OBJECTIVES:Granulomatosis with polyangiitis (GPA) is an autoimmune disease marked by relapses and treatment-related morbidity. CD4⁺ T cells play a central role in GPA pathogenesis, but their molecular profile remains incompletely defined. This study aimed to characterise CD4⁺ T cells in patients with proteinase 3-antineutrophil cytoplasmic antibody (ANCA)-positive GPA (PR3-GPA) using single-cell multiomics to identify molecular signatures associated with disease activity. METHODS:Peripheral blood mononuclear cells were obtained from patients with PR3-GPA during active disease, remission, and healthy controls. Single-cell RNA sequencing (scRNA-seq) and Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) were used to define transcriptional and protein expression profiles of CD4⁺ T cell subsets. Findings were validated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and flow cytometry-based assays, including phospho-flow cytometry. Serum cytokines were measured by a Luminex assay, and CD4⁺ T-cell responses to Pim kinase inhibition were assessed. Spatial transcriptomics data from kidney biopsies from patients with PR3-ANCA-positive-associated crescentic glomerulonephritis were analysed. RESULTS:scRNA-seq and CITE-seq revealed upregulated PIM1 expression in CD4⁺ T-cell subsets in active GPA compared with controls. RT-qPCR and flow cytometry-based analyses confirmed elevated expression of PIM1, SOCS3, and STAT3 in CD4⁺ T cells from patients with active disease. STAT3 phosphorylation and serum interleukin-6 (IL-6) were increased in active GPA. Pim kinase inhibition reduced CD4⁺ T-cell activation, proliferation, and cytokine production. Spatial transcriptomics showed upregulated STAT3 and PIM kinase expression in inflamed regions of PR3-ANCA-associated glomerulonephritis biopsies. CONCLUSIONS:These findings indicate that circulating CD4⁺ T cells in active PR3-GPA are characterised by activation of the STAT3-PIM1 axis, potentially driven by upstream IL-6 signalling. This supports further investigation of Pim kinase inhibition and Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway modulation as potential therapeutic strategies in PR3-GPA.

Pemphigus vulgaris (PV) is a severe autoimmune blistering disease characterized by pathogenic autoantibodies primarily targeting desmoglein-3. B-cell depletion by rituximab (anti-CD20) is an effective first-line treatment for PV; however, relapses are frequently observed. Evidence suggests that PV is associated with immune dysregulation beyond the B-cell compartment. So far, existing studies have largely focused on selected immune components or populations, or specific clinical states such as active disease, remission, or immediate post-rituximab, leaving it unclear how prior rituximab treatment shapes the overall circulating immune landscape during active PV, including relapse.In this study, we performed high-dimensional immune profiling to characterize circulating immune-cell composition and cytokine/chemokine signatures using a 40-colour spectral flow cytometry panel and a 46-plex Luminex assay, respectively. We compared active PV patients with healthy controls and performed a subgroup analysis of rituximab-naive versus rituximab-experienced relapsed PV patients.High-dimensional immune profiling showed that active PV is associated with a redistribution of circulating immune cells, with increased monocytes and decreased lymphocytes and plasmacytoid dendritic cells, together with reduced frequencies of double-negative (CD4−CD8−) T cells and T follicular helper cells compared with healthy controls. When comparing rituximab-naive with rituximab-experienced PV patients, in rituximab-experienced patients, the cellular composition changes were confined to the B-cell compartment, showing a shift toward a naive-dominant B-cell subset distribution. In parallel, PV was characterized by elevated serum concentrations of CD40L, CCL11/eotaxin, G-CSF, IL-1RA, IL-7, CCL20/MIP-3α, PD-L1/B7-H1/CD274, PDGF-AB/BB, and CCL5/RANTES, while no differences were identified between rituximab-naive patients and rituximab-experienced relapsed PV patients. Correlation analysis revealed that prednisone dosage may increase the frequency of mature B cells, and levels of CCL11/eotaxin, and IL-7, while decreasing the frequency of plasmacytoid dendritic cells and T follicular helper cells.Together, these findings reveal a systemic immune signature of active PV involving changes in circulating monocyte, T-cell, and soluble immune compartments, whereas prior rituximab exposure in relapsed patients is associated with sustained remodelling confined to B-cell subset composition, without long-term restructuring of non–B-cell immune lineages.

