Intraclonal Complexity Of CLL Fractions In Cell Proliferation Rates, Gene Expression Signatures, and Responses To Autologous T-Cell Help In Peripheral blood and Secondary Lymphoid Tissues

Chronic lymphocytic leukemia (CLL) is a disease of resting and dividing B cells. In order to understand the trafficking and transition of proliferative and resting fractions between periphery and lymphoid tissues, we have tried to [1] define the relative levels of CLL B-cell proliferation in distinct anatomic sites, [2] identify and characterize CLL intraclonal fractions from these sites that differ in time since last replication, and [3] model the abilities of these subsets to repopulate immune deficient mice. In the blood, resting and recently-divided CLL cells can be distinguished by surface expression of CXCR4 and CD5. CXCR4^dimCD5^br (DIM) cells are an activated subset, CXCR4^intCD5^int (INT) cells are the clonal bulk, and CXCR4^brCD5^dim (BR) cells are older and more quiescent. Here we compared in vivo proliferation rates, gene expression profiles, and differences in xenografting of these 3 fractions. In addition, we studied the same parameters in 3 other fractions, CXCR4^dimCD5^dim (dDIM), CXCR4^intCD5^br (INT/BR) and CXCR4^brCD5^br (dBR).

CLL B-cell kinetics was analyzed in 7 treatment-naïve patients who drank deuterated “heavy” water (²H₂O). At day 13, peripheral blood (PB), lymph node (LN), and bone marrow (BM) samples were collected from each patient, and cells from these 3 sites were analyzed for ²H-labeled CD5⁺CD19⁺ cells after FACS sorting into 6 fractions: DIM, dDIM, INT, INT/BR, BR and dBR. Overall, more CLL B-cell proliferation was found in LN than PB and BM; only small numbers of divided cells were found in BM. Interestingly, the DIM, dDIM, and INT/BR had the highest ²H-incorporation in LN and PB; and DIM cells from LN and PB showed similar levels of ²H-incorporation.

Gene expression profiling using Illumina Human HT12 BeadChips was then performed on the same 6 fractions from PB and LN. Expression value ratios for fractions from each patient were analyzed using R, and sets of significant genes (fold change >1.5 and P<0.05) were determined. Unsupervised hierarchical clustering grouped together the DIM and dDIM fractions from all other fractions. Although the INT/BR had high ²H-incorporation levels, it clustered with the BR, INT and dBR fractions. Genes upregulated in DIM and dDIM included genes involved in cell proliferation and survival, such as DUSP1, PRKCD and BMF, and chemokine genes CCL3, CCL3L1, CCL3L3, CCL4L2. Genes changed in BR, dBR, and INT/BR included negative regulators of cell survival and proliferation, e.g. PRICKLE1 and GRAP.

Finally, functional differences in the CLL fractions were analyzed in vivo using NOD/SCID/γc^null (NSG) mice. 3-5x10⁶ PB B cells were injected with 1-1.5x10⁵ resting, autologous T cells. Compared to BR, dBR and INT/BR fractions, DIM and dDIM cells led to more extensive T-cell growth and B-cell expansion in the spleen and BM at week 6. In contrast, INT injected mice exhibited only minimal or no CLL B or T cells. Adding INT cells suppressed DIM and dDIM induced T-cell expansion. This function, which was resistant to irradiation, was associated with greater immunological synapse impairment in vitro.

Thus, in vivo kinetics analysis using D2O identified levels of divided cells based on the following ranking: DIM ≥ INT/BR> dDIM > INT> BR=dBR. Although the INT/BR fraction contains recently-divided cells based on ²H-labeling, its genetic signature is similar to fractions containing lesser numbers of recently activated cells, suggesting INT/BR cells have divided but are being inactivated in the microenvironment. Therefore, these cells may represent a transition population between proliferating and resting cells. Because PB and LN DIM fractions are very similar in proliferation rates and gene expression profiles and because little CLL B-cell proliferation occurs in the blood, the circulating DIM fraction accurate reflects LN proliferation. When combining kinetics and gene expression profiling, the DIM and dDIM fractions appear as the most activated intraclonal fractions. In xenografted mice, PB DIM and dDIM cells are better at activating T cells and consequently receiving T-cell help for engraftment and growth. The INT fraction, which constitutes the tumor bulk, inhibits T-cell activation. Finally, the BR and dBR fraction are the older, most quiescent cells in CLL clones. Overall, these results inform about trafficking and transition of CLL cells between lymphoid tissues and periphery, and provide a rationale for preferential therapeutic targeting of these fractions.