A transitional B-cell cytokine biomarker for risk stratifying renal transplant patients with borderline rejection.

Borderline allograft rejection can promote acute rejection and graft loss in some, but not all, patients. In this issue, Cherukuri et al. use a novel test based on peripheral blood transitional T1 B cells producing interleukin-10 and tumor necrosis factor-α, which identifies patients at high risk for poor outcomes. The potential mechanisms by which transitional T1 B cells might modulate alloreactivity need exploration, but following appropriate validation, this biomarker could risk stratify patients in need of early intervention.

The Clinical Utility of Post-Transplant Monitoring of Donor-Specific Antibodies in Stable Renal Transplant Recipients: A Consensus Report With Guideline Statements for Clinical Practice.

Solid phase immunoassays improved the detection and determination of the antigen-specificity of donor-specific antibodies (DSA) to human leukocyte antigens (HLA). The widespread use of SPI in kidney transplantation also introduced new clinical dilemmas, such as whether patients should be monitored for DSA pre- or post-transplantation. Pretransplant screening through SPI has become standard practice and DSA are readily determined in case of suspected rejection. However, DSA monitoring in recipients with stable graft function has not been universally established as standard of care. This may be related to uncertainty regarding the clinical utility of DSA monitoring as a screening tool. This consensus report aims to appraise the clinical utility of DSA monitoring in recipients without overt signs of graft dysfunction, using the Wilson & Junger criteria for assessing the validity of a screening practice. To assess the evidence on DSA monitoring, the European Society for Organ Transplantation (ESOT) convened a dedicated workgroup, comprised of experts in transplantation nephrology and immunology, to review relevant literature. Guidelines and statements were developed during a consensus conference by Delphi methodology that took place in person in November 2022 in Prague. The findings and recommendations of the workgroup on subclinical DSA monitoring are presented in this article.

Update on the ethical, legal and technical challenges of translating xenotransplantation.

This manuscript reports on a landmark symposium on the ethical, legal and technical challenges of xenotransplantation in the UK. King's College London, with endorsement from the British Transplantation Society (BTS), and the European Society of Organ Transplantation (ESOT), brought together a group of experts in xenotransplantation science, ethics and law to discuss the ethical, regulatory and technical challenges surrounding translating xenotransplantation into the clinical setting. The symposium was the first of its kind in the UK for 20 years. This paper summarises the content of the expert lectures showcasing the progress which has been made in xenotransplantation including-the history of xenotransplantation, advances in gene edited animals and progress towards clinical xenotransplantation. We then set out the ethical and legal issues still to be resolved. Finally, we report the themes of the roundtable discussion highlighting areas of consensus and controversy. While the detail of the legal discussion was directed towards the UK, the principles and summary reported here are intended to be applicable to any jurisdiction seeking to implement clinical xenotransplantation.

Regulatory B cells: Development, phenotypes, functions, and role in transplantation.

The interest in regulatory B cells (Bregs) began in the 1970s with the evidence that B cells could downregulate the immune system by the production of "inhibitory" antibodies. Subsequently, a series of results from different studies have emphasized that B cells have antibody-independent immunoregulatory functions. Since then, different subsets of B cells with regulatory functions and their development and mechanisms of action have been identified both in human and in animal models of inflammation, transplantation, and autoimmunity. The present review outlines the suggested pathways by which Bregs develop, describes the different subsets of Bregs with their phenotypes and function as well as their role in transplantation, highlighting the differences between human and animal studies throughout.

Regulation of T- and B-cell interactions determines the clinical phenotype associated with donor-specific antibodies.

The cellular mechanisms that regulate donor-specific antibody formation and antibody-mediated rejection remain unknown. In this issue, Louis et al. report that specific T-regulatory cell and B-regulatory transitional cell subsets are concomitantly diminished in patients with donor-specific antibody and consequent antibody-mediated rejection and advance alterations in specific cytokines and costimulatory molecules as important mechanisms by which these cells may suppress donor-specific antibody formation and, independently, progression to antibody-mediated rejection.

The phenotype of HLA-binding B cells from sensitized kidney transplant recipients correlates with clinically prognostic patterns of interferon-γ production against purified HLA proteins.