Background. B cells play a crucial role in kidney transplantation through antibody production and cytokine secretion. To better understand their impact on kidney transplantation, this retrospective study aimed to characterize circulating B-cell phenotypes and cytokine production in a cohort of kidney transplant patients to identify whether pretransplant donor-specific antibodies (DSAs) or biopsy-proven rejection is associated with different B-cell profiles. Methods. Pretransplant cryopreserved peripheral blood mononuclear cells were obtained from 96 kidney transplant recipients, of whom 42 had pretransplant DSAs. The cells underwent surface marker staining using a 33-color spectral flow cytometry panel for B-cell phenotyping. Simultaneously, cells were stimulated for interleukin-10, tumor necrosis factor-α, and interleukin-6 production, and analyzed with a 6-color panel. Results. Rejection was linked to decreased naive B cells and increased plasmablasts, CD27+ memory B cells, and memory B-cell subsets (all P < 0.04) compared with no rejection. Cytokine-producing B cells and immune regulatory molecule expression showed no significant differences. Multivariate analysis identified resting memory B cells (CD27+CD21+) and pretransplant DSAs as significantly associated with rejection (P = 0.01; odds ratio [OR], 1.07; P = 0.02; OR, 3.10, respectively). Cox regression analysis revealed resting memory B cells were associated with early antibody-mediated rejection (P = 0.04; OR, 1.05). Conclusions. B-cell subset distributions differed between patients with and without rejection. Resting memory B-cell frequency was associated with increased early antibody-mediated rejection risk, whereas cytokine production and immune checkpoint expression did not influence rejection. The results suggest that B-cell subset composition could aid in rejection risk assessment and serve as a potential pretransplant diagnostic parameter.

Granulomatosis with polyangiitis (GPA) is a B-cell-mediated, relapsing, autoimmune disease. There is a need for novel therapeutic approaches and relapse markers to achieve durable remission. B cells express immune regulatory molecules that modulate their activation and maintain tolerance. While recent studies show dysregulation of these molecules in other autoimmune diseases, data on their expression in GPA are limited. This study aimed to map the expression of surface immune regulatory molecules on circulating B-cell subsets in GPA and correlate their expression with clinical parameters. Immune regulatory molecule expression on circulating B-cell subsets was comprehensively examined in active GPA (n = 16), GPA in remission (n = 16), and healthy controls (n = 16) cross-sectionally using a 35-color B-cell-specific spectral flow cytometry panel. Our supervised and unsupervised in-depth analysis revealed differential expression of inhibitory and stimulatory immune molecules on distinct B-cell populations in GPA, with the most notable differences observed in active GPA. These differences include the upregulation of Fc gamma RIIB on nonmature B cells, downregulation of CD21 and upregulation of CD86 on antigen-experienced B cells, and elevated CD22 expression on various populations. Additionally, we found a strong association between Fc gamma RIIB, BTLA, and CD21 expression on specific B-cell populations and disease activity in GPA. Together, these findings provide novel insights into the immune regulatory molecule expression profile of B cells in GPA and could potentially form the foundation for new therapeutic approaches and disease monitoring markers.

