Multiomic profiling of transplant glomerulopathy reveals a novel T-cell dominant subclass.

Kidney transplant (KTx) biopsies showing transplant glomerulopathy (TG) (glomerular basement membrane double contours (cg) > 0) and microvascular inflammation (MVI) in the absence of C4d staining and donor-specific antibodies (DSAs) do not fulfill the criteria for chronic active antibody-mediated rejection (CA-AMR) diagnosis and do not fit into any other Banff category. To investigate this, we initiated a multicenter intercontinental study encompassing 36 cases, comparing the immunomic and transcriptomic profiles of 14 KTx biopsies classified as cg+MVI DSA-/C4d- with 22 classified as CA-AMR DSA+/C4d+ through novel transcriptomic analysis using the NanoString Banff-Human Organ Transplant (B-HOT) panel and subsequent orthogonal subset analysis using two innovative 5-marker multiplex immunofluorescent panels. Nineteen genes were differentially expressed between the two study groups. Samples diagnosed with CA-AMR DSA+/C4d+ showed a higher glomerular abundance of natural killer cells and higher transcriptomic cell type scores for macrophages in an environment characterized by increased expression of complement-related genes (i.e., C5AR1) and higher activity of angiogenesis, interstitial fibrosis tubular atrophy, CA-AMR, and DSA-related pathways when compared to samples diagnosed with cg+MVI DSA-/C4d-. Samples diagnosed with cg+MVI DSA-/C4d- displayed a higher glomerular abundance and activity of T cells (CD3+, CD3+CD8+, and CD3+CD8-). Thus, we show that using novel multiomic techniques, KTx biopsies with cg+MVI DSA-/C4d- have a prominent T-cell presence and activity, putting forward the possibility that these represent a more T-cell dominant phenotype.

The enigma of sclera-specific autoimmunity in scleritis.

Scleritis is a severe and painful ophthalmic disorder, in which a pathogenic role for collagen-directed autoimmunity was repeatedly suggested. We evaluated the presence of sclera-specific antibodies in a large cohort of patients with non-infectious scleritis. Therefore, we prospectively collected serum samples from 121 patients with non-infectious scleritis in a multicenter cohort study in the Netherlands. In addition, healthy (n = 39) and uveitis controls (n = 48) were included. Serum samples were tested for anti-native human type II collagen antibodies using a validated enzyme-linked immunosorbent assay (ELISA). Further, sclera-specific antibodies were determined using indirect immunofluorescence (IIF) on primate retinal/scleral cryosections. Lastly, human leukocyte antigen (HLA) typing was performed in 111 patients with scleritis. Anti-type II collagen antibodies were found in 13% of scleritis patients, in 10% of healthy controls and in 11% of uveitis controls (p = 0.91). A specific reaction to scleral nerve tissue on IIF was observed in 33% of patients with scleritis, which was higher than in healthy controls (11%; p = 0.01), but similar to uveitis controls (25%; p = 0.36). Reactivity to the scleral nerve tissue was significantly associated with earlier onset of scleritis (48 versus 56 years; p < 0.001), bilateral involvement (65% versus 42%; p = 0.01), and less frequent development of scleral necrosis (5% versus 22%; p = 0.02). HLA-B27 was found to be twice as prevalent in patients with scleritis (15.3%) compared to a healthy population (7.2%). In conclusion, scleral nerve autoantibody reactivity was more common in scleritis and uveitis patients in contrast to healthy controls. Further research is needed to characterize these scleral-nerve directed antibodies and assess their clinical value.

Diagnosis and treatment of allograft rejection in islet transplantation.

