Cees van Kooten

Epstein-Barr virus (EBV) reprograms autoreactive B cells into activated antigen-presenting cells that drive pathogenic T- and B-cell responses in autoimmune disease. ^1–4 Whether eliminating these cells contributes to durable remission after CD19 chimeric antigen receptor (CAR) T-cell therapy ^5–7 is unknown. Here, using EBV B-cell sequencing (EBV-seq) ³ , we profiled EBV ⁺ B cells from patients treated with CD19-CAR T cells (systemic lupus erythematosus, n=6; inflammatory myopathy, n=3; systemic sclerosis, n=3) or rituximab (n=15) and recombinantly expressed their antibodies to define antigen specificity. At baseline, EBV ⁺ B cells were enriched relative to healthy donors, class-switched and hypermutated, expressed the latency III program with heightened antigen-presentation signatures, and encoded autoantibodies. CAR T cells, but not rituximab, depleted EBV ⁺ B cells and shifted the reconstituted repertoire towards IgM ⁺ , minimally mutated, largely non-autoreactive cells, consistent with infection of newly generated B cells. After rituximab, EBV ⁺ B cells retained a class-switched, hypermutated and autoreactive with an activated antigen-presenting phenotype. Sustained drug-free remission occurred in 8/12 CAR T-cell-treated versus 0/15 rituximab-treated patients. In vitro , EBV ⁺ B cells resisted anti-CD20 and anti-CD19 antibodies but were killed by CAR T cells and a CD19 T-cell engager. Our findings suggest resetting the EBV ⁺ autoreactive B-cell repertoire contributes to sustained drug-free remission.

BK polyomavirus (BKPyV) infection may lead to BKPyV‐associated nephropathy (BKPyVAN) and subsequent graft loss in kidney transplant recipients. Experimental studies have suggested that glucocorticoids (GCs) may enhance BKPyV replication through a steroid‐responsive element within the viral genome. However, clinical observations have not consistently supported this association, prompting re‐investigation of the effect of GCs on BKPyV replication in primary human renal epithelial cells (PTECs). PTECs were infected with BKPyV and subsequently exposed to increasing concentrations of methylprednisolone (MPS) or dexamethasone (Dexa). Viral replication was quantified by quantitative polymerase chain reaction (qPCR). Relative differences in viral load were analyzed using univariate analysis of variance. Activation of the GC pathway was assessed by measuring FKBP5 and PER1 gene expression. Inflammatory responses were evaluated by enzyme‐linked immunosorbent assays (ELISA) for IL‐6, IL‐8, and TNF‐α. Treatment of BKPyV‐infected PTECs with increasing concentrations of MPS or Dexa did not affect viral replication compared to untreated controls. While treatment‐related changes in gene expression of FKBP5 and PER1 were observed, confirming GC‐signaling pathway activation in BKPyV‐infected cells. Cytokine production (IL‐6 and IL‐8) was unaffected. In primary human PTECs infected with BKPyV, treatment with MPS or Dexa did not enhance BKPyV replication. These findings contrast with earlier in vitro studies and suggest that glucocorticoids do not directly promote BKPyV replication under the conditions tested. Further studies are needed to determine whether these findings extend to other cell models and in vivo.

Background: The immunomodulatory activity of normothermic machine perfusion (NMP) on donor organs may be reflected by the release of immune‐regulating factors into the perfusate. Dendritic cells (DCs) are potent antigen‐presenting cells and are able to sense these factors. We hypothesized that activation of DCs could be used to monitor the release of these mediators into the perfusate. Methods: Human monocytes from buffy coats were differentiated into monocyte‐derived DCs (moDCs). Perfusate was obtained from human donor kidneys deemed unsuitable for transplantation ( n = 15) that underwent 6 h of NMP. MoDCs were incubated for 24 h with perfusate collected at different timepoints. DC supernatant was analyzed with Luminex and ELISA. MoDCs were analyzed with flow cytometry for maturation markers and co‐cultured with allogenic T cells. Results: Incubation with perfusate resulted in moDCs with a reduced surface expression of CD86, without affecting other maturation markers. Analysis of DC supernatant showed there was a specific increase of IL‐10 production upon incubation with perfusate. As a consequence, moDCs incubated with perfusate showed lower T cell stimulatory capacity. Conclusion: This study showed that the activation status of DCs can be used to monitor the release of factors into the perfusate during NMP with an anti‐inflammatory DC phenotype and function. It demonstrates the feasibility of a cell‐based model to determine immune regulatory effects during ex vivo organ perfusion.

Background.: Brain death (BD) results in an inflammatory response, including complement activation. The clinical impact of prolonged BD duration on the graft-to-be is still unclear. We investigated how BD duration impacts complement activation levels, both systemically and locally within donor kidneys. Methods.: EDTA plasma samples and kidney biopsies were obtained from the Quality in Organ Donation biobank (n = 120). Samples were routinely taken at 3 fixed points during BD management and donors were grouped according to short (≤14 h), medium (15–22 h), or long (≥23 h) duration of BD. ELISAs were used for quantification of complement in plasma, and immunohistochemistry was performed to determine complement activation at tissue level. Results.: Plasma levels of C4d, Bb, C3c, and C5b-9 were significantly elevated compared with living donor samples taken at similar timepoint. Complement activation was already observed at the start of donor management and remained elevated. Prolonged BD duration was associated with reduced complement activation, with significantly lower levels of C4d and Bb, and trends toward lower C3c. Elevated levels of Bb were associated with increased delayed graft function (DGF), while increased C4d levels showed trends toward higher DGF and lower eGFR at 3 mo posttransplantation. Also, renal biopsies taken just before reperfusion, showed local complement activation, with more intense complement staining (C3d and C5b-9) in the vascular pole in kidneys that developed DGF. Conclusions.: The complement system is already activated in BD donors early on during donor management. Prolonged BD duration was associated with reduced systemic complement activation. Increased systemic and local complement activation appears to negatively impact short-term kidney function.

