Gregor Bartel

Lymphatic endothelial progenitor cells contribute to de novo lymphangiogenesis in human renal transplants.

De novo lymphangiogenesis influences the course of different human diseases as diverse as chronic renal transplant rejection and tumor metastasis. The cellular mechanisms of lymphangiogenesis in human diseases are currently unknown, and could involve division of local preexisting endothelial cells or incorporation of circulating progenitors. We analyzed renal tissues of individuals with gender-mismatched transplants who had transplant rejection and high rates of overall lymphatic endothelial proliferation as well as massive chronic inflammation. Donor-derived cells were detected by in situ hybridization of the Y chromosome. We compared these tissues with biopsies of essentially normal skin and intestine, and two rare carcinomas with low rates of lymphatic endothelial proliferation that were derived from individuals with gender-mismatched bone marrow transplants. Here, we provide evidence for the participation of recipient-derived lymphatic progenitor cells in renal transplants. In contrast, lymphatic vessels of normal tissues and those around post-transplant carcinomas did not incorporate donor-derived progenitors. This indicates a stepwise mechanism of inflammation-associated de novo lymphangiogenesis, implying that potential lymphatic progenitor cells derive from the circulation, transmigrate through the connective tissue stroma, presumably in the form of macrophages, and finally incorporate into the growing lymphatic vessel.

Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

Posttransplant HLA Alloreactivity in Stable Kidney Transplant Recipients—Incidences and Impact on Long-Term Allograft Outcomes

Humoral alloreactivity is well established to predict adverse allograft outcomes. However, in some recipients, alloantibodies may also occur in the absence of graft dysfunction. We evaluated if and how often complement‐ and noncomplement‐fixing alloantibodies are detectable in stable recipients and whether, in this context, they affect long‐term outcomes. Sera obtained from 164 kidney transplant recipients at 2, 6 and 12 months were evaluated by FlowPRA screening and single‐antigen testing for detection of IgG‐ or C4d‐fixing HLA panel reactivity and donor‐specific antibodies (DSA). Applying stringent criteria, we selected 34 patients with an uneventful 1‐year course (no graft dysfunction or rejection) and excellent graft function at 12 months [estimated glomerular filtration rate (eGFR) ≥60 mL/min and proteinuria ≤0.5 g/24 h]. Nine (27%) and 5 (15%) of these recipients tested positive by [IgG] and [C4d]FlowPRA screening, respectively. In five cases, DSA were identified. Frequencies of positive test results and DSA binding intensities were not significantly lower than those documented for patients who did not fulfill the above criteria. In recipients with an excellent 1‐year course, FlowPRA reactivity was not associated with lower eGFR or increased protein excretion during 68‐month median follow‐up. Our results suggest cautious interpretation of antibody monitoring in patients with normal‐functioning grafts.

In Vitro Detection of C4d‐Fixing HLA Alloantibodies: Associations With Capillary C4d Deposition in Kidney Allografts

Capillary C4d deposition is a valuable marker of antibody‐mediated rejection (AMR). In this analysis, flow cytometric detection of alloantibody‐triggered C4d deposition to HLA antigen‐coated microparticles ([C4d]FlowPRA) was evaluated for its value as a marker for C4d deposition in renal allografts. For comparative analysis, 105 first renal biopsies performed for graft dysfunction and an equal number of concurrent sera were subjected to immunohistochemistry and [C4d] plus standard [IgG]FlowPRA, respectively. C4d deposition/fixation was detected in 17 biopsies and, applying [C4d]FlowPRA HLA class I and II screening, also in a small number of corresponding sera (N = 20). IgG reactivity detected by standard [IgG]FlowPRA was more frequent (49% of sera). Comparing [C4d]FlowPRA screening with capillary C4d staining, we found a high level of specificity (0.92 [95% confidence interval: 0.86–0.98]), which far exceeded that calculated for [IgG]FlowPRA (0.60 [0.50–0.70]). [IgG]FlowPRA screening, however, turned out to be superior in terms of sensitivity (0.94 [0.83–1.05] vs. 0.76 [0.56–0.97] calculated for C4d‐fixing panel reactivity). Remarkably, posttransplant single antigen testing for identification of complement‐fixing donor‐specific alloreactivities failed to improve the predictive value of FlowPRA‐based serology. In conclusion, our results suggest that detection of complement‐fixing HLA panel reactivity could provide a specific tool for monitoring of C4d‐positive AMR.