B cells play crucial roles in cell-mediated alloimmune responses. In vitro, B cells can support or regulate indirect T-cell alloreactivity in response to donor antigens on ELISpot and these patterns associate with clinical outcome. Previous reports of associations between B-cell phenotype and function have examined global phenotypes and responses to polyclonal stimuli. We hypothesized that studying antigen-specific B cells, using samples from sensitized patients, would inform further study to identify novel targets for intervention. Using biotinylated HLA proteins, which bind HLA-specific B cells via the B-cell receptor in a dose-dependent fashion, we report the specific phenotype of HLA-binding B cells and define how they associated with patterns of anti-HLA response in interferon-γ ELISpot. HLA-binding class-switched and IgM+CD27+ memory cells associated strongly with B-dependent interferon-γ production and appeared not suppressible by endogenous Tregs. When the predominant HLA-binding phenotype was naïve B cells, the associated functional ELISpot phenotype was determined by other cells present. High numbers of non-HLA-binding transitional cells associated with B-suppressed interferon-γ production, especially if Tregs were present. However, high frequencies of HLA-binding marginal-zone precursors associated with B-dependent interferon-γ production that appeared suppressible by Tregs. Finally, non-HLA-binding marginal zone precursors may also suppress interferon-γ production, though this association only emerged when Tregs were absent from the ELISpot. Thus, our novel data provide a foundation on which to further define the complexities of interactions between HLA-specific T and B cells and identify new targets for intervention in new therapies for chronic rejection.

A multicenter randomized controlled trial indicates that paclitaxel-coated balloons provide no benefit for arteriovenous fistulas.

The role of paclitaxel-coated balloons has been established in the coronary and peripheral arterial circulations with recent interest in the use of paclitaxel-coated balloons to improve patency rates following angioplasty of arteriovenous fistulas. To assess the efficacy of paclitaxel-coated angioplasty balloons to prolong the survival time of target lesion primary patency in arteriovenous fistulas, we designed an investigator-led multi-center randomized controlled trial with follow up time variable for a minimum of one year. Patients with an arteriovenous fistula who were undergoing an angioplasty for a clinical indication were included but patients with one or more lesions outside the treatment segment were excluded. Following successful treatment with a high-pressure balloon, 212 patients were randomized. In the intervention arm, the second component was insertion of a paclitaxel-coated balloon. In the control arm, an identical procedure was followed, but using a standard balloon. The primary endpoint was time to loss of clinically driven target lesion primary patency. Primary analysis showed no significant evidence for a difference in time to end of target lesion primary patency between groups: hazard ratio 1.18 with a 95% confidence interval of 0.78 to 1.79. There were no significant differences for any secondary outcomes, including patency outcomes and adverse events. Thus, our study demonstrated no evidence that paclitaxel-coated balloons provide benefit, following standard care high-pressure balloon angioplasty, in the treatment of arteriovenous fistulas. Hence, in view of the benefit suggested by other trials, the role of paclitaxel-coated angioplasty balloons remains uncertain.

Effect of delayed graft function on longer-term outcomes after kidney transplantation from donation after circulatory death donors in the United Kingdom: A national cohort study.

Kidneys from donation after circulatory death (DCD) donors are utilized variably worldwide, in part due to high rates of delayed graft function (DGF) and putative associations with adverse longer-term outcomes. We aimed to determine whether the presence of DGF and its duration were associated with poor longer-term outcomes after kidney transplantation from DCD donors. Using the UK transplant registry, we identified 4714 kidney-only transplants from controlled DCD donors to adult recipients between 2006 and 2016; 2832 recipients (60·1%) had immediate graft function and 1882 (39·9%) had DGF. Of the 1847 recipients with DGF duration recorded, 926 (50·1%) had DGF < 7 days, 576 (31·2%) had DGF 7-14 days, and 345 (18·7%) had DGF >14 days. After risk adjustment, the presence of DGF was not associated with inferior long-term graft or patient survivals. However, DGF duration of >14 days was associated with an increased risk of death-censored graft failure (hazard ratio 1·7, p = ·001) and recipient death (hazard ratio 1·8, p < ·001) compared to grafts with immediate function. This study suggests that shorter periods of DGF have no adverse influence on graft or patient survival after DCD donor kidney transplantation and that DGF >14 days is a novel early biomarker for significantly worse longer-term outcomes.