Granulomatosis with polyangiitis (GPA) is an autoimmune disorder characterized by recurrent relapses that can cause severe tissue damage and life-threatening organ dysfunction. Multiple immune cells and cytokines/chemokines are involved in the different stages of the disease. Immune profiling of patients may be useful for tracking disease activity, however, reliable immune signatures for GPA activity are lacking. In this study, we examined circulating immune profiles in GPA patients during active and remission disease states to identify potential immune patterns associated with disease activity. The distribution and phenotypic characteristics of major circulating immune cells, and the profiles of circulating cytokines/chemokines, were studied on cryopreserved peripheral blood mononuclear cells from GPA patients (active, n = 20; remission, n = 20) and healthy controls (n = 20) leveraging a 40-color optimized multicolor immunofluorescence panel (OMIP-69) and in serum using a 46-plex Luminex multiplex assay, respectively. Deep phenotyping uncovered a distinct composition of major circulating immune cells in active GPA and GPA in remission, with the most significant findings emerging within the monocyte compartment. Our detailed analysis revealed circulating monocyte diversity beyond the conventional monocyte subsets. We identified eight classical monocyte populations, two intermediate monocyte populations, and one non-classical monocyte population. Notably, active GPA had a higher frequency of CD45RA + CCR5 + CCR6 - CCR7 +/low CD127 - HLA-DR + CD2 - classical monocytes and a lower frequency of CD45RA - CCR5 -/low CCR6 - CCR7 - CD127 - HLA-DR + CD2 +/- classical monocytes, which both strongly correlated with disease activity. Furthermore, serum levels of CXCL1, CXCL2, and CCL20, all linked to monocyte biology, were elevated in active GPA and correlated strongly with disease activity. These findings shed light on the circulating immune profile of GPA and may lead to immune signature profiles for assessing disease activity. Monocytes in particular may be studied further as potential markers for monitoring GPA.

B lineage cells are critically involved in ANCA-associated vasculitis (AAV), evidenced by alterations in circulating B cell subsets and beneficial clinical effects of rituximab (anti-CD20) therapy. This treatment renders a long-term, peripheral B cell depletion, but allows for the survival of long-lived plasma cells. Therefore, there is an unmet need for more reversible and full B lineage cell targeting approaches. To find potential novel therapeutic targets, RNA sequencing of CD27+ memory B cells of patients with active AAV was performed, revealing an upregulated NF-κB-associated gene signature. NF-κB signaling pathways act downstream of various B cell surface receptors, including the BCR, CD40, BAFFR and TLRs, and are essential for B cell responses. Here we demonstrate that novel pharmacological inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signaling) and inhibitor-of-κB-kinase-β (IKKβ, canonical NF-κB signaling) can effectively inhibit NF-κB signaling in B cells, whereas T cell responses were largely unaffected. Moreover, both inhibitors significantly reduced B cell proliferation, differentiation and production of antibodies, including proteinase-3 (PR3) autoantibodies, in B lineage cells of AAV patients. These findings indicate that targeting NF-κB, particularly NIK, may be an effective, novel B lineage cell targeted therapy for AAV and other autoimmune diseases with prominent B cell involvement.

Background B cells are crucially important in the pathogenesis of many autoimmune diseases, including ANCA-associated vasculitis (AAV), which is further demonstrated by the beneficial clinical effects of rituximab (anti-CD20) B cell targeted therapy. Despite its advantages, this treatment strategy results in long-term B cell depletion and fails to target long-lived plasma cells, rendering an unmet need for more reversible and combined B and plasma cell targeting approaches to achieve long-term disease remission. This aim may be reached by targeting signalling pathways essential for different B cell functions. The canonical and non-canonical NF-κB signalling pathways regulate fundamental B and/or plasma cell responses downstream of various B cell surface receptors, including the B cell receptor, CD40, and TLRs. Objectives To study potential alterations in NF-κB signalling in B lineage cells and to evaluate the effects of targeting NF-κB on B cell responses in general, and more specifically on proliferation, plasmablast differentiation and (auto)antibody production in B cells from AAV patients. Methods Memory B cells were obtained from AAV patients and healthy donors (HD), and gene expression profiles were generated by RNA-sequencing. Functional assays were performed by culturing PMBCs from AAV patients and HD with stimuli mimicking T cell-dependent (anti-CD40/IL-21) and T cell-independent (CpG/IL-2) conditions. Pharmacological inhibitors of Inhibitor-of-κB-kinase-β (IKKβ, canonical pathway) or NF-κB inducing kinase (NIK, non-canonical pathway) were added to the cultures. Downstream NF-κB signalling was determined by Western blot. After 6-day cultures, B cell proliferation and differentiation were determined by flow cytometry, and ELISA was performed for the detection of (auto)antibody production. Results Memory B cells from AAV patients with active disease exhibited an increased expression of NF-κB-associated genes compared to patients in remission and HD. Targeting IKKβ or NIK in AAV and HD B cells effectively blocked downstream canonical or non-canonical NF-κB signalling, respectively. IKKβ and NIK inhibition significantly reduced B cell proliferation in both stimulatory conditions of the functional assay. In addition, IKKβ and NIK inhibitors attenuated B cell differentiation into plasmablasts and antibody production, including anti-proteinase-3 (PR3) autoantibodies. Interestingly, the effects of NIK inhibition appeared to be B cell-specific, as T cell proliferation was largely unaffected. Conclusion These data reveal that NF-κB-associated genes are upregulated in memory B cells from AAV patients with active disease and that targeting of NF-κB signalling inhibits B cell responses that are important contributors to the autoimmune process, including autoantibody production. These findings suggest that targeting NF-κB, and particularly NIK, may be developed into a novel more reversible and B cell directed treatment modality for AAV. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests Ana Merino-Vico: None declared, Jan Piet Van Hamburg: None declared, Carlo Bonasia: None declared, Peter Heeringa: None declared, Paul Tuijnenburg: None declared, Boy Helder: None declared, Maaike Jansen: None declared, Aram Al-Soudi: None declared, Henric K. Olsson Employee of: Astrazeneca, Niek de Vries: None declared, Coen Stegeman: None declared, Jan-Stephan Sanders: None declared, Abraham Rutgers: None declared, Wayel Abdulahad: None declared, Paul Lyons: None declared, Theo Bijma: None declared, Aldo Jongejan: None declared, Taco W. Kuijpers: None declared, Sander Tas: None declared.