Clinical relevance of preformed C4d-fixing and non-C4d-fixing HLA single antigen reactivity in renal allograft recipients

Donor‐specific alloantibodies (DSA), especially those fixing complement, may pose a particular immunologic risk to transplant recipients. To assess the clinical impact of C4d‐ or non‐C4d‐fixing (IgG) HLA sensitization, pretransplant sera obtained from 338 kidney allograft recipients prescreened by FlowPRA were retrospectively evaluated by Luminex single antigen (SA) testing using a novel fluorescent‐labeled anti‐C4d reagent for detection of antibody‐triggered C4d deposition in addition to IgG binding. Recipients with [IgG]DSA (n = 39) showed a substantially higher rate of C4d positive rejection (33%) than 16 patients with [IgG] non‐DSA (0%) or 283 antibody‐negative patients (4%, multivariate analysis excluding retransplantation because of high co‐linearity: P < 0.0001), and adversely affected 5‐year death‐censored graft survival (74% vs. 81% and 90%, respectively, multivariate model: P < 0.05). [C4d] DSA (n = 21) and [C4d] non‐DSA (n = 25) increased rates of C4d positive rejections to a similar extent (24% and 28% vs. 4% in recipients without C4d‐fixing reactivity; multivariate analysis: P ≤ 0.002) with a trend towards adverse 5‐year graft survival (76% and 76% vs. 90%; P ≤ 0.2). In conclusion, Luminex‐based characterization of HLA sensitization may be a useful strategy for risk stratification. Possibly as a result of intensified immunosuppression in presensitized recipients, identification of C4d‐fixing DSA was not associated with a further increase of rejection and graft loss rates.

Peritransplant immunoadsorption for positive crossmatch deceased donor kidney transplantation.

Various desensitization protocols were shown to enable successful living donor kidney transplantation across a positive complement‐dependent cytotoxicity crossmatch (CDCXM). Positive crossmatch transplantation, however, is less well established for deceased donor transplantation. We report a cohort of 68 deceased donor renal allograft recipients who, on the basis of broad sensitization (lymphocytotoxic panel reactivity ≥40%), were subjected to a protocol of peritransplant immunoadsorption (IA). Treatment consisted of a single session of immediate pretransplant IA (protein A) followed by posttransplant IA and antilymphocyte antibody therapy. Twenty‐one patients had a positive CDCXM, which could be rendered negative by pretransplant apheresis. Solid phase HLA antibody detection revealed preformed donor‐specific antibodies (DSA) in all 21 CDCXM‐positive and in 30 CDCXM‐negative recipients. At 5 years, overall graft survival, death‐censored graft survival and patient survival were 63%, 76% and 87%, respectively, without any differences between CDCXM‐positive, CDCXM‐negative/DSA‐positive and CDCXM‐negative/DSA‐negative recipients. Furthermore, groups did not differ regarding rates of antibody‐mediated rejection (24% vs. 30% vs. 24%, p = 0.84), cellular rejection (14% vs. 23% vs. 18%, p = 0.7) or allograft function (median 5‐year serum creatinine: 1.3 vs. 1.8 vs. 1.7 mg/dL, p = 0.62). Our results suggest that peritransplant IA is an effective strategy for rapid desensitization in deceased donor transplantation.

Determinants of the complement-fixing ability of recipient presensitization against HLA antigens.

Background. The presence of preformed alloantibodies with the ability to activate complement may pose a particular risk for kidney allograft rejection. The aim of this study was to evaluate variables that determine the complement-fixing capability of human leukocyte antigen (HLA) sensitization. Methods. Sixty-five sensitized patients with ≥10% pretransplant panel-reactive antibody (PRA) levels uncovered by immunoglobulin G [IgG]FlowPRA HLA class I and/or class II screening were included. Applying modified FlowPRA screening, sera were evaluated for patterns of alloreactive IgG subclasses and IgM, and, in parallel, for their complement-activating ability assessed by flow cytometric detection of human complement split product deposition ([C4d]FlowPRA). Results. Approximately two-thirds (68%) of tested sera were found to contain complement-fixing alloreactivity (≥10%[C4d]FlowPRA). IgG1 type panel reactivity was predominant (detectable HLA class I and II reactivity in 93% and 91% of IgG-positive sera), followed by IgG3 (49%/44%), IgG2 (44%/27%), and IgG4 (19%/11%). Applying partial correlation we found an independent correlation of both %[IgG1]FlowPRA and %[IgG3]FlowPRA with %[C4d]FlowPRA reactivities (P≤0.01). In addition, for IgG1 a contribution of the amount of bound alloantibody to complement-fixation was observed. Complement-fixation was also favored by the simultaneous presence of alloreactive IgG1, IgG3, and IgM. Previous grafting, but not pregnancy and transfusion, was independently associated with complement-fixing sensitization (P<0.05), presumably due to increased IgG1 type reactivity. Conclusions. Anti-HLA antibody-triggered complement activation is dependent on both the pattern of Ig reactivities and the amount of bound antibody. Previous transplantation represents a major risk factor for the development of complement-fixing sensitization.