Islet transplantation stabilizes glycemic control in patients with complicated diabetes mellitus. Rapid functional decline could be due to islet allograft rejection. However, there is no reliable method to assess rejection, and treatment protocols are absent. We aimed to characterize diagnostic features of islet allograft rejection and assess effectiveness of high-dose methylprednisolone treatment. Over a median follow-up of 61.8 months, 22% (9 of 41) of islet transplant recipients experienced 10 suspected rejection episodes (SREs). All first SREs occurred within 18 months after transplantation. Important features were unexplained hyperglycemia (all cases), unexplained C-peptide decrease (ΔC-peptide, 77.1% [-59.1% to -91.6%]; ΔC-peptide:glucose, -76.3% [-49.2% to -90.4%]), predisposing event (5 of 10 cases), and increased immunologic risk (5 of 10 cases). At 6 months post-SRE, patients who received protocolized methylprednisolone (n = 4) had significantly better islet function than untreated patients (n = 4), according to C-peptide (1.39 ± 0.59 vs 0.14 ± 0.19 nmol/L; P = .007), Igls score (good [4 of 4 cases] vs failure [3 of 4 cases] or marginal [1 of 4 cases]; P = .018) and ß score (6.0 [6.0-6.0] vs 1.0 [0.0-3.5]; P = .013). SREs are prevalent among islet transplant recipients and are associated with loss of islet graft function. Timely treatment with high-dose methylprednisolone mitigates this loss. Unexplained hyperglycemia, unexpected C-peptide decrease, a predisposing event, and elevated immunologic risk are diagnostic indicators for SRE.

Chronic Pancreas Allograft Rejection Followed by Successful HLA-Incompatible Islet Alloautotransplantation: A Novel Strategy?

The purpose of pancreas or islet transplantation is to restore glycemic control in order to mitigate diabetes-related complications and prevent severe hypoglycemia. Complications from chronic pancreas allograft rejection may lead to transplantectomy, even when the endocrine function remains preserved. We present first evidence of a successful HLA incompatible islet re-transplantation with islets isolated from a rejecting pancreas allograft after simultaneous kidney pancreas transplantation. The pancreas allograft was removed because of progressively painful pancreatic panniculitis from clinically uncontrolled chronic rejection. The endocrine function was preserved. Induction treatment for this "islet alloautotransplantation" consisted of plasmapheresis, IVIg and alemtuzumab. At 1 year, the patient retained islet graft function with good glycemic control and absence of severe hypoglycemia, despite persistent low-grade HLA donor-specific antibodies. His panniculitis had resolved completely. In our point of view, islet alloautotransplantation derived from a chronically rejecting pancreas allograft is a potential option to salvage (partial) islet function, despite preformed donor-specific antibodies, in order to maintain stable glycemic control. Thereby it protects against severe hypoglycemia, and it potentially mitigates kidney graft dysfunction and other diabetes-related complications in patients with continued need for immunosuppression and who are otherwise difficult to retransplant.

Activated CD4+ T Cells and Highly Differentiated Alloreactive CD4+ T Cells Distinguish Operationally Tolerant Liver Transplantation Recipients.

Spontaneous operational tolerance to the allograft develops in a proportion of liver transplantation (LT) recipients weaned off immunosuppressive (IS) drugs. Several studies have investigated whether peripheral blood circulating T cells could play a role in the development or identify operational tolerance, but never characterized alloreactive T cells in detail due to the lack of a marker for these T cells. In this study, we comprehensively investigated phenotypic and functional characteristics of alloreactive circulating T cell subsets in tolerant LT recipients (n = 15) using multiparameter flow cytometry and compared these with LT recipients on IS drugs (n = 23) and healthy individuals (n = 16). Activation-induced CD137 was used as a marker for alloreactive T cells upon allogenic stimulation. We found that central and effector memory CD4+ T cells were hyporesponsive against donor and third-party splenocyte stimulation in tolerant LT recipients, whereas an overall hyperresponsiveness was observed in alloreactive terminally differentiated effector memory CD4+ T cells. In addition, elevated percentages of circulating activated T helper cells were observed in these recipients. Lastly, tolerant and control LT recipients did not differ in donor-specific antibody formation. In conclusion, a combination of circulating hyperresponsive highly differentiated alloreactive CD4+ T cells and circulating activated T helper cells could discriminate tolerant recipients from a larger group of LT recipients.

Tacrolimus Monotherapy is Safe in Immunologically Low-Risk Kidney Transplant Recipients: A Randomized-Controlled Pilot Study.