B lymphocytes contribute to indirect pathway T cell sensitization via acquisition of extracellular vesicles.

B cells have been implicated in transplant rejection via antibody-mediated mechanisms and more recently by presenting donor antigens to T cells. We have shown in patients with chronic antibody-mediated rejection that B cells control the indirect T cell alloresponses. To understand more about the role of B cells as antigen-presenting cells for CD4+ T cell with indirect allospecificity, B cells were depleted in C57BL/6 mice, using an anti-CD20 antibody, prior to receiving MHC class I-mismatched (Kd ) skin. The absence of B cells at the time of transplantation prolonged skin graft survival. To study the mechanisms behind this observation, T cells with indirect allospecificity were transferred in mice receiving a Kd skin transplant. T cell proliferation was markedly inhibited in the absence of recipient B cells, suggesting that B cells contribute to indirect pathway sensitization. Furthermore, we have shown that a possible way in which B cells present alloantigens is via acquisition of MHC-peptide complexes. Finally, we demonstrate that the addition of B cell depletion to the transfer of regulatory T cells (Tregs) with indirect alloresponse further prolonged skin graft survival. This study supports an important role for B cells in indirect T cell priming and further emphasizes the advantage of combination therapies in prolonging transplant survival.

C3d-positive donor-specific antibodies have a role in pretransplant risk stratification of cross-match-positive HLA-incompatible renal transplantation: United Kingdom multicentre study.

Anti-HLA-antibody characteristics aid to risk-stratify patients and improve long-term renal graft outcomes. Complement activation by donor-specific antibody (DSA) is an important characteristic that may determine renal allograft outcome. There is heterogeneity in graft outcomes within the moderate to high immunological risk cases (cross-match-positive). We explored the role of C3d-positive DSAs in sub-stratification of cross-match-positive cases and relate to the graft outcomes. We investigated 139 cross-match-positive living-donor renal transplant recipients from four transplant centres in the United Kingdom. C3d assay was performed on serum samples obtained at pretreatment (predesensitization) and Day 14 post-transplant. C3d-positive DSAs were found in 52 (37%) patients at pretreatment and in 37 (27%) patients at Day 14 post-transplant. Median follow-up of patients was 48 months (IQR 20.47-77.57). In the multivariable analysis, pretreatment C3d-positive DSA was independently associated with reduced overall graft survival, the hazard ratio of 3.29 (95% CI 1.37-7.86). The relative risk of death-censored five-year graft failure was 2.83 (95% CI 1.56-5.13). Patients with both pretreatment and Day 14 C3d-positive DSAs had the worst five-year graft survival at 45.5% compared with 87.2% in both pretreatment and Day 14 C3d-negative DSA patients with the relative risk of death-censored five-year graft failure was 4.26 (95% CI 1.79, 10.09). In this multicentre study, we have demonstrated for the first time the utility of C3d analysis as a distinctive biomarker to sub-stratify the risk of poor graft outcome in cross-match-positive living-donor renal transplantation.

Clinical-pathological correlations in post-transplant thrombotic microangiopathy.

AIMS: Post-transplant thrombotic microangiopathy (TMA) is a rare and clinically challenging finding in renal transplant biopsies. In addition to recurrent atypical haemolytic uraemic syndrome, TMA in renal transplants is associated with various conditions, such as calcineurin inhibitor (CNI) treatment, antibody-mediated rejection (ABMR), viral infections, sepsis, pregnancy, malignancies, and surgery. The therapeutic implications of this diagnosis are considerable. In order to better understand post-transplant TMA and to identify histological or clinical differences between associated causes, we conducted a multicentre retrospective study. METHODS AND RESULTS: Clinical parameters and transplant renal biopsy findings from 81 patients with TMA were analysed. Biopsies from 38 patients were also analysed with electron microscopy. On the basis of clinical-pathological correlation, TMA was attributed to a main aetiology, whenever possible. TMA occurred at a median of 30 days post-transplantation. Systemic features of TMA were present in only 18% of cases. Twenty-two per cent of cases were attributed to CNI and 11% to ABMR. Although other potentially contributing factors were found in 56% of patients, in most cases (63%) no clearly attributable cause of TMA was identified. Histological differences between groups were minimal. The detection of ultrastructural features that are usually associated with ABMR may help to establish ABMR as the cause of TMA. CONCLUSIONS: Although CNI and ABMR appear to be the main contributors to post-transplant TMA, the aetiology of most cases is probably multifactorial, and TMA cannot be unequivocally attributed to a single underlying aetiology. Morphological features of TMA are not discriminating, but electron microscopy may help to identify ABMR-associated TMA.

