant groups without specificity. This study provides a specific method for the in vivo monitoring of the immunological status of patients after skin grafting. This method can quickly and accurately detect the immunological status of recipients following the injection of a mixed splenocyte suspension, thereby indicating the strength of immune rejection by the immune systems of the recipients.

Renal cancer treatment with recipient lymphocyte infusion enhanced the antitumor effect of nonmyeloablative allogeneic stem cell transplantation

BackgroundNonmyeloablative allogeneic stem cell transplantation (SCT) is an option for the treatment of metastatic renal cancer. Mature donor T cells cause graft versus-host disease (GVHD) although they are also the main mediators of the beneficial graft-versus-tumor (GVT) activity associated with this treatment. Hence, the segregation of GVT activity from GVHD is an important challenge in managing the clinical course of treatment. We previously reported a series of studies regarding the allograft tolerance induced by allogeneic spleen cells (with bone marrow cells) and cyclophosphamide in mice. MethodsThe aim of the present study was to modify the cyclophosphamide-using cell therapy to reduce the risk of GVHD while preserving the antitumor activity against RENCA, a murine carcinogen-induced renal cell carcinoma with recipient lymphocyte infusion (RLI). ResultsRegarding the in vivo antitumor effect, there was a significant difference between RLI and no lymphocyte infusion after the cyclophosphamide treatment, whereas the histologic findings of the small intestine showed that the cyclophosphamide-using cell therapy with RLI decreased the risk of GVHD as compared with donor lymphocyte infusion. In addition, the acquired immunity against RENCA was clearly observed in RLI-treated mice. ConclusionsOur results show that RLI during cyclophosphamide-using nonmyeloablative cell therapy can dissociate GVT effects from GVHD by reducing the risk of GVHD. We considered that this was the first report to provide the evidence of nonmyeloablative allogeneic SCT with RLI for the treatment of renal cell carcinoma which never induce complete chimerism.

Allogeneic Splenocyte Transfer and Lipopolysaccharide Inhalations Induce Differential T Cell Expansion and Lung Injury: A Novel Model of Pulmonary Graft-versus-Host Disease

BackgroundPulmonary GVHD (pGVHD) is an important complication of hematopoietic cell transplant (HCT) and is thought to be a consequence of the HCT conditioning regimen, allogeneic donor cells, and posttransplant lung exposures. We have previously demonstrated that serial inhaled lipopolysaccharide (LPS) exposures potentiate the development of pGVHD after murine allogeneic HCT. In the current study we hypothesized that allogeneic lymphocytes and environmental exposures alone, in the absence of a pre-conditioning regimen, would cause features of pGVHD and would lead to a different T cell expansion pattern compared to syngeneic cells.MethodsRecipient Rag1−/− mice received a transfer of allogeneic (Allo) or syngeneic (Syn) spleen cells. After 1 week of immune reconstitution, mice received 5 daily inhaled LPS exposures and were sacrificed 72 hours after the last LPS exposure. Lung physiology, histology, and protein levels in bronchoalveolar lavage (BAL) were assessed. Lung cells were analyzed by flow cytometry.ResultsBoth Allo and Syn mice that undergo LPS exposures (AlloLPS and SynLPS) have prominent lymphocytic inflammation in their lungs, resembling pGVHD pathology, not seen in LPS-unexposed or non-transplanted controls. Compared to SynLPS, however, AlloLPS have significantly increased levels of BAL protein and enhancement of airway hyperreactivity, consistent with more severe lung injury. This injury in AlloLPS mice is associated with an increase in CD8 T cells and effector CD4 T cells, as well as a decrease in regulatory to effector CD4 T cell ratio. Additionally, cytokine analysis is consistent with a preferential Th1 differentiation and upregulation of pulmonary CCL5 and granzyme B.ConclusionsAllogeneic lymphocyte transfer into lymphocyte-deficient mice, followed by LPS exposures, causes features of pGVHD and lung injury in the absence of a pre-conditioning HCT regimen. This lung disease associated with an expansion of allogeneic effector T cells provides a novel model to dissect mechanisms of pGVHD independent of conditioning.