Objectives: Regulatory T cells (Tregs) are frequently functionally impaired in patients with granulomatosis with polyangiitis (GPA). However, the mechanism underlying their impaired function is unknown. Here, we hypothesized that Treg dysfunction in GPA is due to altered microRNA (miRNA) expression. Methods: RNA isolated from FACS-sorted memory (M) Tregs (CD4+CD45RO+CD25+CD127-) of 8 healthy controls (HCs) and 8 GPA patients without treatment was subjected to miRNA microarray analysis. Five differentially expressed miRNAs were validated in a larger cohort by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). An miRNA target gene database search revealed targets that were tested with RT-qPCR in MTregs from patients and HCs. cAMP levels were measured using flow cytometry. Results: Microarray analysis revealed 19 differentially expressed miRNAs, of which miR-142-3p was confirmed to be significantly upregulated in MTregs from GPA patients compared to those from HCs (1.9-fold, p = 0.03). In vitro overexpression of miR-142-3p lowered the suppressive capacity of MTregs (2.1-fold, p = 0.03), and miR-142-3p expression correlated negatively with the suppressive capacity (rho = -0.446, p = 0.04). Overexpression of miR-142-3p significantly decreased cAMP levels (p = 0.02) and tended to decrease the mRNA levels of a predicted target gene, adenylate cyclase 9 (ADCY9; p = 0.06). In comparison to those from HCs, MTregs from GPA patients had lower ADCY9 mRNA levels (2-fold, p = 0.008) and produced significantly less cAMP after stimulation. Importantly, induction of cAMP production in miR-142-3p overexpressed MTregs by forskolin restored their suppressive function in vitro. Conclusion: Overexpression of miR-142-3p in MTregs from GPA patients might cause functional impairment by targeting ADCY9, which leads to the suppression of cAMP production.

Background:Early detection of renal involvement in anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is of major clinical importance to allow prompt initiation of treatment and limit renal damage. Urinary soluble cluster of differentiation 163 (usCD163) has recently been identified as a potential biomarker for active renal vasculitis. However, a significant number of patients with active renal vasculitis test negative using usCD163. We therefore studied whether soluble CD25 (sCD25), a T cell activation marker, could improve the detection of renal flares in AAV.Methods:sCD25 and sCD163 levels in serum and urine were measured by enzyme-linked immunosorbent assay in 72 patients with active renal AAV, 20 with active extrarenal disease, 62 patients in remission and 18 healthy controls. Urinary and blood CD4+ T and CD4+ T effector memory (TEM) cell counts were measured in 22 patients with active renal vasculitis. Receiver operating characteristics (ROC) curves were generated and recursive partitioning was used to calculate whether usCD25 and serum soluble CD25 (ssCD25) add utility to usCD163.Results:usCD25, ssCD25 and usCD163 levels were significantly higher during active renal disease and significantly decreased after induction of remission. A combination of usCD25, usCD163 and ssCD25 outperformed all individual markers (sensitivity 84.7%, specificity 95.1%). Patients positive for sCD25 but negative for usCD163 (n = 10) had significantly higher C-reactive protein levels and significantly lower serum creatinine and proteinuria levels compared with the usCD163-positive patients. usCD25 correlated positively with urinary CD4+ T and CD4+ TEM cell numbers, whereas ssCD25 correlated negatively with circulating CD4+ T and CD4+ TEM cells.Conclusion:Measurement of usCD25 and ssCD25 complements usCD163 in the detection of active renal vasculitis.

