ALBERT

Description: Introduction: FL is generally responsive to conventional-dose chemotherapy but long term disease-free survival (DFS) is uncommon. High-dose chemo-radiotherapy followed by ASCT has the potential to induce remission in this disease but the long-term benefit of this modality remains to be determined. Methods: Between 1990 and 2003, we transplanted 52 pts originally diagnosed with low-grade FL (31 grade 1, 21 grade 2). Twenty-five (48%) had biopsy-proven large cell transformation (FL grade 3 or diffuse large cell lymphoma) before ASCT. The median number of prior therapies was 2 (range: 1 to 7). Prior to ASCT, 45 pts (87%) were responsive to salvage therapy with 20 pts (38%) in CR. Five pts (10%) had chemo-resistant disease at the time of ASCT. High-dose regimens included BCNU-cyclophosphamide-etoposide (31%), melphalan/TBI (27%), and cyclophosphamide/TBI (25%). Thirty-eight pts (73%) received peripheral stem cells (PSCT) and 14 pts (27%) received autologous bone marrow (BM) with 4-hydroxyperoxycyclophosphamide (4-hc) purging in 9 cases (17%). The median age was 49 yrs (range: 29–65). Results: There was 1 treatment-related death during the first 100 days. After ASCT, 36 pts (69%) achieved a CR, 2 (4%) had a PR, and 7 (13%) had stable disease. Among those in CR, 20 (56%) had a CR pre-ASCT, 14 (41%) had a lesser response, and 1 (3%) was chemo-resistant. Median follow-up (f/u) of survivors was 5.3 yrs (range: 1.7 months to 12.4 yrs). The median overall survival (OS) has not yet been reached. The median event-free survival (EFS) is 3.4 yrs (range: 1.7 months to 12.4 yrs). Among complete responders, more than 50% are disease free at last follow-up (range 1.7 months to 12.1 yrs). Variables favorably affecting EFS and OS are age 〈 60 yrs (p = 0.007, 0.015 respectively), achievement of a CR after ASCT (p = 0.002, 0.001), absence of transformation (p = 0.038, 0.017), BM vs. PSCT (p = 0.042, 0.086), and 4-hc BM purging (p = 0.044, 0.059). Number of prior regimens, response prior to ASCT, type of preparative regimen, and addition of TBI, were not significantly associated with EFS, DFS, or OS. In multivariable analysis, achievement of CR after ASCT and age 〈 60 yrs are the only significant predictors of EFS and OS. Adjusted for age, 53% of pts with a CR after ASCT are alive and event-free at last f/u (range: 2.4 months to 12.4 yrs) (Figure 1). In contrast, the median EFS among pts without a CR is 0.5 yrs (range: 1.7 months to 5.3 yrs). Conclusion: ASCT is a reasonable therapeutic approach to FL, resulting in long term EFS for some pts, even with relapsed, refractory and/or transformed disease. In our experience, significant predictors of EFS and OS after ASCT are complete response and age

Description: CD4+CD25+ T-regulatory (Treg) cells have been shown to critically regulate self- and allograft tolerance in several model systems. Studies of human Treg cells have been restricted by the small number present in peripheral blood and their naturally hypoproliferative state. To better characterize Treg suppressor cell function, we determined methods for the isolation and expansion of these cells. Stringent magnetic microbead-based purification was required for potent suppressor cell line generation. Culture stimulation with cell-sized Dynabeads coated with anti-CD3 and anti-CD28 monoclonal antibodies, CD4+ feeder cells, and interleukin 2, provided for marked expansion in cell number (100-fold), with retention and enhancement of suppressor function. The potent Treg cell lines suppressed proliferation in dendritic cell-driven allo-mixed lymphocyte reaction (MLR) cultures by more than 90%. The Treg-derived suppressor cells functioned early in allo-MLR because expression of activation antigens and accumulation of cytokines was nearly completely prevented. Importantly, cultured Treg cells also suppressed activated and matured dendritic cell-driven responses. These results demonstrate that short-term suppressor cell lines can be generated, and they can express a very potent suppressive activity. This approach will enable more detailed biologic studies of Treg cells and facilitate the evaluation of cultured Treg cells as a novel form of immunosuppressive therapy. (Blood. 2004;104:453-461)

