Adoptive transfer of donor-B lymphocytes: a phase I/IIa study for patients after allogeneic stem cell transplantation.

Immune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is slow and patients carry a high and prolonged risk for opportunistic infections. We hypothesized that the adoptive transfer of donor B cells can foster post-HSCT immuno-reconstitution. Here we report on the results of a first-in-human phase I/IIa study aimed to evaluate the feasibility and safety of adoptively transferred donor B cells and to test their activity upon recall vaccination. GMP-grade B-cell products were generated from donor apheresis products using a two-step magnetic cell separation. 15 allo-HSCT patients were enrolled and treated after taper of immunosuppression (median day +148, range 130 to 160). Patients received four different doses of B cells (0.5x106 - 4.0x106 B-cells per kg body weight (BW)). To test the activity of infused donor memory B cells in vivo patients were vaccinated with a pentavalent vaccine 7 days after B cell transfer. We observed mobilization of plasmablasts and an increase of serum titers against vaccine antigens with a stronger response in patients receiving higher B cell numbers. The analysis of immunoglobulin VH-sequences by next-generation-sequencing revealed that plasmablasts responding to the vaccination originated from memory B cell clones from the donor. Donor B cell transfer was safe as no EBV reactivation was observed, and only low-grade GvHD occurred in 4 out of 15 patients. This pilot trial may pave the way for further studies exploring the adoptive transfer of memory B cells to reduce the frequency of infections after allo-HSCT. This trial was registered at ClinicalTrial.gov NCT02007811.

Correlation of Fc Receptor Polymorphisms with Pneumococcal Antibodies in Vaccinated Kidney Transplant Recipients.

Several polymorphisms within Fc receptors (FCR) have been described, some of which correlate with allograft function. In the current study, we determined three Fcγ receptor and five Fcα receptor dimorphisms in 47 kidney transplant recipients who had been vaccinated against Streptococcus pneumoniae. We analyzed if FCR genotypes correlated with pneumococcal antibodies and their serotype-specific opsonophagocytic function, tested prior to and at months 1 and 12 post-vaccination. In parallel, we assessed antibodies against HLA and MICA and determined kidney function. We observed that IgG2 antibodies against pneumococci at months 1 and 12 after vaccination and IgA antibodies at month 1 differed significantly between the carriers of the three genotypes of FCGR3A rs396991 (V158F, p = 0.02; 0.04 and 0.009, respectively). Moreover, the genotype of FCGR3A correlated with serotype-specific opsonophagocytic function, reaching statistical significance (p < 0.05) at month 1 for 9/13 serotypes and at month 12 for 6/13 serotypes. Heterozygotes for FCGR3A had the lowest antibody response after pneumococcal vaccination. On the contrary, heterozygotes tended to have more antibodies against HLA class I and impaired kidney function. Taken together, our current data indicate that heterozygosity for FCGR3A may be unfavorable in kidney transplant recipients.

A noninvasive diagnostic approach to retrospective donor HLA typing in kidney transplant patients using urine.

Antibody-mediated rejection (AMR) is a major obstacle to long-term kidney transplantation. AMR is mostly caused by donor specific HLA antibodies, which can arise before or any time after transplantation. Incomplete donor HLA typing and unavailability of donor DNA regularly preclude the assessment of donor-specificity of circulating anti-HLA antibodies. In our centre, this problem arises in approximately 20% of all post-transplant HLA-antibody assessments. We demonstrate that this diagnostic challenge can be resolved by establishing donor renal tubular cell cultures from recipient´s urine as a source of high-quality donor DNA. DNA was then verified for genetic origin and purity by fluorescence in situ hybridization and short tandem repeat analysis. Two representative cases highlight the diagnostic value of this approach which is corroborated by analysis of ten additional patients. The latter were randomly sampled from routine clinical care patients with available donor DNA as controls. In all 12 cases, we were able to perform full HLA typing of the respective donors confirmed by cross-comparison to results from the stored 10 donor DNAs. We propose that this noninvasive diagnostic approach for HLA typing in kidney transplant patients is valuable to determine donor specificity of HLA antibodies, which is important in clinical assessment of suspected AMR.