DCs sensitized with mPD-L1-Ig fusion protein improve the effect of heart transplantation in mice by promoting the generation of T-reg cells

PurposeTo detect the effects of DCs sensitized by mPD-L1-Ig fusion protein in heart transplantation in mice as well as its mechanisms. MethodThe mPD-L1-IgG1 construct was used to build a yeast expression system, and the fusion protein was expressed by secretion after the transfection of the GS115 yeast strain, purified by affinity chromatography and ion exchange chromatography, and assayed by SDS–PAGE and Western blot. The ability of the fusion protein to bind to the acceptor PD-1 was tested by ELISA, and the ability of the fusion protein to inhibit the function of T cells was tested by mixed lymphocyte reaction (MLR). ResultsWe used the new PD-L1-IgG1 fusion protein to sensitize imDCs and maintained the immature state of DCs, so as to induce stable and effective immune tolerance to heart transplantation. After the treatment of DCs by mPD-L1-Ig in vitro, the levels of CD80, CD40 and I-Ab expression on DCs are relatively weaker, the ability of DCs to stimulates the proliferation of allogeneic spleen T cells was significantly decreased (P<0.01), and the levels of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) secreted by induced allogeneic T cells were significantly decreased (P<0.01). An in vivo experiment also revealed that DCs sensitized by mPD-L1-IgG1 could prolong the survival time of a transplanted heart to 17.8±1.12days, and alleviate the pathological change of the cardiac allografts compared with other three groups. ConclusionDCs sensitized by the yeast-expressed mPD-L1-Ig fusion protein are shown to alleviate the cardiac allograft rejection in mice.

Selective Allodepletion: Have We Finally Found the Holy Grail?

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment option for patients with hematologic malignancies and nonmalignant disorders of hematopoiesis. Outcomes of allogeneic HSCT have improved steadily over the decades [1]. However, significant challenges remain. One of these challenges is the so-called Holy Grail of stem cell transplantation immunology: to preserve the beneficial effects of donor T cells against the tumor and against pathogenswhile avoidingallogeneic graft-versus-host disease (GVHD) [2]. To find the Holy Grail, we must develop methods or treatments that selectively deplete Tcells that react against host alloantigens, yet preserve tumor-specific and pathogenreactive T cells. Experiments published by Ross et al. in this issue of Biology of Blood and Marrow Transplantation [3] suggest that post-transplantation cyclophosphamide (PTC), a novel form of GVHD prophylaxis, may do just that. PTC is celebrating its golden anniversary. In October 1963, Berenbaum and Brown reported that cyclophosphamide (Cy) treatment of rats prolonged the survival of skin allografts, and that graft survival was longest when the drug was given 1 to 3 days after, rather than before, transplantation [4]. The road to the use of PTC in allogeneic HSCT is long, but significant credit must be given to Hisanori Mayumi et al. in the laboratory of Kikuo Nomoto [5]. These investigators developed a method for establishing hematopoietic microchimerism and robust tolerance to minor histocompatibility antigens in mice by i.v. infusion of allogeneic spleen cells followed in 2 or 3 days by intraperitoneal administration of high-dose Cy [6]. They showed that a high dose of cells was required for tolerance induction, and that the dose and timing of Cy were critical to the outcome [7]. For example, tolerance and chimerismwere induced if the drugwere given on day 2 or 3 after cell infusion, but not if the drugwere given on day 1 or on day 4. Interestingly, Cy-induced tolerance was blocked by peritransplantation treatment of the recipient with cyclosporine A [8]. Finally, they showed that tolerance of the microchimeric donor cells was mediated initially by Cy-induced destruction of alloreactive host T cells, then by intrathymic clonal deletion of donor-reactive host cells, and

Preservation of the native spleen in multivisceral transplantation

The native spleen is usually removed in patients undergoing MTV. The consequential asplenic state is associated with a high risk of sepsis, especially in immunosuppressed children. In contrast, the inclusion of an allogeneic spleen in multivisceral grafts has been associated with a high incidence of GVHD. We propose an alternative technique for patients undergoing MTV, consisting of the preservation of the native spleen. This approach avoids the additional risk of infection that characterizes the asplenic state without the detrimental side effects of the allogeneic spleen.