Description: IPI-504 is a novel inhibitor of Hsp90 based on the geldanamycin pharmacophore. When placed in rat, monkey, and human blood, IPI-504 rapidly converts to the known and well-studied compound 17-allylamino-17-demethoxy-geldanamycin (17-AAG). 17-AAG is the subject of multiple clinical trials for the treatment of hematologic and solid tumors. However, 17-AAG suffers from poor aqueous solubility necessitating the use of sub-optimal formulations to deliver this agent to patients. IPI-504 is over 1000-fold more soluble than 17-AAG in aqueous solution. In vitro, both 17-AAG and IPI-504 bind tightly to, and selectively inhibit Hsp90 derived from cancer cells. The cytotoxic effect of IPI-504, as well as its ability to stimulate the degradation of Hsp90 client proteins and increase the intracellular levels Hsp70, were monitored in two human multiple myeloma cells lines (RPMI-8226 and MM1.S). The effects of IPI-504 were compared to 17-AAG. We demonstrate that the actions of IPI-504 are bioequivalent to 17-AAG and that both compounds induce apoptosis in these cells and stimulate the degradation of HER2 and c-Raf. In addition, both agents stimulate Hsp70 protein levels. In all cases the EC50s are virtually the same for both molecules (~200–400 nM). Furthermore, IPI-504 inhibits the secretion of immunoglobulin light chain from the RPMI-8226 multiple myeloma cells (EC50 ~300 nM). Importantly, IPI-504 is active in tumor xenograft models of multiple myeloma. The data indicate that active metabolites of IPI-504 accumulate in these xenografts long after these metabolites are cleared from the plasma compartment, suggesting that they preferentially accumulate in tumor cells based on their increased affinity to Hsp90 derived from tumor cells. In conclusion, we have developed IPI-504 as a novel, potent inhibitor of Hsp90 with greatly increased solubility over 17-AAG, and that IPI-504 is an active anti-tumor agent in vitro and in vivo.

Description: Plasma hemoglobin (Hb) is a scavenger of nitric oxide (NO), which is a likely contributor to the pathogenesis and treatment of clinical abnormalities such as pulmonary hypertension and vasocclusive episodes in sickle cell anemia (SCA) and possibly in thalassemia syndromes, where pulmonary hypertension has been described most frequently in splenectomised patients with thalassemia intermedia (TI). Since plasma Hb consists of both free Hb and RBC-derived microvesicle Hb (Greenwalt. Vox Sang1991;61:14), the relationship between plasma Hb and circulating vesicles may be significant. We have measured plasma Hb and plasma vesicle levels in adults with SCA and thalassemia intermedia (TI). Patients with SCA were all untransfused and TI patients had no transfusions during the previous 3 months. Plasma Hb values in healthy adult controls (1.77±0.2, n=7) were significantly lower than in SCA (11.21±2.08mg/dl, n=15, p=0.003) or in splenectomised TI patients (48.46 ±3.66mg/dl, n=5, p=0.0038). Plasma Hb levels were significantly greater in TI as compared to levels in SCA (p=0.001). Vesicle numbers in SCA were 12.59±3.65 x103/ul (n=21, p〈 0.001, SCA v control) and in TI were 24.19 ±12.23 (n=7, p〈 0.001, TI v control). Thus both plasma Hb and circulating vesicle levels in SCA and TI were significantly greater than healthy controls. Plasma Hb was significantly greater in TI than in SCA and circulating vesicles markedly greater in TI than in SCA. Furthermore there was a significant correlation between plasma Hb and vesicle numbers in SCA, TI and normal controls (n=26, R2=0.59, p=0.0015). An analysis of splenectomised versus non-splenectomised TI patients revealed a further trend; both plasma Hb and vesicle numbers were significantly higher in splenectomised (n=5, n=7 respectively) than in non-splenectomised patients (n=4, n=4 respectively). Plasma Hb and vesicle numbers were respectively 48.5±3.6 and 24.19±12.2 in splenectomised patients compared to 17.18±5.58 (p=0.014) and 4.36±0.81 (p=0.008) in non-splenectomized TI patients. These findings show that raised plasma Hb levels are related to increments in vesicle numbers in TI and SCA. Splenectomy in TI, which is associated with increased risk of pulmonary hypertension and thrombosis, is associated with increased vesicle numbers and plasma Hb. We suggest that the scavenger characteristics of Hb containing vesicles for NO may differ from the scavenger characteristics of free Hb and thus require detailed study..