For the many: permitting deceased donor kidney transplantation across low-titre blood group antibodies can reduce wait times for blood group B recipients, and improve the overall number of 000MM transplants - a multicentre observational cohort study.

Blood group O or B recipients wait longer for a kidney transplant. We studied the distribution of anti-ABO blood group antibody titres in patients awaiting a kidney transplant, and modelled the effect of altering the UK National Kidney Allocation Scheme to allow for patients with 'LOW' titres (≤1:8, ≤3 dilutions) to receive a deceased donor ABOi (ddABOi) transplant. In a prospective study of 239 adult patients on the waiting list for a transplant in 2 UK centres, ABO-antibody titres (anti-A and anti-B) were measured. Based on the proportions of 'LOW' anti-A or anti-B antibodies, four simulations were performed to model the current allocation rules compared with variations allowing ddABOi allocation under various conditions of blood group, HLA matching, and waiting time. The simulations permitting ddABOi resulted in more blood group B recipients being transplanted, with median waiting time reduced for this group of recipients, and more equitable waiting times across blood groups. Additionally, permitting ddABOi resulted in greater numbers of 000MM allocations overall in compatible transplants under modelled conditions. Changing allocation in the UK to permit ddABOi in patients with 'LOW' titres would not change the total number of transplants, but redistributes allocation more equitably amongst blood groups, altering waiting times accordingly.

Endothelial cell-specific anticoagulation reduces inflammation in a mouse model of acute lung injury.

Tissue factor (TF)-dependent coagulation contributes to lung inflammation and the pathogenesis of acute lung injury (ALI). In this study, we explored the roles of targeted endothelial anticoagulation in ALI using two strains of transgenic mice expressing either a membrane-tethered human tissue factor pathway inhibitor (hTFPI) or hirudin fusion protein on CD31+ cells, including vascular endothelial cells (ECs). ALI was induced by intratracheal injection of LPS, and after 24 h the expression of TF and protease-activated receptors (PARs) on EC in lungs were assessed, alongside the extent of inflammation and injury. The expression of TF and PARs on the EC in lungs was upregulated after ALI. In the two strains of transgenic mice, expression of either of hTFPI or hirudin by EC was associated with significant reduction of inflammation, as assessed by the extent of leukocyte infiltration or the levels of proinflammatory cytokines, and promoted survival after LPS-induced ALI. The beneficial outcomes were associated with inhibition of the expression of chemokine CCL2 in lung tissues. The protection observed in the CD31-TFPI-transgenic strain was abolished by injection of an anti-hTFPI antibody, but not by prior engraftment of the transgenic strains with WT bone marrow, confirming that the changes observed were a specific transgenic expression of anticoagulants by EC. These results demonstrate that the inflammation in ALI is TF and thrombin dependent, and that expression of anticoagulants by EC significantly inhibits the development of ALI via repression of leukocyte infiltration, most likely via inhibition of chemokine gradients. These data enhance our understanding of the pathology of ALI and suggest a novel therapeutic strategy for treatment.

Post-listing survival for highly sensitised patients on the UK kidney transplant waiting list: a matched cohort analysis.

