Jifu Jiang

Regulatory T-cell generation and kidney allograft tolerance induced by mesenchymal stem cells associated with indoleamine 2,3-dioxygenase expression.

Background. The immunoregulatory properties of mesenchymal stem cells (MSCs) have been observed in vitro and in vivo. However, the underlying mechanisms of this immunomodulation remain undefined. Recent research demonstrated that MSCs express the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO), known to suppress T-cell responses. This study was designed to address whether MSCs induce kidney allograft tolerance and whether IDO contributes to the immunoregulatory functions of MSCs in vivo. Methods. MSCs (1×106, intravenously) from wild-type (WT-MSCs) or IDO knockout (IDO−/−-MSCs) C57BL/6 mice were injected into BALB/c recipients 24 hr after receiving a life-supporting orthotopic C57BL/6 renal graft. Results. WT-MSC-treated recipients achieved allograft tolerance with normal histology and undetectable antidonor antibody levels. Tolerant recipients demonstrated increased circulating kynurenine levels and significantly high frequencies of tolerogenic dendritic cells. They also exhibited significantly impaired CD4+ T-cell responses consisting of decreased donor-specific proliferative ability and a Th2-dominant cytokine shift. In addition, high frequencies of CD4+CD25+Foxp3+ regulatory T cells (Tregs) were found in recipient spleens and donor grafts, with antibody-induced CD25+ cell depletion confirming the critical role of Tregs in the MSC-induced tolerance. Interestingly, renal allograft recipients treated with WT MSCs concomitant with the IDO inhibitor 1-methyl-tryptophan, or those treated with IDO−/−-MSCs alone, were unable to achieve allograft tolerance—revealing that functional IDO was necessary for the immunosuppression observed with WT-MSC treatment. Conclusions. IDO secreted by MSCs was responsible, at least in part, for induction of kidney allograft tolerance through generation of Tregs. This study supports the clinical application of MSCs in transplantation.

Vascular Smooth Muscle Cells of Recipient Origin Mediate Intimal Expansion after Aortic Allotransplantation in Mice

Intimal expansion by vascular smooth muscle cells (SMCs) is a characteristic feature of graft vascular disease. Whether graft intimal SMCs arise from donor or recipient tissue is not well established but has important pathogenetic implications. We examined for the presence of male cells in the expanded intima of sex-mismatched mouse aortic allografts (C57BL/6-to-BALB/c) at 30 or 60 days after transplant by in situ hybridization using a Y-chromosome probe. Study groups included male-to-female allografts, female-to-male allografts, and female-to-female allografts in recipients previously engrafted with male bone marrow. Although intimal expansion developed in all allografts, male-to-female allografts lacked Y-chromosome-positive intimal cells. In contrast, such cells were abundant in female-to-male allografts and most of these cells co-labeled for smooth muscle alpha-actin by immunostain. Female-to-female allografts in recipients with male bone marrow showed a limited number of intimal Y-chromosome-positive cells. However, none of these clearly co-labeled for smooth muscle alpha-actin and their numbers declined throughout time, consistent with graft-infiltrating inflammatory cells. We conclude that intimal expansion of mouse aortic allografts is mediated by SMCs that originated from the recipient. There was little evidence of their derivation from the bone marrow, suggesting instead the adjacent host aorta as the primary source of intimal SMCs.

Inhibition of Terminal Complement Components in Presensitized Transplant Recipients Prevents Antibody-Mediated Rejection Leading to Long-Term Graft Survival and Accommodation

Ab-mediated rejection (AMR) remains the primary obstacle in presensitized patients following organ transplantation, as it is refractory to anti-T cell therapy and can lead to early graft loss. Complement plays an important role in the process of AMR. In the present study, a murine model was designed to mimic AMR in presensitized patients. This model was used to evaluate the effect of blocking the fifth complement component (C5) with an anti-C5 mAb on prevention of graft rejection. BALB/c recipients were presensitized with C3H donor skin grafts 7 days before heart transplantation from the same donor strain. Heart grafts, transplanted when circulating anti-donor IgG Abs were at peak levels, were rejected in 3 days. Graft rejection was characterized by microvascular thrombosis and extensive deposition of Ab and complement in the grafts, consistent with AMR. Anti-C5 administration completely blocked terminal complement activity and local C5 deposition, and in combination with cyclosporine and short-term cyclophosphamide treatment, it effectively prevented heart graft rejection. These recipients achieved permanent graft survival for >100 days with normal histology despite the presence of systemic and intragraft anti-donor Abs and complement, suggesting ongoing accommodation. Furthermore, double-transplant experiments demonstrated that immunological alterations in both the graft and the recipient were required for successful graft accommodation to occur. These data suggest that terminal complement blockade with a functionally blocking Ab represents a promising therapeutic approach to prevent AMR in presensitized recipients.

