Histological Evidence Of Chronic Rejection Research Articles

Circulating Exosomes with Lung Self-Antigens, Kα1Tubulin and Collagen V, and Immune Responses to Lung Self-Antigens Precedes Histological Evidence of Chronic Rejection Following Orthotopic Murine Lung Transplantation

Purpose Lung transplantation (LTx) is a treatment option for end-stage pulmonary diseases. Despite improvements in surgical techniques and post-transplant (Tx) immunosuppression, LTx continues to have one of the poorest long-term outcomes compared to other solid organ Tx, with five year survival of about 50%. Methods Using an orthotopic single LTx model of B6D2F1/J to DBA/2J strain combination, we demonstrate that >80% of transplanted animals developed histological signs of chronic rejection by day 30 of the transplanted lung but not native. We collected blood at days 7, 14, 28, and 30 and serum was tested by ELISA for development of antibodies to lung self-antigens (SAgs), Collagen V (Col-V) and K-alpha 1 Tubulin (Kα1T). Results Significantly elevated levels of antibodies to Col-V and Kα1T were demonstrable on day 14 when there was no histological evidence for chronic rejection of the transplanted lungs. Circulating exosomes were isolated from sera collected on days 7 and 14 which demonstrated increased levels of lung SAgs by western blots in the exosomes isolated from day 14 sera. These results clearly demonstrate that not only antibodies to lung SAgs are present in sera from day 14, but also circulating exosomes with Col-V and Ka1T are easily demonstrable in sera collected on day-14. Semi-quantitation of western blot by densitometry further demonstrated significantly increased levels of exosomes with lung SAgs starting from day 14 until sacrifice on day 30. Splenocytes isolated following sacrifice demonstrated specific reactivity to lung SAgs with significantly high frequency of IFN-γ (Col-V 109±2.5, Kα1T 101±5.5) and TNF-α (Col-V 39±1.5, Kα1T 53±5.5) secreting cells by ELISPOT. Conclusion Taken together our results demonstrate that circulating exosomes with lung SAgs and de novo development of antibodies to lung SAgs occurs prior to histological evidence of chronic rejection in this murine model of chronic rejection following orthotopic single LTx suggesting that circulating exosomes with lung SAgs can contribute in the pathogenesis of chronic rejection following LTx. Further, our data suggests that circulating exosomes with lung SAgs could be a potential biomarker for patients at risk for developing chronic rejection following human LTx.

Donor HLA-C Genotype Has a Profound Impact on the Clinical Outcome Following Liver Transplantation

Late allograft dysfunction is a significant problem following liver transplantation and its pathogenesis is uncertain. HLA-C is the major inhibitory ligand for killer immunoglobulin-like receptors (KIRs) that regulate the cytotoxic activity of natural killer (NK) cells. HLA-C alleles can be allocated into two groups, termed HLA-C1 and HLA-C2, based on their KIR specificity. HLA-C2 interactions are more inhibiting to NK cell activation. We studied the clinical importance of HLA-C genotype in a large liver transplant cohort and found that possession of at least one HLA-C2 allele by the donor allograft was associated with less histological evidence of chronic rejection and graft cirrhosis, a 16.2% reduction in graft loss (p = 0.003) (hazard ratio: 2.7, 95% CI 1.4-5.3) and a 13.6% improvement in patient survival (p = 0.01) (hazard ratio: 1.9, 95% CI 1.1-3.3) at 10 years. Transplantation of an HLA-C2 homozygous allograft led to a particularly striking 26.5% reduction in graft loss (p < 0.001) (hazard ratio: 7.2, 95% CI 2.2-23.0) at 10 years when compared to HLA-C1 homozygous allografts. Donor HLA-C genotype is therefore a major determinant of clinical outcome after liver transplantation and reveals the importance of NK cells in chronic rejection and graft cirrhosis. Modulation of HLA-C and KIR interactions represents an important novel approach to promote long-term graft and patient survival.