Molecular targeting of hepatocyte growth factor by an antagonist, NK4, in the treatment of rheumatoid arthritis

IntroductionHepatocyte growth factor (HGF) is a potent proangiogenic molecule that induces neovascularization. The HGF antagonist, NK4, competitively antagonizes HGF binding to its receptor. In the present study, we determined the inhibitory effect of NK4 in a rheumatoid arthritis (RA) model using SKG mice.MethodsArthritis was induced in SKG mice by a single intraperitoneal injection of β-glucan. Recombinant adenovirus containing NK4 cDNA (AdCMV.NK4) was also injected intravenously at the time of or 1 month after β-glucan injection. Ankle bone destruction was examined radiographically. The histopathologic features of joints were examined using hematoxylin and eosin and immunohistochemical staining. Enzyme-linked immunosorbent assays were used to determine the serum levels of HGF, interferon γ (IFN-γ, interleukin 4 (IL-4) and IL-17 production by CD4+ T cells stimulated with allogeneic spleen cells.ResultsThe intravenous injection of AdCMV.NK4 into SKG mice suppressed the progression of β-glucan-induced arthritis. Bone destruction was also inhibited by NK4 treatment. The histopathologic findings of the ankles revealed that angiogenesis, inflammatory cytokines and RANKL expression in synovial tissues were significantly inhibited by NK4 treatment. Recombinant NK4 (rNK4) proteins inhibited IFN-γ, IL-4 and IL-17 production by CD4+ T cells stimulated with allogeneic spleen cells.ConclusionsThese results indicate that NK4 inhibits arthritis by inhibition of angiogenesis and inflammatory cytokine production by CD4+ T cells. Therefore, molecular targeting of angiogenic inducers by NK4 can potentially be used as a novel therapeutic approach for the treatment of RA.

Activated Apoptotic Cells Induce Dendritic Cell Maturation via Engagement of Toll-like Receptor 4 (TLR4), Dendritic Cell-specific Intercellular Adhesion Molecule 3 (ICAM-3)-grabbing Nonintegrin (DC-SIGN), and β2 Integrins

Dendritic cells (DCs) are professional antigen-presenting cells playing a central role in connecting innate and adaptive immunity. Maturation signals are, however, required for DCs to undergo phenotypic and functional changes to acquire a fully competent antigen-presenting capacity. We previously reported that activated apoptotic peripheral lymphocytes (ActApo) provide activation/maturation signals to human monocyte-derived DCs. In this paper, we have characterized the signaling pathways and molecules involved in ActApo-mediated DC maturation. We found that both cellular and supernatant fractions from ActApo are required for DC maturation signaling. ActApoSup-induced CD80 and CD86 expression was significantly blocked in the presence of neutralizing antibodies against tumor necrosis factor-α (TNF-α). Cell-cell contact-dependent signaling involved β2 integrins, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and TLR4 because ActApo-induced up-regulation of the maturation markers CD80 and CD86 was significantly inhibited in the presence of neutralizing antibodies against CD18, CD11a, CD11b, and DC-SIGN as well as TLR4. The role of TLR4 was further confirmed by silencing of TLR4 in DCs. In addition, the endogenous adjuvant effect exerted by activated apoptotic splenocytes (ActApoSp) was reduced after immunization with human serum albumin in TLR4(-/-) mice. We detected activation of multiple signaling pathways and transcription factors in DCs upon co-culture with ActApo, including p38, JNK, PI3K-Akt, Src family kinases, NFκB p65, and AP1 transcription factor family members c-Jun and c-Fos, demonstrating the complex interactions occurring between ActApo and DCs. These studies provide important mechanistic insight into the responses of DCs during encounter with cells undergoing immunogenic cell death.

Th17 Alloimmunity Prevents Neonatal Establishment of Lymphoid Chimerism in IL-4-Deprived Mice

Immune responses in newborn mice are known to be biased toward the helper type 2 phenotype. This may account for their propensity to develop tolerance. Herein, we evaluated the effects of IL-4 deprivation on CD4(+) T-cell activities elicited by neonatal exposure to allogeneic spleen cells. We showed that chimerism, Th2-type polarization and pathology, as well as skin allograft acceptance were inhibited in BALB/c mice immunized at birth with (A/J x BALB/c) F(1) spleen cells upon in vivo IL-4 neutralization. While IL-4 neutralization inhibited the development of Th2 cells in this model, it led to the accumulation of IL-17A, IL-17F, IL-22, IL-6 and RORγt mRNA in the spleen or graft tissues. Moreover, IL-4 deprivation led to the differentiation of donor-specific Th17 cells with a concomitant Th1 response characterized by IFN-γ production. The Th17-type response emerging in IL-4-deprived mice was found to mediate both intragraft neutrophil infiltration and the abrogation of B-cell chimerism. Neutralization of this Th17 response failed however to restore functional skin graft acceptance. Collectively, our observations indicate that the neonatal Th2 response opposes the development of Th17 cells, and that Th17 cells are responsible for controlling lymphoid chimerism in mice neonatally injected with semiallogeneic cells.