Targeting of canonical WNT signaling ameliorates experimental sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation. The molecular mechanisms underlying cGVHD remain poorly understood, and targeted therapies for clinical use are not well established. Here, we examined the role of the canonical WNT pathway in sclerodermatous cGVHD (sclGVHD). WNT signaling was activated in human sclGVHD with increased nuclear accumulation of the transcription factor ß-catenin and a WNT-biased gene expression signature in lesional skin. Treatment with the highly selective tankryase inhibitor G007-LK, the CK1α agonist pyrvinium, or the LRP6 inhibitor salinomycin abrogated the activation of WNT signaling and protected against experimental cGVHD, without a significant impact on graft-versus-leukemia effect (GVL). Treatment with G007-LK, pyrvinium, or salinomycin almost completely prevented the development of clinical and histological features in the B10.D2 (H-2d) → BALB/c (H-2d) and LP/J (H-2b) → C57BL/6 (H-2b) models of sclGVHD. Inhibition of canonical WNT signaling reduced the release of extracellular matrix from fibroblasts and reduced leukocyte influx, suggesting that WNT signaling stimulates fibrotic tissue remodeling by direct effects on fibroblasts and by indirect inflammation-dependent effects in sclGVHD. Our findings may have direct translational potential, because pyrvinium is in clinical use, and tankyrase inhibitors are in clinical trials for other indications.

Role of Donor Clonal Hematopoiesis in Allogeneic Hematopoietic Stem-Cell Transplantation.

PURPOSE: Clonal hematopoiesis of indeterminate potential (CHIP) occurs in the blood of approximately 20% of older persons. CHIP is linked to an increased risk of hematologic malignancies and of all-cause mortality; thus, the eligibility of stem-cell donors with CHIP is questionable. We comprehensively investigated how donor CHIP affects outcome of allogeneic hematopoietic stem-cell transplantation (HSCT). METHODS: We collected blood samples from 500 healthy, related HSCT donors (age ≥ 55 years) at the time of stem-cell donation for targeted sequencing with a 66-gene panel. The effect of donor CHIP was assessed on recipient outcomes, including graft-versus-host disease (GVHD), cumulative incidence of relapse/progression (CIR/P), and overall survival (OS). RESULTS: A total of 92 clonal mutations with a median variant allele frequency of 5.9% were identified in 80 (16.0%) of 500 donors. CHIP prevalence was higher in donors related to patients with myeloid compared with lymphoid malignancies (19.2% v 6.3%; P ≤ .001). In recipients allografted with donor CHIP, we found a high cumulative incidence of chronic GVHD (cGVHD; hazard ratio [HR], 1.73; 95% CI, 1.21 to 2.49; P = .003) and lower CIR/P (univariate: HR, 0.62; 95% CI, 0.40 to 0.97; P = .027; multivariate: HR, 0.63; 95% CI, 0.41 to 0.98; P = .042) but no effect on nonrelapse mortality. Serial quantification of 25 mutations showed engraftment of 24 of 25 clones and disproportionate expansion in half of them. Donor-cell leukemia was observed in two recipients. OS was not affected by donor CHIP status (HR, 0.88; 95% CI, 0.65 to 1.321; P = .434). CONCLUSION: Allogeneic HSCT from donors with CHIP seems safe and results in similar survival in the setting of older, related donors. Future studies in younger and unrelated donors are warranted to extend these results. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that donor CHIP might foster cGVHD development and reduce relapse/progression risk.

Analysis of Luminex-based Algorithms to Define Unacceptable HLA Antibodies in CDC-crossmatch Negative Kidney Transplant Recipients.

