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BACKGROUND Immunoglobulin-cytokine fusion molecules have been shown to be the new generation of immunomodulating agents in transplantation tolerance induction. In the present study, we tested whether immunoregulatory cytokine fusion proteins of IL-10/Fc, TGF-β/Fc, or IL-2/Fc would enhance allogeneic bone marrow cell (BMC) engraftment and promote tolerance induction. METHODS B6 (H2) mice were conditioned with anti-CD154 (MR1) and rapamycin (Rapa) plus 100 cGy total body irradiation (MR1/Rapa/100 cGy) and transplanted with allogeneic B10.D2 (H2) BMC. Recipients were treated with lytic IL-2/Fc, nonlytic IL-2/Fc, TGF-β/Fc, or IL-10/Fc fusion proteins to promote chimerism to induce tolerance. RESULTS Donor chimerism was achieved in 20% of recipients conditioned with MR1/Rapa/100 cGy. The addition of TGF-β/Fc (5- or 10-day treatment) or nonlytic IL-2/Fc (10-day treatment) fusion proteins to the conditioning resulted in engraftment in nearly 100% of recipients. In contrast, lytic IL-2/Fc or IL-10/Fc had no effect. The combination of nonlytic IL-2/Fc and TGF-β/Fc had a synergistic effect to promote engraftment and resulted in significantly higher donor chimerism compared with recipients conditioned with TGF-β/MR1/Rapa/100 cGy. Engraftment was durable in the majority of chimeras and increased over time. The chimeras accepted donor skin grafts and promptly rejected third-party skin grafts. Moreover, specific T cell receptor-Vβ5.½ and TCR-Vβ11 clonal deletion was detected in host T cells in chimeras, suggesting central tolerance to donor alloantigens. CONCLUSIONS Allogeneic BMC engraftment is enhanced with TGF-β/Fc fusion protein treatment. TGF-β/Fc and nonlytic IL-2/Fc exert a synergistic effect in promotion of alloengraftment and donor-specific transplant tolerance, significantly decreasing the minimum total body irradiation dose required.

Background. This study investigated whether mesenchymal stem cells (MSCs) combined with bone marrow transplantation (BMT), irradiation, or short-term immunosuppressant therapy could prolong composite tissue allotransplant survival in a swine hind-limb model. Methods. Heterotopic hind-limb transplantation was performed in outbred miniature swine. Group I (n= 5) was the untreated control. Group II (n=3) received MSCs alone (given on days ―1, +3, +7, +14, +21). Group III (n=6) received cyclosporine A (CsA days 0 to +28). Group IV (n=4) received preconditioning irradiation (day ―1), BMT (day + 1), and CsA (days 0 to +28). Group V (n=5) received irradiation (day ―1), BMT (day + 1), CsA (days 0 to +28), and MSCs (days + 1, +7,+ 14). The expression and localization of CD4 + /CD25 + T cells and MSCs were assessed using flow cytometry and immunohistochemistry. Results. The allografts survival with MSCs alone revealed a significant prolongation, when compared with the controls (P=0.02). Allografts with CsA treatment exhibited delayed rejection. Irradiation and BMT-CsA treatment revealed no significant allograft survival benefit when compared with the CsA treatment group, but graft-versus-host disease (GVHD) was evident. However, combination of MSCs-BMT-CsA treatment demonstrated significant prolongation of allograft survival (>200 days, P