Dominant Tolerance to Kidney Allografts Induced by Anti-Donor MHC Class II Antibodies: Cooperation between T and Non-T CD103+ Cells

Allograft acceptance can be induced in the rat by pretransplant infusion of donor blood or spleen cells. Although promoting long-term acceptance, this treatment is also associated with chronic rejection. In this study, we show that a single administration of anti-donor MHC class II alloimmune serum on the day of transplantation results in indefinite survival of a MHC-mismatched kidney graft. Long-term recipients accept a donor-type skin graft and display no histological evidence of chronic rejection. The kidney grafts of tolerant animals display an accumulation of TCR Cbeta, FoxP3, and IDO transcripts. Moreover, as compared with syngeneic recipients, tolerant recipients harbor a large infiltrate of MHC class II(+) cells and CD103(+) cells. In vitro, splenocytes from tolerant recipients exhibit decreased donor-specific proliferation, which is restored by depletion of non-T cells and partially restored by the blockade of IDO. Finally, splenocytes from tolerant recipients, but not purified T cell splenocytes, transfer donor-specific infectious tolerance without chronic rejection, after infusion into naive recipients, over two generations. However, splenocytes depleted of T cells or splenocytes depleted of CD103(+) cells fail to transfer tolerance. Collectively, these data show that a single administration of anti-donor MHC class II alloimmune serum induces a tolerant state characterized by an infiltration of the kidney graft by regulatory T cells and CD103(+) cells. These data also show that the transfer of tolerance requires the presence of both T cells and CD103(+) dendritic cells. The precise mechanism of cooperation of these two cell subsets remains to be defined.

ANTI-CD28 ANTIBODIES-MEDIATED SUPPRESSION OF ACUTE AND CHRONIC ALLOGRAFT REJECTION IS ASSOCIATED WITH THE PRESENCE OF REGULATORY CELLS AND WITH ANTI-DONOR CLASS II ANTIBODIES

P913 Aims: B7/CD28 interactions participate in the initiation of T cell activation and B7/CTLA-4 in the termination. CTLA-4 interaction on B7 molecules on APC also results in the production of indoleamine deoxygenase (IDO), an enzyme that catabolizes tryptophan which, in turn, inhibits T cell proliferation. The activation of IDO has been associated with tolerance induction in rodents. Therefore, our hypothesis is that selectively inhibiting the B7/CD28 pathway without blocking that of B7/CTLA-4 may facilitate tolerance induction. Methods: We monitored the immune responses in a model of acute kidney graft rejection (LEW 1W -RT1Au- to fully mismatched LEW 1A -RT1Aa-), after the selective inhibition of CD28/B7 interaction using the modulating anti-rat monoclonal antibody JJ319. This antibody was previously shown to prevent rejection in the F344 to LEW model of chronic rat kidney graft rejection. Results: A short term treatment with 8 doses of 4mg/kg of JJ319 (day 0 to 7) resulted in 55% of grafts surviving long term (>150 days). Three to four months after transplantation, kidney graft function was normal and stable (Urea: 10 mmol/L, Creatinine: 39μmol/L) and no histological evidence of chronic rejection. Nonetheless, recipients presented an alloantibody response skewed towards a Th-2 type (IgG1 and IgG2a isotypes) and specifically directed against donor MHC II molecules. Alloantibodies were occasionally found deposited onto the graft endothelium but not into the glomeruli. This contrasted with the antibody response of the Th1-type (IgG2b) directed against both MHC I and II molecules deposited onto the endothelium and the glomeruli found in untreated rejecting recipients and in recipients treated on the short term with cyclosporine A, a treatment which prevents acute but not chronic rejection in this combination. In anti-CD28 treated functionally tolerant animals, PBMC and spleen cells were unable to proliferate against donor cells in mixed lymphocyte reactions but proliferated against third party cells. The blockade of IDO (using 1D methyl tryptophan) and NO generation (using N-Methyl-L-Arginin) fully restored the anti-donor reactivity. Moreover, whereas T cells purified from the same PBMC and splenocytes were fully reactive, depletion of either OX42+ (CD11b/c), OX62+, IgM+, HIS24+, or CD8+ cell subsets did not restore proliferation. Conclusions: The selective blockade of CD28 does not generates classical regulatory T cells but regulatory APCs as well as an anti-class II antibody response of the Th2-type. These regulatory mechanisms are associated with a normal kidney graft function in the long term without histological signs of chronic rejection.

Humoral rejection of human organ transplants.

