Lymphoid neogenesis in skin of human hand,nonhuman primate,and rat vascularized composite allografts

The mechanisms of skin rejection in vascularized composite allotransplantation (VCA) remain incompletely understood. The formation of tertiary lymphoid organs (TLO) in hand transplantation has been recently described. We assess this phenomenon in experimental and clinical VCA rejection. Skin biopsies of human (n = 187), nonhuman primate (n = 11), and rat (n = 15) VCAs were analyzed for presence of TLO. A comprehensive immunohistochemical assessment (characterization of the cell infiltrate, expression of adhesion molecules) including staining for peripheral node addressin (PNAd) was performed and correlated with rejection and time post‐transplantation. TLO were identified in human, nonhuman primate, and rat skin samples. Expression of PNAd was increased in the endothelium of vessels upon rejection in human skin (P = 0.003) and correlated with B‐ and T‐lymphocyte numbers and LFA‐1 expression. PNAd expression was observed at all time‐points after transplantation and increased significantly after year 5. In nonhuman primate skin, PNAd expression was found during inflammatory conditions early and late after transplantation. In rat skin, PNAd expression was strongly associated with acute rejection and time post‐transplantation. Lymphoid neogenesis and TLO formation can be uniformly found in experimental and human VCA. PNAd expression in vascular endothelium correlates with skin rejection and T‐ and B‐cell infiltration.     

The Role of Lymphoid Neogenesis in Allografts

De novo induction of organized lymphoid aggregates at nonlymphoid sites has been observed in many chronic inflammatory conditions where foreign antigens such as infectious agents, autoantigens or alloantigens, persist. The prevailing opinion in the field of transplantation is that lymphoid neogenesis within allografts is detrimental to the establishment of immune tolerance. These structures, commonly referred to as tertiary lymphoid organs (TLOs), are thought to contribute to graft rejection by generating and propagating local alloimmune responses. However, recent studies have shown that TLOs rich in regulatory Foxp3⁺ cells are present in long‐term accepting allografts. The notion that TLOs can contribute to the local downregulation of immune responses has been corroborated in other chronic inflammation models. These findings suggest that contrary to previous suggestions that the induction of TLOs in allografts is necessarily harmful, the induction of “tolerogenic” TLOs may prove advantageous. In this review, we discuss our current understanding of how TLOs are induced and how they regulate immune responses with a particular focus on alloimmunity.     

Tertiary Lymphoid Tissues Generate Effector and Memory T Cells That Lead to Allograft Rejection

Tertiary lymphoid tissues are lymph node‐like cell aggregates that arise at sites of chronic inflammation. They have been observed in transplanted organs undergoing chronic rejection, but it is not known whether they contribute to the rejection process by supporting local activation of naïve lymphocytes. To answer this question, we established a murine transplantation model in which the donor skin contains tertiary lymphoid tissues due to transgenic expression of lymphotoxin‐α(RIP‐LTα), whereas the recipient lacks all secondary lymphoid organs and does not mount primary alloimmune responses. We demonstrate in this model that RIP‐LTα allografts that harbor tertiary lymphoid tissues are rejected, while wild‐type allografts that lack tertiary lymphoid tissues are accepted. Wild‐type allografts transplanted at the same time as RIP‐LTα skin or 60 days later were also rejected, suggesting that tertiary lymphoid tissues, similar to secondary lymphoid organs, generate both effector and memory immune responses. Consistent with this observation, naive T cells transferred to RIP‐LTα skin allograft but not syngeneic graft recipients proliferated and differentiated into effector and memory T cells. These findings provide direct evidence that tertiary lymphoid structures perpetuate the rejection process by supporting naïve T‐cell activation.     

