Selective Loss of a Putative Precursor Population of B-Chronic Lymphocytic Leukemia Cells Following Immunization with hCD40L/IL-2 Expressing Autologous Tumor Cells
Authors: Foster Aaron , Okur Fatma , Dotti Gianpietro , Shafer Jessica , Lu An , Biagi Ettore , Yvon Eric , Bollard Catherine M , Savoldo Barbara , Andreeff Michael , Goodell Margaret A , Rooney Cliona M , Brenner Malcolm K 
Published: April 2020

Abstract
Side-population (SP) cells, defined on the basis of their ability to rapidly expel Hoechst dye, may be precursor cells for normal and malignant tissues and are more resistant to chemotherapy. We have now characterized a malignant SP subset in the peripheral blood of B-CLL patients and show how vaccination with CD40L/IL-2 expressing autologous tumor cells can induce a T cell immune response that reduces or eliminates this tumor subpopulation. Hoechst 33342 staining combined with CD5/CD19 antibody labeling of peripheral blood mononuclear cells showed a distinct CD5+CD19+ SP phenotype (median=0.22%, range; 0.2–2.17%) in 85% (18/21) of B-CLL patients (Stage I–III). We sorted SP and non-SP (NSP) cells and extracted RNA and performed a comprehensive gene expression analysis by microarray (n=9 donor SP and NSP pairs), finding 85 genes overexpressed (&gt;2-fold log ratio) in SP compared to NSP, and 228 genes higher in NSP versus SP. The analysis showed that compared to NSP cells from the same patient, the SP subset consistently and significantly overexpressed genes associated with cellular activation, proliferation and survival including eIF4A1 (p&lt;.05), Wnt10a (p&lt;.05), Bcl2L12 (p&lt;.05) and Rel A (p=.07), a difference confirmed by qPCR. Conversely, NSP significantly overexpressed GSK3β (p&lt;.05) compared to SP. CD40 mediated activation of B-CLL tumor cells further enhanced relative expression of these genes in the SP fraction, and induced overexpression of the proliferation-associated genes Ki-67 and PCNA in SP compared to NSP cells by 8-fold and 132-fold, respectively. We found SP cells were more resistant to exposure to chemotherapeutic agents including 50 mM fludarabine than NSP. Analysis of results in patients who had received an autologous B-CLL cell tumor vaccine expressing CD40L and IL-2, however, showed that B-CLL SP cells were vulnerable to a T cell mediated immune response. Hence, 3–8 subcutaneous injections CD40L/IL-2 B-CLL cells had no immediate effect on total B-CLL cell numbers in the peripheral blood, but Hoechst analysis showed a disappearance of SP cells in 7/8 immunized patients, associated with a rise in B-CLL-reactive T cells. CD8+ T cell clones isolated and expanded with CD40-activated autologous B-CLL tumor cells specifically responded to autologous SP cells, suggesting that SP depletion is mediated vaccine induced B-CLL-specific T cells. In 6/7 patients SP cells returned within a few weeks of completing the course of vaccination, coincident with a decline in T cell immunity. In one patient, however, immunity was longer lived and in this patient control of SP B-CLL cells persisted for more that 12 months. In this individual there was a delayed (by 6 months), but progressive disappearance of the bulk (non-SP) tumor cells. These results are consistent with the existence of an SP tumor precursor population of B-CLL, that can be targeted by immune T cells in vivo, and removal of which leads to later disappearance of their non-SP progeny.