Analysis of CD45 Isoforms Displayed By Human Peripheral Blood Mononuclear Cells

Abstract

CD45 is the most prevalent glycoprotein on the surface of mature leukocytes and hematopoietic stem/progenitor cells (HSPCs). Accordingly, this glycoprotein has been intensively interrogated since its discovery, and antibody-drug-conjugates (ADCs) and radioimmunoconjugates targeting the extracellular domain of CD45 have been explored in pre-clinical and clinical trials as a potential replacement of, or supplement to, existing myeloablative regimens for hematopoietic stem cell transplant (HSCT) preparation. Importantly, human CD45 consists of five distinct isoforms, resulting from assortment of three alternatively spliced exons (known as "A", "B", and "C"). Though monoclonal antibodies (mAbs) directed to each of these exons, and to the CD45 isoform bearing no such exons (i.e., the "CD45RO" isoform) are available, our understanding of the display of these distinct exons is grossly incomplete. To date, the most well-characterized leukocyte subset differentially expressing CD45 isoforms is the human T cell. By contrast, the human myeloid compartment's expression of specific CD45 isoforms has been largely unstudied. Given this, we sought to comprehensively interrogate CD45 isoform expression on human peripheral blood mononuclear cells following defined in vitro manipulations of the cells. CD4+ and CD8+ human T cells were isolated from peripheral blood of healthy donors and activated with anti-CD3 and anti-CD28 antibodies for 5 days. Before and after activation, we performed flow cytometry combined with western blot analysis to assess shifts to CD45RA, CD45RB, CD45RC, and CD45RO expression. We isolated baseline human CD14+ monocytes and utilized the same techniques to ascribe CD45 isoforms to resting human monocytes. Upon activation of human T cells, our analyses recapitulated the expected transition of these cells from CD45RA+ to CD45RO+. However, strikingly, all native and in vitro-activated human T cells universally react with anti-CD45RB mAb as determined by both flow cytometry and western blotting. Similarly, 100% of human CD14+ monocytes display CD45RB and CD45RO, as determined by flow cytometry and western blotting. The universal reactivity of human T cells with anti-CD45RB illustrates that canonical CD45RA-/CD45RO+ memory and activated T cells co-express the CD45RB isoform. In a similar manner, baseline human CD14+ monocytes co-express CD45RO and CD45RB isoforms. The capability to unveil the ubiquity of CD45RB isoform expression was enabled by concurrent use of exon-specific mAbs. These data indicate a previously unappreciated complexity in human CD45 isoform display, draw caution on simply stratifying CD45 expression on the basis of CD45RA or CD45RO phenotypes, and highlight a broader significance of the CD45RB isoform among human T cells and monocytes, and within the greater human hematologic system.