Marco Lanzillotta

Immunology of IgG4-related disease.

Immunoglobulin G4‐related disease (IgG4‐RD) is a fibroinflammatory condition that derives its name from the characteristic finding of abundant IgG4+ plasma cells in affected tissues, as well as the presence of elevated serum IgG4 concentrations in many patients. In contrast to fibrotic disorders, such as systemic sclerosis or idiopathic pulmonary fibrosis in which the tissues fibrosis has remained largely intractable to treatment, many IgG4‐RD patients appear to have a condition in which the collagen deposition is reversible. The mechanisms underlying this peculiar feature remain unknown, but the remarkable efficacy of B cell depletion in these patients supports an important pathogenic role of B cell/T cell collaboration. In particular, aberrant T helper type 2 (Th2)/regulatory T cells sustained by putative autoreactive B cells have been proposed to drive collagen deposition through the production of profibrotic cytokines, but definitive demonstrations of this hypothesis are lacking. Indeed, a number of unsolved questions need to be addressed in order to fully understand the pathogenesis of IgG4‐RD. These include the identification of an antigenic trigger(s), the implications (if any) of IgG4 antibodies for pathophysiology and the precise immunological mechanisms leading to fibrosis. Recent investigations have also raised the possibility that innate immunity might precede adaptive immunity, thus further complicating the pathological scenario. Here, we aim to review the most recent insights on the immunology of IgG4‐RD, focusing on the relative contribution of innate and adaptive immune responses to the full pathological phenotype of this fibrotic condition. Clinical, histological and therapeutic features are also addressed.

IgG4-related disease in Italy: clinical features and outcomes of a large cohort of patients

Objectives: To describe the clinical features, treatment response, and follow-up of a large cohort of Italian patients with immunoglobulin (Ig)G4-related disease (IgG4-RD) referred to a single tertiary care centre. Method: Clinical, laboratory, histological, and imaging features were retrospectively reviewed. IgG4-RD was classified as ‘definite’ or ‘possible’ according to international consensus guidelines and comprehensive diagnostic criteria for IgG4-RD. Disease activity was assessed by means of the IgG4-RD Responder Index (IgG4-RD RI). Results: Forty-one patients (15 females, 26 males) were included in this study: 26 with ‘definite’ IgG4-RD and 15 with ‘possible’ IgG4-RD. The median age at diagnosis was 62 years. The median follow-up was 36 months (IQR 24–51). A history of atopy was present in 30% of patients. The pancreas, retroperitoneum, and major salivary glands were the most frequently involved organs. Serum IgG4 levels were elevated in 68% of cases. Thirty-six patients were initially treated with glucocorticoids (GCs) to induce remission. IgG4-RD RI decreased from a median of 7.8 at baseline to 2.9 after 1 month of therapy. Relapse occurred in 19/41 patients (46%) and required additional immunosuppressive drugs to maintain long-term remission. Multiple flares occurred in a minority of patients. A single case of orbital pseudotumour did not respond to medical therapy and underwent surgical debulking. Conclusions: IgG4-RD is an elusive inflammatory disease to be considered in the differential diagnosis of isolated or multiple tumefactive lesions. Long-term disease control can be achieved with corticosteroids and immunosuppressive drugs in the majority of cases.

Antineutrophil cytoplasmic antibody positivity in IgG4-related disease: A case report and review of the literature.

