Generation Of Anti-Human Delta-42PD1 Monoclonal Antibodies And Preliminary Study On Immune Function Of Delta-42PD1

Programmed cell death 1 (PD1), an immunoreceptor belonging to the CD28/CTLA-4 family, negatively regulates T cell receptor and B cell receptor signaling upon interacting with either of two ligands, PD-L1 or PD-L2. The biological significance of PD1/PD-L axis pervades almost every aspect of immune responses including autoimmunity, tumor immunity, infectious immunity, transplantation immunity, allergy and immunological privilege. Four alternatively spliced PD1 isoforms with highly induced expression have been reported from other group with unknown biological function. Ascertainment of immunoregulatory function of PD1 isoforms is the key to fully understand immunoregulatory function of PD1 and helpful to evolve new thoughts for disease treatment and vaccine development.Our group have very recently identified a new isoform of human PD1, termed Δ42PD1. This isoform does not bind to PD-L1 or PD-L2, and is not recognized by PD1-specific monoclonal antibodies. Soluble forms of recombinant Δ42PD1 could induce release of proinflammation from PBMCs in vitro and enhance CD8+ T cell immunity in vivo, indicating a crucial functional immune regulatory molecular. Monoclonal antibody specific recognition of Δ42PD1 but not PD1 is the key to explore biological characteristics of the newly discovered PD1 isoform.The present study aimed to develop mouse anti-human A42PD1 monoclonal antibodies to facilitate functional exploration of the newly discovered human A42PD1. For antibody screening, human PD1 and Δ42PD1 stable-expression 293T cell lines (293T-PD1 and 293T-A42PD1) were established by stable transfection. Monoclonal populations were selected by limiting serial dilutionand the expression of gene of interested in these cell lines was confirmed by flow cytometry, RT-PCR and Western blot. For immunogen preparation,293F protein expression system was used to produce recombinant protein of sΔ42PD1Fc (fusion of human sΔ42PD1 and rabbit IgG1 Fc) and recombinant protein G was used for purification of recombinant protein. BALB/c mice were immunized through DNA prime protein boost immunization regimen, using human sΔ42PD1Fc recombinant plasmid DNA and protein. Serums of immunized mice were used to bind 293T-PD1 and 293T-Δ42PD1 cells and analyzed by flow cytometry to assess the immunogenic difference between human PD1 and Δ42PD1. And result showing the binding of serumfrom immunized mice to 293T-A42PD1 was significantly higher than to 293T-PD1, indicate astriking immunogenic difference between human PD1 and Δ42PD1. After the final immunization, mouse was sacrificed and then spleen cells were separated and fused with SP2/0 cells using conventional technology. Two clones of hybridoma secreting anti-human Δ42PD1 monoclonal antibody designated CH34 and CH101 were selected by indirect ELISA. CH34 and CH101 both specifically recognize human A42PD1 but not PD1 on cell surface by Flow cytometry, and engage both denatured PD1 and Δ42PD1 but did not cross react with mouse PD1 by Western blot. The binding epitope of CHI01 was defined using yeast surface display and intracellular staining of truncated Δ42PD1 protein and peptide ELISA. Heavy and light chain variable regions of CH34 and CH101 were cloned by RT-PCR and sequenced.By intracellular staining and flow cytometry, we identified that CD 14+ monocytes product proinflammatory cytokine response to recombinant sΔ42PD1Fc. Anti-Δ42PD1 monoclonal antibody CH34 and CH101 could not block proinflammatory cytokine production induced by recombinant sΔ42PD1Fc. Membrane-bound A42PD1 signaling research based on 293T-Δ42PD1 cell system revealed that Δ42PD1, like PD1, could inhibit AKT signal pathway in case of engagement with their ligands expressed on PBMCs. Binding of monoclonal antibody CH34 or CH101 with 293T-Δ42PD1 cells did not trigger Δ42PD1 signaling. And monoclonal antibody CH34 or CH101 could not block Δ42PD1 signaling triggered by engagement of Δ42PD1 with its unknown ligand on PBMCs. On the contrary, CH101 seems to augment Δ42PD1 signaling triggered by binding with its unknown ligand. Based on monoclonal antibody CH34 and CH101, we developed an indirect double-antibody sandwich enzyme-linked immunosorbent assay, which sensitivly and specifically recognize human sΔ42PD1 but not sPD1. Using this method, we found that compare to healthy human, plasma from HIV infected people contain signidicantly higher level of sΔ42PD1. This result indicated a potential rule of sΔ42PD1 in HIV infection.PD1 is inducedly expressed in activated T cells and B cells, and regulates adaptive immune response by inhibition of TCR and BCR signaling. Similarly, Δ42PD1 seemed constitutively expressed in myeloid-derived immunocytes, indicating a potential function of regulate innate immune response by inhibitory signaling. Meaningfully, generation of Δ42PD1 specific monoclonal antibody paved the way for further function exploration of the newly identified immune regulatory molecule.