ALBERT

Description: Abstract 315 Adoptive transfer of primary patient CLL cells into NOD/SCID/γcnull(NSG) mice results in engraftment and proliferation of CLL cells if autologous T cells are present. Formation of splenic follicles consisting of B cells interspersed and surrounded by T cells indicates engraftment. However, ultimately these CD20+ cells are lost several weeks later. We describe one of the mechanisms for this apparent loss: differentiation to plasma cells. Peripheral blood cells from 9 IgM+ CLL patients (6 U-CLL and 3 M-CLL) were adoptively transferred into NSG mice with enriched autologous CD3+ cells pre-activated with anti-CD3/28 beads. B and T cell engraftment and subset distributions were analyzed for 47 mice by immunohistochemistry (IHC) and flow cytometry (FC) at the time of sacrifice. The earliest and latest times of assessment were 12 and 124 days, respectively, after CLL cell injection. In some cases, CLL cells were labeled with CFSE to track cell division. At sacrifice, 3 engraftment patterns were observed. Pattern 1 (observed up to day 56) showed small follicles of CD20+ cells with low-moderate numbers of surrounding T cells. Intensely positive CD38 cells were inconspicuous. FC showed CD19+CD5+ cells with no increase in CD38 and variable CFSE dilution indicating lower levels of proliferation. Pattern 2 (observed throughout the study period) showed much higher T and B cell numbers. CD20+ cells were interspersed with and surrounded by principally CD4+ cells which were activated and functional as indicated by expression of Ki-67, PD-1, CD57, and T cell derived cytokines IFNγ and IL5 in plasma. Follicles contained CD20 and cytoplasmic Ig+ (cIg+) cells that double stained for IRF-4 and Blimp-1, transcription factors required for B cell differentiation. While Bcl-6 staining in these cells was minimal or absent, follicles from all 9 patients contained activation-induced deaminase (AID)+ cells. Cells with dim IgM expression localized to follicles; however, cells with intense IgM, IgA, or IgG were present both within, surrounding, and outside follicles matched by similar CD38 staining. Smaller populations of CD138+ cells surrounded follicles and were interspersed throughout non-follicular splenic areas. FC showed a novel CD19+CD5-CFSE-CD38++ population containing a CD138+ subset. Pattern 3 (observed in a limited subset of cases not before day 63) had minimal CD20+ cells by IHC, but noticeable populations of cIg+CD38+ and CD138+ cells interspersed amongst plentiful T cells. Such cells corresponded with cells with plasma cell morphology. Confirmation that differentiated cells were from the patient clone was achieved in 3 ways. First, in FACS sorted CD19+CD5+ and CD19+CD5-38++ cells from a subset of pattern 2 cases, RT-PCR revealed that all fractions contained both IGHC unswitched and switched clones identical to those found in the patients. Second, cases with pattern 3 engraftment generated CLL clonal switched and unswitched cDNA sequences. Finally, adoptive transfer of highly purified CD5+CD19+ patient cells generated IRF-4+Blimp-1+CD138+ cells. The generation of switched cells from all 9 patients indicated functional AID. In one U- CLL case, ultra-deep sequencing on pre-transfer and post-transfer human cells taken from mouse spleen revealed a significant number of new IGHVDJ mutations in spleen-derived cells. Such mutations targeted nucleotides typical for AID's action. In conclusion, CLL cells can diversify, switch, and differentiate in NSG mice in response to autologous T cell signals. The extent of this maturation is a function of T cell numbers and activity and the duration of the experiment. Differentiation without significant Bcl-6 expression suggests that follicles in NSG mice are not recapitulating classic germinal center reactions, possibly giving clues to the origin of CLL. Several features of poor prognosis disease were demonstrated (e.g., increased CD38 and AID expression with the development of clonally related switched transcripts) that might mirror clinical disease features. AID expressed by CLL cells is fully functional as indicated by de novo somatic hypermutation and class