Although T-cell mediated rejection has remained the most common form of acute rejection, humoral rejection now accounts for a substantial fraction in patients with kidney or heart allografts, and probably causes the majority of acute graft losses. The frequency, variously estimated at 20-30%, is attributed to improved methods of detection, including staining for C4d in tissues, which is more sensitive and specific than histological features. Detection of circulating anti-donor reactive antibody (usually to donor HLA antigens) confirms the diagnosis. The clinico-pathological entity of acute humoral rejection is well accepted in kidney and increasingly in heart transplantation. Recent evidence points to a new category of chronic humoral rejection, which accounts for about 60% of chronic rejection of kidneys. Importantly, the hallmark of humoral rejection, C4d, can be detected in the grafts before development of histological evidence of chronic rejection. Humoral rejection is generally not responsive to the usual anti-T cell immunosuppressive agents, but small, non-controlled trials suggest humoral rejection can be reversed with plasmapheresis, intravenous immunoglobulin, anti-CD20 and other treatments, all of which deserve formal clinical evaluation. Prophylaxis for chronic rejection is expected to require donor-specific serological monitoring and protocol biopsies.

Predominant expression of the Th2 response in chronic cardiac allograft rejection.

Chronic rejection is the main cause of late allograft failure in patients. CD4+ T cells activated by indirect recognition of alloantigens are implicated in this rejection reaction. However, the type of T cell response (Th1 vs Th2) that contributes to chronic rejection has not been fully investigated. The purpose of this study is to examine whether chronic rejection is associated with a polarized T-cell response in a rat cardiac allograft model, where long-term graft survival is achieved by intrathymic immunomodulation with donor class I, RT1.Aa, allopeptides. All long-surviving allografts showed histological evidence of chronic rejection. Chronic rejection was associated with high levels of intragraft Th2 cytokines and the Th2-regulated alloantibodies. The Th2 response was systemic, since long-surviving allografts with chronic rejection had high levels of serum IL-10. The predominance of the Th2 cytokines demonstrates that the Th2 response was not sufficient for the prevention of chronic rejection in this model. The predominant expression of Th2 cytokines, together with the presence of Th2-regulated alloantibodies, suggests that the Th2 response may play a role in the development of chronic rejection.

A RATIONALLY DESIGNED IMMUNOMODULATORY PEPTIDE UPPREGULATES EXPRESSION OF HEMEOXYGENASE-1 AND ATTENUATES CHRONIC REJECTION IN A RAT RENAL ALLOGRAFT MODEL

982 Upregulation of hemeoxygenase-1 (HO-1) has been shown to be associated with long term graft survival in various transplant models. In addition, modulation of HO-1 was shown to inhibit cell proliferation of several immune effector functions. Recently, a novel rationally designed immunomodulatory peptide (RDP1258) was shown to induce HO-1 expression in vivo. The present study was undertaken to determine whether peptide RDP1258 would attenuate the pathological changes seen in chronic renal allograft rejection. Orthotopic renal transplantations were performed using F344 (RT11v1) rats as donors and Lewis (RT11) rats as recipients. Recipients were treated briefly (10 days) with low dose cyclosporine (CsA, 0.75mg/kg sc) to reverse the initial acute rejection. Animals that survived > 140 days demonstrated histological evidence of chronic rejection characterized by vascular obliteration, tubular atrophy, glomerulosclerosis as well as interstitial and cortical fibrosis. Recipients were randomly allocated into one of four groups: 1) CsA alone (n=11); 2) CsA + RDP1258, day 0-20 (n=10); 3) CsA + RDP1258, day 50-70 (n=8); and 4) CsA + RDP1258 day 100-120 (n=9). Renal function was assessed by serum creatinine levels at day 140 and pathology was graded by two blinded pathologists. Survival rate > 140 days was 58.3%, 50%, 87.5% and 62.5% for groups 1-4 respectively. Mean serum creatinine levels were all within normal range (66.4-130.1 μmol/L). Grafts treated with CsA and RDP1258 from day 50-70 had a marked reduction in both intimal thickening and cortical scarring compared to grafts treated with CsA alone. (Table)TableLew-Lew controls had 100% survival rate and had no histological evidence of chronic rejection. We conclude that peptide RDP1258 can inhibit transplant vasculopathy in rat renal allografts. Although RDP1258 was shown to upregulate HO-1 expression and to decrease TNFα production, further studies will be necessary to elucidate the peptide's mechanism of action in this chronic rejection model.

Role of the thymus in transplantation tolerance in miniature swine: II. Effect of steroids and age on the induction of tolerance to class I mismatched renal allografts.