Lymphoid neogenesis and lymphangiogenesis: two newcomers in the pathophysiology of chronic rejection

Chronic rejection is one of the main causes of late allograft failure and no therapy is currently available to prevent efficiently its development. Improving the comprehension of the mechanisms involved in the pathophysiology of chronic rejection is a mandatory step to propose innovative therapies that would prolong grafts' survival. Using the rat aortic interposition model of chronic vascular rejection, we have demonstrated that the intragraft inflammatory infiltrate progressively organized itself into a functional ectopic lymphoid tissue (tertiary lymphoid organ) supporting the local synthesis of alloantibody. Thus, during chronic rejection the graft is at the same time the target and the site of elaboration of the humoral allo-immune response. This hypothesis has been confirmed in the clinical setting by the analysis of human grafts (kidneys, hearts and lungs) removed for terminal failure due to chronic rejection. This lymphoid neogenesis process, previously identified in other chronic inflammatory diseases, occurs with a strikingly high frequency in chronically rejected grafts, suggesting that an additional mechanism synergizes to initiate the development of tertiary lymphoid organs during chronic rejection. We propose that the defective lymphatic drainage of chronically rejected organs triggers lymphoid neogenesis and we discuss the complex crosstalk between lymphoid neogenesis and lymphangiogenesis that takes place during chronic rejection.     

Cytomegalovirus Latency Promotes Cardiac Lymphoid Neogenesis and Accelerated Allograft Rejection in CMV Naïve Recipients

Human cytomegalovirus (HCMV) infection is associated with the acceleration of transplant vascular sclerosis (TVS) and chronic allograft rejection (CR). HCMV‐negative recipients of latently HCMV infected donor grafts are at highest risk for developing CMV disease. Using a rat heart transplant CR model, we have previously shown that acute rat CMV (RCMV) infection following transplantation significantly accelerates both TVS and CR. Here, we report that RCMV‐naïve recipients of heart allografts from latently RCMV‐infected donors undergo acceleration of CR with similar kinetics as acutely infected recipients. In contrast to acutely infected recipients, treatment of recipients of latently infected donor hearts with ganciclovir did not prevent CR or TVS. We observed the formation of tertiary lymphoid structures (TLOs) containing macrophages and T cells in latently infected hearts prior to transplantation but not in uninfected rats. Moreover, pathway analysis of gene expression data from allografts from latently infected donors indicated an early and sustained production of TLO‐associated genes compared to allografts from uninfected donors. We conclude that RCMV‐induced TLO formation and alteration of donor tissue T cell profiles prior to transplantation in part mediate the ganciclovir‐insensitive rejection of latently infected donor allografts transplanted into naïve recipients by providing a scaffold for immune activation.     

IL‐22 is required for the induction of bronchus‐associated lymphoid tissue in tolerant lung allografts

Long‐term survival after lung transplantation remains profoundly limited by graft rejection. Recent work has shown that bronchus‐associated lymphoid tissue (BALT), characterized by the development of peripheral nodal addressin (PNAd)‐expressing high endothelial venules and enriched in B and Foxp3⁺ T cells, is important for the maintenance of allograft tolerance. Mechanisms underlying BALT induction in tolerant pulmonary allografts, however, remain poorly understood. Here, we show that the development of PNAd‐expressing high endothelial venules within intragraft lymphoid follicles and the recruitment of B cells, but not Foxp3⁺ cells depends on IL‐22. We identify graft‐infiltrating gamma‐delta (γδ) T cells and Type 3 innate lymphoid cells (ILC3s) as important producers of IL‐22. Reconstitution of IL‐22 at late time points through retransplantation into wildtype hosts mediates B cell recruitment into lymphoid follicles within the allograft, resulting in a significant increase in their size, but does not induce PNAd expression. Our work has identified cellular and molecular requirements for the induction of BALT in pulmonary allografts during tolerance induction and may provide a platform for the development of new therapies for lung transplant patients.     

Secondary Lymphoid Organs Are Important But Not Absolutely Required for Allograft Responses

The role of secondary lymphoid organs in adaptive immune responses following transplantation is controversial. To examine the requirement for peripheral lymphoid organs in mounting immune responses to transplantation antigens, lymphotoxin alpha-deficient (LTalpha-/-) and LTbeta-receptor-deficient (LTbetaR-/-) mice that lack lymph nodes and Peyer's patches were used as recipients of fully allogeneic heart and skin grafts. Splenectomized LTalpha-/- and LTbetaR-/- mice effectively rejected skin and cardiac allografts, although with delayed kinetics when compared with wild-type controls. In addition, initial skin allograft challenge in splenectomized LTbetaR-/- mice resulted in accelerated rejection of subsequent donor cardiac allografts when compared with heart rejection in nonsensitized controls. Thus, although peripheral lymphoid organs play an important role in allowing allograft responses to occur, they do not appear to be absolutely required for either acute allograft rejection, or T-cell priming. These results suggest that immunologic events capable of leading to allograft rejection can successfully occur at sites other than classical secondary lymphoid organs.     

