Hyperlipidemia Promotes Anti‐Donor Th17 Responses That Accelerate Allograft Rejection

Hyperlipidemia occurs in 95% of organ transplant recipients, however its effect on organ allograft rejection has not been investigated. We found that induction of hyperlipidemia in mice caused a significant acceleration of rejection of cardiac allografts. Accelerated rejection was associated with an aggressive T cell infiltrate that mediated significant tissue damage as well as increased serum levels of the proinflammatory cytokines IL‐2, IL‐6, and IL‐17. Hyperlipidemic mice had an increased number of Th17 cells in their periphery and rejecting allografts from hyperlipidemic mice contained significant numbers of IL‐17 producing T cells that were not detectable in transplants harvested from controls. Neutralization or genetic ablation of IL‐17 prolonged survival of cardiac allografts transplanted into hyperlipidemic recipients, suggesting that IL‐17 production promotes accelerated rejection. Analysis of alloreactive T cell frequencies directly ex vivo in naïve mice revealed that the frequency of donor reactive IL‐17 producing cells in hyperlipidemic was increased prior to antigen exposure, suggesting that hyperlipidemia was sufficient to alter T cell alloreactivity and promote anti‐donor Th17 responses on first exposure to antigen. Together, our data suggest that hyperlipidemia alters rejection by altering the types of T cell subsets that respond to donor antigen by promoting Th17 biased anti‐donor reactivity.     

Galectin‐1 Prolongs Survival of Mouse Liver Allografts From Flt3L‐Pretreated Donors

Liver allografts are spontaneously accepted across MHC barriers in mice. The mechanisms underlying this phenomenon remain poorly understood. Galectin‐1, an endogenous lectin expressed in lymphoid organs, plays a vital role in maintaining central and peripheral tolerance. This study was to investigate the role of galectin‐1 in spontaneous tolerance of liver allografts in mice, and to evaluate the therapeutic effects of galectin‐1 on liver allograft rejection induced by donor Flt3L pretreatment. Blockade of the galectin‐1 pathway via neutralizing antigalectin‐1 mAb did not affect survival of the liver allografts from B6 donors into C3H recipients. Administration of rGal‐1 significantly prolonged survival of liver allografts from Flt3L‐pretreated donors and ameliorated Flt3L‐triggered liver allograft rejection. This effect was associated with increased apoptosis of T cells in both allografts and spleens, decreased frequencies of Th1 and Th17 cells, decreased expression of Th1‐associated cytokines (IL‐12, IL‐2 and IFN‐γ), Th17‐associated cytokines (IL‐23 and IL‐17) and granzyme B, in parallel with selectively increased IL‐10 expression in liver allografts. In vitro, galectin‐1 inhibited Flt3L‐differentiated DC‐mediated proliferation of allo‐CD4⁺ T cells and production of IFN‐γ and IL‐17. These data provide new evidence of the potential regulatory effects of galectin‐1 in alloimmune responses in a murine model of liver transplantation.     

IL‐17 Deficiency Attenuates Allograft Injury and Prolongs Survival in a Murine Model of Fully MHC‐Mismatched Renal Allograft Transplantation

IL‐17 is a pro‐inflammatory cytokine implicated in the pathogenesis of inflammatory and autoimmune diseases. However the role of IL‐17 in renal allograft rejection has not been fully explored. Here, we investigate the impact of IL‐17 in a fully MHC‐mismatched, life‐sustaining, murine model of kidney allograft rejection using IL‐17 deficient donors and recipients (IL‐17^?/? allografts). IL‐17^?/? allografts exhibited prolonged survival which was associated with reduced expression of the Th1 cytokine IFN‐γ and histological attenuation of acute and chronic allograft rejection, as compared to wild‐type allograft recipients. Results were confirmed in WT allograft recipients treated with an IL‐17 blocking antibody. Subsequent experiments using either donors or recipients deficient in IL‐17 showed a trend towards prolongation of survival only when recipients were IL‐17^?/?. Administration of a depleting anti‐CD25 antibody to IL‐17^?/? recipients abrogated the survival advantage conferred by IL‐17 deficiency, suggesting the involvement of a CD4⁺CD25⁺ T cell regulatory mechanism. Therefore, IL‐17 deficiency or neutralization was protective against the development of kidney allograft rejection, which may be mediated by impairment of Th1 responses and/or enhanced protection by Tregs.

