Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: Evaluating possible antigens in Lupus Nephritis

Background: Lupus glomerulonephritis (LN) represent one of the most severe and most frequent organ manifestations and one the major cause of morbidity and death among Systemic Lupus Erythematosus (SLE) patients. Kidney biopsy is fundamental to determine the grade of organ involvement, the prognosis, the disease activity and to adopt the correct therapeutic approach. Different studies affirmed that the presence of tubule-interstitial infiltrate (TII) represents an independent prognostic factors of renal outcome, irrespective of glomerular involvement. Lymphocytic aggregates resembling ectopic lymphoid structures (ELSs) were found in LN specimens with TII but their characterization is still unclear. Moreover, in a recent study, vimentin in the TII was found to act as an in situ auto-antigen. One of the emerging pathogenetic mechanism in LN is the extrusion of NETs from neutrophils in response to inflammatory stimuli. Objective: The principal aim of these project was to evaluate the inflammatory infiltrates organization in kidney biopsies obtained from LN patients and to investigate possible autoantigens for in situ immune response. Patients and methods: The research includes a retrospective and a prospective study. Paraffin embedded kidney specimens collected since 1999 from SLE patients who underwent a renal biopsy for diagnostic purpose were re-evaluated. Clinical, laboratory and histological data were collected in a standardized, computerized and electronically filled form, including demographics and past medical history. Disease activity was assessed by using SLEDAI-2K and remission in response to therapy was defined as the absence of renal impairment and as a score 0 of renal item of the SLEDAI . The cellular infiltrate and the degree of lymphoid organization were assessed by hematoxylin-eosin (HE) and by immunohistochemistry (IHC) with a staining of sequential sections for monoclonal antibodies to CD3, CD20, CD21. Staining for detections of LL-37, vimentin and citrulline was made. Serological levels of CXCL13 and anti-vimentin antibodies (AVAs) were evaluated in a subgroup of patients. Results: Fifty-three kidney samples from LN patients (F:M = 51:2, median age at biopsy-IQR years 37- 14; median disease duration at date of biopsy-IQR 8±15 years) were re-evaluated. TII were found in 33 kidney specimens and a well-defined infiltrate pattern resembling ELSs was found in 13 renal samples. In these samples, the presence of organized infiltrates was confirmed by IHC, with evidence of segregated T and B cells areas in most of them. In one sample, the CD21 staining was positive confirming the presence of ELSs. Eighteen paraffin embedded renal specimens, from LN patients were evaluated from 2017 until 2019 (F:M = 17:1, median age at biopsy-SD years 37-23; median disease duration at date of biopsy-IQR 3-4 years). A histo- morphologic grading score was performed based on the total count of TI lymphocytes and the presence of ELSs structures. Grade 1 aggregates were found in 12/18 of LN patients (66.7%), grade 2 structures in 10/18 patients (55.6%), and grade 3 foci (figure 4) were observed in 6/18 patients (33.4%). A correlation was found between grade 3 structures and the absence of renal remission with conventional immunosuppressive therapy at the last follow up (P=0.0026). A staining for LL37 was performed on the 18 kidney specimens. Samples showing a grade 3 foci were the specimens with significantly higher intensity of LL37 (P=0.013) and LL37 co- localization (P=0.006) compare to the other lymphocytic infiltrates. A staining for the detection of vimentin and citrulline was made . Any correlation was found among the intensity of vimentin and citrulline and the grade of lymphoid aggregates. Serological levels of CXCL13 and AVAs were investigated in a subgroup of 10 LN patients with TII from the prospective (6 patients) and retrospective (4 patients) cohorts. A statistically significant inverse correlation between AVAs serum levels and response to therapy was found (P=0.0048). Moreover, higher level of AVAs and CXCL13 were found in patients with grade 3 structures. Conclusions: The study demonstrated that tubule-interstitium (TI) involvement is associated with the presence of lymphocytic aggregation and poor renal outcome. It has been demonstrated for the first time that patients with grade 3 structures had a significant decreased response to immunosuppressant conventional therapy compared to those without ELSs. These results suggest a possible photogenetic and prognostic role of lymphocytic aggregates. To evaluate whether the presence of LL37, and thus NETosis, could represent a promoting factor of lymphocytes aggregation in LN with TII, a staining for detection of LL37 was made in the scored biopsies. LL37 was significantly colocalized with grade 3 foci and showed a more intense positivity in specimens with grade 3 structures compared to grade 1 and 2. Hence, LL37 have a possible role in inducing the formation of lymphocytic structures. In the small cohort of patients enrolled, high levels of AVAs were associated to a poor renal outcome. Moreover, patients showing a grade 3 foci presented a trend of higher serological levels of AVAs as well as CXCL13 lending to AVAs and CXCL13 a role as biomarkers of the presence of grade 3 structures and, thus, promoting their prognostic role. In addition, three out the 71 patients enrolled underwent to anti-CD20 treatment: two showed a grade 3 foci and one a grade 1. Notably, renal remission was achieved only in the two patients displaying a grade 3 structure. This preliminary premises might encourage the therapeutic effect of anti-CD20 monoclonal antibodies in patients with LN and TII, supporting the importance of evaluating TII.