Background:IgG4-related disease (IgG4-RD) is a fibroinflammatory condition characterized by serum IgG4 elevation and tissue infiltration of IgG4-positive plasma cells. Substantial overlap between IgG4-RD and antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV) exists in terms of organ involvement and histopathological features. A positive ANCA assay is regarded as a highly specific finding in favor of an AAV, and generally influences away from a diagnosis of IgG4-RD. Recent reports, however, have raised the possibility that some patients with IgG4-RD are ANCA positive, thus suggesting reconsideration of the role of ANCA in the diagnostic workup. In the present work, we describe the first case of concomitant biopsy-proven IgG4-RD and granulomatosis with polyangiitis (GPA), demonstrating antiproteinase 3 (PR3) ANCA of the IgG4 subclass in the patients serum. We also review the literature in order to provide clinicians with tools for interpreting ANCA positivity in IgG4-RD patients. Case summary:A 51-year-old woman was referred for left exopthalmos due to lacrimal gland enlargement and increased serum IgG4 concentration. IgG4-RD was suspected and further imaging studies disclosed multiple pulmonary masses in the right lung. Histological analysis of the left lacrimal gland was diagnostic for IgG4-RD, but lung biopsy showed typical features of GPA. ANCA assay was positive for anti-PR3 antibodies. Further immunofluorescence studies demonstrated anti-PR3 antibodies of IgG1 and IgG4 subclass. Treatment with rituximab induced swift remission of both IgG4-RD and GPA manifestations. We identified 9 other reports of patients with IgG4-RD and positive ANCA in the English literature, 5 cases with biopsy-proven IgG4-RD and 4 cases in whom IgG4-RD was diagnosed presumptively. Four patients had also histological evidence of concomitant AAV. Conclusion:The present work demonstrates that ANCA positivity in patients with biopsy-proven IgG4-RD should prompt the exclusion of a concomitant vasculitic process; a positive ANCA does not exclude the diagnosis of IgG4-RD; confirmation through immunoenzymatic assays of the ANCA specificity, clinical-pathological correlation, and histopathological evaluation remain crucial steps for the differential diagnosis between AAV and IgG4-RD.

Quantitative measurement of 18F-FDG PET/CT uptake reflects the expansion of circulating plasmablasts in IgG4-related disease

Objective [18F]Fluorodeoxyglucose (18F-FDG) PET/CT is increasingly used to assess organ involvement and response to treatment in IgG4-related disease (IgG4-RD), but clear correlations between 18F-FDG uptake and disease activity have not been established yet. We aimed to correlate the intensity and distribution of 18F-FDG uptake with validated clinical, serological and immunological parameters of IgG4-RD activity. Methods Twenty patients with active IgG4-RD underwent a baseline 18F-FDG PET/CT. Ten patients repeated 18F-FDG PET/CT after immunosuppressive treatments. 18F-FDG tissue uptake was measured using the standardized uptake value corrected for the partial volume effect (PVC-SUV) and the total lesion glycolysis (TLG) with (TLGtot+ln) and without (TLGtot-ln) lymph nodes. Disease activity was assessed by means of clinical parameters [IgG4-RD Responder Index (RI)], serological (ESR and CRP) and immunological (serum IgG4 and circulating plasmablasts) biomarkers. The enhanced liver fibrosis score was exploited as a biomarker for fibroblast activation. Results Thirteen (65%) patients had two or more organs affected by IgG4-RD. All patients had active IgG4-RD as defined by a median IgG4-RD RI value of 9 (range 6-15; normal < 3). Serum IgG4 and plasmablasts were elevated in 85% of patients. Circulating plasmablasts positively correlated with PVC-SUV (P = 0.027), inversely correlated with TLGtot-ln (P = 0.023) and did not correlate with TLGtot+ln (P > 0.05). No statistically significant correlation was found between PVC-SUV or TLG and IgG4-RD RI, ESR, CRP, serum IgG4 or enhanced liver fibrosis score (P > 0.05). Clinical response to immunosuppressive therapies was associated with a consensual reduction of circulating plasmablasts, PVC-SUV, TLGtot+ln and TLGtot-ln values (P < 0.05 for all comparisons). Conclusions 18F-FDG uptake of IgG4-RD lesions reflects immunological perturbations of the B cell compartment rather than fibroblast activation and extracellular matrix deposition. Conventional biomarkers of disease activity, namely IgG4-RD RI, ESR, CRP and serum IgG4 levels, do not appear to correlate with the radiometabolic activity of IgG4-RD lesions. In light of our results PET/CT represents a reliable instrument for assessing IgG4-RD activity, although lymph-node uptake deserves careful interpretation.

Roles of Plasmablasts and B Cells in IgG4-Related Disease: Implications for Therapy and Early Treatment Outcomes.

High serum IgG4 concentrations are a striking feature of many patients with IgG4-related disease (IgG4-RD). Blood levels of IgG4 often reach ten, twenty, and even thirty or more times higher than the upper limit of normal. Under the proper clinical circumstances, the finding of an elevated serum IgG4 concentration serves as a useful biomarker for the diagnosis of this condition. This serum IgG4 elevation quickly called attention to the possibility of therapies targeting cells of the B lymphocyte lineage. In addition, a greater understanding of the cellular mechanisms that underpin IgG4-RD has identified peripheral blood plasmablasts as a promising biomarker for this disease. The roles of plasmablasts and B cells in IgG4-RD are discussed in this chapter.

A CD8α− Subset of CD4+SLAMF7+ Cytotoxic T Cells Is Expanded in Patients With IgG4-Related Disease and Decreases Following Glucocorticoid Treatment

An unconventional population of CD4+ signaling lymphocytic activation molecule family member 7–positive (SLAMF7+) cytotoxic effector memory T (TEM) cells (CD4+ cytotoxic T lymphocytes [CTLs]) has been linked causally to IgG4‐related disease (IgG4‐RD). Glucocorticoids represent the first‐line therapeutic approach in patients with IgG4‐RD, but their mechanism of action in this specific condition remains unknown. We undertook this study to determine the impact of glucocorticoids on CD4+ CTLs in IgG4‐RD.

Deconstructing IgG4-related disease involvement of midline structures: Comparison to common mimickers

Abstract Objective: A series of destructive and tumefactive lesions of the midline structures have been recently added to the spectrum of IgG4-related disease (IgG4-RD). We examined the clinical, serological, endoscopic, radiological, and histological features that might be of utility in distinguishing IgG4-RD from other forms of inflammatory conditions with the potential to involve the sinonasal area and the oral cavity. Methods: We studied 11 consecutive patients with erosive and/or tumefactive lesions of the midline structures referred to our tertiary care center. All patients underwent serum IgG4 measurement, flow cytometry for circulating plasmablast counts, nasal endoscopy, radiological studies, and histological evaluation of tissue specimens. The histological studies included immunostaining studies to assess the number of IgG4 + plasma cells/HPF for calculation of the IgG4+/IgG + plasma cell ratio. Results: Five patients with granulomatosis with polyangiitis (GPA), three with cocaine-induced midline destructive lesions (CIMDL), and three with IgG4-RD were studied. We found no clinical, endoscopic, or radiological findings specific for IgG4-RD. Increased serum IgG4 and plasmablasts levels were not specific for IgG4-RD. Rather, all 11 patients had elevated blood plasmablast concentrations, and several patients with GPA and CIMDL had elevated serum IgG4 levels. Storiform fibrosis and an IgG4+/IgG + plasma cell ratio >20% on histological examination, however, were observed only in patients with IgG4-RD. Conclusions: Histological examination of bioptic samples from the sinonasal area and oral cavity represents the mainstay for the diagnosis of IgG4-RD involvement of the midline structures.

SAT0011 Mertk+ monocytes are expanded in the peripheral blood of patients with active igg4-related disease and infiltrate affected organs