Renal Tubular Epithelial Cell Self-Injury Through Fas/Fas Ligand Interaction Promotes Renal Allograft Injury

Tubular epithelial cells (TECs) coexpress Fas and Fas ligand (FasL), which could influence renal allograft injury. While TECs can resist apoptosis by Fas antibody, TEC apoptosis by contact with adjacent TECs has not been studied. Fas expression increased in TECs with cytokine treatment (IFN‐γ, TNF‐α) while abundant FasL levels were not altered. Apoptosis (Annexin‐V, DNA fragmentation) occurred in cytokine‐treated TECs monolayers from C3H‐HeJ mice by 24 h, but was absent in similarly treated TECs from Fas‐deficient (lpr) or FasL‐mutant (gld) mice, suggesting that ‘self injury’ occurred through Fas/FasL. Membrane labeling of TECs in cocultures confirmed that FasL‐bearing TECs induced apoptosis when in contact with Fas‐bearing TECs. Culturing TECs with allogeneic C57BL/6 (H‐2b) splenocytes resulted in apoptosis and elimination of C3H‐HeJ TECs by 48 h, with enhanced survival and reduced apoptosis using lpr or gld TECs. In a renal allograft model, survival of C57BL/6 recipients was greater (p < 0.05) and renal function improved (p < 0.001) using C3H‐lpr or C3H‐gld (H‐2 k) donor kidneys compared with C3H‐HeJ kidneys. These data demonstrate for the first time that cytokine‐activated TECs can injure TECs through expression of functional FasL and Fas. We suggest that inhibition of TEC–TEC ‘self injury’ may be a novel strategy to augment renal allograft survival.

Induction of kidney allograft tolerance by soluble CD83 associated with prevalence of tolerogenic dendritic cells and indoleamine 2,3-dioxygenase.

Background. Tolerogenic dendritic cells (Tol-DCs) play a critical role in inducing and maintaining tolerance. Recognizing that both T-cell inactivation and activation are contingent on signals provided by DCs and that graft-specific activated T cells are major mediators of transplant rejection, we aimed to create an environment favoring Tol-DCs with a novel reagent, human soluble CD83 (hsCD83). Methods. Life-supporting orthotopic kidney transplantation was performed in a C57BL/6-to-BALB/c mouse model. The study group was treated with hsCD83 (100 &mgr;g/mouse/day, postoperative days −1 to +7, intravenously) and compared with untreated controls. Results. Treatment with hsCD83 achieved kidney allograft tolerance (>100 days), with negligible antidonor antibody detected. In contrast, kidney grafts in untreated recipients demonstrated severe rejection after 35 days, characterized by cellular infiltration, interstitial hemorrhage and edema, and glomerular and tubular necrosis, as well as high antidonor antibody titers. In addition, splenic DCs of tolerant recipients exhibited significantly decreased levels of surface major histocompatibility complex class II, CD40, CD80, and intracellular interleukin-12, as well as reduced allogeneic stimulatory capacity. Adoptive transfer of CD11c+ DCs from tolerant hsCD83-treated animals induced kidney allograft tolerance in syngeneic recipients. Blocking indoleamine 2,3-dioxygenase with 1-methyl-tryptophan (15 mg/mouse/day; gavage) prevented the immunosuppressive effect of hsCD83, abrogating hsCD83-induced Tol-DCs and graft tolerance, and leading to acute kidney graft rejection in 22 days. Conclusion. hsCD83 alone was capable of inducing kidney allograft tolerance through a mechanism involving Tol-DC generation and, at least in part, indoleamine 2,3-dioxygenase activity. Because sCD83 is of human origin, the therapeutic approach used in our mouse transplant model holds significant promise for clinical transplantation.