switch recombination. Both U-CLL and M-CLL clones respond in a similar manner in this model, suggesting the importance of T– B cell interactions in all types of CLL. Finally, the demonstration that cells can differentiate when appropriately induced may lead to novel therapeutic options for CLL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 975 We recently described a xenograft model of chronic lymphocytic leukemia (CLL) using NOD/SCID/γcnull (NSG) mice. Adoptive transfer of primary patient PBMCs into these mice results in engraftment and proliferation of CLL cells if autologous activated T cells are present. To date, such CLL-derived T cell proliferation has been achieved by co-transfer of third party antigen presenting cells (APCs). Unfortunately, in this setting, mice succumb to graft-versus-host disease, due to complex interactions between CLL cells, alloAPCs, T cells, and the xenogeneic host. We hypothesized that alternative strategies of autologous T cell activation might refine the model, ultimately providing longer CLL cell engraftment and animal survival. We describe 3 approaches to achieving engraftment and activation of CLL-derived T cells that support B cell growth in vivo. First, we activated CLL CD3+ cells isolated from PBMCs of 4 patients with anti-CD3/28 beads for 72 hours in vitro. Cells were then mixed with CFSE-labeled PBMCs from the same patient at varying ratios (1:50 to 1:1000 CD3+ cells:CLL PBMCs) and injected intraorbitally (io) into a total of 17 mice. CD4, CD8 and CD19 cell engraftment, identified by a human CD45 lymphocyte gate (hCD45), and proliferation, assessed by CFSE dilution of labeled cells, were monitored weekly. All mice demonstrated detectable CD3+ and CD5+CD19+ cells from week (wk) 1. The percent (%) CD3+ cells, as a proportion of hCD45, increased weekly in all mice receiving anti-CD3/28-activated cells. While overall % CD5+CD19+ cells decreased weekly, the % proliferating increased and strongly correlated with increasing % of T cells (r2=0.7799, p

Description: Lenalidomide (Revlimid®), a thalidomide analogue, is an orally administered second generation immunomodulator with anti-angiogenic and anti-neoplastic properties. Initial studies treating patients with chronic lymphocytic leukemia (CLL) suggest that lenalidomide can have considerable efficacy and that its mode of action is mainly indirect, affecting non-malignant cells in the microenvironment, in particular T lymphocytes. Because a recently described xenograft model for CLL has highlighted the importance of CLL-derived, autologous T cells in promoting leukemic B-cell engraftment and growth in vivo, we have studied the influence of lenalidomide on the expansion of CLL B- and T-lymphocytes in this model. After an initial 12 day culture of FACS-isolated CLL-derived T cells with or without anti-CD3/CD28 beads plus IL-2 (30 IU/ml), T lymphocytes were transferred into alymphoid NSG mice via the retro-orbital plexus (day 0). On day 7, CLL cells were delivered retro-orbitally. These recipient animals are referred to as “T + PBMC mice”. Mice that did not receive T cells on day 0 but were given CLL PBMCs at day 7, with or without lenalidomide, served as controls (“PBMC only mice”). Recipient mice received lenalidomide (10mg/kg/day) or vehicle control daily by gavage starting at day 0. All mice were sacrificed at day 28 (28 days after T-cell and 21 days after B-cell transfer), and blood, spleen, and bone marrow were collected. On this material, four analyses were performed: [1] level of human CD45+ cell engraftment; [2] numbers and types of CLL-derived T cells; [3] numbers of CLL B cells; and [4] levels of cytokines reflective of Th1 and Th2 immune responses. There was a clear enhancement in human hematopoietic (CD45+) cell engraftment in those mice exposed to lenalidomide. This was most marked for the PBMC only mice (vehicle: 10.64%; lenalidomide: 38.53%), although it was also evident for T + PBMC mice (vehicle: 55.96%; lenalidomide: 69.65%). T-cell phenotyping was carried out, before and after cell culture and also at sacrifice. Prior to culture, CLL samples contained on average ∼96% CD5+CD19+ cells and ∼3% CD5+CD19- cells; for