BACKGROUND: HLA-specific antibodies detected by solid phase assays are increasingly used to define unacceptable HLA antigen mismatches (UAM) before renal transplantation. The accuracy of this approach is unclear. METHODS: Day of transplant sera from 211 complement-dependent cytotoxicity crossmatch-negative patients were retrospectively analyzed for donor-specific anti-HLA antibodies (DSA) using Luminex technology. HLA were defined as UAM if DSA had mean fluorescence intensity above (I) 3000 (patients retransplanted and those with DSA against HLA class I and II) or 5000 (all other patients), (II) 5000 for HLA-A, -B, and -DR and 10 000 for HLA DQ or (III) 10 000 (all HLA). We then studied the accuracy of these algorithms to identify patients with antibody-mediated rejection (AMR) and graft loss. UAM were also determined in 256 transplant candidates and vPRA levels calculated. RESULTS: At transplantation, 67 of 211 patients had DSA. Of these, 31 (algorithm I), 24 (II) and 17 (III) had UAM. Nine (I and II) and 8 (III) of 11 early AMR episodes and 7 (I), 6 (II) and 5 (III) of 9 graft losses occurred in UAM-positive patients during 4.9 years of follow-up. Algorithms I and II identified patients with persistently lower glomerular filtration rate even in the absence of overt AMR. Of the waiting list patients, 22-33% had UAM with median virtual panel reactive antibody of 69.2% to 79.1%. CONCLUSIONS: Algorithms I and II had comparable efficacy but were superior to Algorithm III in identifying at-risk patients at an acceptable false-positive rate. However, Luminex-defined UAM significantly restrict the donor pool of affected patients, which might prolong waiting time.

Characteristics of donor-specific anti-HLA antibodies and outcome in renal transplant patients treated with a standardized induction regimen.

Background: Pre-transplant donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) have been associated with antibody-mediated rejection (AMR) and early kidney allograft loss. Uncertainties remain regarding the general applicability of these findings and the optimal induction therapy in DSA-positive patients. Methods: Pre-transplant sera from 174 patients receiving a crossmatch-negative kidney transplant were retrospectively analysed for DSA using Luminex technology. DSA with mean-fluorescence intensity (MFI) values above 500 were considered positive. All recipients received basiliximab induction and tacrolimus-based maintenance immunosuppression. DSA were monitored post-transplantation in patients with pre-transplant DSA. Antibody results were correlated with the incidence of rejection and graft loss. Results: In total, 61/174 patients had pre-transplant DSA. We found a strong correlation between the presence of DSA against class I and II HLA and DSA MFI greater than 10 000. Both DSA patterns independently predicted an increased risk of early AMR (odds ratio 4.24 and 4.75, respectively, P < 0.05). The risk for AMR in patients with intermediate MFI (3000-10 000) gradually increased with increasing MFI but group sizes were too small to allow for final conclusions. The risk for AMR was comparable to nonsensitized patients in patients with only class I or II HLA-DSA or MFI below 3000. 5-year allograft survival was lowest in patients with simultaneous presence of class I and II HLA-DSA and MFI above 10 000 (45%) but was comparable between patients with only HLA class I or II or no DSA (90.0, 90.0 and 88.1%, respectively). AMR was the only independent predictor of graft loss. Undetectable DSA 14 days post-transplant predicted excellent long-term outcome. Conclusion: . The favourable outcome in the majority of DSA-positive patients despite non-depleting antibody induction and the poor outcome in patients with class I and II HLA-DSA and high DSA strength call for a differentiated therapeutic approach in this patient population.

Presence of HLA-B27 is associated with changes of serum levels of mediators of the Wnt and hedgehog pathway.

BACKGROUND: HLA-B27 is present in 5% of the Caucasian population and is strongly associated with the development of spondyloarthritis (SpA), a disease characterized by inflammation and substantial bone changes. We hypothesized that the presence of HLA-B27 in itself is associated with alterations of key regulatory of bone homeostasis. METHODS: Sera of 241 individuals were assessed for the serum levels of Wnt pathway regulators, sclerostin and dickkopf (Dkk)-1 as well as Indian hedgehog (IHH) and collagen type I cleavage products (CTX1). Of the 151 HLA-B27+ subjects, 31 had SpA, 30 had anterior uveitis, 30 were healthy individuals and 60 healthy siblings of patients with SpA. RESULTS: Sclerostin levels were significantly (P<0.001) lower in HLA-B27+ subjects (314±21pg/mL) compared to HLA-B27 negative controls (mean±SEM: 492±30pg/mL), no matter if subjects were either healthy, or affected by SpA or uveitis. Similar results were found for Dkk-1. No differences between the groups with respect to the bone resorption marker CTX1 were found. In contrast, IHH levels were significantly (P<0.001) higher in the carriers of HLA-B27 than in the negative controls. CONCLUSIONS: Changes in key regulators of the Wnt pathway as well as IHH, a molecule regulating endochondral ossification, are found in HLA-B27 carriers, independent if they were healthy or affected by uveitis or SpA.