Highly sensitised kidney transplant candidates face substantial barriers to transplantation due to limited donor compatibility. Delisting unacceptable antigens with detectable HLA‐specific antibodies can improve transplant access. It is, however, challenging to determine which specificities are safe to delist. Assessment of circulating HLA‐specific memory B‐cells may support risk stratification. However, current methodologies are limited by the predominant localisation of memory B‐cells in secondary lymphoid organs and thus by potential false negativity. BAFF inhibitors, such as belimumab, mobilise memory B‐cells into the circulation, which could reduce false‐negative evaluations and improve pretransplant risk stratification. We administered off‐label treatment with 200 mg of subcutaneous belimumab weekly for 4 weeks to seven highly sensitised patients with limited allocation probability. The clinical aim was to safely increase allocation probability by selectively delisting unacceptable HLA‐specificities without detectable B‐cell memory. HLA‐specific memory B‐cell profiles were assessed before and after treatment, revealing significantly broader HLA‐specific memory B‐cell profiles after belimumab in addition to a significant increase of HLA‐antibody MFI in the eluate of stimulated memory B‐cells. This strategy may pave the way for a new paradigm in pretransplant immunological risk‐stratification, allowing improved assessment of HLA‐specific memory B‐cell profiles, which could potentially limit the risk of memory responses.

Repeat transplantations represent up to 20% of all kidney transplants. Whereas repeat transplantations in the presence of circulating donor‐specific HLA‐antibodies are generally avoided, the risk of repeated HLA‐mismatches (RMM) without detectable antibodies remains debated. This multicenter study evaluated the hazard of RMM, stratified by HLA‐class, on transplant outcomes in the absence of preformed donor‐specific antibodies. We included repeat kidney transplant recipients from January 2009 onward with available HLA typing for HLA‐A, ‐B, ‐C, ‐DRB1, ‐DRB3/4/5 and ‐DQB1 from current and previous donors. RMM were defined at the split serological antigen level, excluding patients with only HLA‐DP or HLA‐DRB3/4/5 RMM. Patients were included if: (a) preformed donor‐specific HLA‐antibodies (DSA) had never been detected and (b) this was confirmed by Luminex assays within 6 months pre‐transplantation. A competing risk model, adjusting for demographic factors and total HLA‐mismatch load while accounting for death as a competing risk, showed that HLA‐DRB1 and/or HLA‐DQB1 RMM significantly increased the risk of graft loss within 1 year post‐transplant (HR 3.75 [95% CI 1.51–9.34], p = 0.004). Cox proportional hazard models further linked these HLA‐class II RMM to higher risks of biopsy‐proven rejection (HR 1.98 [95% CI 1.04–3.76], p = 0.037) and DSA development (HR 9.89 [95% CI 1.92–50.99], p = 0.006), while no significant risks were observed for HLA‐class I RMM. Sensitivity analyses in patients screened for pretransplant DSA via single antigen bead assays, those with similar immunosuppression, and those with allelic RMM further confirmed these findings. These results suggest that avoiding HLA‐DRB1 and HLA‐DQB1 RMM, when feasible, may improve transplant outcomes.

Background: Infusion of mesenchymal stromal cells (MSCs) has been proposed as immune-modulatory therapy in solid organ transplantation. The use of allogenic MSCs could improve standardization and allow for direct availability of the product.Method: The nonrandomized phase Ib Neptune clinical trial provided safety and feasibility data on the use of allogenic bone-marrow-derived MSCs, infused in 10 patients at week 25 and 26 post kidney transplantation. Here, we performed detailed analysis on the peripheral blood immune cell composition of these patients up to 52 weeks post transplantation. We used a 40 marker antibody panel with mass cytometry to assess potential effects of MSC therapy on the immune system.Results: We showed minor changes in major immune lineages at week 27, 34 and 52 post kidney transplantation after MSC infusion at week 25 and week 26, confirming previous data with regular flow cytometry. However, in a direct comparison between pre- and post MSC infusion, as soon as 4 hours after MSC infusion, we observed a significant increase in cell numbers of B cell and T cell subsets that shared a unique expression of CD11b, CD11c, CD38, CD39, and Ki-67.Conclusion: Exploring these CD11b⁺CD11c⁺CD38⁺CD39⁺Ki-67⁺ B cells and T cells in the context of MSC infusion after kidney transplantation may be a promising avenue to better understand the immunological effects of MSC therapy.

Systemic lupus erythematosus (SLE) is marked by excessive complement activation, contributing to tissue damage. Complement activation can be detected in many organs including the skin, kidney, and brain. The involvement of the central nervous system is particularly relevant to understanding neuropsychiatric SLE (NPSLE), one of the poorest understood manifestations of SLE for which no biomarkers are available. We studied the levels of complement inhibitors in SLE in relation to disease activity and as possible biomarkers to identify NPSLE. Serum levels of complement inhibitors C1‐inhibitor (C1‐INH), C4b‐binding protein (C4BP), Factor I, and Factor H were measured in 345 SLE patients (including 102 with NPSLE) and 108 healthy controls. Compared with controls, SLE patients had higher C1‐INH and C4BP but lower Factor I and H levels. All inhibitors positively correlated with total C3 and C4 levels. While correlating with the SLE Disease Activity Index (SLEDAI), no distinction in inhibitor levels was found between SLE and NPSLE patients. Over time, C1‐INH and Factor H levels normalized, but no significant changes were observed for C4BP and Factor I. In SLE the levels of circulating complement inhibitors are inversely correlated to complement consumption but do not serve as biomarkers for NPSLE.