BACKGROUND: More than 40% of patients awaiting a kidney transplant in the UK are sensitised with human leucocyte antigen (HLA) antibodies. Median time to transplantation for such patients is double that of unsensitised patients at about 74 months. Removing antibody to perform an HLA-incompatible (HLAi) living donor transplantation is perceived to be high risk, although patient survival data are limited. We compared survival of patients opting for an HLAi kidney transplant with that of similarly sensitised patients awaiting a compatible organ. METHODS: From the UK adult kidney transplant waiting list, we selected crossmatch positive living donor HLAi kidney transplant recipients who received their transplant between Jan 1, 2007, and Dec 31, 2013, and were followed up to Dec 31, 2014 (end of study). These patients were matched in a 1:4 ratio with similarly sensitised patients cases listed for a deceased-donor transplant during that period. Data were censored both at the time of transplantation (listed only), and at the end of the study period (listed or transplant). We used Kaplan-Meier curves to compare patient survival between HLAi and the matched cohort. FINDINGS: Of 25 518 patient listings, 213 (1%) underwent HLAi transplantation during the study period. 852 matched controls were identified, of whom 41% (95% CI 32-50) remained without a transplant at 58 months after matching. We noted no difference in survival between patients who were in the HLAi group compared with the listed only group (log rank p=0·446), or listed or transplant group (log rank p=0·984). INTERPRETATION: Survival of sensitised patients undergoing HLAi in the UK is comparable with those on dialysis awaiting a compatible organ, many of whom are unlikely to be have a transplant. Choosing a direct HLAi transplant has no detrimental effect on survival, but offers no survival benefit, by contrast with similar patients studied in a North American multicentre cohort. FUNDING: UK National Health Service Blood & Transplant and Guy's & St Thomas' National Institute for Health Research Biomedical Research Centre.

Clinical risk stratification of paediatric renal transplant recipients using C1q and C3d fixing of de novo donor-specific antibodies.

INTRODUCTION: We have previously shown that children who developed de novo donor-specific human leukocyte antigen (HLA) antibodies (DSA) had greater decline in allograft function. We hypothesised that patients with complement-activating DSA would have poorer renal allograft outcomes. METHODS: A total of 75 children developed DSA in the original study. The first positive DSA sample was subsequently tested for C1q and C3d fixing. The primary event was defined as 50% reduction from baseline estimated glomerular filtration rate and was analysed using the Kaplan-Meier estimator. RESULTS: Of 65 patients tested, 32 (49%) and 23 (35%) tested positive for C1q and C3d fixing, respectively. Of the 32 C1q-positive (c1q+) patients, 13 (41%) did not show concomitant C3d fixing. The mean fluorescence intensity values of the original immunoglobulin G DSA correlated poorly with complement-fixing positivity (C1q: adjusted R 2 0.072; C3d: adjusted R 2 0.11; p < 0.05). C1q+ antibodies were associated with acute tubulitis [0.75 ± 0.18 (C1q+) vs. 0.25 ± 0.08 (C1q-) episodes per patient (mean ± standard error of the mean; p < 0.05] but not with worse long-term renal allograft dysfunction (median time to primary event 5.9 (C1q+) vs. 6.4 (C1q-) years; hazard ratio (HR) 0.74; 95% confidence ratio (CI) 0.30-1.81; p = 0.58]. C3d-positive (C3d+) antibodies were associated with positive C4d histological staining [47% (C3d+) vs. 20% (C3d-); p = 0.04] and with significantly worse long-term allograft dysfunction [median time to primary event: 5.6 (C3d+) vs. 6.5 (C3d-) years; HR 0.38; 95% CI 0.15-0.97; p = 0.04]. CONCLUSION: Assessment of C3d fixing as part of prospective HLA monitoring can potentially aid stratification of patients at the highest risk of long-term renal allograft dysfunction.

Transitional B cell subsets-a convincing predictive biomarker for allograft loss?

In this issue, Cherukuri and colleagues describe a convincing association between the proportion of transitional B lymphocyte subsets in kidney transplant recipients and long-term outcomes, and present a biologically plausible mechanism, based on differential ability of T1 and T2 cells to regulate in vitro T cell responses to explain the link. Further work is clearly needed to validate their claim that measurement of T1/T2 ratios may represent a reliable and reproducible predictive biomarker of transplant outcomes.

Graft dysfunction in chronic antibody-mediated rejection correlates with B-cell-dependent indirect antidonor alloresponses and autocrine regulation of interferon-γ production by Th1 cells.