Association of a coding polymorphism in Fc gamma receptor 2A and graft survival in re-transplant candidates.

The family of Fc gamma receptors (FcγRs) is involved in mediating immunological effector functions. FcγRs are differentially expressed on immune cells and can act either activating or inhibitory, with FcγR2A belonging to the first group. The polymorphism H131R (rs1801274) in FCGR2A has been associated with acute rejection and can shift the overall balance between activating and inhibitory FcγRs. Anti-HLA allo-antibodies in transplant recipients have been identified as risk factor for organ survival after transplantation. In this study we genotyped FCGR2A H131R in 200 patients who had undergone kidney transplantation and experienced loss of graft function. FCGR2A polymorphism was related to graft survival and anti-HLA antibodies. Graft survival was calculated as the time interval between transplantation and return to chronic dialysis after transplantation. The gene frequency of FCGR2A R/R131 was found significantly more often in patients with earlier (⩽60months) compared to patients with later (>60months) graft failure. Overall patients homozygous for R/R131 had a significantly shorter graft survival, compared to H/H131 or H/R131 which is even more pronounced, when anti-HLA antibodies were present. These data suggest, that FCGR2A polymorphisms constitute a risk factor for graft loss following kidney transplantation and that this effect is related to anti-HLA antibodies.

Monitoring of hematopoietic chimerism by real-time quantitative PCR of micro insertions/deletions in samples with low DNA quantities.

BACKGROUND: Sensitive and accurate methods to detect hematopoietic chimerism after hematopoietic stem cell transplantation (HSCT) are essential to evaluate engraftment and to monitor response to therapeutic procedures such as donor lymphocyte infusion. Continuous long-term follow up, however, requires large amounts of pre-HSCT samples limiting the application of many widely used techniques for sensitive chimerism monitoring. METHODS: DNAs from 42 normal healthy donors and 16 HSCT donor/recipient pairs were employed to validate the use of allele-specific insertion/deletion (indel) quantitative real-time polymerase chain reaction (qPCR) to quantify chimerism in samples with low amounts of DNA. Consequently, indel-qPCR analyses of samples from 16 HSCT patients were compared to short-tandem repeat (STR) specific PCR analyses. RESULTS: Typing with reduced amounts of input DNA (15 vs. 60 ng) allowed for the reliable distinction of positive (mean threshold cycle (ct) 28.05) and negative (ct >36) signals. The high informativity of primer/probe sets, with 12 out of 19 markers exceeding 20% informativity, was confirmed in our cohort (n = 74). Importantly, a fourfold reduction of input DNA compared to published protocols did not alter PCR efficiencies and allowed for a more sensitive detection of chimerism in 7 of 16 HSCT patients compared to results obtained by STR-PCR. CONCLUSIONS: Our data suggest that indel-qPCR is a more sensitive technique for the detection of hematopoietic chimerism compared to STR-PCR and works efficiently for samples with low amounts of DNA.

Identification of HLA-C restricted, HIV-1-specific CTL epitopes by peptide induced upregulation of HLA-C expression.

HIV-1 negative regulatory factor (Nef) can inhibit CTL recognition by downregulation of HLA-A and HLA-B on the cell surface. In contrast, HLA-C is not affected by Nef and a growing number of studies demonstrate an important role of HLA-C for the control of HIV-1. So far, only a limited number of HLA-C restricted CTL epitopes are known. As the mapping of new CTL epitopes is time and labor intensive, we investigated a novel method for the identification of HLA-C restricted CTL epitopes. B-lymphoblastoid cell lines (B-LCLs) and T2-cells were incubated with HIV-1 specific peptides and subsequently stained for HLA-C surface expression using the HLA-C specific antibody DT9. Peptides that led to increased HLA-C surface expression were used for stimulation of PBMC from HIV-1-infected patients. Subsequently, outgrowing cells were tested for peptide recognition in IFN-γ ELISPOT assays and HLA restriction of the recognized peptides was analyzed in ELISPOT assays using HLA-matched B-LCL. We observed that known HLA-C binding peptides increase HLA-C surface expression on T2-cells and on HLA-C*0102 and HLA-C*0702 homozygous B-LCL. Moreover, screening of HIV-1 Nef with overlapping peptides for potential C*0702 restricted epitopes using this method revealed a total of 8 peptides which considerably increased cell surface expression of HLA-C. By epitope mapping and functional analysis of peptide-stimulated T-cell lines we were able to define the peptide YPLTFGWCY as a new C*0702-restricted CTL epitope. These results show that the analysis of peptide induced HLA-C upregulation on B-LCL and T2-cells enables the efficient identification of new HLA-C restricted CTL epitopes.

