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 7

Th1 cells are involved in CLL B cell division and maturation.

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Th1 cells are involved in CLL B cell division and maturation. (A) FC plo...
(A) FC plots of pretransfer in vitro–activated chronic lymphocytic leukemia (CLL) CD4+ cells (upper) compared with spleen-residing CD4+ cells (lower) stained for ICOS, CXCR5, CD57 and PD1. Red, isotype control; blue, CD5+CD4+ cells. Numbers represent percentage of cells expressing each surface protein over isotype control. (B) IH of human tonsil and CLL cells from NSG spleen stained for ICOS, CD57, and PD1. Scale bar: 250 μm. Representative IH and FC from 5 (PD1), 6 (CD57), and 5 (ICOS) independent experiments. (C) IFNγ is the most frequent and abundant T cell–derived cytokine, and its levels correlate with T cell numbers and degree of expansion in mice. Better correlation exists for IFNγ than for IL-5. Data from 7 independent experiments, n = 77 mice. IFNγ becomes detectable (>10 pg/ml) 2 weeks following cell transfer and increases thereafter. Data from 30 mice for U-CLL1122, with 5 mice euthanized at each time point. Representative of 2 independent experiments. Dotted line indicates plasma level of 10 pg/ml. (D) Spleen-residing CD4+ cells express IFNγ from 2 weeks after transfer. Data from 2 independent experiments with 2 CLL cases (M-CLL0827 and M-CLL1024), 5 mice euthanized per case at 1 and 2 weeks after transfer, unpaired t test result, mean and SEM shown. Red, isotype control; blue, CD5+CD4+IFNγ+ cells. (E) T-bet expression increases in CD4+ cells and B cells and is maintained in CD8+ cells following xenografting. Pretransfer data from 8 CLL cases subsequently xenografted in 8 independent experiments. Wilcoxon test result. At euthanasia, all cases showed evidence for plasma cell maturation. 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; FC, flow cytometry; IH, immunohistology; NSG, NOD/Shi-scid,γcnull.