Chronic lymphocytic leukemia cells diversify and differentiate in vivo via a nonclassical Th1-dependent, Bcl-6–deficient process
Piers E.M. Patten, Gerardo Ferrer, Shih-Shih Chen, Rita Simone, Sonia Marsilio, Xiao-Jie Yan, Zachary Gitto, Chaohui Yuan, Jonathan E. Kolitz, Jacqueline Barrientos, Steven L. Allen, Kanti R. Rai, Thomas MacCarthy, Charles C. Chu, Nicholas Chiorazzi
Piers E.M. Patten, Gerardo Ferrer, Shih-Shih Chen, Rita Simone, Sonia Marsilio, Xiao-Jie Yan, Zachary Gitto, Chaohui Yuan, Jonathan E. Kolitz, Jacqueline Barrientos, Steven L. Allen, Kanti R. Rai, Thomas MacCarthy, Charles C. Chu, Nicholas Chiorazzi
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Research Article Hematology Immunology

Chronic lymphocytic leukemia cells diversify and differentiate in vivo via a nonclassical Th1-dependent, Bcl-6–deficient process

Abstract

Xenografting primary tumor cells allows modeling of the heterogeneous natures of malignant diseases and the influences of the tissue microenvironment. Here, we demonstrate that xenografting primary chronic lymphocytic leukemia (CLL) B lymphocytes with activated autologous T cells into alymphoid mice results in considerable CLL B cell division and sizable T cell expansion. Nevertheless, most/all CD5+CD19+ cells are eventually lost, due in part to differentiation into antibody-secreting plasmablasts/plasma cells. CLL B cell differentiation is associated with isotype class switching and development of new IGHV-D-J mutations and occurs via an activation-induced deaminase-dependent pathway that upregulates IRF4 and Blimp-1 without appreciable levels of the expected Bcl-6. These processes were induced in IGHV-unmutated and IGHV-mutated clones by Th1-polarized T-bet+ T cells, not classical T follicular helper (Tfh) cells. Thus, the block in B cell maturation, defects in T cell action, and absence of antigen-receptor diversification, which are often cardinal characteristics of CLL, are not inherent but imposed by external signals and the microenvironment. Although these activities are not dominant features in human CLL, each occurs in tissue proliferation centers where the mechanisms responsible for clonal evolution operate. Thus, in this setting, CLL B cell diversification and differentiation develop by a nonclassical germinal center–like reaction that might reflect the cell of origin of this leukemia.

Authors

Piers E.M. Patten, Gerardo Ferrer, Shih-Shih Chen, Rita Simone, Sonia Marsilio, Xiao-Jie Yan, Zachary Gitto, Chaohui Yuan, Jonathan E. Kolitz, Jacqueline Barrientos, Steven L. Allen, Kanti R. Rai, Thomas MacCarthy, Charles C. Chu, Nicholas Chiorazzi

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Figure 4

Xenografted chronic lymphocytic leukemia (CLL) B cells demonstrate clonally related IgM and IgG IGHV-D-J mutations that exhibit hallmarks of AID action.

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Xenografted chronic lymphocytic leukemia (CLL) B cells demonstrate clona...
(A) AID hotspot and coldspot mutation frequencies were calculated from new IGHV-D-J mutations in 19 xenografted samples and log2 transformed for statistical analysis by Wilcoxon test (P = 0.0002, left). This indicated that AID hotspot mutation frequencies are significantly higher. The same was observed among IgM subclones (n = 13; P = 0.0007, center) and among Ig isotype-switched subclones (n = 6; P = 0.0355, right). (B) Representative phylogenetic relationships of new subclones are illustrated using a polar tree layout, with each branch tip representing a distinct subclone. The length that each branch extends from the circle is roughly proportional to the number of mutations (K80 phylogenetic distance, scale bar: 0.03). Subclones with single mutations are indicated by branch tips extending a short distance from the circle, while multiple-mutated subclones are represented by tips extending farther out. The branches connecting each tip illustrate the sequence-relatedness between subclones, with closely related sequences clustered together. Total number of subclones, defined by changes in DNA sequence from that of the initial clone and from those of subclones present in vivo at the time of sampling can be determined by counting the number of terminal branch tips at various lengths from the circle’s center. U-CLL515-1 IgM subclone IGHV-D-J sequences describe a tree with large multibranched relationships (386 subclones with 1–25 mutations). M-CLL1623-1 IgM subclone IGHV-D-J sequences define a tree with few branched relationships (91 subclones with 1–7 mutations).(C) Comparison of IgM and IgG phylogenetic relationships from same sample are shown as in B. IgM and IgG relationships are shown for U-CLL1122-1 (173 subclones with 1–26 mutations and 225 subclones with 1–28 mutations, respectively) and M-CLL1164-2 (135 subclones with 1–35 mutations and 144 subclones with 1–32 mutations, respectively) with shared sequences indicated as red branches. U-CLL, CLL clone with IGHV sequence differing ≤2% from most similar germline gene; M-CLL, CLL clone with IGHV sequence differing >2% from most similar germline gene.