Immunosuppression Involving Soluble CD83 Induces Tolerogenic Dendritic Cells That Prevent Cardiac Allograft Rejection

Background. Dendritic cells (DCs) are crucial regulators of immunity and important in inducing and maintaining tolerance. Here, we investigated the potential of a novel DC-immunomodulating agent, soluble CD83 (sCD83), in inducing transplant tolerance. Methods. We used the C3H-to-C57BL/6 mouse cardiac transplantation model that exhibits a combination of severe cell-mediated rejection and moderate antibody-mediated rejection and investigated whether sCD83 could augment a combination therapy consisting of Rapamycin (Rapa) and anti-CD45RB monoclonal antibody (&agr;-CD45) to prolong allograft survival. Results. Monotherapies consisting of Rapa and &agr;-CD45 were incapable of preventing rejection. However, all treatments involving sCD83 were capable of (1) down-modulating expression of various DC surface molecules, such as major histocompatibility complex class II and costimulatory molecules, (2) reducing the allogeneic stimulatory capacity of the DCs, and (3) significantly inhibiting antidonor antibody responses. Most striking results were observed in the triple therapy-treated group, sCD83+Rapa+&agr;-CD45, where cell-mediated rejection and antibody-mediated rejection were abrogated for over 100 days. Donor-specific tolerance was achieved in long-term surviving recipients, because donor skin transplants were readily accepted for an additional 100 days, whereas third-party skin grafts were rejected. Success of triple therapy treatment was accompanied by enhancement of tolerogenic-DCs that conferred antigen-specific protection on adoptive transfer to recipients of an allogeneic heart graft. Conclusions. Our study revealed that sCD83 is capable of attenuating DC maturation and function, and inducing donor-specific allograft tolerance, in the absence of toxicity. Thus, sCD83 seems to be a safe and valuable counterpart to current DC-modulating agents.

Influence of macrophage depletion on bacterial translocation and rejection in small bowel transplantation

Rejection and sepsis can be intimately related following small bowel transplantation when rejection compromises normal intestinal barrier mechanisms and bacterial translocation results. Macrophages play a role in controlling the egress of intestinal luminal bacteria--and they have also been implicated in allograft rejection. In this study, the role of macrophages in rejection and bacterial translocation was evaluated by depleting macrophages in donors and/or recipients of rat small bowel allografts with injection of liposome-encapsulated dichloromethylene diphosphonate (CL2MDP). In preliminary studies, we demonstrated that a single intraperitoneal injection of liposome-encapsulated CL2MDP (350 mg/kg) depleted ED2-positive macrophages by > 90% in the liver mesenteric lymph nodes and proximal and distal small bowel, and by approximately 50% in the spleen. ED1-positive macrophages were depleted by > 90% in the liver and by approximately 50% at the other sites. ED3-positive macrophages were completely depleted. Dendritic cells were > 90% depleted in the spleen and mesenteric lymph nodes, but were not depleted in the small bowel. Macrophage depletion in the donor resulted in increased translocation of bacteria to the peritoneal cavity (P = 0.03) if recipient macrophages were present. With histopathologic analysis, a significantly milder rejection with less arteritis was seen in the allografts of the recipient macrophage-depleted group compared with nondepleted controls (P = 0.045). This suggests that recipient macrophages play an important role in rejection. With macrophage depletion in both the donor and the recipient, graft survival was prolonged significantly (13.2 +/- 1.9 days) compared with non-macrophage-depleted controls (9.2 +/- 1.3 days) (P = 0.003). These studies suggest that strategies targeting recipient macrophages may be useful in controlling small bowel allograft rejection without increasing bacterial translocation.

Chemokine-binding viral protein M-T7 prevents chronic rejection in rat renal allografts.