Background. Early detection of renal involvement in antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is of major clinical importance to allow prompt initiation of treatment and limit renal damage. Urinary soluble cluster of differentiation 163 (usCD163) has recently been identified as a potential biomarker for active renal vasculitis. However, a significant number of patients with active renal vasculitis test negative using usCD163. We therefore studied whether soluble CD25 (sCD25), a T cell activation marker, could improve the detection of renal flares in AAV. Methods. sCD25 and sCD163 levels in serum and urine were measured by enzyme-linked immunosorbent assay in 72 patients with active renal AAV, 20 with active extrarenal disease, 62 patients in remission and 18 healthy controls. Urinary and blood CD4þ T and CD4þ T effector memory (TEM) cell counts were measured in 22 patients with active renal vasculitis. Receiver operating characteristics (ROC) curves were generated and recursive partitioning was used to calculate whether usCD25 and serum soluble CD25 (ssCD25) add utility to usCD163. Results. usCD25, ssCD25 and usCD163 levels were significantly higher during active renal disease and significantly decreased after induction of remission. A combination of usCD25, usCD163 and ssCD25 outperformed all individual markers (sensitivity 84.7%, specificity 95.1%). Patients positive for sCD25 but negative for usCD163 (n1⁄4 10) had significantly higher C-reactive protein levels and significantly lower serum creatinine and proteinuria levels compared with the usCD163positive patients. usCD25 correlated positively with urinary CD4þ T and CD4þ TEM cell numbers, whereas ssCD25 correlated negatively with circulating CD4þ T and CD4þ TEM cells. Conclusion. Measurement of usCD25 and ssCD25 complements usCD163 in the detection of active renal vasculitis.

Background Circulating regulatory T cells (Tregs) in anti-neutrophil cytoplasmic antibody associated vasculitis (AAV) are frequently functionally deficient. The mechanism behind their impaired function is however unknown. Small non-coding microRNA (miR) are post-transcriptional regulators of protein synthesis and previous studies have shown that differently expressed miRs in T cells are associated with auto immunity. Objectives To investigate whether the dysfunctionality of Tregs in AAV is due to altered microRNA (miR) expression. Methods Tregs (CD4+CD45RO+CD25+CD127-) of healthy controls (HC) and AAV patients in remission without treatment (AAV-REM) were FACS-sorted, and total RNA was isolated. Samples from 8 HCs and 8 AAV-REMs were subjected to miRNA microarray analysis. Based on relative expression and fold change, 5 differentially expressed miRs were validated in an independent cohort using qRT-PCR and a database and literature search was performed to identify potential targets. Results Nineteen miRs differentially expressed were detected by microarray analysis, of which Let-7g, miR-20a-5p, miR-26a-5p, miR-142–3 p, miR-146a-5p were validated in an independent cohort. Of these, miR-142–3 p was confirmed to be significantly upregulated (2-fold, p=0.03) in Tregs from AAV-REM patients compared to HC Tregs (n=23, n=22). To study the functional impact of miR-142–3 p overexpression, HC Tregs were transfected using either a mimic-miR-142–3 p or a scrambled (SCR)-control. After transfection, live Tregs were co-cultured with T effectors (CD4+CD25-) in a suppression assay to test their suppressive capacity. Transfection with mimic-miR-142–3 p significantly increased the miR-142–3 p levels (2.4 fold, p=0.03) and reduced the suppressive capacity compared to SCR-transduced Tregs (1.9 fold reduction, p=0.02). Moreover, miR-142–3 p levels tended to correlate to the suppressive function of Tregs (p=0.06, rho=−0.591). A database and literature search identified adenylyl cyclase 9 (AC9) as a promising target of miR-142–3 p. mRNA levels of AC9 tended to be lower in AAV-REM patients compared to HC (3.8 fold, p=0.07). In addition, cAMP levels, which are partly produced by AC9, were significantly lower in Tregs from AAV-REM patients after 48 hour of stimulation with anti-CD3 and anti-CD28 (1.7 fold, p=0.003). Conclusions Increased expression of miR-142–3 p in Tregs of AAV-REM patients is associated with their functional impairment, potentially by targeting the AC9/cAMP mediated suppression. Disclosure of Interest G. Dekkema: None declared, T. Bijma: None declared, W. Abdulahad Grant/research support from: European Union’s Horizon 2020 research and innovation program under grant No 6 68 036., P. Jellema: None declared, A. Van Den Berg: None declared, B. J. Kroesen: None declared, C. Stegeman: None declared, P. Heeringa Grant/research support from: European Union’s Horizon 2020 research and innovation program under grant No 6 68 036., J.-S. Sanders Grant/research support from: Nierfonds grant no. 13OKJ39