     

Subcutaneous administration of a neutralizing IL‐1β antibody prolongs limb allograft survival

Cytokine‐expression profiles revealed IL‐1ß highly upregulated in rejecting skin of limb allografts. We investigate the effect of intragraft treatment with a neutralizing IL‐1β antibody in limb transplantation. Following allogenic hind‐limb transplantation, Lewis rats were either left untreated 1 or treated with anti‐lymphocyte serum + tacrolimus (baseline) 2 ; baseline immunosuppression + anti‐IL‐1β (1 mg/kg once/week, 6‐8 subcutaneous injections) into the transplanted 3 or contralateral 4 limb. Endpoint was rejection grade III or day 100. Graft rejection was assessed by histology, immunohistochemistry, flow cytometry phenotyping of immune cells, and monitoring cytokine expression. Anti‐IL‐1β injections into the allograft or contralateral limb resulted in a significant delay of rejection onset (controls: 58.60 ± 0.60; group 3: 75.80 ± 10.87, P = .044; group 4: 73.00 ± 6.49, P = .008) and prolongation of graft survival (controls: 64.60 ± 0.87; group 3: 86.60 ± 5.33, P = .002; group 4: 93.20 ± 3.82, P = .002), compared to controls. Although the phenotype of the graft infiltrating immune cells did not differ between groups, significantly decreased skin protein levels of IL‐1β, IL‐4, IL‐13, IP‐10, MCP‐1, and MCP‐3 in long‐term‐survivors indicate an overall decrease of chemoattraction and infiltration of immune cells as the immunosuppressive mechanism of anti‐IL‐1β. Inhibition of IL‐1β with short‐term systemic immunosuppression prolongs limb allograft survival and represents a promising target for immunosuppression in extremity transplantation.     

T Cell‐mediated Rejection of Kidney Transplants: A Personal Viewpoint

In kidney allografts, T cell mediated rejection (TCMR) is characterized by infiltration of the interstitium by T cells and macrophages, intense IFNG and TGFB effects, and epithelial deterioration. Recent experimental and clinical studies provide the basis for a provisional model for TCMR. The model proposes that the major unit of cognate recognition in TCMR is effector T cells engaging donor antigen on macrophages. This event creates the inflammatory compartment that recruits effector and effector memory CD4 and CD8 T cells, both cognate and noncognate, and macrophage precursors. Cognate T cells cross the donor microcirculation to enter the interstitium but spare the microcirculation. Local inflammation triggers dedifferentiation of the adjacent epithelium (e.g. loss of transporters and expression of embryonic genes) rather than cell death, via mechanisms that do not require known T‐cell cytotoxic mechanisms or direct contact of T cells with the epithelium. Local epithelial changes trigger a response of the entire nephron and a second wave of dedifferentiation. The dedifferentiated epithelium is unable to exclude T cells, which enter to produce tubulitis lesions. Thus TCMR is a cognate recognition‐based process that creates local inflammation and epithelial dedifferentiation, stereotyped nephron responses, and tubulitis, and if untreated causes irreversible nephron loss.     

Blockade of Macrophage Colony-Stimulating Factor Reduces Macrophage Proliferation and Accumulation in Renal Allograft Rejection

Macrophage accumulation within an acutely rejecting allograft occurs by recruitment and local proliferation. To determine the importance of M-CSF in driving macrophage proliferation during acute rejection, we blocked the M-CSF receptor, c-fms, in a mouse model of acute renal allograft rejection. C57BL/6 mouse kidneys (allografts, n = 20) or BALB/c kidneys (isografts, n = 5) were transplanted into BALB/c mice. Anti-c-fms antibody (AFS98) or control Ig (50 mg/kg/day, i.p.) was given daily to allografts from days 0-5. All mice were killed day 6 postoperatively. Expression of the M-CSF receptor, c-fms, was restricted to infiltrating CD68+ macrophages. Blockade of c-fms reduced proliferating (CD68+/BrdU+) macrophages by 82% (1.1 v 6.2%, p < 0.001), interstitial CD68+ macrophage accumulation by 53% (595 v 1270/mm2, p < 0.001), and glomerular CD68+ macrophage accumulation by 71% (0.73 V 2.48 CD68+ cells per glomerulus, p < 0.001). Parameters of T-cell involvement (intragraft CD4+, CD8+ and CD25+ lymphocyte numbers) were not affected. The severity of tubulointerstitial rejection was reduced in the treatment group as shown by decreased tubulitis and tubular cell proliferation. Macrophage proliferation during acute allograft rejection is dependent on the interaction of M-CSF with its receptor c-fms. This pathway plays a significant and specific role in the accumulation of macrophages within a rejecting renal allograft.     