&NA; M‐T7 is a myxoma virus‐encoded protein that has been found to bind and disrupt human chemokine gradients. This study examined whether purified M‐T7 could prevent chronic rejection in a rat renal allograft model. Fisher F344 renal allografts were transplanted into Lewis rats. Recipients were randomly grouped into two groups: control animals treated with cyclosporine alone and animals treated with cyclosporine combined with low‐, medium‐ and high‐dose M‐T7 viral protein. The survival rate was not significantly different between allograft groups. Renal allografts treated with high‐dose M‐T7 demonstrated a significant reduction in tubular atrophy, glomerular atrophy, vascular hyalinization, cortical scarring, and lymphocyte infiltration. Morphometric analyses demonstrated that the high‐dose M‐T7 group also showed a significantly decreased amount of glomerulosclerosis and transplant arteriosclerosis. These data demonstrate for the first time that the immunoregulatory viral protein M‐T7 can effectively attenuate chronic rejection in rat renal allografts.

Reduction of Foxp3-expressing regulatory T cell infiltrates during the progression of renal allograft rejection in a mouse model

BACKGROUND Regulatory T (Treg) cells are the immune suppressors in the maintenance of immune homeostasis and tolerance to self and non-self antigens, and may have therapeutic potential in the treatment of transplant rejection in patients. However, Treg cell development and action are poorly understood in transplantation. In this study, the association of CD4(+)Foxp3(+) infiltrates within renal allograft tissue with graft survival was investigated in a mouse model. METHODS Kidney donors from C57BL/6J mice (H-2(b)) were transplanted to bilaterally nephrectomized Balb/c recipient mice (H-2(d)). Treg cells were examined with FACS and immunohistochemical staining. RESULTS Here we showed that without any immunosuppressive regimen, kidney allografts were mostly rejected from 20 to 60 days after transplantation. During the progression of allograft rejection Foxp3(+) Treg phenotype infiltrates were significantly diminished, while intragraft expression of TGF-beta1, IL-6, IL-17 and IL-23 was up-regulated. The regulatory function of CD4(+)CD25(+) infiltrates was confirmed by their suppressive activity in mixed lymphocyte reaction. Further in vitro studies indicated that primary renal tubular epithelial cell (TEC) cultures produced high levels of IL-6 in response to allogeneic lymphocyte or IL-17 stimulation, and neutralization of IL-6 increased CD4(+)CD25(+)Foxp3(+) cells in co-cultures with TEC. CONCLUSION Diminution of Foxp3(+) Treg infiltrates associates with renal allograft rejection, and neutralization of IL-6 activity enhances Foxp3(+) Treg cell differentiation. Our findings suggest that increase in intragraft IL-6 may down-regulate infiltrating Foxp3(+) Treg cells.

Induction of indefinite cardiac allograft survival correlates with toll-like receptor 2 and 4 downregulation after serine protease inhibitor-1 (serp-1) treatment

Background. Innate immunity provides obstacles to successful organ transplantation, which cannot be prevented by cyclosporine (CsA). Here we have determined the potential of a myxoma viral serpin, Serp-1, with proven anti-inflammatory and antiatherogenic actions, to modulate innate immunity and contribute synergistically with CsA in the prevention of acute cardiac allograft rejection. Methods. Brown-Norway rat hearts were heterotopically transplanted into Lewis rats and given either a monotherapy treatment of Serp-1, a subtherapeutic dose of CsA, or the two drugs in combination. Results. A brief treatment of Serp-1 alone, or a subtherapeutic dose of CsA, resulted in a marked decrease in intragraft macrophage infiltration and downregulation of toll-like receptor (TLR)-2, TLR4 and MyD88 at 48 hours posttransplantation, which was associated with significantly reduced numbers of mature dendritic cells. A significant reduction in intragraft T-lymphocyte infiltration was observed with both Serp-1 monotherapy and Serp-1 and CsA combination therapy, with the combination treatment achieving indefinite graft survival (>100 days) with normal histology. The CsA monotherapy group displayed partially reduced lymphocyte infiltration compared to the untreated controls, but failed to inhibit early innate immune graft recognition events such as macrophage infiltration and TLR 2, TLR4, and MyD88, and was ultimately unsuccessful in preventing rejection (36.3±7.8 days). Conclusion. Observed suppressive effects of Serp-1 on early innate immune response components such as TLR-2 and 4, and on adaptive responses such as T-cell intragraft infiltration suggests that Serp-1 may modulate the transition from innate to adaptive immunity, exhibiting a synergistic effect on allograft survival when used in combination with a subtherapeutic dose of CsA.