Background IgG4-Related Disease (IgG4-RD) is a multi-organ fibro-inflammatory disorder characterised by tumefactive lesions with well-defined histological features including lymphoplasmacitic infiltrate rich in IgG4+ plasma cells, storiform fibrosis and obliterative phlebitis1. Alternatively-activated macrophages (M2) have also been reported to abundantly infiltrate IgG4-RD lesions, but their role in IgG4-RD pathogenesis remains elusive. M2 macrophages have been recently shown to modulate innate and adaptive immune responses as well as tissue fibrosis by direct interaction with stromal cells2–4. Both these roles can be mediated by Mer tyrosine kinase (MerTK), a member of the TAM – Tyro3, Axl and MerTK- receptor family, which is highly expressed on M2 macrophages5. The relevance of MerTK and of its ligands Protein S (ProS) and Growth arrest-specific protein 6 (Gas6) in IgG4-RD has never been assessed before. Objectives To assess a pathogenetic relevance of the MerTK-ProS/Gas6 axis in IgG4-RD. Methods Immunohistochemical studies for CD68, CD163 and MerTK were performed on 8 cases of IgG4-RD involving different organs. MerTK expression within the different circulating monocyte subsets was quantified by flow cytometry both in 11 active untreated IgG4-RD patients and in 10 healthy controls (HC). Plasma levels of ProS, Gas6 and of their decoy receptor – soluble Mer (sMer) – were measured by ELISA in 34 IgG4-RD patients and 20 HC. Results MerTK was abundantly expressed in IgG4-RD lesions both within the inflammatory infiltrate and in the newly formed fibrous tissue. The pattern of MerTK expression was similar to that of the M2 macrophage marker CD163 (figure 1). Total circulating monocytes and their subsets were not expanded in active untreated IgG4-RD compared to HC. However, MerTK+ monocytes were significantly increased in the peripheral blood of patients with IgG4-RD compared to HC (p<0.001). Plasma levels of Gas6 and ProS were significantly higher in IgG4-RD patients compared to HC (p=0.003 and p=0.01, respectively), while the plasma level of sMer was comparable to that of HC. The number of MerTK+ monocytes correlated positively with the number of circulating plasmablasts (r=0.6; p<0.05) and negatively with Gas6 plasmatic concentration (r=−0.7; p<0.05).Abstract SAT0011 – Figure 1 Distribution of macrophages and MerTK-expressing cells in the lung (A-B), pancreas (C-D) and biliary tree (E-F) of IgG4-RD patients. Each set of sections was stained with MerTK, CD68 as a marker of both M1 and M2 macrophages and CD163 as a marker of M2 macrophages. Conclusions A subset of MerTK+ M2 macrophages abundantly infiltrates IgG4-RD fibrotic lesions and their MerTK+ monocyte precursors are expanded in the peripheral blood of patients with active IgG4-RD. MerTK ligands are also increased in IgG4-RD, suggesting an augmented activation of MerTK signalling pathways. Further studies are needed to better characterise this monocyte/macrophage subpopulation, to understand its role in IgG4-RD and to identify possible biomarkers and therapeutic targets. References [1] Deshpande V, et al. Mod. Pathol2012. [2] Furukawa S, et al. Clin. Immunol2015. [3] Furukawa S, et al. Sci. Rep2017. [4] Ohta M. et al. Med2016. [5] Rothlin CV, et al. Annu. Rev. Immunol2015. Disclosure of Interest None declared

OP0035 Circulating Plasmablasts Levels Reflect Inflammatory Activity in IGG4-Related Disease Lesions as Assessed by Quantitative Positron Emission Tomography

Background IgG4-Related Disease (IgG4-RD) is a systemic inflammatory condition characterized by fibrous swelling of affected organs, serum IgG4 elevation, and IgG4+ plasmacells tissue infiltration. 18-Fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) scan is emerging as a promising imaging technique to detect organs involved by IgG4-RD and to assess disease response to treatment. The relationship between FGD-PET findings and immunological perturbations occurring in IgG4-RD has never been evaluated. Objectives To correlate the intensity and distribution of FDG-PET uptake with clinical and immunological parameters in patients with active untreated IgG4-RD. Methods Patients with active, untreated, biopsy proven IgG4-RD were included in the study. Disease activity was assessed through clinical (IgG4-RD Responder Index (RI)) and immunological (erythrocyte sedimentation rate (ESR), C reactive protein (CRP), serum IgG4, and circulating plasmablasts) parameters. Plasmablasts, a recently characterized disease biomarker, were identified as CD19+CD20-CD27+CD38bright cells on flow cytometry. FDG-PET/CT was performed in all patients at diagnosis. Quantitative assessment of FDG uptake was measured using the mean Standardized Uptake Value corrected for the Partial Volume Effect (PVC-SUV). Lymph nodes <1 cm of diameter were excluded from the analysis because of the risk of PVC-SUV over/under-estimation. In patients with multiorgan involvement, the IgG4-RD lesion with the highest PVC-SUV was selected to correlate FGD uptake with clinical and serological parameters. Results We studied 15 patients (7 males, 8 females) with a mean age of 63 years (range, 30–77 years). Twelve (80%) patients had multiorgan IgG4-RD involving lymphnodes (7 patients); aorta (5 patients); parotids glands and pancreas (3 cases each); bones, skin, thyroid, lung, submandibular and lachrymal glands (2 cases each); meninges, nasal cavity, oropharynx, palate, liver, CNS, and retrorbital space (one case each). The median IgG4-RD RI was 9 (range 6–16; normal =0). The median levels of ESR, CRP, serum IgG4 and plasmablasts at baseline were 30 mm/h (range 6–121 mm/h, normal <20 mm/h), 11.0 mg/L (range 0.0–48.0 mg/L; normal <6mg/L), 284.0 mg/dL (range 45–2100 mg/dL, normal <121 mg/dL), and 3870 cells/mL (range 1000–10000 cells/mL, normal <690 cells/mL), respectively. The median PCV-SUV was 6.24 (range 2.48–16.39). Significant positive correlation was found between PVC-SUV and serum plasmablasts levels (r=.84, p=.004). No correlation was found between PVC-SUV and either CRP, ESR, serum IgG4 levels, number of organs involved, and IgG4-RD RI at baseline (p>.05). Conclusions Our study demonstrates for the first time a positive correlation between circulating plasmablasts and inflammatory activity in IgG4-RD lesions as assessed by PVC-SUV on FDG-PET. Our results further strengthen the utility of circulating plasmablasts as a biomarker of disease activity. Conventional inflammatory markers, serum IgG4 levels, and IgG4-RD RI do not appear to correlate with metabolic activity in IgG4-RD lesions. References Ebbo M et al. Arthritis Care Res. 2014 Jan;66(1):86–96. Campochiaro C et al. Scand J Rheumatol. 2015 Sep 23:1–11. Disclosure of Interest None declared