In this randomized-controlled pilot study, the feasibility and safety of tacrolimus monotherapy in immunologically low-risk kidney transplant recipients was evaluated [NTR4824, www.trialregister.nl]. Low immunological risk was defined as maximal 3 HLA mismatches and the absence of panel reactive antibodies. Six months after transplantation, recipients were randomized if eGFR >30 ml/min, proteinuria <50 mg protein/mmol creatinine, no biopsy-proven rejection after 3 months, and no lymphocyte depleting therapy given. Recipients were randomized to tacrolimus/mycophenolate mofetil (TAC/MMF) or to taper and discontinue MMF at month 9 (TACmono). 79 of the 121 recipients were randomized to either TACmono (n = 38) or TAC/MMF (n = 41). Mean recipient age was 59 years and 59% received a living donor transplant. The median follow-up was 62 months. After randomization, 3 TACmono and 4 TAC/MMF recipients experienced a biopsy-proven rejection. At 5 years follow-up, patient survival was 84% in TACmono versus 76% in TAC/MMF with death-censored graft survival of 97% for both groups and no differences in eGFR and proteinuria. Eleven TACmono recipients had an infectious episode versus 22 TAC/MMF recipients (p < 0.03). Donor-specific anti-HLA antibodies were not detected during follow-up in both groups. Tacrolimus monotherapy in selected immunologically low-risk kidney transplant recipients appears safe and reduces the number of infections.

Autologous bone marrow-derived mesenchymal stromal cell therapy with early tacrolimus withdrawal: The randomized prospective, single-center, open-label TRITON study.

After renal transplantation, there is a need for immunosuppressive regimens which effectively prevent allograft rejection, while preserving renal function and minimizing side effects. From this perspective, mesenchymal stromal cell (MSC) therapy is of interest. In this randomized prospective, single-center, open-label trial, we compared MSCs infused 6 and 7 weeks after renal transplantation and early tacrolimus withdrawal with a control tacrolimus group. Primary end point was quantitative evaluation of interstitial fibrosis in protocol biopsies at 4 and 24 weeks posttransplant. Secondary end points included acute rejection, graft loss, death, renal function, adverse events, and immunological responses. Seventy patients were randomly assigned of which 57 patients were included in the final analysis (29 MSC; 28 controls). Quantitative progression of fibrosis failed to show benefit in the MSC group and GFR remained stable in both groups. One acute rejection was documented (MSC group), while subclinical rejection in week 24 protocol biopsies occurred in seven patients (four MSC; three controls). In the MSC group, regulatory T cell numbers were significantly higher compared to controls (p = .014, week 24). In conclusion, early tacrolimus withdrawal with MSC therapy was safe and feasible without increased rejection and with preserved renal function. MSC therapy is a potentially useful approach after renal transplantation.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor-specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA-specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA-DR-specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA-DR antigen-reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA-DRB1*01:01, HLA-DRB1*04:01, and HLA-DRB1*07:01 antigen-reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA-DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.

Human leukocyte antigen selected allogeneic mesenchymal stromal cell therapy in renal transplantation: The Neptune study, a phase I single-center study.

Mesenchymal stromal cells (MSC) hold promise as a novel immune-modulatory therapy in organ transplantation. First clinical studies have used autologous MSCs; however, the use of allogeneic "off-the-shelf" MSCs is more sustainable for broad clinical implementation, although with the risk of causing sensitization. We investigated safety and feasibility of allogeneic MSCs in renal transplantation, using a matching strategy that prevented repeated mismatches. Ten patients received two doses of 1.5 × 106 /kg allogeneic MSCs 6 months after transplantation in a single-center nonrandomized phase Ib trial, followed by lowering of tacrolimus (trough level 3 ng/mL) in combination with everolimus and prednisone. Primary end point was safety, measured by biopsy proven acute rejection (BPAR) and graft loss 12 months after transplantation. Immune monitoring was performed before and after infusion. No BPAR or graft loss occurred and renal function remained stable. One patient retrospectively had DSAs against MSCs, formed before infusion. No major alterations in T and B cell populations or plasma cytokines were observed upon MSC infusion. Administration of HLA selected allogeneic MSCs combined with low-dose tacrolimus 6 months after transplantation is safe at least in the first year after renal transplantation. This sets the stage to further explore the efficacy of third-party MSCs in renal transplantation.

Allocation to highly sensitized patients based on acceptable mismatches results in low rejection rates comparable to nonsensitized patients.