Recent studies in young (5-7 months) miniature swine have demonstrated that the thymus is involved in the rapid induction of stable tolerance to class I mismatched renal allografts after a 12-day course of Cyclosporine (CyA). Because both steroids and age are known to influence the structure and function of the thymus, we have now studied the effects of these two parameters on tolerance induction in this model. In young swine, the administration of methylprednisolone (MP) during the standard tolerance-inducing regimen (a 12-day course of CyA) produced severe renal dysfunction and acute cellular rejection histologically. However, the renal allografts recovered and were accepted for >100 days with histological evidence of chronic rejection. To test the effect of age, two relatively old swine (55 and 71 months) received transplants of class I mismatched renal allografts and the standard 12-day course of CyA. One animal rejected the allograft acutely on postoperative day 22, and the second also rejected, but more slowly, with manifestations of chronic rejection. These findings suggest that both MP and old age interfere with the induction of stable tolerance in a fashion similar to the previously described effect of thymectomy. These results may have important implications for the mechanism of thymic-dependent tolerance, for the use of steroids in clinical protocols for the induction of allograft tolerance, and for the application of such protocols to adult patients.

Porcine and bovine cartilage transplants in cynomolgus monkey: I. A model for chronic xenograft rejection.

Transplantation of discordant xenograft tissues usually results in antibody-mediated hyperacute rejection response. It has been speculated that because cartilage has a limited vascular, neural, and lymphatic supply, it might be immunologically privileged and may not undergo hyperacute or chronic rejection. Moreover, porcine and bovine cartilage were found to express very low amounts of alpha-galactosyl epitopes (Gal alpha1-3Gal beta1-4GlcNAc-R). To evaluate animal cartilage for possible human transplantation, xenograft meniscal cartilage was transplanted from pigs and cows into the suprapatellar pouches of six cynomolgus monkeys (group 1). In a second group of six monkeys (group 2), porcine meniscal cartilage and porcine articular cartilage plugs were evaluated. During the 2-month evaluation period in group 1, all monkeys displayed an extensive humoral response to the xenograft, as indicated by the increase in production of antibodies against bovine and porcine cartilage. Upon explant, all meniscal cartilage samples in this group demonstrated histological evidence of chronic rejection, including fibroplasia, encapsulation, mononuclear infiltrates, foreign body giant cells, and eosinophilic infiltrates. There was no difference between the response seen in untreated tissues and that seen in tissues treated with UV irradiation or ozone oxidation. In group 2, the menisci explanted after 1 month displayed extensive infiltration of eosinophils alone or eosinophils mixed with mononuclear cells. The mononuclear infiltrates consisted primarily of CD4+ and CD8+ T cells and of macrophages. The articular cartilage plugs demonstrated only a small area of fibrous encapsulation and leukocyte infiltration at the periphery. This study suggests that xenograft cartilage tissue does not appear to be immunoprivileged and is unsuitable for human implantation due to a chronic rejection mechanism, which is evident already within 1 month after transplantation. In addition, this study may serve as a general model for the primate immune response against xenografts in the absence of hyperacute rejection.

THE ALLOANTIBODY RESPONSE AND ACTIVE ENHANCEMENT OF SOME RAT RENAL ALLOGRAFTS

Lewis rats that were given injections of 10(6) to 10(8) (Lewis X Brown Norway) F1 hybrid bone marrow cells produce predominantly, if not exclusively, 19S lymphocytotoxic antibodies. A number of Lewis rats that received transplants of perfused renal allografts from bone marrow donors at, or near, the peak of IgM response survival for well over 200 days with good renal function and no histological evidence of chronic rejection. All long-surviving rats had detectable lymphocytotoxic antibodies up to 120 days after allografting; late enhancing antibodies had the restricted specificity possibly identical or similar to anti-I region antisera. All rats bearing prolonged renal allografts were unable to accept donor-specific skin grafts or to respond with specific lymphocytotoxic antibodies following skin grafting. The possible involvement of non-complement-fixing 19S alloantibodies in active enhancement of rat renal allografts is discussed.

Effect of organ culture on the survival of thyroid allografts in mice.

Mouse thyroid can be maintained in organ culture for 4 weeks. Uncultured BALB/c thyroid is rejected 10-15 days after transplantation under the kidney capsule of H-2 disparate recipients (C57BL, CBA). Organ culture of thyroid tissue prior to transplantation prolongs allograft survival. This prolongation of graft survival increases with increasing time in culture and 80-90% of BALB/c thyroids maintained in culture for 26 days survive in allogeneic CBA recipients for a 60- to 70-day test period. These allografts show normal function as measured by 125I uptake, and show no histological evidence of chronic rejection. Cultured allografts can be rejected if the host's immune system is stimulated with viable leukocytes of donor origin. Host animals carrying a functioning allograft are not tolerant of donor tissues and will reject a second uncultured allograft from the same donor strain.