Purpose: The use of proteasome inhibitors, such as bortezomib (BTZ), to target anti-HLA antibody (aHLA Ab)-producing plasma cells (PCs) is a promising emerging therapeutic to combat antibody-mediated rejection (AMR) and to reduce HLA sensitization via desensitization regimens. However, as with all anti-humoral therapies to date, BTZ-based desensitization is associated with post-treatment rebound in aHLA Ab levels, correlated with a lack of depletion of PCs within the bone-marrow (BM) niche, and our single-cell RNA-sequencing (scRNAseq) data show that the remaining BMPCs have an increased autophagy signature. Thus, we hypothesize that long-lived plasma cells (LLPCs) in the BM are uniquely capable of adapting their unfolded protein response (UPR) and autophagy induction to cope with their high-demand antibody synthesis amidst proteasome inhibition.
Methods: A retrospective analysis of aHLA Ab levels was performed on a patient who received BTZ for early aggressive AMR post solid organ transplant. Also, single-cell RNA-sequencing (scRNAseq) of BMPCs from three BTZ-treated patients as part of a desensitization trial was performed and BMPC subsets were defined by label transfer of existing public BMPC gene expression data. Finally, mice with an inducible, PC-specific expression of Cre-recombinase (JchainCre-ERT2) were used to define PC-specific roles of autophagy genes in controlling sensitivity to BTZ.
Results: In response to BTZ therapy for AMR, denovoaHLA Ab made by newly formed PCs (NFPCs) dramatically fell below the limit of detection. In contrast, pre-formed aHLA Ab made by LLPCs, while increased during AMR, returned to pre-transplant levels, suggesting selective survival of LLPCs post-BTZ. GSEA analysis of human LLPCs remaining after treatment with BTZ revealed an upregulation of a UPR gene signature that was not evident in NFPCs. Interestingly, a trio of genes related to the handling of unfolded proteins was significantly upregulated in LLPCs relative to NFPCs, including HSPA5 (BiP), VCP (p97) and SQSTM1 (p62). In mice, PC-specific loss of p62 combined with BTZ treatment or haploinsufficiency of BiP resulted in substantial PC depletion.
Conclusions: Our findings indicate that LLPCs are programmed to adapt to cellular stresses of high protein synthesis by upregulating autophagic UPR pathways to deal with misfolded proteins. Mouse studies suggest that a combinatorial treatment targeting multiple UPR pathways may lead to effective LLPC depletion. Further studies will define these pathways with the goal of designing more effective treatments to specifically target LLPCs for AMR treatment and desensitization.
Methods: A retrospective analysis of aHLA Ab levels was performed on a patient who received BTZ for early aggressive AMR post solid organ transplant. Also, single-cell RNA-sequencing (scRNAseq) of BMPCs from three BTZ-treated patients as part of a desensitization trial was performed and BMPC subsets were defined by label transfer of existing public BMPC gene expression data. Finally, mice with an inducible, PC-specific expression of Cre-recombinase (JchainCre-ERT2) were used to define PC-specific roles of autophagy genes in controlling sensitivity to BTZ.
Results: In response to BTZ therapy for AMR, denovoaHLA Ab made by newly formed PCs (NFPCs) dramatically fell below the limit of detection. In contrast, pre-formed aHLA Ab made by LLPCs, while increased during AMR, returned to pre-transplant levels, suggesting selective survival of LLPCs post-BTZ. GSEA analysis of human LLPCs remaining after treatment with BTZ revealed an upregulation of a UPR gene signature that was not evident in NFPCs. Interestingly, a trio of genes related to the handling of unfolded proteins was significantly upregulated in LLPCs relative to NFPCs, including HSPA5 (BiP), VCP (p97) and SQSTM1 (p62). In mice, PC-specific loss of p62 combined with BTZ treatment or haploinsufficiency of BiP resulted in substantial PC depletion.
Conclusions: Our findings indicate that LLPCs are programmed to adapt to cellular stresses of high protein synthesis by upregulating autophagic UPR pathways to deal with misfolded proteins. Mouse studies suggest that a combinatorial treatment targeting multiple UPR pathways may lead to effective LLPC depletion. Further studies will define these pathways with the goal of designing more effective treatments to specifically target LLPCs for AMR treatment and desensitization.