Delayed therapy with clopidogrel and everolimus prevents progression of transplant arteriosclerosis and impairs humoral alloimmunity in murine aortic allografts.

OBJECTIVES: It was previously shown that the combination of clopidogrel and everolimus reduced the development of transplant arteriosclerosis. The aim of this study was to investigate whether delayed onset of treatment, similar to the clinical situation after heart transplantation, inhibits progression of transplant arteriosclerosis. METHODS: Fully allogeneic C57BL/6 (H2-b) donor aortas were transplanted into CBA.J (H2-k) recipients treated with clopidogrel and everolimus alone or in combination starting on Days 1, 7 or 14. Grafts were analysed by histology and alloantibodies were detected by fluorescence activated cell sorting after transplantation. RESULTS: Delayed platelet inhibition with clopidogrel reduced the development of transplant arteriosclerosis [neointima formation (Day 14): 50±4 vs 84±9% (control)]. The combination of clopidogrel and everolimus almost abolished formation of transplant arteriosclerosis when therapy was started on Day 1 [neointima formation (Day 1): 14±5 vs 84±9% (control)] and also showed a remarkable reduction in both delayed treatment groups [neointima formation (Day 7): 24±7 vs 84±9% (control); neointima formation (Day 14): 28±11 vs 84±9% (control)]. Platelet inhibition alone and in combination with everolimus resulted in reduced alloantibody production. CONCLUSIONS: These results demonstrate that delayed treatment with clopidogrel and everolimus-representative of a clinical setting-prevents the progression of transplant arteriosclerosis and impairs humoral immunity in this experimental model.

Donor-specific HLA antibodies: evaluating the risk for graft loss in renal transplant recipients with isotype switch from complement fixing IgG1/IgG3 to noncomplement fixing IgG2/IgG4 anti-HLA alloantibodies.

Human leukocyte antigen alloantibodies have a multitude of damaging effects on the allograft, both complement (C') activation and Fc-independent ones. To date, the clinical significance of non-C' fixing (NCF) HLA donor-specific antibodies (DSA) is still unclear. In this study, we investigated whether renal transplant recipients with NCF-DSA subclasses (IgG2/IgG4, IgA1/IgA2) are at higher risk of graft loss compared to patients with exclusively C' fixing (IgG1/IgG3). Blood samples from 274 patients were analyzed for HLA IgG and IgA subclasses using a modified single-antigen bead assay. We identified 50 (18.2%) patients with circulating NCF antibodies either DSA (n=17) or against third-party HLA (n=33). NCF-DSAs were preferentially of IgG2/IgG4 isotype (11/17) and were mainly directed against HLA class II (13/17). NCF DSA were present as a mixture with strong C' fixing IgG1/IgG3. Graft survival was similar between patients with exclusively C' fixing antibodies and those with a mixture panel (log rang test P=0.162), and also among patients with different immunoglobulin isotype and subclasses (long-rank test, P=0.732). We conclude that expansion of DSA to NCF subclasses postrenal transplantation does not seem to be associated with worse graft survival as compared to the presence of exclusive C' fixing subclasses.

Clopidogrel reduces post-transplant obliterative bronchiolitis.