The primary goal in designing new therapies for transplantation is the induction and maintenance of a state of donor antigen (Ag)–specific immunologic tolerance. In organ transplantation, CD4+ T cells play a critical role in initiating the immune response leading ultimately to the effector mechanisms mediating allograft rejection. Interestingly, several reports have pointed out that C8+' T cells could play an important role in graft rejection and particularly in chronic rejection, as C8+S T cell-dependent processes appear to be responsible for costimulation blockade-resistant rejection (1-5). Despite costimulation blockade, engraftment and induction of tolerance in skin, kidney, or heart allografts requires adjunctive treatments depleting CD8+ T cells, such as anti-CD8 monoclonal antibody (mAb) (2, 4) or anti-GMl+CD8+ mAb (5), as these lymphocyte subsets are suspected to be the cells responsible for costimulation blockade-resistant rejection. Therefore, targeting CD8+ T cells appears to be an essential step to control graft survival. Among the cytokines responsible for CD8+ T lymphocyte survival and proliferation, interleukin (IL)-15 appears to be a good candidate as it drives the proliferation and survival of CD8+ T cells and natural killer (NK) cells (6). The phenotype of IL-15−/− (7) and IL-15Rα−/− (8) mice demonstrates that IL-15 selectively provides support for the homeostasis and proliferation of memory CD8+ T cells. The description of IL-15 transgenic mice developing fatal leukemia with a CD8+ T cell phenotype (9) confirms that IL-15 plays an essential role in the homeostatic control of these cells. These findings raise the following question: Can expression of IL-15 be linked to CD8-dependent costimulation resistant rejection? As intragraft gene expression of IL-15 is consistently found in rejected tissues from mice and humans (10, 11) and costimulation blockade cannot act directly to decrease IL-15 expression or inhibit the activation of alloreactive CD8+ T cells (1, 3, 5), we hypothesized that targeting the IL-15/IL-15R system might provide a new tool for the induction of allograft tolerance. We have previously reported on the therapeutic effects of an IL-15 mutant/Fcγ2a protein possessing a prolonged circulating half-life, a high affinity IL-15Rα site specific antagonist function (12) and Fc-related cytocidal potential against IL-15Rα+ cells upon allograft survival (13,14). To determine whether treatment with the IL-15 mutant/Fcγ2a protein inhibits alloreactive CD8+ T cell mediated rejection, we compared the therapeutic activity of the IL-15 related protein to that of a costimulatory blockade agent, CTLA4/Fc, in a CD4-independent, CD8-mediated islet allograft model using CD4 knockout mice as recipients. We analyzed the role of CD8+ T cells in allograft rejection and determined the fate of CD8+ T in vivo by analyzing CD8+ T proliferation in a model mimicking a graft-versus-host (GVH) response in CD4 knockout (CD4KO) mice. Our data suggests that IL-15R+ cells are an important component of allograft rejection and that these cells play a pivotal role in CD8+ T-cell mediated CTLA4/Fc blockade-resistant rejection.

Background. Several studies suggest that MHC-mismatched allografts reject with a Th1 or Th2 immune response, but these models all have low-level expression of the Th1 cytokines interleukin (IL)-2 and interferon-g (IFN-g). Methods. We interbred mice with single targeted gene disruptions for IL-2 and IFN-g to establish IL-2 1 IFN-g double knockout (DKO) mice. Heterotopic cardiac allografts from DBA/2j (H2 d ) donors were transplanted WT, IL-2 knockout (KO), IFN-g KO, and DKO recipients (C57BL/63129; H2 b ). Cytokine transcripts from allografts and DKO splenocytes were analyzed by reverse transcription polymerase chain reaction. Results. DKO mice had a cytokine profile and IgG1/ IgG2a isotype ratio characteristic of Th2 deviation. DKO recipients rejected heterotopic cardiac allografts faster than IL-2 KO mice, but significantly slower than WT and IFN-g KO mice (P

In an attempt to identify potential markers of steroid-resistance in focal segmental glomerulosclerosis (FSGS) we evaluated intra-graft gene expression of IkappaBalpha, nuclear factor-kappaB (NF-kappaB), and angiotensinogen in 60 biopsies from 27 pediatric renal transplant recipients. Intra-graft NF-kappaB expression was significantly elevated in recurrent FSGS (R-FSGS) (218.3 + 55.6 ag/fg versus NON-FSGS 121.1 + 19.9, P=0.04) but not in acute rejection. NF-kappaB:IkappaBalpha ratios were higher in cadaveric donor versus living related donor recipients (15.7 + 2.8 vs. 8.8 + 1.3, respectively, P=0.015), and in African-American versus Caucasian recipients (15.6 + 2.9 vs. 9.1 + 1.3, respectively, P=0.03). Intra-graft angiotensinogen gene expression was significantly elevated in R-FSGS (30.5 + 8.8 ag/fg R-FSGS vs. 16.0 + 4.7 NON-FSGS, P=0.009). We conclude that increased NF-kappaB and angiotensinogen gene expression are associated with R-FSGS. Increased NF-kappaB:IkappaBalpha ratios are associated with cadaveric donor recipients and African-American race.