Lymphoid tissue formation in allografts: innocent until proven guilty

Tertiary lymphoid organs (TLOs) are lymphoid-like structures commonly found at sites of chronic inflammation. Commonly associated with autoimmune responses, they have recently been described within organ allografts. Although TLOs are similar structurally to secondary lymphoid organs, their function remains largely unknown. In this review, we discuss how immune responses within TLOs may influence graft survival.     

Spleen Tyrosine Kinase Modulates Fibrous Airway Obliteration and Associated Lymphoid Neogenesis After Transplantation

Chronic lung allograft dysfunction, the major cause of death following lung transplantation, usually manifests as irreversible airflow obstruction associated with obliterative bronchiolitis (OB), a lesion characterized by chronic inflammation, lymphoid neogenesis, fibroproliferation and small airway obliteration. Spleen tyrosine kinase (Syk), a tyrosine kinase that regulates B cell function and innate immunity, has been implicated in the pathogenesis of chronic inflammation and tissue repair. This study evaluated the role of Syk in development of OB, using an intrapulmonary tracheal transplant model of OB with the conditional Syk‐knockout Syk^flox/flox//rosa26‐CreER^T2 mice and a Syk‐selective inhibitor, GSK2230413. BALB/c trachea allografts were transplanted into Syk‐knockout (Syk^del/del) mice or wild‐type C57BL/6 recipients treated with GSK2230413. At day 28, histological analysis revealed that in the Syk^del/del and GSK2230413‐treated C57BL/6 recipients, the graft lumen remained open compared with allografts transplanted into Syk‐expressing (Syk^flox/flox) and placebo control–treated C57BL/6 recipients. Immunofluorescence showed lymphoid neogenesis with distinct B and T cell zones in control mice. In contrast, lymphoid neogenesis was absent and few B or T cells were found in Syk^del/del and GSK2230413‐treated mice. These observations suggest that inhibition of Syk may be a potential therapeutic strategy for the management of OB following lung transplantation.     

Early intragraft inflammatory events of liver allografts leading to chronic rejection

In this retrospective study, we have investigated the early intragraft inflammatory events of 12 liver allografts leading to chronic rejection. The cytological findings and clinical follow-up were analyzed in detail. Nine patients underwent at least one typical lymphoid activation of acute rejection, and three of them were treated more than once. Diagnosis of rejection was based on biopsy histology, cytology and liver dysfunction. In addition to the acute rejections, cytological analysis demonstrated in 11 of 12 grafts an unidentified lymphoid episode that differed from that of rejection. These lymphoid responses were associated with viral infections; cytomegalovirus (CMV) infection in 10 of 12 patients, hepatitis C virus (HCV) infection in 2 of 12 patients, 1 combined with CMV, and hepatitis B virus (HBV) infection in 1 patient. Graft dysfunction was still seen at the end of the follow-up. Thus, intragraft inflammation caused either by acute rejection or by viral infections may be involved in the induction of chronic rejection.     

Cadaveric versus living-donor livers: differences in inflammatory markers after transplantation

