The immune microenvironment shapes transcriptional and genetic heterogeneity in chronic lymphocytic leukemia

Abstract

In chronic lymphocytic leukemia (CLL), B-cell receptor signaling, tumor-microenvironment interactions and somatic mutations drive disease progression. To better understand the intersection between the microenvironment and molecular events in CLL pathogenesis, we integrated bulk transcriptome profiling of paired peripheral blood (PB) and lymph node (LN) samples from 34 patients. Oncogenic processes were upregulated in LN compared to PB, and in IGHV unmutated compared to mutated cases. Single-cell RNA sequencing distinguished 3 major cell states: quiescent, activated, and proliferating. The activated subpopulation comprised only 2.2% to 4.3% of the total tumor bulk in LN samples. RNA velocity analysis found that CLL cell fate in LN is unidirectional, starts in the proliferating state, transitions to the activated state, and ends in the quiescent state. A 10-gene signature derived from activated tumor cells was associated with inferior treatment-free survival and positively correlated with the proportion of activated CD4+ memory T cells and M2 macrophages in LN. Whole exome sequencing of paired PB and LN samples showed subclonal expansion in LN in approximately half of patients. Since mouse models have implicated activation-induced cytidine deaminase in mutagenesis, we compared AICDA expression between cases with and without clonal evolution, but did not find a difference. In contrast, the presence of a T-cell inflamed microenvironment in LN was associated with clonal stability. In summary, a distinct minor tumor subpopulation underlies CLL pathogenesis and drives clinical outcome. Clonal trajectories are shaped by the LN milieu where T-cell immunity may contribute to suppress clonal outgrowth. The clinical study is registered at clinicaltrials.gov as NCT00923507.

Proliferating CLL cells express high levels of CXCR4 and CD5

“…Here, we combined DNA labelling with Ki67 staining to distinguish cells in G0, G1 and S/G2/M phases and found a small fraction of CLL cells in S/G2/M phase readily detectable in the PB of all patients, regardless of IGHV mutation status or disease course. On average, 0.24±0.17% of CLL cells were in S/G2/M phase, a value surprisingly consistent with previous estimations for daily tumour proliferation rates of 0.1-1% 1 and 0.2% 13 and not much lower than recently reported for LN CLL cells (0.4-1%) 10 . As CLL patients have ≥5*10 9 CLL cells/L in the periphery, proliferation of 0.2% PB CLL cells may represent a substantial part of cell turnover.…”

“…Among PB CLL cells, only a minor subfraction of activated cells in G1 may progress to the proliferating state where the life cycle of a single CLL cell starts over, whilst the majority may exit to G0. This is in line with recent RNA velocity analysis of single cell transcriptomics data indicating LN CLL cells transverse unidirectionally from proliferating to an activated state before entering a final quiescent state 10 . To examine fates after cell division, we tracked proliferation histories in vitro , revealing that the most divided cells by day 9 contained both quiescent and proliferating phenotypes, indicating that cell division may give rise to daughter cells with distinct fates.…”

“…Recent single-cell transcriptomic analysis of both LN and PB CLL cells distinguished three major cell states, quiescent, activated and proliferating 10 , whilst the CXCR4/CD5 model describes recently divided and quiescent cell fractions 6 . Our data demonstrate that actively proliferating CLL cells uniformly express high levels of both CXCR4 and CD5, indicating the CXCR4 hi CD5 hi fraction contains actively proliferating cells, whilst the RAF fraction contains few cells in S/G2/M-phase.…”

“…At the other end, the fraction of cells with high CXCR4 and low CD5 is enriched in quiescent cells. However, a recent study integrating bulk and single-cell RNA sequencing of LN and PB CLL cells identified three major cell states in both compartments: quiescent, activated, and proliferating 10 , suggesting the current CXCR4/CD5 model may not capture all cell states.…”

Recent revelations and future directions using single-cell technologies in chronic lymphocytic leukemia

“…This corroborates previous estimations from in vivo labeling of CLL cells using deuterated water ( 113 ). A portion of the proliferating cells was also shown to proceed in a unidirectional fashion with mitosis followed by activation and subsequently by quiescence ( 114 ). Another study utilizing scRNA-seq on spleen- and LN-derived CLL cells from Eμ-TCL1 Akt-C mice in a model of RT pointed to the importance of sustained Akt signaling for maintaining a pro-proliferative and anti-apoptotic microenvironment through aberrant NOTCH1 activation ( 115 ).…”