Abstract

We have previously reported reliable engraftment of CD19/CD5/CD23/CD45 positive human B-CLL cells in the spleens of NOD/SCID mice (NSS-CLL cells) following transplantation of human peripheral blood-derived B-CLL (PB-CLL) cells. On histology, in the majority of the samples NSS-CLL cells formed focal aggregates which also contained various amounts (range:0–50%) of human CD45/CD3+ T cells (ASH 2005; #52). We now have further characterized these NSS-CLL cells. In our model 1x10^8 MNC are injected i.v. plus i.p. and engraftment in the spleen and other organs is analyzed after 4 (−8) weeks by flow cytometry and immunohistochemistry. In 22 CLL samples investigated in a total of 64 NOD/SCID mice 1.6±0.4x106 NSS-CLL cells representing 10.5±2.3% of total spleen cells were recovered with recovery highly reproducible on repetitive experiments (3.0 vs. 2.0; 1.6 vs. 1.5; 0.2 vs. 1.1; 0.0 vs. 0.0x106 NSS-CLL cells; n=4). In contrast to the low proliferative status reported for human PB-CLL, considerable proliferation was observed in NSS-CLL cells. Following the application of BrdU (1mg/6g d5 i.p., 1mg/ml added to drinking water d6–13; 20–27) BrdU positive NSS-CLL cells ranged from 5.3–25.0% (n=5). Immunohistological analysis showed positive staining for the proliferation marker Ki67 in 18.5±3.5% of NSS-CLL cells. In addition, markers related to proliferation/activation of B-CLL proliferation center cells such as survivin (mean MFI: 5.5±0.8 vs. 2.3±0.2; p=0.028; n=6) and CD100 (semaphorin 4d; mean MFI: 3.3±0.5 vs. 1.1±0.2; p=0.043; n=5) were significantly up-regulated in NSS-CLL vs. PB-CLL cells. When NSS-CLL cells were characterized utilizing additional adhesion/migration, differentiation and/or activation markers, only a subset of NSS-CLL cells (15.7±8.2%, n=6) expressed chemokine coreceptor (CCR) 7, while PB-CLL cells uniformly showed high CCR7 expression. Also CCR5, a migration marker in the context of non-Hodgkin lymphomas, consistently was demonstrated on a fraction of NSS-CLL cells (8.1±3.0%; n=6), whereas here no CCR5 expression was found on PB-CLL cells. No expression of human CD10, CD11c, CD127 or CD49d was detected in B-CLL cells derived from either PB- or NSS-CLL cells, while both sources, as expected, uniformly stained positive for CD40. In summary, NSS-CLL exibit a distinct phenotype as compared to PB-CLL cells and show considerable similarity to B-CLL cells in the proliferation centers of involved human tissues. For these cells not only proliferation but also increased expression of survivin and CD100 as well as down-regulation of CCR7 have been described. Therefore, we think that our model may represent a novel tool to further elucidate biological properties of human B-CLL and, in addition, may serve to devise and evaluate novel treatment strategies.

Disclosure: No relevant conflicts of interest to declare.

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