the latter, ∼67% were CD4+ and ∼33% CD8+. After 12-day culture, these percentages remained largely unchanged. However, the numbers and types of T cells recovered from the spleens at sacrifice were quite different after in vivo exposure to lenalidomide. For the PBMC only, the percentages of CD4+ and CD8+ cells in the spleens differed somewhat based on lenalidomide exposure (CD4: Vehicle 86% vs. Lenalidomide 61%; CD8: Vehicle 10% vs. Lenalidomide 28%). However, this change was dramatic for the T + PBMC mice (CD4: Vehicle 64.1% vs. Lenalidomide 28.9%; CD8: Vehicle 34% vs. Lenalidomide 62%). Furthermore, when the CD8+ cells from these animals were subsetted based on antigen-experience and function, it appeared that lenalidomide exposure had led to the outgrowth of a greater number of effector memory (CD45RO+ CD62L-) than central memory (CD45RO+ CD62L+) T-cells. For CLL-derived B cells, the numbers differed, based not only on lenalidomide exposure but also on prior in vitro activation. Specifically, in PBMC only mice, the addition of lenalidomide led to increased numbers of CLL B cells in the spleen (Vehicle: 7.81% vs. Lenalidomide: 14%). Conversely, in the T + PBMC mice, the numbers of B cells decreased (Vehicle: 2.36% vs. Lenalidomide: 0.34%). An analysis of Th1 and Th2-related cytokines in the plasmas of the mice at sacrifice revealed a fall in IL-4, IL-5, and IL-10 and a marked increase in IFNg, consistent with a Th2 to Th1 transition. The above data suggest that administration of lenalidomide permits greater engraftment of human hematopoietic cells in alymphoid mice. Although this enhancement involves all members of the hematopoietic lineage, T cells, in particular CD8+ effector memory T cells, emerge in excess over time. This CD8 expansion is associated with diminished levels of CLL B cells suggesting that the decrease is due to T-cell mediated cytolysis. In contrast, in the absence of prior T-cell activation, CLL T cells appear to support better CLL B-cell growth. These findings suggest that lenalidomide alters B-cell expansion in vivo depending on the activation and differentiation state of the autologous T-cell compartment. They also implicate the generation of cytolytic T cells as one mechanism whereby lenalidomide leads to clinical improvement in CLL. Disclosures: Allen: Celgene Corporation: Honoraria.

Description: Background. Chronic lymphocytic leukemia (CLL) is a prototypic microenvironment-dependent B-cell malignancy, in which neoplastic B cells co-evolve with a supportive tissue microenvironment to promote leukemia cell survival, growth, and drug-resistance. Within the microenvironment, hematopoietic and non-hematopoietic stromal and tumor cells produce factors that recruit circulating monocytes to tumor sites and induce differentiation into macrophages. Mirroring the Th1/Th2 paradigm, cells of monocyte-macrophage lineage reprogram their functions in response to environmental signals, undergoing M1 (classical) or M2 (alternative) activation, which represent extremes of a broad continuum of functional states. Classical M1 cells (activated by IFNs and TLR) are involved as inducer and effectors cells in polarized Th1 responses and as effectors of resistance against intracellular parasites and tumors. In contrast, M2 cells (activated by IL4 and IL13) are poor at antigen presentation, suppress Th1 adaptive immunity, actively scavenge debris, contribute to the dampening of inflammation, promote angiogenesis and tissue remodeling, and support tumor progression. One way to distinguish the two types of macrophages is based on surface antigen expression: M1-like cells up-regulate Fcg receptors I, II, III such as CD16, CD32 and CD64, whereas M2-like macrophages display abundant levels of CD23, CD163, and scavenger receptors (e.g. MCR1). Understanding the microenvironment and the crosstalk between B-CLL cells and their tissue neighbors can give insight into disease biology and for therapy. Aim. To investigate if the CLL milieu, contained within serum, influences monocyte-to-macrophage differentiation, promoting an anti (M2)- or pro (M1)- inflammatory microenvironment. Methods. Monocytes from healthy