Abstract
Alemtuzumab, a monoclonal antibody targeting the glycophosphatidylinositol (GPI) anchored CD52 protein, is used for in vivo and/or in vitro T cell depletion before allogeneic stem cell transplantation (alloSCT) to reduce the risk of graft rejection and graft versus host disease. Following profound lymphodepletion, we observed a rapid recovery of T cell numbers early after transplantation despite presence of lytic levels of residual alemtuzumab (Halkes et al. ASH 2011). In the majority of patients, a substantial portion of these T cells completely lack CD52 membrane expression, explaining why these cells escaped alemtuzumab induced cytotoxicity. The aim of the current study was to further characterize these cells and unravel the mechanism underlying the loss of CD52 membrane expression. To study the functionality of the CD52 negative T cells, tetramer staining, cytokine production analysis, and cytotoxicity assays were performed. These analyses showed that the CD52 negative T cells which were present early after alloSCT contain functional T cells specific against multiple viral targets and that their lytic capacity was comparable to that of CD52 positive counterparts, demonstrating that the function of these cells is not impaired. To investigate whether absence of CD52 expression was the result of loss of CD52 gene expression, we performed mRNA expression analysis on CD52 negative T cells purified from peripheral blood samples taken from alloSCT recipients at three months post transplantation. No loss of CD52 mRNA expression was observed. Since CD52 is tethered to the membrane via the GPI anchor, we analyzed whether loss of CD52 membrane expression resulted from loss of GPI anchor expression by flow cytometry using counterstaining with a fluorescently labeled GPI-specific aearolysin FLAER. This analysis on CD52 negative T cells from 3 alloSCT recipients revealed that loss of CD52 expression generally resulted from loss of GPI anchor expression. To study whether loss of GPI anchor expression was due to active genetic down regulation, GPI positive and GPI negative CD8+ T cell populations (n=2) were purified by fluorescent activated cell sorting followed by gene expression analysis of the 26 genes that comprise the GPI anchor biosynthesis pathway. No overall loss of expression was observed for any of the 26 genes. Since loss of GPI anchor expression in paroxysmal nocturnal hemoglobinuria and aplastic anemia has been described to be the result of mutations in PIGA, one of the 26 GPI anchor biosynthesis genes and unique for its location on the X chromosome, we performed mutation analysis on clonally isolated and expanded CD52/GPI negative (CD4+ n=53, CD8+ n=13) and CD52/GPI positive (CD4+ n=8, CD8+ n=7) populations from 3 alloSCT patients. mRNA was isolated from each clone and Sanger sequencing was performed covering the protein coding region twice, with opposing primers. Mutations were only scored when observed in both reads. Using this strategy we were able to detect mutations in 35/53 CD4+ and in 8/13 CD8+ CD52/GPI negative clones, which included point mutations (n=7), non-coding mutations (n=5), small deletions <18bp (n=13), small insertions <5bp (n=6), and exon skipping (n=12). None of the individual mutations was found more than twice within clones from the same recipient, demonstrating a highly polyclonal mutational landscape. Additionally, in CD52/GPI positive clones no mutations in the PIGA coding region where found. To investigate whether these mutations in PIGA were sufficient to induce loss of GPI anchor expression, we analyzed 35 CD52/GPI negative CD4+ T cell clones by retroviral transduction with constructs encoding wtPIGA or an empty vector. Restored GPI anchor expression and coinciding CD52 membrane expression was observed in all 35 clones upon transduction with PIGA, but not with empty vector.
We conclude that loss of CD52 membrane expression in T cells isolated early after alemtuzumab-based T cell depleted alloSCT is the result of various mutations in the PIGA gene and consequential loss of GPI anchor expression. We showed that these CD52 negative populations contain functional virus-specific T cells and may therefore be essential in immune protection early after transplantation.
Disclosures
Off Label Use: Alemtuzumab, conditioning of graft and/or recipient before allogeneic stem cell transplantation.
Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy