Search

Abstract 3714

Abstract 1460

The exopeptidase CD26 (also known as DPPIV/dipeptidylpeptidase IV) cleaves dipeptides from the N-terminus of proteins that contain the required X-Pro or X-Ala motif. We have previously reported that inhibition or loss of CD26 activity results in a deficiency in normal granulocyte-colony stimulating factor (G-CSF) induced mobilization, suggesting that CD26 is a necessary component of mobilization (Christopherson, et al Blood2003 and Christopherson, et al Exp Hematol2003). The chemokine CXCL12 (SDF-1, stromal cell derived factor-1) contains the appropriate recognition sequence for CD26 induced cleavage. This combined with the importance of CXCL12 in the trafficking of hematopoietic stem and progenitor cells (HSC/HPC) suggests CXCL12 as a likely functional target of CD26 during G-CSF induced mobilization. For this reason we therefore decided to investigate whether genetically engineered mice lacking CD26 (CD26−/−) could be mobilized utilizing the CXCR4 antagonist, AMD3100. To evaluate this, ten week old C57BL/6 and CD26−/−mice (also on a C57BL/6 background) received a single subcutaneous injection of AMD3100 (1mg/1kg). One hour following injection the mice were euthanized by CO2inhalation. Peripheral blood was then obtained by heart stick with a 1.2 ml syringe containing EDTA as an anticoagulant. A complete blood count was taken for each peripheral blood sample. Following red blood cell lysis, cells were plated for myeloid colony formation in a standard 1% methylcellulose colony assay containing the appropriate cytokines. Following 7 days of incubation at 5% O2, 5% CO2and 37°C plates were scored for colony-forming units-granulocyte macrophage (CFU-GM), burst-forming units-erythroid (BFU-E), and colony-forming units-granulocyte, erythroid, macrophage, and megakaryocytic (CFU-GEMM). Data is presented as the number of colonies per femur for the bone marrow and as the number of colonies per ml of whole blood for the peripheral blood. AMD3100 treatment resulted in an increase in white blood cell (WBC) counts from 5.05±0.48 × 106/ml in untreated mice to 10.21±0.88×106/ml in treated mice (p≤0.01). An increase in WBC counts was also observed during AMD3100 treatment in CD26−/−mice from 7.77±1.28×106/ml in untreated mice to 16.7 ±2.11 × 106/ml in treated mice (p<0.01). AMD3100 treatment resulted in an increase in circulating myeloid progenitors in the peripheral blood of C57BL/6 and CD26−/−mice as compared to untreated C57BL/6 and CD26−/−mice respectively (p≤0.01). Specifically, a 2.38, 3.75, 12.33 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of C57BL/6 mice after treatment. A 2.63, 5.48, 14.29 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of CD26−/−mice after treatment. Existing pre-clinical and clinical data suggest that the CXCR4 antagonist, AMD3100, rapidly mobilizes hematopoietic progenitor cells from the bone marrow into the periphery. The results presented here provide pre-clinical evidence that disruption of the interaction between the CXCR4 chemokine receptor and CXCL12, via sub-cutaneous injection of AMD3100, mobilizes significant numbers of myeloid progenitors in mice, even in the absence of CD26. These results support the notion that CD26 is downstream of G-SCF treatment. Additionally, these results support the potential use of AMD3100 to treat patients that may have an altered ability to respond to G-CSF treatment as a result of a reduction or loss in CD26 activity. Future studies are warranted to evaluate potential variations in CD26 levels or activity in the general population, in differing patient populations, and during different treatment regimens.