donors were isolated using Monocytes Isolation Kit II (Miltenyi) and cultured in Ultra-Low Attachment plates with 10% normal human AB serum or 10% CLL-derived serum -/+ IL4 or IFNg for 3 days. Macrophages were stained for CD23, CD64, CD32, MRC1, CD14, CD16, and data were acquired with a BD LSRII flow cytometer and analyzed by FlowJo V7.2.4 software. Results. Normal monocytes were differentiated to macrophages in vitro in the presence of sera from 24 untreated CLL patients with different prognostic factors (genomic aberrations, % CD38 and IGHVmutational status). About 45% of the CLL sera (N=10; 6 M-CLL, 4 U-CLL) drove macrophage maturation toward an M2-like phenotype, as assessed by surface expression of CD23, CD64, CD32, CD36, MRC1, etc. These 10 sera induced higher CD23 expression after 3 days in culture compared to AB human serum, whereas the levels of M1-specific markers (CD64 and CD32) did not change relative to the control. Interestingly all of these 10 CLL sera came from patients bearing 13q14 Δ (N=5), 17p13 Δ (N=3) or a combination of these (13q14 Δ + 17p13 Δ; N=1) and 17p13 Δ + trisomy12; N=1)). On the contrary, no increase in CD23 expression was detected in presence of sera from patients with 11q22 Δ (N=1) alone or in combination with 13q14 Δ (13q14 + 11q22 Δ; N=5). Of note, treatment with a neutralizing mAb specific for IL-4 did not block the CLL serum induced up-regulation of CD23 (N=2). In a parallel set of studies, normal monocytes were incubated with each of the 24 CLL sera in combination with the M1 promoting cytokine, IFNg or the M2 promoting cytokine, IL4. In all cases IL4 induced CD23 up-regulation and an M2 phenotype. Paradoxically, IFNg, which normally induces an M1 phenotype, also induced an M2 phenotype (i.e., enhanced CD23 expression) when co-cultured with sera from a subset patients (N=8; 6 M-CLL and 2 U-CLL). Of note, the IFNg stimulatory effect on CD23 expression was observed with a different set of sera from those that directly stimulated CD23 expression. Furthermore, CD64 expression did change after incubation with IFNg + CLL serum in 6 of 8 cases, yielding another unusual (CD23+CD64+) macrophage phenotype. The 2 sera that did not yield such hybrids were from M-CLL patients. Conclusions. Sera from CLL patients contain two apparently novel activities that mature normal monocytes to M2-like macrophages. The first acts directly by an action that is apparently independent of IL-4 and associates with 13q14 Δ or 17p13 Δ abnormalities. The second acts indirectly through IFNg and leads to macrophages with a hybrid M2/M1 phenotype (CD23+CD64+), suggestive of a new type of macrophage. Disclosures No relevant conflicts of interest to declare.

Description: Chronic lymphocytic leukemia (CLL) is an incurable adult disease of unknown etiology. Understanding the biology of CLL cells, particularly cell maturation and growth in vivo, has been impeded by lack of a reproducible adoptive transfer model. We report a simple, reproducible system in which primary CLL cells proliferate in nonobese diabetes/severe combined immunodeficiency/γcnull mice under the influence of activated CLL-derived T lymphocytes. By cotransferring autologous T lymphocytes, activated in vivo by alloantigens, the survival and growth of primary CFSE-labeled CLL cells in vivo is achieved and quantified. Using this approach, we have identified key roles for CD4+ T cells in CLL expansion, a direct link between CD38 expression by leukemic B cells and their activation, and support for CLL cells preferentially proliferating in secondary lymphoid tissues. The model should simplify analyzing kinetics of CLL cells in vivo, deciphering involvement of nonleukemic elements and nongenetic factors promoting CLL cell growth, identifying and characterizing potential leukemic stem cells, and permitting preclinical studies of novel therapeutics. Because autologous activated T lymphocytes are 2-edged swords, generating unwanted graph-versus-host and possibly autologous antitumor reactions, the model may also facilitate analyses of T-cell populations involved in immune surveillance relevant to hematopoietic transplantation and tumor cytoxicity.