BACKGROUND: Prolonged cold storage of organs for transplantation may lead to inflammatory damage upon reperfusion. The aim of this study was to investigate whether organs from living donors experience less damage upon reperfusion than those retrieved from cadaver donors, where cold ischemia times are significantly longer. METHODS: Biopsies were obtained from cadaveric (n=23) and living-related donor (LRD) (n=10) liver transplants before and 2 hours after reperfusion. Cryosections were stained with antibodies against neutrophils, platelets, activated platelets, and endothelium. RESULTS: LRD liver allografts showed minimal changes postreperfusion. In contrast, after reperfusion of cadaver allografts, neutrophil infiltration was detected in 22% and increased expression of von Willebrand factor (vWF), CD41, and P-selectin in 48%, 30%, and 13% of allografts, respectively. In cadaver allografts with deposition of activated platelets expressing either P-selectin or vWF, the cold ischemia time was significantly longer (885 +/- 123 min vs. 608 +/- 214 min, P=0.04; 776.8 +/- 171 min vs. 559.3 +/- 216 min, P=0.01, respectively). Increases in neutrophils and platelets after reperfusion were not significantly associated with clinical events posttransplant. However, in cadaver transplants that experienced early acute rejection, the mean cold ischemia time was significantly longer than in allografts with no rejection (732 +/- 174 min vs. 480 +/- 221 min, P=0.006). CONCLUSIONS: This study demonstrates that in the clinical situation, cold ischemia causes platelet deposition and neutrophil infiltration after reperfusion of cadaveric liver allografts. These early inflammatory events may contribute to make the graft more susceptible to acute rejection.     

The absence of chronic rejection in pediatric primary liver transplant patients who are maintained on tacrolimus-based immunosuppression: a long-term analysis

BACKGROUND: Although the outcome of liver transplantation has improved significantly during the past two decades, graft loss caused by chronic rejection after liver transplantation still occurs in 2% to 20% of recipients. The overall incidence of chronic rejection is also reported to be low in adult recipients, and risk factors have been identified. Chronic rejection is associated with the inability to maintain baseline immunosuppression. Additionally, the diagnoses of primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune hepatitis, hepatitis B virus, and hepatitis C virus, common indications for liver transplantation in adults, are associated with a higher incidence of chronic rejection. Fortunately, these diagnoses are rarely seen in children. Little is known about chronic rejection in long-term pediatric liver transplant survivors. The purpose of this longitudinal study was to examine the incidence of biopsy-proven chronic rejection in long-term survivors of primary pediatric liver transplantation under tacrolimus-based immunosuppression. METHODS: From October 1989 to December 1992, 166 children (boys=95, girls=71; mean age=5.0+/-2.9 years) received a primary liver transplant. These patients were followed until March 2000 with a mean follow-up of 9+/-0.8 (range, 7.4-10.4) years. All liver biopsy specimens and explanted grafts were evaluated for evidence of chronic rejection using the International Banff Criteria. RESULTS: The mortality rate during the follow-up period was 15% (n=25). Retransplantation was required in 11% (n=18) of recipients. Actuarial patient and graft survival rates at 10 years were 84.9% and 80.1%, respectively. There were 535 liver biopsy samples available for evaluation, including the 18 explanted allografts. Biopsy specimens of three other functioning allografts showed evidence of chronic rejection. Immunosuppression had been discontinued or drastically reduced in these recipients because of life-threatening infections, noncompliance, or both. On restoring baseline immunosuppression, all three children had normalized liver function and the allografts were maintained; the liver transplant patients who are alive currently have normal liver functions. CONCLUSION: The findings of this study suggest that chronic rejection does not occur in pediatric liver transplant recipients receiving tacrolimus-based immunosuppression, provided baseline immunosuppression is maintained.     

Asialo GM1 positive CD8+ T cells induce skin allograft rejection in the absence of the secondary lymphoid organs

BACKGROUND: Secondary lymphoid organs are considered to be the only organs in which APCs and na?ve T cells interact to initiate adaptive immune responses. Aly/aly mice are autosomal recessive mutants of C57BL/6 mice, and lack lymph nodes and Peyer's patches. In this study, we investigated immune responses to skin allografts in splenectomized aly/aly mice, which lack secondary lymphoid organs completely, and examined the effect of anti-asialo GM1 (AsGM1) antibodies on these responses. METHODS: Skin allografts were transplanted to 1) heterozygous aly/+ mice, which had normal secondary lymphoid organs, 2) splenectomized aly/+ mice, 3) aly/aly mice, and 4) splenectomized aly/aly mice, with and without anti-AsGM1 antibody treatment. Graft survival time and alloreactive antibody production were investigated. RESULTS: Heterozygous aly/+ mice and splenectomized aly/+ mice rejected skin allografts acutely. Aly/aly mice also rejected skin allografts, but at a later time than aly/+ mice. Sixty percent of splenectomized aly/aly mice rejected skin allografts within 120 days. Serial administration of anti-AsGM1 antibodies prevented skin allograft rejection in splenectomized aly/aly mice during the same 120-day period of observation. After ceasation of anti-AsGM1 antibody treatment, skin allografts were rejected; we observed a simultaneous increase in AsGM1 expression on CD8+ T cells. Alloreactive antibodies were detected in both splenectomized aly/aly mice that rejected skin allografts and in splenectomized aly/aly mice that accepted skin allografts after treatment with anti-AsGM1 antibodies. CONCLUSIONS: Cytotoxic and humoral immune responses to skin allografts could be initiated despite the absence of secondary lymphoid organs. AsGM1+ cells were important effector cells in secondary lymphoid organ-independent skin allograft rejection.     