Background. We have shown previously that heightened expression of the cytotoxic lymphocyte (CL) effector genes perforin (P), granzyme B (GB), and Fas ligand (FasL), is closely correlated with acute allograft rejection, particularly when two or more target genes are up-regulated. Methods. We used quantitative reverse transcription-polymerase chain reaction to analyze CL gene expression from peripheral blood leukocytes (PBLs) and renal allograft biopsies in 31 paired samples of PBLs and renal tissue from 25 renal allograft recipients. Our aims were (1) to determine whether the expression of CL gene expression in PBLs correlates with expression of these genes in renal allograft biopsy tissue and (2) to determine whether CL gene expression in PBLs correlates with the histological diagnosis. Results. Coordinate gene expression in PBLs and acutely rejecting allografts was found in 9/11 (82%) for P, 07/11 (64%) for GB, and 10/11 (91%) for FasL. Coordinate absence was found in 15/20 (75%) for P, 17/20 (85%) for GB, and 16/20 (80%) for FasL in nonrejecting allografts. Furthermore, up-regulation of any two genes in PBLs correlated with pathological diagnosis of rejection with excellent positive (100%) and negative (95%) predictive values. Conclusion. Coordinate CL gene expression in PBLs and the allograft is usually detected. CL gene expression in PBLs is closely associated with a pathologic diagnosis of rejection. CL gene expression in PBLs may serve as a noninvasive method of monitoring for renal allograft rejection. Despite the growing array of immunosuppressive therapies available to transplant recipients, acute allograft rejection is a common event and is a major factor in determining both short-term and long-term outcomes for the transplant recipient (1). The occurrence of acute rejection is a significant risk factor for hastened development of chronic rejection (2), and therefore prompt diagnosis and treatment of acute rejection episodes is of utmost importance. Currently, acute renal allograft rejection is suspected only when the serum creatinine level rises, after the adverse effect of immunological and inflammatory processes on graft function. Surveillance allograft biopsies may prove to be beneficial in predicting rejection, but clinical application is limited by the invasive nature of this procedure. The early phases of T cell activation and the early immune activation events that precede fixed graft tissue injury can now be detected by quantitative reverse transcription-polymerase chain reaction (RT-PCR*) in preclinical models (3). Using human renal allograft biopsy specimens, we have shown previously that quantitative RT-PCR analysis of intragraft gene expression of the cytotoxic cell (CL) effector molecules perforin (P), granzyme B (GB), and Fas ligand (FasL) correlates with the pathological diagnosis with extraordinary sensitivity and specificity, particularly when any two of the three genes are simultaneously up-regulated (4). In this study, we are testing the hypotheses that cytotoxic lymphocyte (CL) effector genes are coordinately expressed in peripheral blood leukocytes (PBLs) and renal allografts and CL effector molecule gene expression in PBLs correlates with the histological diagnosis.

Background Interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15, all T-cell growth factors (TCGFs), utilize the common IL-2 receptor gammac chain as a critical signaling component in their receptor complexes. We have bred IL-2-/- and IL-4-/- double knockout (DKO) mice and showed vigorous islet allograft rejection by DKO hosts. The identity of TCGFs that support the IL-2- and IL-4-independent allograft rejection is unclear. Methods We analyzed IL-9 gene expression in rejecting islet allografts in wild-type and in DKO mice, as well as in human renal transplant biopsy specimens, by reverse transcriptase polymerase chain reaction and compared the expression of IL-9 with that of other TCGFs. Results IL-9 gene expression was not detected in rejecting murine islet allografts in either wild-type or DKO recipient mice despite robust expression of other TCGFs, including IL-7 and IL-15. IL-9 transcripts were also not expressed in any of the human renal transplant biopsies obtained 4 to 251 days after transplantation, regardless of the presence or absence of histological evidence of rejection. Despite expression of IL-9 by DKO splenic cells upon in vitro mitogenic stimulation, IL-9 alone was unable to stimulate the proliferation of concanavalin A-activated splenic leukocytes harvested from DKO mice. Conclusion IL-9 is conspicuously absent despite vigorous expression of IL-2, IL-4, IL-7, and IL-15 genes during acute allograft rejection.

