Immunoglobulin Gene Repertoire in Ocular Adnexa Lymphomas (OAL): Hints on the Nature of the Antigenic Stimulation
Authors: Dagklis Antonis , Ponzoni Maurilio , Ferreri Andres JM , Cangi Maria Giulia , Pecciarini Lorenza , Dolcetti Riccardo , Stamatopoulos Kostas , Doglioni Claudio , Ghia Paolo 
Published: April 2020

Abstract
OALs arising in the ocular adnexae account for 15% of all extranodal lymphomas and are mostly represented by mucosa-associated lymphoid tissue (MALT)-type lymphomas (OAML). OAML have been described to be significantly associated, although with a variable geographic distribution, with Chlamydiophila psittaci (CP) infection, the etiologic agent of human psittacosis. Though the role played by Chlamydiae in OAML pathogenesis is also strongly supported by the observation of clinical remissions after bacterial eradication alone, the actual mechanisms have not been yet elucidated.
Previous analysis of the Immunoglobulin Heavy Chain Variable (IGHV) genes in OAML showed a restricted repertoire with mutated genes and ongoing somatic mutations, thereby alluding to an origin from an antigen–experienced B cell, which has undergone antigen-selection; however, data on the potential antigenic elements stimulating OAML B lymphocytes are not available.
In order to get hints on the potential ligands, we aimed at investigating the Immunoglobulin structure and, in particular, the molecular features of the antigen binding site (i.e. the HCDR3) of IG genes expressed by OAML. We analyzed the monoclonal IGHV-D-J rearrangements in a series of 30 OAML cases. IGHV3 subgroup genes predominated (24/30, 80%), followed by IGHV4 (4/30, 13.3%) and IGHV1 (2/30, 6,7%) subgroup genes, in keeping with the normal B cell IG gene repertoire. According to the 98% identity cut-off value, 23/30 sequences (76,7%) were defined as “mutated”, with a percentage of identity ranging between 89.3% and 97.7%, whereas the remainder (7/30 sequences; 23.3%) had “unmutated” IGHV genes. At individual gene level, IGHV3-23 and IGHV3-43 were the most frequently used genes (6/30 sequences, 20%, and 4/30 sequences, 13,3%, respectively); interestingly, the latter is rarely used in the normal repertoire. No significant correlations were found between IGHV gene usage and CP infection. HCDR3 length ranged from 9 to 30 amino acids (median 16) and 20/28 HCDR3 sequences showed an isoelectric point (pI) value &gt;6.0, (in 10/28 pI&gt;8.0), a feature shared with antibodies with anti–DNA reactivity. Shared epitope recognition was excluded by cluster analysis of HCDR3 sequences, also among OAML cases using the same IGHV genes. When we aligned HCDR3 sequences to a panel of 8214 unique IGHVD- J sequences from various types of normal, autoreactive or malignant B cell clones, we observed significant similarity between 3 OAML cases and 2 auto-reactive antibodies, present in rheumatoid arthritis and Sjoegren’s syndrome, respectively, and 1 antibody with anti-thyroid peroxidase (TPO) reactivity. The latter case showed a HCDR3 similarity also with the IG gene of a Chronic Lymphocytic Leukemia case, a disease characterized by frequent expression of auto-reactive Igs. Interestingly, the same search performed using anti-Chlamydia antibodies did not show any significant similarity.
In conclusion, these results suggest that OAML express a distinctive IG gene repertoire and may originate from B cells selected for their capacity to bind to auto-antigens in a similar way to what reported for gastric MALT lymphomas, where malignant B cells, instead of directly reacting against Helicobacter Pylori, are actually stimulated by tissue auto-antigens exposed during the inflammatory reaction, as confirmed by molecular analyses of antigen binding sites of the monoclonal IG. Our findings support the hypothesis that stimulation by auto-antigens, likely generated within an inflammatory background promoted by Chlamydial infection, may be the driving force also in OAML pathogenesis, rather than a direct action of the bacteria on B cells.