Prevention and treatment of alloantibody-mediated kidney transplant rejection.

Summary Antibody‐mediated rejection (AMR), which is commonly caused by preformed and/or de novo HLA alloantibodies, has evolved as a leading cause of early and late kidney allograft injury. In recent years, effective treatment strategies have been established to counteract the deleterious effects of humoral alloreactivity. One major therapeutic challenge is the barrier of a positive pretransplant lymphocytotoxic crossmatch. Several apheresis‐ and/or IVIG‐based protocols have been shown to enable successful crossmatch conversion, including a strategy of peritransplant immunoadsorption for rapid crossmatch conversion immediately before deceased donor transplantation. While such protocols may increase transplant rates and allow for acceptable graft survival, at least in the short‐term, it has become evident that, despite intense treatment, many patients still experience clinical or subclinical AMR. This reinforces the need for innovative strategies, such as complementary allocation programs to improve transplant outcomes. For acute AMR, various studies have suggested efficiency of plasmapheresis‐ or immunoadsorption‐based protocols. There is, however, no established treatment for chronic AMR and the development of strategies to reverse or at least halt chronic active rejection remains a big challenge. Major improvements can be expected from studies evaluating innovative therapeutic concepts, such as proteasome inhibition or complement blocking agents.

Solid phase detection of C4d‐fixing HLA antibodies to predict rejection in high immunological risk kidney transplant recipients

Protocols for recipient desensitization may allow for successful kidney transplantation across major immunological barriers. Desensitized recipients, however, still face a considerable risk of antibody‐mediated rejection (AMR), which underscores the need for risk stratification tools to individually tailor treatment. Here, we investigated whether solid phase detection of complement‐fixing donor‐specific antibodies (DSA) has the potential to improve AMR prediction in high‐risk transplants. The study included 68 sensitized recipients of deceased donor kidney allografts who underwent peritransplant immunoadsorption for alloantibody depletion (median cytotoxic panel reactivity: 73%; crossmatch conversion: n = 21). Pre and post‐transplant sera were subjected to detection of DSA‐triggered C4d deposition ([C4d]DSA) applying single‐antigen bead (SAB) technology. While standard crossmatch and [IgG]SAB testing failed to predict outcomes in our desensitized patients, detection of preformed [C4d]DSA (n = 44) was tightly associated with C4d‐positive AMR [36% vs. 8%, P = 0.01; binary logistic regression: odds ratio: 10.1 (95% confidence interval: 1.6–64.2), P = 0.01]. Moreover, long‐term death‐censored graft survival tended to be worse among [C4d]DSA‐positive recipients (P = 0.07). There were no associations with C4d‐negative AMR or cellular rejection. [C4d]DSA detected 6 months post‐transplantation were not related to clinical outcomes. Our data suggest that pretransplant SAB‐based detection of complement‐fixing DSA may be a valuable tool for risk stratification.

Significance of peritubular capillary, glomerular, and arteriolar C4d staining patterns in paraffin sections of early kidney transplant biopsies.

Background. Although diffuse linear C4d deposition in peritubular capillaries (PTCs) is a well-established criterion of alloantibody-mediated kidney transplant rejection, the actual relevance of focal or granular C4d deposits or staining outside PTC (glomeruli and arterioles) has yet to be established. Methods. This study was designed to evaluate the diagnostic significance of such nontypical C4d staining patterns. A total of 539 early indication biopsies (329 kidney transplants) were analyzed by immunohistochemistry using a polyclonal anti-C4d antibody. Results. We found a close interrelationship between diffuse or focal linear C4d deposition in PTC, linear endothelial deposition in glomeruli, and arteriolar C4d. These specific patterns were also related to transplant glomerulitis and recipient presensitization. No such associations, however, were observed for other patterns, such as granular C4d in PTC. Detection of diffuse but not focal linear C4d in PTC was found to be associated with adverse allograft survival (5-year death-censored graft survival: 48% vs. 82%, 89%, or 84% in patients with focal, minimal, or no C4d, respectively; P<0.0001). Univariate analysis also revealed inferior graft survival in recipients with linear C4d in glomeruli (P=0.02). Applying multivariate Cox regression analysis, however, only diffuse linear PTC staining was found to be predictive of graft loss (hazard ratio 3.95 [95% confidence interval 1.62–9.60]; P=0.002). Conclusion. There might be a relationship between humoral alloimmunity and distinct less established staining patterns, such as focal linear C4d in PTC, endothelial C4d in glomeruli, or arteriolar C4d. Nevertheless, our results reemphasize the prognostic value of diffuse linear PTC staining.