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

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 CXCR4^DIMCD5^BRIGHT (CXCR4^DIM) and contain higher numbers of CD38⁺ and Ki-67⁺ cells. Circulating “resting” CLL cells express CXCR4^BRIGHTCD5^DIM (CXCR4^BR) and genetic signatures of older, quiescent cells that need to home to lymphoid tissues or die. CXCR4^DIM and CXR4^BR 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 (CXCR4^INT). Based on these differences, we proposed a model of transitioning CXCR4^DIM → CXCR4^INT → CXCR4^BR 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 CXCR4^DIM and CXCR4^BR 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 (CXCR4^DIM>CXCR4^INT>CXCR4^BR), in vivo apoptosis and transmigration in vitro (both CXCR4^DIM< CXCR4^INT< CXCR4^BR). Thus, while more CXCR4^BR cells undergo apoptosis, CXCR4^BR cells can migrate better to tissues to receive survival signals.

In vivo functional differences were then studied in a NOD/SCID/γc^null (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 CXCR4^BR, CXCR4^INT or CXCR4^DIM 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 CXCR4^DIM than in those injected with CXCR4^BR or CXCR4^INT. 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 CXCR4^BR 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 CXCR4^BR and CXCR4^DIM cells, CXCR4^DIM injected mice had more extensive T cell growth starting from week 2. Mice injected with CXCR4^BR 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 CXCR4^BR or CXCR4^DIM. Notably, no human B or T cells were detected in CXCR4^INT injected mice. In fact, adding CXCR4^INT cells to CXCR4^DIM mice suppressed CXCR4^DIM induced T cell expansion and cytokine production. Specifically, mice receiving both CXCR4^DIM and CXCR4^INT 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 CXCR4^BR cells with activated T cell support, and identify a greater ability of CXCR4^DIM cells to activate autologous T cells, although some U-CLL CXCR4^BR cells could do so to a lesser degree. Superior activation of T cells by CXCR4^DIM B cells may be due to higher numbers of CD23⁺, CD25⁺, CD27⁺, CD29⁺ and CD44⁺ cells in CXCR4^DIM fraction that facilitate cellular interactions. Finally, unlike CXCR4^BR and CXCR4^DIM cells, the major fraction in patient blood, CXCR4^INT, 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.