Cell Therapy With Autologous Tolerogenic Dendritic Cells Induces Allograft Tolerance Through Interferon‐Gamma and Epstein‐Barr Virus‐Induced Gene 3

Innovative therapeutic strategies are needed to diminish the impact of harmful immunosuppression in transplantation. Dendritic cell (DC)‐based therapy is a promising approach for induction of antigen‐specific tolerance. Using a heart allograft model in rats, we analyzed the immunoregulatory mechanisms by which injection of autologous tolerogenic DCs (ATDCs) plus suboptimal immunosuppression promotes indefinite graft survival. Surprisingly, we determined that Interferon‐gamma (IFNG), a cytokine expected to be propathogenic, was threefold increased in the spleen of tolerant rats. Importantly, its blockade led to allograft rejection [Mean Survival Time (MST) = 25.6 ± 4 days], showing that IFNG plays a critical role in immunoregulatory mechanisms triggered by ATDCs. IFNG was expressed by TCRαβ⁺CD3⁺CD4^?CD8^?NKRP1^? cells (double negative T cells, DNT), which accumulated in the spleen of tolerant rats. Interestingly, ATDCs specifically induced IFNG production by DNT cells. ATDCs expressed the cytokinic chain Epstein‐Barr virus‐induced gene 3 (EBI3), an IL‐12 family member. EBI3 blockade or knock‐down through siRNA completely abolished IFNG expression in DNT cells. Finally, EBI3 blockade in vivo led to allograft rejection (MST = 36.8 ± 19.7 days), demonstrating for the first time a role for EBI3 in transplantation tolerance. Taken together our results have important implications in the rationalization of DC‐based therapy in transplantation as well as in the patient immunomonitoring follow‐up.     

Lentivirus IL‐10 Gene Therapy Down‐Regulates IL‐17 and Attenuates Mouse Orthotopic Lung Allograft Rejection

The purpose of the study was to examine the effect of lentivirus‐mediated IL‐10 gene therapy to target lung allograft rejection in a mouse orthotopic left lung transplantation model. IL‐10 may regulate posttransplant immunity mediated by IL‐17. Lentivirus‐mediated trans‐airway luciferase gene transfer to the donor lung resulted in persistent luciferase activity up to 6 months posttransplant in the isograft (B6 to B6); luciferase activity decreased in minor‐mismatched allograft lungs (B10 to B6) in association with moderate rejection. Fully MHC‐mismatched allograft transplantation (BALB/c to B6) resulted in severe rejection and complete loss of luciferase activity. In minor‐mismatched allografts, IL‐10‐encoding lentivirus gene therapy reduced the acute rejection score compared with the lentivirus‐luciferase control at posttransplant day 28 (3.0 ± 0.6 vs. 2.0 ± 0.6 (mean ± SD); p = 0.025; n = 6/group). IL‐10 gene therapy also significantly reduced gene expression of IL‐17, IL‐23, and retinoic acid‐related orphan receptor (ROR)‐γt without affecting levels of IL‐12 and interferon‐γ (IFN‐γ). Cells expressing IL‐17 were dramatically reduced in the allograft lung. In conclusion, lentivirus‐mediated IL‐10 gene therapy significantly reduced expression of IL‐17 and other associated genes in the transplanted allograft lung and attenuated posttransplant immune responses after orthotopic lung transplantation.     

Pilot Study Exploring Lung Allograft Surfactant Protein A (SP‐A) Expression in Association With Lung Transplant Outcome

Primary graft failure and chronic lung allograft dysfunction (CLAD) limit lung transplant long–term outcomes. Various lung diseases have been correlated with surfactant protein (SP) expression and polymorphisms. We sought to investigate the role of SP expression in lung allografts prior to implantation, in relation to posttransplant outcomes. The expression of SP‐(A, B, C, D) mRNA was assayed in 42 allografts. Posttransplant assessments include pulmonary function tests, bronchoscopy, broncho‐alveolar lavage fluid (BALF) and biopsies to determine allograft rejection. BALF was assayed for SP‐A, SP‐D in addition to cytokines IL‐8, IL‐12 and IL‐2. The diagnosis of CLAD was evaluated 6 months after transplantation. Lung allografts with low SP‐A mRNA expression prior to implantation reduced survival (Log‐rank p < 0.0001). No association was noted for the other SPs. Allografts with low SP‐A mRNA had greater IL‐2 (p = 0.03) and IL‐12 (p < 0.0001) in the BALF and a greater incidence of rejection episodes (p = 0.003). Levels of SP‐A mRNA expression were associated with the SP‐A2 polymorphisms (p = 0.015). Specifically, genotype 1A1A⁰ was associated with lower SP‐A mRNA expression (p < 0.05). Lung allografts with low levels of SP‐A mRNA expression are associated with reduced survival. Lung allograft SP‐A mRNA expression appears to be associated with SP‐A gene polymorphisms.