Background The interaction between CD40 and its ligand CD40L is essential for the development and maintenance of vigorous immunity. We have sought to determine: (i) whether a heightened level of CD40L transcripts is evident during acute allograft rejection and (ii) the kinetics of CD40L gene expression during allograft rejection. Methods. By using quantitative reverse transcriptase-assisted polymerase chain reaction techniques, we found that heightened CD40L gene expression is a correlate of acute human renal allograft rejection (P

We developed and applied a quantitative competitive polymerase chain reaction method to study the expression of various cytokine genes in Con A-stimulated murine splenocytes. This method relies on the use of competitive templates that differ from the target cytokine cDNA templates only by the introduction of a unique restriction endonuclease site in the center of each competitive template. After same-tube amplification using a single pair of oligonucleotide primers for both the target and competitive templates, restriction with the unique endonuclease yields 2 species that are readily resolved on agarose gel electrophoresis: full-length target and half-length competitor fragments. Using this method, we studied the time course and effects of CsA and rapamycin on IL-2, IFN-gamma, IL-4, and IL-10 gene expression following Con A stimulation. IL-2, IFN-gamma, and IL-10 share a common pattern of gene expression peaking at approximately 6 hr, while IL-4 gene expression peaks later, at approximately 20 hr. CsA very effectively inhibits expression of IL-2, IFN-gamma, and IL-4, but it inhibits IL-10 expression only 65%. Rapamycin inhibits IL-10 gene expression 100% and is less effective inhibiting the other cytokines. This pattern of inhibition is consistent with a calcium and protein kinase C independent pathway for IL-10 gene regulation and supports the notion that CsA and rapamycin may be used together to advantage.

Background. The development of microsurgical techniques has facilitated the establishment of vascularized composite tissue transplant models in small mammals. Because the mouse would be the ideal model to study various composite tissue allotransplantation (CTA)-related problems, we designed two new surgical techniques for orthotopic (ORT) and heterotopic (HET) hind limb transplantation. Methods. BALB/c hind limbs were transplanted to BALB/c or C57BL6 recipients using a nonsuture cuff technique. ORT: donor femoral vessels were anastomosed to recipient femoral vessels, the sciatic nerve approximated end-to-end and osteosynthesis was performed using an intramedullary rod. HET/cervical: Donor femoral vessels of a reduced size osteomyocutaneous hind limb CTA were anastomosed to recipient common carotid artery and external jugular vein without nerve approximation. Results. Both procedures could be performed with a high success rate (ORT: 62%; HET: 90%). Donor operation lasted for 100±12 min and recipient operation 114±27 min (ORT) and 54±16 min (HET). Complication rates in terms of bleeding, and thrombosis at the cuff side was slightly higher in the ORT group. All syngeneic grafts survived long term (>100 days). FK506 (2 mg/kg) significantly prolonged graft survival (87±22 days) when compared with untreated controls (6±1 day). Functional evaluation of ORT grafts by means of video gait kinematics and CatWalk analysis revealed specific differences of gait parameters when compared with nontransplanted controls (P

Background. As transcriptional profiling techniques appear to provide a useful means to evaluate the allograft response, we have now initiated an attempt to use proteomics to examine the allograft response. Methods. To this end, we have evaluated the use of Protein Chip technology, coupled with bioinformatics analysis towards discovery of allograft response biomarkers in a mouse skin transplant model. To compare samples obtained from acutely rejecting allograft recipients at days 7,9, and 21, we treated one group with a potent antirejection regimen. Mean survival time in the fully MHC-mismatched skin graft model using this protocol is more than 100 days. We also studied recipients of nontreated syngenetic grafts. We applied Protein Chip technology toward discovery of allograft response markers in this model. Results. At days 7 and 9, before the clinical appearance of rejection at day 10, several protein biomarker candidates were detected, based on their molecular mass that clearly differentiated between rejection and the nonrejection groups. Conclusions. Protein profiling of serum as a means to characterize the allografts response of a given host deserves further testing in clinical studies.

Background. Combined treatment of allograft recipients with anti-CD40 ligand and CTLA-4Ig (costimulation blockade) is a powerful promising albeit not consistently tolerizing therapy. It would be desirable to use an effective conventional immunosuppressive regimen in low doses or for a short course as an adjunct; however, cyclosporine treatment drastically blunts the ability of costimulation blockade to produce long-term engraftment. Methods. Short courses of cyclosporine or rapamycin were compared as adjuncts to costimulation blockade in the murine BALB/c to C3H/He heterotopic cardiac allograft model. Results. Although cyclosporine therapy blocked the capacity of costimulation blockade to produce permanent engraftment, combined rapamycin and costimulation blockade treatment produced permanent engraftment. Conclusion. A theoretical basis for the differing effects of cyclosporine and rapamycin upon the outcome of costimulation blockade is forwarded. Combined use of costimulation blockade and rapamycin may provide a means to bring costimulation blockade into the clinic.