Description: Abstract 2326 Poster Board II-303 B-cell type chronic lymphocytic leukemia (B-CLL), an incurable disease of unknown etiology, results from the clonal expansion of a CD5+CD19+ B lymphocyte. Progress into defining the cell of origin of the disease and identifying a stem cell reservoir has been impeded because of the lack of a reproducible model for growing B-CLL cells in vivo. At least one possible cause for this is the murine microenvironment's inability to support B-CLL survival and proliferation. To overcome this barrier, we reconstituted NOD/SCID/γnull mice by intrabone (ib) or intravenous (iv) injection of 1 × 105 CD34+ cord blood cells along with ∼106 bone marrow-derived human mesenchymal stem cells (hMSCs) by ib injection. After human cellular engraftment, a total of 108 CFSE-labeled PBMCs from individual B-CLL patients were injected into the same bones or iv. Every two weeks thereafter, blood from the mice was examined for the presence of cells bearing CFSE, human CD45, and various human lineage markers by flow cytometry. In the presence of a human hematopoietic microenvironment derived from hHSCs, CFSE+CD5+CD19+ cells were readily detected in the blood of mice and many of these leukemic cells underwent at least 6 cell doublings. In contrast, the numbers of leukemic cells circulating in the blood of mice reconstituted with hMSCs without CD34+ cells was much less and these failed to proliferate. Thus, hMSCs were not essential in the model. Moreover, the percentage of leukemic cells expressing CD38 in the CFSE+CD5+CD19+ cell fraction was similar to that in the donor patient inoculum only in the mice in which B-CLL cell proliferation occurred. The percentage and intensity of CD38-expressing B-CLL cells was higher in the spleen and bone marrow (BM), far exceeding that in the blood and peritoneum. Of note, B-CLL cells formed follicular structures in the spleen that contained larger B cells expressing the same Ig H and Ig L chains as the CLL MNCs. CLL cells from these spleens exhibited the same IGHV/D/J rearrangement as in the donor leukemic cells, indicating their leukemic origin. These follicular structures are reminiscent of proliferation centers/pseudofollicles seen in patient lymph nodes and BM, in that they contained leukemic B cells of intermediate and large size. Finally, B-CLL cells adoptively transferred into these mice exhibit kinetics similar to those observed in patients in vivo, with birth rates calculated from CFSE-dilution data of (X-Y% per day). Robust T-cell expansion occurred in mice receiving CD34+ cells and occasionally in mice (10-20%) not receiving hCD34+ cells. Based on detailed SNP analyses, the expanded T cells were of B-CLL patient origin and not from hCD34+ cells. Notably when T cells were eliminated by injecting an anti-CD3 mAb (OKT3), B-CLL cell proliferation was inhibited. These latter two findings clearly indicate a need for autologous T lymphocytes in the successful adoptive transfer of B-CLL cells in this model and also highlight the fact that another cell type, derived from the normal allogeneic CD34+ cells, is needed. Because CD34+ cells could be substituted for by mature allogeneic monocytes or B lymphocytes from the blood of normal individuals, antigen-presenting cells may be the key cells that develop from the normal hHSCs. This would suggest that an allogeneic mixed lymphocyte reaction is needed for successful B-CLL cell survival and proliferation. This is consistent with finding that most of the mice with significant T-cell overexpansion died within 6 weeks of B-CLL cell injection from apparent graft vs. host disease. Finally, preliminary data using Rituxan (anti-CD20 mAb) to eliminate B-CLL cells from recipient mice suggest that this model is a good tool to perform pre-clinical studies on the efficacy of action of novel therapeutics. In summary, these studies indicate that allogeneic human antigen-presenting cells and autologous human T cells permit adoptive xenogeneic transfer and clonal expansion of B-CLL cells in immune deficient mice. This model will be useful in discovering and understanding non-genetic factors promoting B-CLL expansion because it recapitulates several features of human B-CLL. Finally, the model may help in the study of the basic biology of this disease, such as if leukemic stem cells exist, and also in conducting pre-clinical tests on possible new therapeutics. Disclosures: No relevant conflicts of interest to declare.