Soluble HLA antigens, anti-HLA antibodies, and antiidiotypic antibodies in the circulation of renal transplant recipients

Chronic rejection represents the major threat to long-term survival of organ allografts. It is presumed that this form of rejection is mediated by antibodies against mismatched HLA antigens of the graft. The presence and specificity of anti-HLA-antibodies in posttransplantation sera are, however, difficult to document. We have explored the possibility that anti-HLA antibodies form immune complexes with soluble HLA antigens released from the injured graft and/or that they are blocked by antiidiotypic, anti-anti-HLA-antibodies. Our data demonstrate that the long-term survival of renal allografts is significantly lower in patients who develop anti-HLA-antibodies following transplantation than in patients who do not form antibodies. Following depletion of soluble HLA antigens by magnetic immunoaffinity, we could identify anti-HLA-antibodies in 57% of the sera obtained from patients undergoing chronic rejection of kidney allografts, compared with 41% prior to antigen depletion. In patients tolerating the graft for 4 years or more, the corresponding frequencies of antibody-positive sera was 2% and 5% prior and following depletion of HLA antigens. The presence of HLA antigen/anti-HLA-antibody immune complexes in patients' sera was positively associated with chronic humoral rejection (P less than 0.0001). Patients who tolerated the graft in spite of having developed antibodies against one of its mismatched HLA antigens show specific antiidiotypic (anti-anti-HLA-antibodies). Such antiidiotypic antibodies were not found in sera from patients with chronic rejection (P = 0.005). This indicates that antiidiotypic antibodies may delay the progression of chronic humoral rejection.     

Differential expression of tissue factor (TF) in calcineurin inhibitor-induced nephrotoxicity and rejection--implications for development of a possible diagnostic marker

Deposition of fibrin in the form of fibrinoid necrosis is a common feature of severe acute renal allograft rejection. The role of the coagulation system and its initiator tissue factor (TF) during this process is, however, still poorly understood. In this study, we analyzed the expression of TF in 88 renal transplants afflicted with different forms of rejection and calcineurin inhibitor-induced nephrotoxicity, to see whether there was differential expression of this protein. TF immunoreactivity was evaluated semiquantitatively in six different renal structures: the podocytes, Bowman epithelium, the endothelium of the glomeruli, the brush border of tubular cells, the thin ascending loop of Henle, and small arteries/arterioles. The TF expression of normal renal tissue (n=6) was restricted to the glomerular podocytes and Bowman epithelium, and to some extent the ascending loop of Henle. Renal allografts undergoing acute rejection (AR) of grades I-III, (n=13, n=17 and n=12, respectively) did not show any altered TF expression in the glomeruli or vascular endothelium. In the ascending loop of Henle, a reduced expression could be seen (ARI, p=0.015; and ARII, p=0.043). TF staining of the brush border of renal transplants undergoing acute cyclosporin A (CsA) nephrotoxicity (n=18) was significantly higher than in normal kidneys (p=0.0003), as well as in transplants undergoing various degrees of acute rejection (ARI, p=0.027; ARII, p=0.0012; and ARIII, p=0.0001). Tubular brush border-expressed TF was also evident in 10 of 15 allografts suffering from chronic CsA nephrotoxicity, compared to 4 out of 13 cases with chronic allograft vasculopathy (CAV), but the increase was not statistically significant relative to normal kidneys. The majority of the grafts afflicted with either of the two chronic conditions displayed a TF-positive arterial endothelium (CAV, p=0.0034; and chronic CsA nephrotoxicity, p=0.0026) relative to controls. In conclusion, these results indicate that vascular TF expression is not altered during acute rejection, but may be of importance in chronic allograft nephropathy. Furthermore, TF immunoreactivity in the tubular brush border may be specific to acute CsA nephrotoxicity and might be used as a biomarker for this condition. Further studies are required to evaluate the possible role of brush border-expressed TF in the pathogenesis of CsA nephrotoxicity.     