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The role for autoimmunity in the pathogenesis of COPD is controversial, and the identity of putative autoantigens is subject to debate. In order to examine the role for autoantibodies in COPD, we cloned the complete variable light and heavy chains from single-cell sorted IgG or IgA-positive activated memory B cells isolated from COPD and control lung tissue, and expressed these as complete human Ig monoclonal antibodies (HumAbs). These HumAbs were tested for autoreactivity by histology using lung tissue sections of COPD patients and healthy controls and lung cell lines. In addition, serum of COPD patients and healthy controls was used to determine binding of antibodies to epithelial, fibroblast, and endothelial cell lines. The HumAbs stained multiple cells and structures in lung tissue sections such as smooth muscles cells, epithelial cells, and the adventitia. The cell lines examined thus far, two epithelial cell lines, were also stained by the HumAbs. Serum IgA and IgG antibody titers against epithelial, fibroblast, and endothelial lung cell lines overlapped considerably between patients and controls. As a consequence, the differences between titers of patients and controls were marginally significant. Interestingly, however, some COPD patients had a high serum antibody titer against one cell line, but a low titer against another cell line. Our results illustrate the complexity of autoreactivity in COPD in that concomitant with the preferential binding of different types of lung cells by antibodies in serum of COPD patients, individual antibodies may also target multiple cells and structures in the lung.

Gemtuzumab ozogamicin (GO, Mylotarg) is a targeted therapeutic agent in which an anti-CD33 antibody is chemically coupled to a highly cytotoxic calicheamicin derivative through a hydrolysable linker. GO has improved the treatment outcome for a subgroup of acute myeloid leukemia (AML) patients, but its use is associated with severe myelosuppression and hepatotoxicity. Here, we report on a novel anti-leukemia agent, designated scFvCD33:sTRAIL, in which an anti-CD33 single chain fragment of variable regions (scFv) antibody fragment is genetically linked to soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). Normal CD33-positive monocytes were fully resistant to prolonged treatment with scFvCD33:sTRAIL, whereas treatment with GO resulted in substantial cytotoxicity. The activity of scFvCD33:sTRAIL towards AML cells was up to 30-fold higher than GO. The CD33-restricted anti-leukemia activity of scFvCD33:sTRAIL remained stable during prolonged storage at 37 °C, whereas GO showed a rapid increase in CD33-independent cytotoxicity. Moreover, scFvCD33:sTRAIL showed potent anti-leukemia activity towards CD33+ CML cells when treatment was combined with the Bcr-Abl tyrosine kinase inhibitor, Gleevec. Importantly, ex vivo treatment of patient-derived CD33+ AML tumor cells with scFvCD33:sTRAIL resulted in potent apoptosis induction that was enhanced by valproic acid, mitoxantrone and 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG). Taken together, scFvCD33:sTRAIL is superior to GO in terms of tumor selectivity, activity and stability, warranting its further development for the treatment of CD33-positive leukemias.