Highly Restricted Usage of Immunoglobulin Light Chain IGKV3-20 with Stereotyped Sequence in Primary Cold-Agglutinin Disease (CAD)
Notice bibliographique
Résumé
Abstract Primary cold agglutinin disease (CAD) is a type of hemolytic anemia mediated by anti-I autoantibodies. Patients suffer from anemia as well as circulatory problems. However, the severity of disease differs greatly between patients. We recently demonstrated that primary CAD is caused by an underlying low grade B cell lymphoproliferative disease of the bone marrow with a typical histology that is different from lymphoplasmacytic lymphoma and, accordingly, does not display the MYD88 L265P mutation (Randen et al., Haematologica, 2013). The majority of patients display circulating monoclonal antibodies encoded by the immunoglobulin heavy chain gene IGHV4-34. The disease severity does not correlate with antibody titers, but seems to be determined by the thermal amplitude, i.e., the highest temperature at which the cold agglutinin binds to the antigen. The framework region 1 of IGHV4-34 encodes for a sequence that binds to I antigen. However, this does not explain the molecular basis of disease heterogeneity. We studied 27 patients with well-characterized primary CAD and sequenced immunoglobulin heavy as well as immunoglobulin light chains to find additional consensus regions that may determine anti-I reactivity. Bone marrow aspirates, or frozen bone marrow trephine biopsies and blood from 27 patients with well-documented primary CAD were collected. Monoclonal B cells were isolated by flow sorting (FACS Aria Ilu High speed sorter, Becton Dickinson). Viable cells were detected using the forward scatter versus side scatter dot plot. Subsequently, CD45 bright events with low side scatter features representing lymphocytes, were selected. Then, CD5 positive and CD19 negative events, i.e. T cells, were gated out using a CD5 versus CD19 dot plot leaving only B cells. Finally, monoclonal B cells were selected using the immunoglobulin light chain gate, either k or l. Clonally rearranged IGH genes were detected using the Somatic Hypermutation Assay v2.0 (Invivoscribe) and were then sequenced. Immunoglobulin light chain genes (IGL) were amplified by an in-house diagnostic protocol based on Biomed-2 primers (van Dongen et al., Leukemia, 2003). All sequences were analyzed using the IMGT database (www.imgt.org). Productive IGHV4-34 gene rearrangements were identified in 22/27 patients. In 4 patients, no productive rearrangement was identified, while in one patient a productive IGHV3-23 was seen. No significant homology of complementarity determining region 3 (CDR3) regions was found between IGHV sequences. The N-glycosylation sequence within the CDR2 region, affecting antigen-binding, was mutated in 8 patients whereas no mutations were present in 7 patients and mutations in flanking residues were seen in 6 patients. The latter mutations may modulate glycosylation efficacy. Clonal rearrangement of the IGKV3-20 was detected in 16/27 patients, clonal IGKV3-15 gene rearrangements were identified in 4/27 patients whereas other IGL genes were rearranged in 4/27 patients. No clonal IGL gene rearrangement was found in 3/27 patients. Of interest, 7 of the patients with IGKV3-20 rearrangement displayed highly homologous CDR3 regions. The latter was highly associated with an un-mutated N-glycosylation sequence of the respective IGHV4-34 sequence. In conclusion, our data show that in addition to IGHV, also IGLV usage is highly restricted in CAD. Furthermore, stereotyped IGLV sequences are seen that are mutually exclusive with mutated N-glycosylation sequences in the IGHV CDR2 sequence. These data indicate that multiple regions within the immunoglobulin heavy chain as well as immunoglobulin light chain contribute to I-antigen binding. The data suggest that subtle differences in these multiple binding sequences may contribute to the differences in thermal amplitude of I antigen binding of the antibody. The highly restricted usage of IGKV3-20 provides a rationale for vaccination with IGKV3-20 proteins, known to be immunogenic and being considered for treatment in other lymphoproliferative diseases (Martorelli et al., Clin Cancer Res, 2012). Disclosures No relevant conflicts of interest to declare.
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,001 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».