Chronic antibody-mediated rejection, a common cause of renal transplant failure, has a variable clinical phenotype. Understanding why some with chronic antibody-mediated rejection progress slowly may help develop more effective therapies. B lymphocytes act as antigen-presenting cells for in vitro indirect antidonor interferon-γ production in chronic antibody-mediated rejection, but many patients retain the ability to regulate these responses. Here we test whether particular patterns of T and B cell antidonor response associate with the variability of graft dysfunction in chronic antibody-mediated rejection. Our results confirm that dynamic changes in indirect antidonor CD4+ T-cell responses correlate with changes in estimated glomerular filtration rates, independent of other factors. Graft dysfunction progressed rapidly in patients who developed unregulated B-cell-driven interferon-γ production. However, conversion to a regulated or nonreactive pattern, which could be achieved by optimization of immunosuppression, associated with stabilization of graft function. Functional regulation by B cells appeared to activate an interleukin-10 autocrine pathway in CD4+ T cells that, in turn, impacted on antigen-specific responses. Thus, our data significantly enhance the understanding of graft dysfunction associated with chronic antibody-mediated rejection and provide the foundation for strategies to prolong renal allograft survival, based on regulation of interferon-γ production.

Expression of human tissue factor pathway inhibitor on vascular smooth muscle cells inhibits secretion of macrophage migration inhibitory factor and attenuates atherosclerosis in ApoE-/- mice.

BACKGROUND: Tissue factor (TF) and coagulation proteases are involved in promoting atherosclerosis, but the molecular and cellular bases for their involvement are unknown. METHODS AND RESULTS: We generated a new strain (ApX4) of apolipoprotein E-deficient mice expressing a membrane-tethered human tissue factor pathway inhibitor fusion protein on smooth muscle actin-positive cells, including vascular smooth muscle cells (SMCs). ApX4 mice developed little atherosclerosis on either a normal chow or high-fat diet. Lipid levels were similar to those in parental ApoE(-/-) mice, and there was no detectable difference in systemic (circulating) tissue factor pathway inhibitor levels or activity. The small lipid-rich lesions that developed had markedly reduced leukocyte infiltrates, and in contrast to ApoE(-/-) mice, SMCs did not express macrophage migratory inhibitory factor (MIF), including at sites distant from atheromatous lesions. Low levels of circulating MIF in ApX4 mice normalized to levels seen in ApoE(-/-) mice after injection of an inhibitory anti-human tissue factor pathway inhibitor antibody, which also led to MIF expression by tissue factor-positive medial SMCs. MIF production by SMCs in ApoE(-/-) mice in vitro and in vivo was shown to be dependent on tissue factor and protease-activated receptor signaling, which were inhibited in ApX4 mice. CONCLUSIONS: Our data indicate that tissue factor plays a hitherto unreported role in the generation of MIF by SMCs in atherosclerosis-prone ApoE(-/-) mice, inhibition of which significantly prevents the development of atherosclerosis, through inhibition of leukocyte recruitment. These data significantly enhance our understanding of the pathophysiology of this important pathology and suggest new potential translational strategies to prevent atheroma formation.

B-lymphocytes support and regulate indirect T-cell alloreactivity in individual patients with chronic antibody-mediated rejection.

We explored how B-lymphocytes influence in vitro T-cell alloresponses in patients with antibody-mediated rejection (AMR), testing whether B-cells would be preferentially involved in this group of patients. Peripheral blood mononuclear cells were collected from 65 patients having biopsy: 14 patients with AMR and 5 with no pathology on protocol; 38 with AMR and 8 with nonimmunologic damage on 'for cause'. Using enzyme-linked immunosorbent spot assays, we found interferon-γ production by indirect allorecognition in 45 of 119 total samples from the 65 patients. B-cells preferentially processed and presented donor alloantigens in samples from AMR patients. In a further 25 samples, B-cell-dependent allo-specific reactivity was shown by depletion of CD25(+) cells and these individuals had higher percentages of CD4CD25hi cells. In 21 samples, reactivity was shown by depletion of CD19(+) cells, associated with polarized cytokine production toward IL-10 after polyclonal activation by IgG/IgM. Overall, this shows a significant contribution by B-cells to indirect donor-specific T-cell reactivity in vitro in patients with AMR. Active suppression by distinct phenotypes of T- or B-cells in approximately half of the patients indicates that chronic AMR is not characterized by a universal loss of immune regulation. Thus, stratified approaches that accommodate the heterogeneity of cell-mediated immunity might be beneficial to treat graft dysfunction.