Whereas regular allocation avoids unacceptable mismatches on the donor organ, allocation to highly sensitized patients within the Eurotransplant Acceptable Mismatch (AM) program is based on the patient's HLA phenotype plus acceptable antigens. These are HLA antigens to which the patient never made antibodies, as determined by extensive laboratory testing. AM patients have superior long-term graft survival compared with highly sensitized patients in regular allocation. Here, we questioned whether the AM program also results in lower rejection rates. From the PROCARE cohort, consisting of all Dutch kidney transplants in 1995-2005, we selected deceased donor single transplants with a minimum of 1 HLA mismatch and determined the cumulative 6-month rejection incidence for patients in AM or regular allocation. Additionally, we determined the effect of minimal matching criteria of 1 HLA-B plus 1 HLA-DR, or 2 HLA-DR antigens on rejection incidence. AM patients showed significantly lower rejection rates than highly immunized patients in regular allocation, comparable to nonsensitized patients, independent of other risk factors for rejection. In contrast to highly sensitized patients in regular allocation, minimal matching criteria did not affect rejection rates in AM patients. Allocation based on acceptable antigens leads to relatively low-risk transplants for highly sensitized patients with rejection rates similar to those of nonimmunized individuals.

Antibodies against ARHGDIB are associated with long-term kidney graft loss.

The clinical significance of non-HLA antibodies on renal allograft survival is a matter of debate, due to differences in reported results and lack of large-scale studies incorporating analysis of multiple non-HLA antibodies simultaneously. We developed a multiplex non-HLA antibody assay against 14 proteins highly expressed in the kidney. In this study, the presence of pretransplant non-HLA antibodies was correlated to renal allograft survival in a nationwide cohort of 4770 recipients transplanted between 1995 and 2006. Autoantibodies against Rho GDP-dissociation inhibitor 2 (ARHGDIB) were significantly associated with graft loss in recipients transplanted with a deceased-donor kidney (N = 3276) but not in recipients of a living-donor kidney (N = 1496). At 10 years after deceased-donor transplantation, recipients with anti-ARHGDIB antibodies (94/3276 = 2.9%) had a 13% lower death-censored covariate-adjusted graft survival compared to the anti-ARHGDIB-negative (3182/3276 = 97.1%) population (hazard ratio 1.82; 95% confidence interval, 1.32-2.53; P = .0003). These antibodies occur independently from donor-specific anti-HLA antibodies (DSA) or other non-HLA antibodies investigated. No significant relations with graft loss were found for the other 13 non-HLA antibodies. We suggest that pretransplant risk assessment can be improved by measuring anti-ARHGDIB antibodies in all patients awaiting deceased-donor transplantation.

A paired kidney analysis on the impact of pre-transplant anti-HLA antibodies on graft survival.

BACKGROUND: Pre-transplant donor-specific anti-human leucocyte antigen (HLA) antibodies (DSAs) are associated with impaired kidney graft survival while the clinical relevance of non-donor-specific anti-HLA antibodies (nDSAs) is more controversial. The aim of the present paired kidney graft study was to compare the clinical relevance of DSAs and nDSAs. METHODS: To eliminate donor and era-dependent factors, a post hoc paired kidney graft analysis was performed as part of a Dutch multicentre study evaluating all transplantations between 1995 and 2005 with available pre-transplant serum samples. Anti-HLA antibodies were detected with a Luminex single-antigen bead assay. RESULTS: Among 3237 deceased donor transplantations, we identified 115 recipient pairs receiving a kidney from the same donor with one recipient being DSA positive and the other without anti-HLA antibodies. Patients with pre-transplant DSAs had a significantly lower 10-year death-censored graft survival (55% versus 82%, P=0.0001). We identified 192 pairs with one recipient as nDSA positive (against Class I and/or II) and the other without anti-HLA antibodies. For the patients with nDSAs against either Class I or II, graft survival did not significantly differ compared with patients without anti-HLA antibodies (74% versus 77%, P = 0.79). Only in patients with both nDSAs Class I and II was there a trend towards a lower graft survival (58%, P = 0.06). Lastly, in a small group of 42 recipient pairs, 10-year graft survival in recipients with DSAs was 49% compared with 68% in recipients with nDSAs (P=0.11). CONCLUSION: This paired kidney analysis confirms that the presence of pre-transplant DSAs in deceased donor transplantations is a risk marker for graft loss, whereas nDSAs in general are not associated with a lower graft survival. Subgroup analysis indicated that only in broadly sensitized patients with nDSAs against Class I and II, nDSAs may be a risk marker for graft loss in the long term.

