B Cell Receptors Of Chlamydophila Psittaci negative MALT Lymphomas Of The Ocular Adnexa Recognize Common Self-Antigens

Ocular adnexal mucosa associated lymphoid tissue lymphomas (OAMALTLs) are the most common lymphomas in the ocular adnexa. The etiology and pathogenesis of OAMALTLs are still controversial. Association with Chlamydophila (C.) psittaci infection demonstrates marked geographic differences. We have previously reported biased IGHV (IGHV4 family and IGHV4-34 gene) and IGKV (IGKV3-20) usage in C. psittaci-negative OAMALTLs (PLoS One. 2011;6(12):e29114; Am J Hematol.2013; 88:379). However, the identity and role of potential antigens (Ags) in OAMALTL pathogenesis are unknown. Herein we evaluated the reactivity of OAMALTLs derived B-cell receptors (BCRs) for bacterial and human antigens.

IGHV and IGKV genes derived from 5 OAMALTLs (tumors 4726 and 4438 expressing IGHV4-34 with and without somatic mutations, respectively, both paired with IGKV3-20; tumor 4968 expressing IGHV3-30 and IGKV1-33; tumor 11274 expressing IGHV3-23 and IGKV3-20; and tumor 5334 expressing IGHV4-59 and IGKV3-1) were cloned into pAH6180 and pAN6714 plasmids, respectively. Irrespective of the original isotype, all recombinant antibodies (rAbs) representing tumor BCRs were expressed on a common IgG3 heavy chain backbone to facilitate the detection and comparability in reactivity assays and to ensure that differences in binding activity could be attributed to specific variable regions. rAbs were produced in HEK293T cells adapted to serum-free suspension cultures. rAbs did not react with bacterial proteins derived from lysates of Staphylococcus aureus, Salmonella enteridis and C. psittaci infected HeLa cells. The rAbs also did not react with I/i Ags. Rheumatoid factor (RF) enzyme-linked immunosorbent assay (ELISA) suggested that rAbs derived from tumors 4726, 4968, 5334, and 11274 exhibit rheumatoid factor activity. However, a competitive inhibition assay using increasing quantities of purified human IgG Fc fragment failed to block the reactivity, indicating non-specific RF reactivity. To test the rAbs for reactivity with self-antigens, the standard indirect immunofluorescence assay (IFA) using permeabilized human HEp-2 cells was performed. All tumor derived rAbs were reactive with HEp-2 cells and exhibited diverse staining patterns; predominantly cytoplasmic staining with rAbs 5334 and 4968 and combined nuclear and cytoplasmic staining with the others. We next examined tumor derived rAbs for polyreactivity based on the commonly used definition of reactivity with at least two of the following diverse self and non-self antigens: ssDNA, dsDNA, insulin, and LPS. Only rAb 4726 showed polyreactivity based on these standard criteria. To identify the self-antigens recognized by the other OAMALTLs derived rAbs, antibody specificity profiling was performed using ProtoArray Human Protein Microarrays v5.0 containing more than 9,000 proteins (Life Technologies), using 0.1 μg/mL and 1.0 μg/mL concentrations of each rAb. The data were processed using Invitrogen’s proprietary ProtoArray Prospector software. These analyses identified 17, 11, 9 and 25 candidate Ags for rAb4438, rAb4726, rAb5334, and rAb11274, respectively. Some of the candidate Ags were unique for specific rAbs (e.g. methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2 (MTHFD2) reactive with rAb4438) while others were recognized by more than one rAb (e.g. galectin-3/LGALS3 was reactive with rAb4438, rAb4726 and rAb11274). Follow up bidirectional co-immunoprecipitation with commercial and tumor derived rAbs confirmed recognition of these, as well as several additional protein microarray identified Ags. We specifically focused on galectin-3, which was recognized by 3 of the tumor BCRs. ELISAs performed with and without lactose confirmed rAbs reactivity with galectin-3. IFA using rAbs and a commercial galectin-3 Ab confirmed colocalization; siRNA-induced galectin-3 knockdown eliminated staining with both commercial and tumor derived rAbs. Immunohistochemistry of primary OAMALTLs showed that galectin-3 is expressed by tumor microenvironment cells but not by the tumor lymphocytes. Our findings demonstrate that the BCR of C. psittaci negative OAMALTLs exhibit oligo-self-reactivity and identified galectin-3 as a common antigen. The role of these antigens in pathogenesis of OAMALTLs is under investigation.