Description: Chronic lymphocytic leukemia (CLL) is a disease commonly associated with an immune disturbance. These alterations of the immune system have been generally considered due to the direct effects of CLL cells. In the last years, myeloid derived suppressor cells (MDSCs) have been found to be expanded in several cancers and to play a major role in helping tumor cells escape from immune surveillance. MDSCs represent a heterogeneous population of HLA-DRlo/CD11b+/CD33+ cells that are subdivided into monocyte-like (CD14+, m-) or granulocyte-like (CD15+, g-) subsets. Here we have investigated the extent that patients with CLL have expansions of MDSCs, their types and functions, and how these correlate with clinical and laboratory characteristics. Using flow cytometry on cryopreserved PBMCs, we did not observe differences in the percentages of HLA-DRlo/CD11b+/CD33+ cells between 17 untreated CLL patients and 10 healthy controls (HC) (3.3% vs. 3.1%). However, the distribution between m-MDSCs and g-MDSCs was dramatically different, with CLL patients exhibiting significantly higher levels of g-MDSCs (79.7% vs. 4.1%, p

Description: Abstract 317 By using reciprocal densities of surface membrane CXCR4 and CD5, chronic lymphocytic leukemia (CLL) B cells can be divided into 3 fractions indicating time since last division (proliferative, intermediate, and resting). It has been suggested that cells in these fractions represent a continuum from resting to intermediate to proliferative. In this study, we made intraclonal gene expression profile (GEP) comparisons of these fractions from 17 CLL patients to try to confirm this notion and interclonal comparisons between U-CLL and M-CLL patients to determine if pathways involved in the actions of these fractions differed between patient subgroups. PBMCs from 8 U-CLL and 9 M-CLL patients were sorted into 3 fractions (CD19+CD3−CD5hiCXCR4lo, PROLIF), (CD19+CD3−CD5intCXCR4int, INTERM), and (CD19+CD3−CD5loCXCR4hi, REST); RNA was purified from each, and gene expression microarrays using Illumina HumanHT12 beadchips performed. To determine differentially expressed genes in intraclonal comparisons, expression value ratios for fractions from each patient were computed, log-transformed, and Student t-test performed using R (www.r-project.org); for interclonal comparisons, raw GEP data between subpopulations were compared: U-PROLIF and M-PROLIF, and U-REST and M-REST. Sets of significant genes (≥1.5 fold change and PINTERM〉REST, or PROLIF

Description: 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.

Description: Abstract 668 Background. In vivo studies of deuterium (2H) incorporation into newly-synthesized DNA of chronic lymphocytic leukemia (B-CLL) B cells indicate that the disease is dynamic, with ongoing birth and death of individual members of a leukemic clone. Therefore, a detailed phenotypic analysis might define the “life cycle” of subpopulations of leukemic cells within a B-CLL clone, thereby identifying recently-born members and members born earlier. Such an extended phenotype could enable structural and genetic characterization of the intra-clonal heterogeneity that birth and death create, and thereby help understand the biology of B-CLL cells and define novel cell surface markers or marker combinations as therapeutic targets. Methods. Our previous studies indicated that B-CLL clones could be subdivided into three populations based on relative densities of expression of CXCR4 and CD5. The CXCR4dimCD5bright subpopulation is enriched in cells with 2H-labeled, newly-synthesized DNA and contains significantly more Ki-67+ and MCM6+ cells than CXCR4intCD5int cells within the same clone. These latter cells in turn are enriched for the same parameters compared to the CXCR4brightCD5dim fraction. We have now identified other markers associated with the CXCR4dimCD5bright subpopulation (“proliferative compartment”) and the CXCR4brightCD5dim (“resting/re-entry compartment”) in B-CLL. PBMC from 20 B-CLL cases were subjected to multi-parameter immunofluorescent, flow cytometric analyses to define the expression of CD11a, CD20, CD23, CD27, CD38, CD47, CD49d, CD52, or Fcƒ×RII on the CD19+CXCR4dimCD5bright and CD19+CXCR4brightCD5dim B-CLL cell subpopulations. Companion studies were performed on Ki-67+ and Ki-67- subpopulations. Finally, gene expression profiling was performed on CXCR4dimCD5bright, CXCR4intCD5int, and CXCR4brightCD5dim subsets of 12 B-CLL cases using the Illumina HumanWG-6 platform. Results. Compared to CXCR4brightCD5dim cells within a clone, the CXCR4dimCD5bright compartment contained significantly more cells expressing CD23, CD52 (both p