Effect of initial immunosuppression on long-term kidney transplant outcome in immunological low-risk patients.

BACKGROUND: Few studies have evaluated the effect of different immunosuppressive strategies on long-term kidney transplant outcomes. Moreover, as they were usually based on historical data, it was not possible to account for the presence of pretransplant donor-specific human-leukocyte antigen antibodies (DSA), a currently recognized risk marker for impaired graft survival. The aim of this study was to evaluate to what extent frequently used initial immunosuppressive therapies increase graft survival in immunological low-risk patients. METHODS: We performed an analysis on the PROCARE cohort, a Dutch multicentre study including all transplantations performed in the Netherlands between 1995 and 2005 with available pretransplant serum (n = 4724). All sera were assessed for the presence of DSA by a luminex single-antigen bead assay. Patients with a previous kidney transplantation, pretransplant DSA or receiving induction therapy were excluded from the analysis. RESULTS: Three regimes were used in over 200 patients: cyclosporine (CsA)/prednisolone (Pred) (n = 542), CsA/mycophenolate mofetil (MMF)/Pred (n = 857) and tacrolimus (TAC)/MMF/Pred (n = 811). Covariate-adjusted analysis revealed no significant differences in 10-year death-censored graft survival between patients on TAC/MMF/Pred therapy (79%) compared with patients on CsA/MMF/Pred (82%, P = 0.88) or CsA/Pred (79%, P = 0.21). However, 1-year rejection-free survival censored for death and failure unrelated to rejection was significantly higher for TAC/MMF/Pred (81%) when compared with CsA/MMF/Pred (67%, P < 0.0001) and CsA/Pred (64%, P < 0.0001). CONCLUSION: These results suggest that in immunological low-risk patients excellent long-term kidney graft survival can be achieved irrespective of the type of initial immunosuppressive therapy (CsA or TAC; with or without MMF), despite differences in 1-year rejection-free survival.

Pretransplant C3d-Fixing Donor-Specific Anti-HLA Antibodies Are Not Associated with Increased Risk for Kidney Graft Failure.

Background Complement-fixing antibodies against donor HLA are considered a contraindication for kidney transplant. A modification of the IgG single-antigen bead (SAB) assay allows detection of anti-HLA antibodies that bind C3d. Because early humoral graft rejection is considered to be complement mediated, this SAB-based technique may provide a valuable tool in the pretransplant risk stratification of kidney transplant recipients.Methods Previously, we established that pretransplant donor-specific anti-HLA antibodies (DSAs) are associated with increased risk for long-term graft failure in complement-dependent cytotoxicity crossmatch-negative transplants. In this study, we further characterized the DSA-positive serum samples using the C3d SAB assay.Results Among 567 pretransplant DSA-positive serum samples, 97 (17%) contained at least one C3d-fixing DSA, whereas 470 (83%) had non-C3d-fixing DSA. At 10 years after transplant, patients with C3d-fixing antibodies had a death-censored, covariate-adjusted graft survival of 60%, whereas patients with non-C3d-fixing DSA had a graft survival of 64% (hazard ratio, 1.02; 95% confidence interval, 0.70 to 1.48 for C3d-fixing DSA compared with non-C3d-fixing DSA; P=0.93). Patients without DSA had a 10-year graft survival of 78%.Conclusions The C3d-fixing ability of pretransplant DSA is not associated with increased risk for graft failure.

Anti-LGI1 encephalitis is strongly associated with HLA-DR7 and HLA-DRB4.

