An Alternative Transcript of the PANE1 Gene Encodes a Minor Histocompatibility Antigen That Is Selectively Expressed in Resting CD19+ Cells and B-CLL.
Authors: Brickner Anthony G. , Mito Jeffrey K. , Feng Xin , Nishida Tetsuya , Fairfull Liane , Ferrell Robert E. , Foon Kenneth A. , Riddell Stanley R. , Warren Edus H. 
Published: April 2020

Abstract
We recently identified an HLA-A3-restricted minor histocompatibility antigen (mHAg) encoded by an alternative transcript (transcript k) of the PANE1 gene within an exon unique from all other PANE1 transcripts. Differential CTL recognition of mHAg+ and mHAg− cells is due to a single nucleotide polymorphism that replaces an arginine codon (CGA) with a translation termination codon (TGA). Cytotoxicity assays revealed robust recognition of HLA-A3+ EBV-LCL, minimal recognition of unfractionated peripheral blood mononuclear cells and PHA-stimulated T-cell blasts (PHA-T), and no recognition of dermal fibroblasts. To determine whether the restricted tissue distribution of the mHAg correlated with transcript k expression, we developed a real-time quantitative PCR assay to specifically detect transcript k. In normal tissues, expression was highest in spleen, low in all other tissues examined, and undetectable in dermal fibroblasts. Analysis of resting and activated peripheral blood cell fractions revealed very high expression levels in resting CD19+ cells, intermediate levels in EBV-LCL and resting CD4+ and CD8+ cells, and very low levels in activated CD19+ cells as well as resting and activated mononuclear cells, CD14+ cells, and activated CD4+ and CD8+ cells. EBV-LCL from mHAg− TGA homozygotes express significantly lower transcript k levels (p=0.043; Student’s t test) than mHAg+ EBV-LCL, suggesting the possibility of nonsense-mediated decay. The preferential expression of transcript k in normal resting CD19+ cells prompted us to investigate expression in malignant CD19+ cells. In a small sample of CD19+ acute lymphoblastic leukemia cells, we observed low-level expression comparable to that of activated normal CD19+ cells. In contrast, we observed high-level expression in CD19+ B-CLL cells comparable to or greater than that of normal resting CD19+ cells, and far greater than the levels in any other cell type tested. CTL cytotoxicity assays of a subset of HLA-A3+, mHAg+ B-CLLs revealed that they uniformly presented the mHAg encoded by PANE1 transcript k. Our finding that transcript k was selectively expressed in resting CD19+ cells and CD19+ B-CLL contradicted a previous report (Bierie et al., Gene Expression Patterns4:389, 2004) that the longest PANE1 transcript (transcript c) is preferentially expressed in activated lymphoid cells. We therefore evaluated transcript c expression in the same panel of samples, and confirmed that it was selectively expressed in activated CD19+ cells, with very low levels in resting CD19+ and primary B-CLL cells. Thus, PANE1 transcripts k and c display reciprocal expression in resting and activated CD19+ cells and primary CD19+ B-CLL cells. To determine if activation of CD19+ cells would increase expression of transcript c and decrease expression of transcript k, we evaluated the effect of CD40L stimulation in 6 primary B-CLL samples. In all samples tested, CD40L stimulation led to a profound decrease in transcript k expression, and a significant increase in transcript c expression. These studies suggest distinct roles for different PANE1 isoforms in resting versus activated CD19+ cells, and identify PANE1 as a potential therapeutic target in B-CLL.