Afzal N. Chaudhry

Genetically Distinct Subsets within ANCA-Associated Vasculitis

BACKGROUND Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a severe condition encompassing two major syndromes: granulomatosis with polyangiitis (formerly known as Wegeners granulomatosis) and microscopic polyangiitis. Its cause is unknown, and there is debate about whether it is a single disease entity and what role ANCA plays in its pathogenesis. We investigated its genetic basis. METHODS A genomewide association study was performed in a discovery cohort of 1233 U.K. patients with ANCA-associated vasculitis and 5884 controls and was replicated in 1454 Northern European case patients and 1666 controls. Quality control, population stratification, and statistical analyses were performed according to standard criteria. RESULTS We found both major-histocompatibility-complex (MHC) and non-MHC associations with ANCA-associated vasculitis and also that granulomatosis with polyangiitis and microscopic polyangiitis were genetically distinct. The strongest genetic associations were with the antigenic specificity of ANCA, not with the clinical syndrome. Anti-proteinase 3 ANCA was associated with HLA-DP and the genes encoding α(1)-antitrypsin (SERPINA1) and proteinase 3 (PRTN3) (P=6.2×10(-89), P=5.6×10(-12,) and P=2.6×10(-7), respectively). Anti-myeloperoxidase ANCA was associated with HLA-DQ (P=2.1×10(-8)). CONCLUSIONS This study confirms that the pathogenesis of ANCA-associated vasculitis has a genetic component, shows genetic distinctions between granulomatosis with polyangiitis and microscopic polyangiitis that are associated with ANCA specificity, and suggests that the response against the autoantigen proteinase 3 is a central pathogenic feature of proteinase 3 ANCA-associated vasculitis. These data provide preliminary support for the concept that proteinase 3 ANCA-associated vasculitis and myeloperoxidase ANCA-associated vasculitis are distinct autoimmune syndromes. (Funded by the British Heart Foundation and others.).

A multicenter survey of rituximab therapy for refractory antineutrophil cytoplasmic antibody–associated vasculitis

OBJECTIVE B cell depletion with rituximab has allowed remissions in relapsing or refractory antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis in small studies. The aim of this study was to determine the efficacy and safety of rituximab for ANCA-associated vasculitis in a larger multicenter cohort. This permitted comparison of rituximab dosing regimens, the value of continuing immunosuppression, and investigation of ANCA and B cell levels as re-treatment biomarkers. METHODS Retrospective, standardized data collection from 65 sequential patients receiving rituximab for refractory ANCA-associated vasculitis at 4 centers in the UK was used. RESULTS All patients achieved B cell depletion. Complete remission occurred in 49 of the 65 patients (75%), partial remission in 15 (23%), and no response in 1 (2%). The prednisolone dosage was reduced from 12.5 mg/day (median) to 9.0 mg/day at 6 months (P = 0.0006). Immunosuppressive therapy was withdrawn in 37 of 60 patients (62%). Twenty-eight of 49 patients who achieved full remission (57%) experienced relapse (median 11.5 months). B cell return preceded relapse in 14 of 27 patients (52%). Although ANCA levels fell after rituximab therapy, relapse was not associated with ANCA positivity or a rise in ANCA levels. Neither the initial rituximab regimen (4 infusions of 375 mg/m(2) each given 1 week apart or 2 infusions of 1 gm each given 2 weeks apart) nor withdrawal of immunosuppressive therapy (37 of 60 patients [62%]) influenced the timing of relapse. Thirty-eight patients received >or=2 courses of rituximab, and complete remission was induced or maintained in 32 of them (84%). IgM levels fell, although IgG levels remained stable. Forty-six serious adverse events occurred, including 2 episodes of late-onset neutropenia, which were attributed to rituximab. CONCLUSION Rituximab was effective remission induction therapy for refractory ANCA-associated vasculitis in this study. There was no difference in efficacy between the 2 main treatment regimens. Continuing immunosuppression did not reduce relapses. Relapses occurred, but re-treatment was effective and safe. There was no clear influence of rituximab on the frequency of serious adverse events. ANCA and B cell levels lacked sufficient sensitivity to guide the timing of re-treatment.

A CD8+ T cell transcription signature predicts prognosis in autoimmune disease

Autoimmune diseases are common and debilitating, but their severe manifestations could be reduced if biomarkers were available to allow individual tailoring of potentially toxic immunosuppressive therapy. Gene expression–based biomarkers facilitating such tailoring of chemotherapy in cancer, but not autoimmunity, have been identified and translated into clinical practice. We show that transcriptional profiling of purified CD8+ T cells, which avoids the confounding influences of unseparated cells, identifies two distinct subject subgroups predicting long-term prognosis in two autoimmune diseases, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), a chronic, severe disease characterized by inflammation of medium-sized and small blood vessels, and systemic lupus erythematosus (SLE), characterized by autoantibodies, immune complex deposition and diverse clinical manifestations ranging from glomerulonephritis to neurological dysfunction. We show that the subset of genes defining the poor prognostic group is enriched for genes involved in the interleukin-7 receptor (IL-7R) pathway and T cell receptor (TCR) signaling and those expressed by memory T cells. Furthermore, the poor prognostic group is associated with an expanded CD8+ T cell memory population. These subgroups, which are also found in the normal population and can be identified by measuring expression of only three genes, raise the prospect of individualized therapy and suggest new potential therapeutic targets in autoimmunity.

Loss of Direct and Maintenance of Indirect Alloresponses in Renal Allograft Recipients: Implications for the Pathogenesis of Chronic Allograft Nephropathy

Chronic allograft nephropathy (CAN) is the principal cause of late renal allograft failure. This complex process is multifactorial in origin, and there is good evidence for immune-mediated effects. The immune contribution to this process is directed by CD4+ T cells, which can be activated by either direct or indirect pathways of allorecognition. For the first time, these pathways have been simultaneously compared in a cohort of 22 longstanding renal allograft recipients (13 with good function and nine with CAN). CD4+ T cells from all patients reveal donor-specific hyporesponsiveness by the direct pathway according to proliferation or the secretion of the cytokines IL-2, IL-5, and IFN-γ. Donor-specific cytotoxic T cell responses were also attenuated. In contrast, the frequencies of indirectly alloreactive cells were maintained, patients with CAN having significantly higher frequencies of CD4+ T cells indirectly activated by allogeneic peptides when compared with controls with good allograft function. An extensive search for alloantibodies has revealed significant titers in only a minority of patients, both with and without CAN. In summary, this study demonstrates widespread donor-specific hyporesponsiveness in directly activated CD4+ T cells derived from longstanding recipients of renal allografts, whether they have CAN or not. However, patients with CAN have significantly higher frequencies of CD4+ T cells activated by donor Ags in an indirect manner, a phenomenon resembling split tolerance. These findings provide an insight into the pathogenesis of CAN and also have implications for the development of a clinical tolerance assay.

Generating an iPSC Bank for HLA-Matched Tissue Transplantation Based on Known Donor and Recipient HLA Types

The likelihood for immunological rejection of Human Leukocyte Antigens (HLA)-mismatched induced pluripotent stem cells (iPSCs) limits their therapeutic potential. Here we show how a tissue bank from 150 selected homozygous HLA-typed volunteers could match 93% of the UK population with a minimal requirement for immunosuppression. Our model provides a practical approach for using existing HLA-typed samples to generate an iPSC stem cell bank that circumvents prospective typing of a large number of individuals.

Rituximab for remission maintenance in relapsing antineutrophil cytoplasmic antibody–associated vasculitis

OBJECTIVE Rituximab is effective induction therapy in refractory or relapsing antineutrophil cytoplasmic antibody-associated vasculitis (AAV). However, further relapse is common, and maintenance strategies are required. The aim of this study was to reduce relapse rates using a fixed-interval rituximab re-treatment protocol. METHODS Retrospective, standardized collection of data from sequential patients receiving rituximab for refractory or relapsing AAV at a single center was studied. Group A patients (n = 28) received rituximab induction therapy (4 infusions of 375 mg/m(2) or 2 infusions 1 gm) and further rituximab at the time of subsequent relapse. Group B patients (n = 45) received routine rituximab re-treatment for 2 years: 2 doses of 1 gm each for remission induction, then 1 gm every 6 months (total of 6 gm). Group C patients (n = 19) comprised patients in group A who subsequently relapsed and began routine re-treatment for 2 years. RESULTS Response (complete/partial remission) occurred in 26 of the 28 patients (93%) in group A, 43 of the 45 patients (96%) in group B, and 18 of the 19 patients (95%) in group C. At 2 years, relapses had occurred in 19 of 26 patients (73%) in group A, 5 of 43 (12%) in group B (P < 0.001), and 2 of 18 (11%) in group C (P < 0.001). At the last followup (median of 44 months), relapses had occurred in 85% of those in group A (22 of 26), 26% of those in group B (11 of 43; P < 0.001), and 56% of those in group C (10 of 18; P = 0.001). Glucocorticoid dosages were decreased and immunosuppression therapy was withdrawn in the majority of patients. Routine rituximab re-treatment was well tolerated, and no new safety issues were identified. CONCLUSION Two-year, fixed-interval rituximab re-treatment was associated with a reduction in relapse rates during the re-treatment period and a more prolonged period of remission during subsequent followup. In the absence of biomarkers that accurately predict relapse, routine rituximab re-treatment may be an effective strategy for remission maintenance in patients with refractory and relapsing AAV.

B-Cell–Depleting Induction Therapy and Acute Cellular Rejection

To the Editor: B-cell depletion is an effective treatment for a number of autoimmune diseases in which B cells were not previously considered to be important, such as multiple sclerosis.1 In renal ...

A Randomized Controlled Trial of Late Conversion from CNI‐Based to Sirolimus‐Based Immunosuppression Following Renal Transplantation

Maintenance immunosuppression with calcineurin inhibitors (CNIs) following renal transplantation is associated with nephrotoxicity and accelerated graft loss. We aimed to assess whether conversion to sirolimus‐based immunosuppression would affect the progression of renal impairment.

Microarray analysis of human leucocyte subsets: the advantages of positive selection and rapid purification.

BackgroundFor expression profiling to have a practical impact in the management of immune-related disease it is essential that it can be applied to peripheral blood cells. Early studies have used total peripheral blood mononuclear cells, and as a consequence the majority of the disease-related signatures identified have simply reflected differences in the relative abundance of individual cell types between patients and controls. To identify cell-specific changes in transcription it would be necessary to profile purified leucocyte subsets.ResultsWe have used sequential rounds of positive selection to isolate CD4 and CD8 T cells, CD19 B cells, CD14 monocytes and CD16 neutrophils for microarray analysis from a single blood sample. We compared gene expression in cells isolated in parallel using either positive or negative selection and demonstrate that there are no significant consistent changes due to positive selection, and that the far inferior results obtained by negative selection are largely due to reduced purity. Finally, we demonstrate that storing cells prior to separation leads to profound changes in expression, predominantly in cells of the myeloid lineage.ConclusionLeukocyte subsets should be prepared for microarray analysis by rapid positive selection.

B-cell depletion with rituximab for refractory head and neck Wegener's granulomatosis: a cohort study.

Objectives:  This study aimed to evaluate the response of refractory Wegener’s granulomatosis affecting the ear, nose and throat and granulomatous eye disease to B‐cell depletion with rituximab.