Leucine-rich glioma-inactivated1 (LGI1) encephalitis is an antibody-associated inflammation of the limbic area. An autoimmune etiology is suspected but not yet proven. We performed human leukocyte antigen (HLA) analysis in 25 nontumor anti-LGI1 patients and discovered a remarkably strong HLA association. HLA-DR7 was present in 88% compared to 19.6% in healthy controls (p = 4.1 × 10-11 ). HLA-DRB4 was present in all patients and in 46.5% controls (p = 1.19 × 10-7 ). These findings support the autoimmune hypothesis. An exploratory analysis was performed in a small group of 4 tumor-LGI1 patients. The strong HLA association seems not applicable in these patients. Therefore, the absence of HLA-DR7 or HLA-DRB4 could raise tumor suspicion in anti-LGI1 patients. Ann Neurol 2017;81:193-198.

Donor-specific anti-HLA antibodies are not associated with nonanastomotic biliary strictures but both are independent risk factors for graft loss after liver transplantation.

Donor-specific alloantibodies (DSA) have been associated with rejection and shorter graft survival after orthotopic liver transplantation (OLT). We examined the role of DSA in nonanastomotic biliary strictures (NAS) after OLT. Patients receiving first OLT who developed NAS (n = 68) and a control group without NAS (n = 83), with pre-OLT and 12 months post-OLT serum samples, were included. DSA were specified using the Luminex single antigen test. Risk factors for NAS and graft survival were analyzed. The presence of preformed DSA was not significantly different between patients with NAS and controls (P = .89). After 12 months, 26.5% of NAS patients and 16.9% of controls had generated de novo DSA (P = .15). Neither de novo class I DSA nor de novo class II DSA were associated with NAS. De novo DSA generally developed after the diagnosis of NAS. Time-dependent regression analysis identified both NAS (aHR 8.05, CI 3.28 - 19.77, P < .01) and de novo class II DSA (aHR 2.84, CI 1.38 - 5.82, P < .01) as independent risk factors for graft loss. Preformed or de novo DSA were not associated with the development of NAS. However, NAS as well as de novo class II DSA were independent risk factors for graft loss after OLT.

Chronic-active antibody-mediated rejection with or without donor-specific antibodies has similar histomorphology and clinical outcome - a retrospective study.

Chronic-active antibody-mediated rejection (c-aABMR) is defined as histological evidence of chronic endothelial injury (cg), also known as transplant glomerulopathy, and either microvascular inflammation (MVI) or positivity for C4d. Importantly, the presence of donor-specific antibodies (DSA) is currently still mandatory for the diagnosis of c-aABMR. This retrospective study of 41 c-aABMR patients investigates whether cases suspicious for c-aABMR (DSA negative, n = 24) differ from cases of c-aABMR (DSA positive, n = 17) with respect to renal histology, allograft function and long-term graft survival. All included patients had progressive loss of allograft function and were diagnosed by for cause biopsy and scored according to the Banff '15 criteria. In all DSApos cases, DSA were de novo and the majority was directed against HLA-II being mostly anti-HLA-DQ antibodies. There were no statistically significant differences in clinical characteristics, decline in allograft function and renal allograft survival in cases with or without DSAs. All cases showed chronic histomorphological damage and inflammation, irrespective of the presence of DSA. Renal histology and clinical outcome of patients suspicious for c-aABMR (DSAneg) do not significantly differ from patients with a diagnosis of c-aABMR (DSApos). We believe that our study adds to the ongoing debate regarding the need for DSAs to be present for the diagnosis of c-aABMR.

Human Lymphoid Tissues Harbor a Distinct CD69+CXCR6+ NK Cell Population.

Knowledge of human NK cells is based primarily on conventional CD56(bright) and CD56(dim) NK cells from blood. However, most cellular immune interactions occur in lymphoid organs. Based on the coexpression of CD69 and CXCR6, we identified a third major NK cell subset in lymphoid tissues. This population represents 30-60% of NK cells in marrow, spleen, and lymph node but is absent from blood. CD69(+)CXCR6(+) lymphoid tissue NK cells have an intermediate expression of CD56 and high expression of NKp46 and ICAM-1. In contrast to circulating NK cells, they have a bimodal expression of the activating receptor DNAX accessory molecule 1. CD69(+)CXCR6(+) NK cells do not express the early markers c-kit and IL-7Rα, nor killer cell Ig-like receptors or other late-differentiation markers. After cytokine stimulation, CD69(+)CXCR6(+) NK cells produce IFN-γ at levels comparable to CD56(dim) NK cells. They constitutively express perforin but require preactivation to express granzyme B and exert cytotoxicity. After hematopoietic stem cell transplantation, CD69(+)CXCR6(+) lymphoid tissue NK cells do not exhibit the hyperexpansion observed for both conventional NK cell populations. CD69(+)CXCR6(+) NK cells constitute a separate NK cell population with a distinct phenotype and function. The identification of this NK cell population in lymphoid tissues provides tools to further evaluate the cellular interactions and role of NK cells in human immunity.

