the role of CD59 in mediating rosette formation in Hodgkin lymphoma through protein-protein interactions
Hodgkin lymphoma(HL) is a lymphatic cancer, the tumor cells in HL are Hodgkin-Reed-Sternberg(HRS) cells, which are significantly outnumbered by immune cells. A unique feature of HL is the formation of CD4+ T-cell rosettes around HRS cells, shielding them from immune response. It is specific to HL and present in most cases, but the mechanisms behind it remain unanswered. Therefore we developed a short-term in vitro co-culture model to study it. In a previous study, we found that CD2-CD58 interplay is important for rosette formation and T cell activation. In this study, we further investigated the role of CD59, an alternative CD2 ligand, in this process.
We used a short-term co-culture model to study rosette generation. HL cell lines (L428(CD58+/CD59+), L1236(CD58+/CD59-) and KMH2(CD58-/CD59+) ) and healthy donor peripheral blood mononuclear cells (PBMCs) were co-cultured. Cytospins were made for visualization and quantification. IL-2 levels in co-cultures were measured by ELISA to determine T cell activation. Proximity ligation assay(PLA) and Co-immunoprecipitation(Co-IP) were used to determine the protein-protein interactions. Immunofluorescence was used to visualize the molecular distribution. A CD59-knock out cell line was generated by Crispr-Cas9 and validated by flowcytometry.
After 30 minutes of co-culture, L428 showed the highest percentage of tumor cells with rosettes, followed by L1236 and KMH2. The same trend of IL-2 production was observed after 18 hours. CD59 knockout also reduced the rosette formation. These results indicate that CD59 expression is related to rosette generation. PLA showed stronger CD2-CD58 signals in L428 than L1236, and CD2-CD59 signals are positive in L428 while negative in KMH2. These demonstrated that CD59 may help to enhance the interaction between CD2-CD58. Co-IP suggested no direct CD58-CD59 interaction while PLA showed CD58-CD59 interaction only in rosetted tumor cells. These imply that CD58-CD59 interaction require the involvement of lymphocytes. During rosette formation, CD58 and CD59 relocalized to the membrane, CD59 followed CD58 to the same site, these supporting the conclusion before.
In short, we found that CD59 increases the rosette formation by enhancing the interaction between CD58 and CD2. The presence of CD58 is necessary for CD59 function.