AB0909 Effects of Combined Therapy with Prednisone and Methotrexate on B Cell Subpopulations in Patients with IGG4-Related Disease

Background IgG4-Related Disease (IgG4-RD) is a systemic fibro-inflammatory condition characterized by oligoclonal expansion of IgG4+ class-switched plasmablasts (PBs) (1). PBs swiftly decline together with clinical improvement after rituximab, suggesting a role for B cells in the pathogenesis of IgG4-RD (2). Studies with alternative effective non B-cell depleting therapies (3) are needed in order to confirm the pathogenic importance of PBs and their utility as disease biomarkers. Objectives To assess the impact of combined therapy with prednisone (PDN) and methotrexate (MTX) on B cell subpopulations in patients with IgG4-RD. Methods Five patients with active biopsy proven IgG4-RD were treated with PDN (0.6–1 mg/kg) and MTX (10–20 mg per week). PDN was gradually tapered and withdrawn in 6 months. Disease activity, partial (PR) and complete response (CR) were assessed through the IgG4-RD Responder Index (IgG4-RD RI) (4). Serological, immunological and radiological studies were performed according to the clinical presentation. Flow cytometry was used to measure PBs (CD19+CD20-CD27+CD38+bright cells), naïve (CD19+CD20+CD27-CD38+ cells), and memory (CD19+CD20+CD27+CD38- cells) B cells at baseline, after 3 and 6 months of therapy. Nine age and sex matched subjects were used as healthy controls (HC). Results At disease onset, the median IgG4-RD RI was 9 (normal <3). The median serum IgG4 level was 534 mg/dl (normal <135mg/dL). Circulating PBs were increased (median 7000 cells/mL) compared to HC (median 605 cells/mL; p<0.004). PBs showed a positive correlation with the number of organs involved (r=0.77), with serum IgG4 level (r=0.87), and with disease activity (r=0.73). The number of circulating memory B cells was comparable between IgG4-RD patients and HC (p=0.57). Naïve B cells were significantly lower in IgG4-RD patients compared to HC (p<0.05). After 6 months of therapy 4 patients achieved CR (IgG4-RD RI <3) and 1 PR. Serum IgG4 level decreased in all patients but normalized only in one patient at 6 months. Circulating PBs declined to levels comparable to HC (median 230 cells/mL, p=0.3). Memory B cells were unaffected by the therapy, while naïve B cells showed a declining trend, yet not statistically significant compared to baseline levels (p>0.05) Conclusions PBs and naïve B cells are expanded and reduced, respectively, in patients with active IgG4-RD compared to HC. Combined therapy with PDN and MTX leads to clinical improvement and to normalization of circulating PBs. Naïve B cells slightly decrease, while memory B cells are not affected by immunosuppressive treatment References Carruthers MN, et al. Rituximab for IgG4-related disease: a prospective, open-label trial. Ann Rheum Dis. 2015 Jun;74(6):1171–7. Wallace ZS, et al. Plasmablasts as a biomarker for IgG4-related disease, independent of serum IgG4 concentrations. Ann Rheum Dis. 2015 Jan;74(1):190–5. Della-Torre E, et al. Methotrexate for maintenance of remission in IgG4-related disease. Rheumatology (Oxford). 2015 Oct;54(10):1934–6. Carruthers MN, et al. Development of an IgG4-RD Responder Index. Int J Rheumatol. 2012;2012:259408. Disclosure of Interest None declared