Project description:Primary lymphoma of the central nervous system (PCNSL) is a diffuse large B cell lymphoma confined to the CNS. In order to elucidate its peculiar organ tropism, we generated recombinant antibodies (recAb) identical with the BCR of a series of 23 PCNSL from immunocompetent patients. While none of the recAb showed self-reactivity upon testing with common autoantigens, they recognized 1547 proteins present on a large-scale protein microarray. Interestingly, proteins recognized by the recAb are physiologically expressed by CNS neurons (GRINL1A, centaurin-α, BAIAP2). Furthermore, 87% (20/23) of the recAb including all antibodies derived from IGHV4 34 using PCNSL recognized galectin-3, which was upregulated on microglia/macrophages, astrocytes, and cerebral endothelial cells upon CNS invasion by PCNSL. Thus, PCNSL Ig may recognize CNS proteins as self-antigens. Their interaction may contribute to BCR signaling with sustained NF-κB activation and, ultimately, may foster tumor cell proliferation and survival. These data may also explain, at least in part, the affinity of the tumor cells of PCNSL to the CNS.
Project description:Primary lymphoma of the central nervous system (PCNSL) is a diffuse large B cell lymphoma confined to the CNS. In order to elucidate its peculiar organ tropism, we generated recombinant antibodies (recAb) identical with the BCR of a series of 23 PCNSL from immunocompetent patients. While none of the recAb showed self-reactivity upon testing with common autoantigens, they recognized 1547 proteins present on a large-scale protein microarray. Interestingly, proteins recognized by the recAb are physiologically expressed by CNS neurons (GRINL1A, centaurin-α, BAIAP2). Furthermore, 87% (20/23) of the recAb including all antibodies derived from IGHV4 34 using PCNSL recognized galectin-3, which was upregulated on microglia/macrophages, astrocytes, and cerebral endothelial cells upon CNS invasion by PCNSL. Thus, PCNSL Ig may recognize CNS proteins as self-antigens. Their interaction may contribute to BCR signaling with sustained NF-κB activation and, ultimately, may foster tumor cell proliferation and survival. These data may also explain, at least in part, the affinity of the tumor cells of PCNSL to the CNS. Recombinant antibodies (recAb) identical with the BCR of a series of 23 PCNSL from immunocompetent patients.
Project description:Introduction: Pediatric systemic lupus erythematosus (pSLE) patients often initially present with more active and severe disease than adults, including a higher frequency of lupus nephritis. Specific autoantibodies, including anti-C1q, anti-DNA and anti-alpha-actinin, have been associated with kidney involvement in SLE, and DNA antibodies are capable of initiating early stage lupus nephritis in severe combined immunodeficiency (SCID) mice. Over 100 different autoantibodies have been described in SLE patients, highlighting the need for comprehensive autoantibody profiling. Knowledge of the antibodies associated with pSLE and proliferative nephritis will increase the understanding of SLE pathogenesis, and may aid in monitoring patients for renal flare. Methods: We used autoantigen microarrays composed of 140 recombinant or purified antigens to compare the serum autoantibody profiles of new-onset pSLE patients (n=45) to healthy controls (n=17). We also compared pSLE patients with biopsy-confirmed class III or IV proliferative nephritis (n=23) and without significant renal involvement (n=18). We performed ELISA with selected autoantigens to validate the microarray findings. We created a multiple logistic regression model, based on the ELISA and clinical information, to predict whether a patient had proliferative nephritis, and used a validation cohort (n=23) and longitudinal samples (88 patient visits) to test its accuracy. Results: Fifty autoantibodies were at significantly higher levels in the sera of pSLE patients compared to healthy controls, including anti-B-cell activating factor (BAFF). High levels of anti-BAFF were associated with active disease. Thirteen serum autoantibodies were present at significantly higher levels in pSLE patients with proliferative nephritis than those without, and we confirmed five autoantigens (dsDNA, C1q, collagens IV & X and aggrecan) by ELISA. Our model, based on ELISA measurements and clinical variables, correctly identified patients with proliferative nephritis with 91% accuracy. Conclusions: Autoantigen microarrays are an ideal platform for identifying autoantibodies associated with both pSLE and specific clinical manifestations of pSLE. Using multiple regression analysis to integrate autoantibody and clinical data permits accurate prediction of clinical manifestations with complex etiologies in pSLE.
Project description:Epitope mapping studies aim to identify the binding sites of antibody-antigen interactions to enhance the development of vaccines, diagnostics and immunotherapeutic compounds. However, mapping is a laborious process employing time- and resource-consuming M-bM-^@M-^Xwet benchM-bM-^@M-^Y techniques or epitope prediction software that are still in their infancy. For polymorphic antigens, another challenge is characterizing cross-reactivity between epitopes, teasing out distinctions between broadly cross-reactive responses, limited cross-reactions among variants and the truly type-specific responses. A refined understanding of cross-reactive antibody binding could guide the selection of the most informative subsets of variants for diagnostics and multivalent subunit vaccines. We explored the antibody binding reactivity of sera from human patients and Peromyscus leucopus rodents infected with Borrelia burgdorferi to the polymorphic outer surface protein C (OspC), an attractive candidate antigen for vaccine and improved diagnostics for Lyme disease. We constructed a protein microarray displaying 23 natural variants of OspC and quantified the degree of cross-reactive antibody binding between all pairs of variants, using Pearson correlation calculated on the reactivity values using three independent transforms of the raw data: (1) logarithmic, (2) rank, and (3) binary indicators. We observed that the global amino acid sequence identity between OspC pairs was a poor predictor of cross-reactive antibody binding. Then we asked if specific regions of the protein would better explain the observed cross-reactive binding and performed in silico screening of the linear sequence and 3-dimensional structure of OspC. This analysis pointed to the C-terminal helix of the structure as a major determinant of type-specific cross-reactive antibody binding. We developed bioinformatics methods to systematically analyze the relationship between local sequence/structure variation and cross-reactive antibody binding patterns among variants of a polymorphic antigen, and this method can be applied to other polymorphic antigens for which immune response data is available for multiple variants. Antibody profiling was performed on sera from Borrelia burgdorferi infected and non-infected humans and Peromyscus leucopus rodents against 23 variants of the surface protein OspC . For infected human serum samples, the OspC type of the infecting B. burgdorferi strain is unknown; for experimentally-infected P. leucopus serum samples, it is known. Of human serum samples, 55 were from infected individuals and 25 from naive controls. Of P. leucopus serum samples, 23 were from infected individuals and 7 were from naive controls.