Donor Lymphoid Organs Are a Major Site of Alloreactive T-Cell Priming Following Intestinal Transplantation

We hypothesized that lymphoid organs within intestinal allografts contribute to their immunogenicity. Consistent with this hypothesis recipient T cells rapidly migrated to the lymph nodes and Peyer's patches of syngeneic and allogeneic intestinal grafts such that at 24 h approximately 50% of the lymphocytes isolated from donor lymphoid organs were of recipient origin. However, only in the lymphoid organs of allografts did recipient T cells display an activated phenotype, proliferate and produce IFNgamma. Rejection of allogeneic intestines lacking lymphoid organs was dramatically impaired in splenectomized, lymph node-deficient recipients compared to lymph node bearing, wild-type allogeneic intestines. This demonstrates the important role of donor lymphoid organs in the rejection process. Furthermore, recipient T cells proliferated more extensively and produced more IFNgamma in donor lymphoid organs than in recipient lymphoid organs, indicating that donor lymphoid organs play a dominant role in initiating the recipient anti-donor immune response following intestinal transplantation.     

Morphological findings in non-episode biopsies of kidney transplant allografts treated with FK506 or cyclosporine

Abstract We conducted an analysis of biopsy specimens of non-episode renal allografts from patients treated with tacrolimus (FK506) or cyclosporine (CsA) to evaluate chronic drug-induced nephropathy in stable allografts. A total of 38 biopsy specimens from stable functioning renal allografts were examined. The patients had been treated with FK506 ( n = 16) or CsA ( n = 18) as main immunosuppressant for 0.3 to 7.4 years. Of the 38 biopsy specimens, 15 showed mild drug-induced arteriolopathy (hyalinosis or insudative change of arterioles and small arteries) with stripeD-form interstitial fibrosis, 10 showed minimum interstitial cellular infiltration (borderline rejection), 2 showed IgA nephropathy, 4 showed evidence of chronic rejection (transplant nephropathy) and 12 showed no abnormal findings. Of 34 renal allograft biopsy specimens with stable function, 22 (65 %) showed pathological evidence of drug-induced nephropathy. There were no significant qualitative or quantitative differences between FK506- and CsA-associated nephropathy.     

Critical Role for IL-6 in Hypertrophy and Fibrosis in Chronic Cardiac Allograft Rejection

Chronic cardiac allograft rejection is the major barrier to long term graft survival. There is currently no effective treatment for chronic rejection except re-transplantation. Though neointimal development, fibrosis, and progressive deterioration of graft function are hallmarks of chronic rejection, the immunologic mechanisms driving this process are poorly understood. These experiments tested a functional role for IL-6 in chronic rejection by utilizing serial echocardiography to assess the progression of chronic rejection in vascularized mouse cardiac allografts. Cardiac allografts in mice transiently depleted of CD4+ cells that develop chronic rejection were compared with those receiving anti-CD40L therapy that do not develop chronic rejection. Echocardiography revealed the development of hypertrophy in grafts undergoing chronic rejection. Histologic analysis confirmed hypertrophy that coincided with graft fibrosis and elevated intragraft expression of IL-6. To elucidate the role of IL-6 in chronic rejection, cardiac allograft recipients depleted of CD4+ cells were treated with neutralizing anti-IL-6 mAb. IL-6 neutralization ameliorated cardiomyocyte hypertrophy, graft fibrosis, and prevented deterioration of graft contractility associated with chronic rejection. These observations reveal a new paradigm in which IL-6 drives development of pathologic hypertrophy and fibrosis in chronic cardiac allograft rejection and suggest that IL-6 could be a therapeutic target to prevent this disease.