Innate and adaptive immunity to human beta cell lines: implications for beta cell therapy.

AIMS/HYPOTHESIS: Genetically engineered human beta cell lines provide a novel source of human beta cells to study metabolism, pharmacology and beta cell replacement therapy. Since the immune system is essentially involved in beta cell destruction in type 1 diabetes and after beta cell transplantation, we investigated the interaction of human beta cell lineswith the immune system to resolve their potential for immune intervention protocol studies. METHODS: Human pancreatic beta cell lines (EndoC-ßH1 and ECi50) generated by targeted oncogenesis in fetal pancreas were assessed for viability after innate and adaptive immune challenges. Beta cell lines were pre-conditioned with T helper type 1 (Th1) cytokines or high glucose to mimic inflammatory and hyperglycaemia-stressed conditions. Beta cells were then co-cultured with auto- and alloreactive cytotoxic T cells (CTL), natural killer (NK) cells, supernatant fraction from activated autoreactive Th1 cells, or alloantibodies in the presence of complement or effector cells. RESULTS: Low HLA expression protected human beta cell lines from adaptive immune destruction, but it was associated with direct killing by activated NK cells. Autoreactive Th1 cell inflammation, rather than glucose stress, induced increased beta cell apoptosis and upregulation of HLA, increasing beta cell vulnerability to killing by auto- and alloreactive CTL and alloreactive antibodies. CONCLUSIONS/INTERPRETATION: We demonstrate that genetically engineered human beta cell lines can be used in vitro to assess diverse immune responses that may be involved in the pathogenesis of type 1 diabetes in humans and beta cell transplantation, enabling preclinical evaluation of novel immune intervention strategies protecting beta cells from immune destruction.

Simultaneous pancreas-kidney transplantation in patients with type 1 diabetes reverses elevated MBL levels in association with MBL2 genotype and VEGF expression.

AIMS/HYPOTHESIS: High levels of circulating mannan-binding lectin (MBL) are associated with the development of diabetic nephropathy and hyperglycaemia-induced vasculopathy. Here, we aimed to assess the effect of glycaemic control on circulating levels of MBL and the relationship of these levels with vascular damage. METHODS: We assessed MBL levels and corresponding MBL2 genotype, together with vascular endothelial growth factor (VEGF) levels as a marker of vascular damage, in type 1 diabetes patients with diabetic nephropathy before and after simultaneous pancreas-kidney (SPK) transplantation. We included diabetic nephropathy patients (n = 21), SPK patients (n = 37), healthy controls (n = 19), type 1 diabetes patients (n = 15) and diabetic nephropathy patients receiving only a kidney transplant (n = 15). Fourteen diabetic nephropathy patients were followed up for 12 months after SPK. RESULTS: We found elevated circulating MBL levels in diabetic nephropathy patients, and a trend towards elevated circulating MBL levels in type 1 diabetes patients, compared with healthy control individuals. MBL levels in SPK patients completely normalised and our data indicate that this predominantly occurs in patients with a polymorphism in the MBL2 gene. By contrast, MBL levels in kidney transplant only patients remained elevated, suggesting that glycaemic control but not reversal of renal failure is associated with decreased MBL levels. In line, levels of glucose and HbA1c, but not creatinine levels and estimated GFR, were correlated with MBL levels. VEGF levels were associated with levels of MBL and HbA1c in an MBL-polymorphism-dependent manner. CONCLUSIONS/INTERPRETATION: Taken together, circulating MBL levels are associated with diabetic nephropathy and are dependent on glycaemic control, possibly in an MBL2-genotype-dependent manner.