Human CLL Intraclonal Fractions Differ in Their Abilities to Respond to, Elicit, and Suppress Pro-Engraftment and Growth Signals From Autologous T Cells in a Murine Adoptive Transfer Model
Authors: Chen Shih-Shih , Patten Piers E.M. , Simone Rita , Marsilio Sonia , Barrientos Jacqueline C. , Kolitz Jonathan E. , Allen Steven L. , Rai Kanti R. , Chiorazzi Nicholas 
Published: October 2019

Abstract
Abstract 316
Chronic lymphocytic leukemia (CLL) clones contain activated/proliferative leukemic cells in lymphoid tissues and resting cells in the periphery. Different subsets of CLL cells have distinct proliferation rates. Recently divided “proliferative” cells have a surface membrane phenotype of CXCR4DIMCD5BRIGHT (CXCR4DIM) and contain higher numbers of CD38+ and Ki-67+ cells. Circulating “resting” CLL cells express CXCR4BRIGHTCD5DIM (CXCR4BR) and genetic signatures of older, quiescent cells that need to home to lymphoid tissues or die. CXCR4DIM and CXR4BR subsets are relatively minor (1–10% of total) components of CLL clones, with the major fraction (≥90%) of CLL cells having intermediate levels of CXCR4 and CD5 (CXCR4INT). Based on these differences, we proposed a model of transitioning CXCR4DIM → CXCR4INT → CXCR4BR CLL cells in the blood. Because higher birth rates correlate with more aggressive disease, and transiting back to solid tissues permits clonal survival and re-activation, this model suggests CXCR4DIM and CXCR4BR subsets as therapeutic targets.
Aiming to further understand functional differences in CLL subsets in vitro and in vivo, we found that CLL subsets differ in cell size (CXCR4DIM>CXCR4INT>CXCR4BR), in vivo apoptosis and transmigration in vitro (both CXCR4DIM< CXCR4INT< CXCR4BR). Thus, while more CXCR4BR cells undergo apoptosis, CXCR4BR cells can migrate better to tissues to receive survival signals.
In vivo functional differences were then studied in a NOD/SCID/γcnull (NSG) mouse model using pre-activated CLL-derived autologous T cells. Primary CLL blood cells from 1 M-CLL and 2 U-CLL patients were sorted for CXCR4BR, CXCR4INT or CXCR4DIM fractions. Each fraction (5×106 cells) was injected into NSG mice with 5×105 CD3/28-activated autologous T cells. At weeks 2–6 post transfer, blood analyses showed more extensive expansion of CLL B and T cells in mice received CXCR4DIM than in those injected with CXCR4BR or CXCR4INT. At weeks 9–12, mice were sacrificed. Although T cells dominated in blood, spleen and bone marrow of all recipients, a larger fraction of CLL B cells existed in CXCR4BR injected mice, suggesting better long-term CLL cell engraftment capacity of this fraction.
Because regulation of T cells plays key roles in CLL cell survival/growth in patients and in the NSG adoptive transfer model, we next analyzed the same fractions for their abilities to activate T cells and elicit help for engraftment and growth. Unactivated CD5+ T cells (1–1.5×105) and B-CLL fractions (3–5×106 cells) were sorted from 6 patient samples (3 U-CLL and 3 M-CLL), injected into mice and followed bi-weekly until week 6. In 5 cases, except one with few CXCR4BR and CXCR4DIM cells, CXCR4DIM injected mice had more extensive T cell growth starting from week 2. Mice injected with CXCR4BR from 2 U-CLL cases also showed T cell expansions, but at comparatively lesser levels and at later time points (from week 4–5). At week 6, CLL B cells were found in spleen and bone marrow in mice with activated T cells; the numbers of CLL B cells correlated with T cell numbers. Also, identical CXCR4 levels were found in CLL cells regardless of origination from CXCR4BR or CXCR4DIM. Notably, no human B or T cells were detected in CXCR4INT injected mice. In fact, adding CXCR4INT cells to CXCR4DIM mice suppressed CXCR4DIM induced T cell expansion and cytokine production. Specifically, mice receiving both CXCR4DIM and CXCR4INT cells had diminished T cell expansion and at least 3 fold reduced serum levels of IFNγ and IL5.
Overall, our data confirm the need for activated T cells for CLL B cell growth in mice; suggest superior long term CLL B cell engraftment by CXCR4BR cells with activated T cell support, and identify a greater ability of CXCR4DIM cells to activate autologous T cells, although some U-CLL CXCR4BR cells could do so to a lesser degree. Superior activation of T cells by CXCR4DIM B cells may be due to higher numbers of CD23+, CD25+, CD27+, CD29+ and CD44+ cells in CXCR4DIM fraction that facilitate cellular interactions. Finally, unlike CXCR4BR and CXCR4DIM cells, the major fraction in patient blood, CXCR4INT, inhibited T cell activation. These results indicate previously unappreciated levels of intraclonal CLL cell heterogeneity that may have important clinical relevance, allow more precise biologic analyses, and provide a rationale for preferential therapeutic targeting of these fractions.

Disclosures:
No relevant conflicts of interest to declare.