A firm understanding of the biology of hematopoietic stem and progenitor cell (HSC/ HPC) trafficking is believed to be critical for the development of methodologies to improve transplant efficiency and subsequently immune reconstitution during hematopoietic stem cell transplantation in the clinical setting. Through the use of CD26 inhibitors and CD26 deficient mice (CD26−/−), we have previously generated data in mice suggesting that suppression of CD26/DPPIV (dipeptidylpeptidase IV) enzymatic activity on the transplant donor cell population can be utilized as a method of increasing transplant efficiency (Christopherson, KW 2nd, et al, Science2004. 305:1000–3). However, the clinical importance of the transplant recipient should not to be overlooked given the potential importance of the bone marrow microenvironment in regulating the transplant process. We therefore investigated here whether inhibition or loss of CD26 activity in recipient mice would have an effect on transplant efficiency utilizing an in vivocongenic mouse model of transplantation. The short-term homing and long-term engraftment of BoyJ donor cells (expressing CD45.1+) into lethally irradiated control C57BL/6, CD26 inhibitor (Diprotin A) treated C57BL/6, or CD26−/−mice (expressing CD45.2+) was monitored by flow cytometric analysis of the bone marrow and peripheral blood at 24 hours and 6 months post-transplant respectively. Twenty-four hours post-transplant of 20×106BoyJ mononuclear cells, we observed 8.85±0.58%, 10.69±1.01%, and 12.45±1.33% donor derived Sca-1+lin−cells in the bone marrow of recipient mice for control, Diprotin A treated, and CD26−/−recipient mice respectively. As compared to control mice, this represents a 20.8% increase (p=0.01) with CD26 inhibitor treatment and a 40.7% increase (p£0.05) resulting from the use of a CD26−/−recipient in short-term homing (N=5 mice per group). Six months post-transplant of 1×105BoyJ mononuclear cells, we observed 39.90± 4.38%, 70.22± 3.72%, and 92.51± 1.04% donor contribution to hematopoiesis in the peripheral blood of control, Diprotin A treated, and CD26−/−recipient mice respectively. This represents a 76.0% increase (p£0.01) with CD26 inhibitor treatment and a 131.9% increase (p£0.01) as a result of the CD26−/−recipient in long-term engraftment as compared to control recipient mice (N=14 mice per group). These results provide pre-clinical evidence of the importance of CD26 expression within the transplant recipient with regard to regulating hematopoietic stem cell homing and engraftment. Our results also support the potential use of CD26 inhibitors to treat transplant patients during hematopoietic stem cell transplantation as a method of improving transplant efficiency. Lastly, our use of inhibitor treated C57BL/6 and CD26−/−recipient mice, which are also on a C57BL/6 background, in conjunction with a congenic model of transplantation provides a accurate and convenient model system for the in vivotesting of the efficacy of existing and new CD26 inhibitors in transplant recipients.

Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disorder manifested by a clonal expansion of mature long-lived functionally defective lymphocytes that are predominantly of B-cell origin (>95% of cases). CLL cells typically express the pan-B-cell antigens CD19 and CD20 as wells as CD5, an antigen of mature T-cells, which is present in 95% of B-CLL patients and is helpful for diagnosis. The disease has a median survival of 8–10 years. Asymptomatic patients (RAI stages 0–2) are often not treated. RAI stages 3 & 4 are generally treated. Current therapies for CLL included a combination of monoclonal antibodies (anti-CD20 monoclonal antibody, rituximab) with purine analogs (fludarabine or pentostatin) and alkylating agents (cyclophosphamide). Current therapies to manage B-cell CLL are not curative and therefore suggest that the current strategy of targeting therapy against B-cells may leave behind residual disease that contributes to progression or relapse. The importance of more primitive Hematopoietic Stem and Progenitor cell populations in B-CLL patients has not been considered. We hypothesized that circulating cells may be spontaneously mobilized into the periphery of CLL patients and would therefore contribute to the peripheral blood microenvironment. To test this hypothesis we examined existing CLL cell populations from un-treated “watch-and-wait” B-CLL patients as compared to normal volunteer donor peripheral blood cell populations utilizing multivariate flow cytometric analysis. We determined CD34+CD38−, CD34+CD38+, and CD10 levels in normal peripheral blood to be 81.0±14.5, 497.3±78.33, and 83.6±17.5 cells/ml respectively. We report here the presence of abnormally high levels of these stem and progenitor cell populations in B-CLL patients (see table). Although the mechanism behind the release of these cells into the periphery in B-CLL patients is unknown, it is reasonable to propose that they may be contributing to the manifestation of the disease and are therefore potential novel target cell populations for future therapies.