Survival after lung transplantation is mainly limited by the development of chronic lung allograft dysfunction (CLAD). The aim of this study was to investigate if platelet inhibition by clopidogrel has an influence on the formation of obliterative bronchiolitis, the histopathological correlate to bronchiolitis obliterans syndrome, present in the majority of patients suffering from CLAD. C57Bl/6(H2(b) ) donor tracheas were orthotopically transplanted into CBA.J(H2(k) ). Mice received different doses of clopidogrel alone or in combination with tacrolimus or everolimus. Grafts were analyzed by histology and immunofluorescence method on postoperative days 15, 30 or 60. Cytokines were analyzed by real-time polymerase chain reaction on postoperative day 21 and alloantibodies by FACS. Mice treated with 20 mg/kg/day clopidogrel for 30 days showed reduced obliteration [34.40 ± 3.76% (20 mg/kg/day clopidogrel) vs. 49.92 ± 2.11% (control), n = 5, P < 0.05]. Platelet inhibition resulted in significant lower infiltration of T cells and macrophages, and we also found significantly lower expression of IL-12, IL-4, IL-6, TNF-α, TGF-ß, PDGFß, MCP1, P-/E-selectin, ICAM1 and CD40L after treatment with clopidogrel. Combination of 1 mg/kg/day clopidogrel and 0.05 mg/kg/day everolimus or 12 mg/kg/day tacrolimus revealed a synergistic effect. Humoral immunity as manifested by donor-specific alloantibody secretion was also impaired after treatment with clopidogrel. Here, we can show that platelet inhibition by clopidogrel as a single treatment and in combination with tacrolimus or everolimus reduced the development of fibrosis and obliteration in tracheal allografts.

Major histocompatibility complex class II polymorphisms are associated with the development of anti-resorptive agent-induced osteonecrosis of the jaw.

The aetiology of anti-resorptive agent-induced osteonecrosis of the jaw (ARONJ) is still under debate. Clinical and genetic risk factors are currently being investigated to help understand its pathogenesis. This case-control study analysed a large number of cancer patients (n = 230) under therapy with intravenous bisphosphonates, half of which were diagnosed with ARONJ. Multiple myeloma, greater patient age and the use of more than one bisphosphonate were identified as clinical risk factors on logistic regression analysis. In addition, 204 patients were genotyped for HLA-DRB1 and DQB1 and the allele frequencies were compared between ARONJ (n = 94) and unaffected cancer patients (n = 110). For the HLA class II alleles, a strong increase in the frequency of DRB1*15, DQB1*06:02, DRB1*01 and DQB1*05:01 was observed in the ARONJ group. These results were reinforced on analysis of the respective haplotypes, with DRB1*15-DQB1*06:02 being significantly associated with the development of ARONJ (odds ratio [OR] 2.5; 95% confidence interval [CI] 1.3-5.0). The presence of at least one of the haplotypes DRB1*15-DQB1*06:02 and DRB1*01-DQB1*05:01 was highly associated with the development of ARONJ (OR 3.0; 95% CI 1.7-5.5). The data in this study of a large number of cancer patients receiving intravenous bisphosphonates suggest that MHC class II polymorphisms represent genetic risk factors for the development of ARONJ. This result supports recent findings that inflammation and infection might play an important role in the pathogenesis of ARONJ.

Attenuation of transplant arteriosclerosis by oral feeding of major histocompatibility complex encoding chitosan-DNA nanoparticles.

One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the alloimmune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation. Therefore, we performed fully allogeneic mouse abdominal aortic transplants using C57BL/6 (H2(b)) mice as donors and CBA.J (H2(k)) mice as recipients. Aortic grafts were analyzed by histology and morphometry on day 30 after transplantation, levels of circulating alloantibodies were detected by FACS analysis. Pre-treatment of recipient mice with chitosan-DNA nanoparticles encoding for K(b), one of the MHC-I molecules of the donor, resulted in a significant reduction of intimal proliferation compared to untreated controls. When Ovalbumin was fed instead of K(b) encoding nanoparticles (K(b)-NP) or Balb/c (H2(d)) grafts were used instead of C57BL/6 (H2(b)) grafts as antigen controls, both groups showed no reduction of intimal thickness indicating an antigen-specific mechanism. In addition, analysis of peripheral blood of the transplanted mice showed significant suppression of alloantibody formation in the K(b)-NP fed group compared to all other allogeneic transplanted groups suggesting modulation of the humoral immune response. These results demonstrate the potential of chitosan-DNA nanoparticles to induce K(b)-specific tolerance and to reduce the development of transplant arteriosclerosis.

Reduction of transplant arteriosclerosis after treatment with mycophenolate mofetil and ganciclovir in a mouse aortic allograft model.

OBJECTIVES: Transplant arteriosclerosis is a major obstacle for long-term allograft survival in heart transplant. The aim of this study was to investigate potential synergistic effects of combined treatment with mycophenolate mofetil and ganciclovir on the development of transplant arteriosclerosis, presence of regulatory T cells, and expression of donor specific alloantibodies. MATERIALS AND METHODS: Donor aortas from C57BL/6 (H2b) mice that were fully mismatched to the major histocompatibility complex were transplanted into CBA (H2k) mouse recipients. Groups of mice received mycophenolate mofetil (100 or 300 mg/kg, oral), ganciclovir (10 or 72 mg/kg, intraperitoneal), or a mycophenolate mofetil and ganciclovir combination. Grafts were analyzed by histology and morphometry on day 30 after transplant. Numbers of regulatory T cells and donor-specific alloantibodies were examined by fluorescence-activated cell sorting analysis of splenic tissue and peripheral blood. RESULTS: Mycophenolate mofetil (100 mg/kg) and ganciclovir (10 mg/kg and 72 mg/kg) did not show effects on transplant arteriosclerosis formation or alloantibody production. However, groups treated with mycophenolate mofetil (300 mg/kg) or a low- or high-dose mycophenolate mofetil and ganciclovir combination had significantly reduced transplant arteriosclerosis and alloantibody levels. Expression of regulatory T cells within the spleen was similar between all experimental groups and untreated controls. CONCLUSIONS: The combination of mycophenolate mofetil and ganciclovir significantly reduced the development of transplant arteriosclerosis in a mouse abdominal aortic allograft model. This effect may be a result of decreased alloantibody production.

Combined inhibition of c-Abl and PDGF receptors for prevention and treatment of murine sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGvHD) is a common complication of allogeneic bone marrow transplantation, and has a major effect on the long-term prognosis. The molecular mechanisms underlying cGvHD have been only partially revealed, and molecular targeted therapies have not yet been established for clinical use. We examined the effects of the combined inhibition of the Abelson kinase (c-Abl) and platelet-derived growth factor receptors (PDGFR) in experimental sclerodermatous cGvHD. Treatment using imatinib or nilotinib abolished the aberrant activation of c-Abl and PDGFR and protected against experimental cGvHD. Preventive therapy using imatinib or nilotinib inhibited the development of sclerodermatous cGvHD. Clinical features such as weight loss, alopecia, and skin ulcers, and histologic features with dermal thickening and accumulation of collagen were significantly reduced in mice that received imatinib or nilotinib therapy, but not in mice that received prednisone therapy. Of note, imatinib and nilotinib were also effective for treatment of experimental cGvHD that had already been clinically manifested. In summary, the combined inhibition of c-Abl and PDGFR is effective for prevention and treatment of experimental sclerodermatous cGvHD. Considering the high morbidity associated with cGvHD, the lack of efficient molecular therapies for clinical use, and first positive signals from uncontrolled studies of imatinib, combined inhibition of c-Abl and PDGFR might be a promising future strategy for treatment of sclerodermatous cGvHD.

Oral gene application using chitosan-DNA nanoparticles induces transferable tolerance.

Oral tolerance is a promising approach to induce unresponsiveness to various antigens. The development of tolerogenic vaccines could be exploited in modulating the immune response in autoimmune disease and allograft rejection. In this study, we investigated a nonviral gene transfer strategy for inducing oral tolerance via antigen-encoding chitosan-DNA nanoparticles (NP). Oral application of ovalbumin (OVA)-encoding chitosan-DNA NP (OVA-NP) suppressed the OVA-specific delayed-type hypersensitivity (DTH) response and anti-OVA antibody formation, as well as spleen cell proliferation following OVA stimulation. Cytokine expression patterns following OVA stimulation in vitro showed a shift from a Th1 toward a Th2/Th3 response. The OVA-NP-induced tolerance was transferable from donor to naïve recipient mice via adoptive spleen cell transfer and was mediated by CD4(+)CD25(+) T cells. These findings indicate that nonviral oral gene transfer can induce regulatory T cells for antigen-specific immune modulation.