Description: High-dose melphalan followed by ASCT is a common component of the early treatment for patients with multiple myeloma. Daily subcutaneous injections of filgrastim (Neupogen) at 5 ug/kg/day until ANC 〉 500/ul are routinely administered at our center from day +4 following ASCT, in order to accelerate hematopoietic recovery and lessen neutropenic complications. Pegfilgrastim (Neulasta) as a single 6 mg fixed dose subcutaneous injection has been shown to have similar efficacy and ease of use when compared to filgrastim in the non-transplant setting, but little data is available in the transplant setting. We began using pegfilgrastim day +1 following ASCT for patients with multiple myeloma and performed a retrospective cohort study comparing those who received filgrastim (n=6) with those who received pegfilgrastim (n=11). Transplants occurred between July 2002 and January 2004 and included all patients transplanted for myeloma in that time period for whom sufficient data was available. All patients had at least 2 x 106 CD34+ cells/kg peripheral stem cells harvested after cytoxan and filgrastim mobilization. Main outcome measures were: days from stem cell infusion to WBC nadir, days to ANC〉500/ul, and days to ANC〉1000/ul. Subjects were excluded if CBCs were drawn less frequently than every four days. There were no significant differences between the filgrastim and pegfilgrastim groups with respect to the following demographic variables: age, gender, hemoglobin, creatinine, calcium, albumin and beta-2 microglobulin at diagnosis. The groups were also balanced with respect to SPEP, UPEP, presence of lytic lesions and number of prior lines of therapy. The median number of CD34+ cells infused was similar: 5.7 x 106 in the filgrastim group vs 4.8 x 106 in the pegfilgrastim group (p=0.28). After transplant, median number of days to WBC nadir in the filgrastim group (FG) was 7 (range 5–9) vs 6 (range 5–8) in the pegfilgrastim group (PG) (p=0.31). However, median number of days to ANC〉500/ul in the FG was 11.5 (range 11–17) vs 10 (range 9–12) for PG (p=0.02). Similarly, median number of days to ANC〉1000/ul was 12 (range 11–17) for FG vs 11 (range 10–13) for PG (p=0.03). Five of six patients in the FG had neutropenic fever after transplant, compared to five of eleven patients in the PG (p=0.30). Currently, no significant differences in infection or relapse rates between groups have been noted and there were no deaths in either group. In this retrospective cohort study, pegfilgrastim was safe and at least equivalent to filgrastim for accelerating hematopoiesis after ASCT for multiple myeloma. Furthermore, there was no significant difference in the incidence of neutropenic fever, infection and survival, suggesting a similar clinical utility.

Description: CD4+CD25+ regulatory T cells (Tr) are negative regulators of immune responses. Studies of human Tr are restricted by their small numbers in peripheral blood and their hypoproliferative state. A recently established method achieved in vitro expansion and generation of Tr cell lines (Godfrey et al; Blood 2004,104:453-61). This approach facilitates the evaluation of cultured Tr cells as a novel form of immunosuppressive therapy and provides a system for molecular analysis of Tr. Activation of Ras and MAP kinases is mandatory for IL-2 production, viability and cell cycle progression of T cells. In anergic T cells activation of these signaling events is impaired, whereas activation of Rap1 is retained. Subsequently, anergic cells have defective IL-2 production, impaired cell cycle progression, and increased susceptibility to apoptosis. In the current study, we sought to determine the signaling and biochemical properties of Tr. Human CD4+CD25+ (Tr) and control CD4+CD25− (Tc) cell lines were generated from human cord blood cells. We examined activation of Ras, Rap1 and MAP kinases as well as cell cycle progression and cell viability, in response to TCR/CD3-plus-CD28 mediated stimulation. Stimulation was done for 15 min, 2 and 16 hrs for assessment of signaling events or for 24, 48 and 72 hrs for assessment of cell cycle progression and viability. Although activation of Rap1 was not affected, activation of Ras was reduced in Tr as compared to Tc. Activation of JNK and Erk1/2 MAP kinases was also significantly impaired. Both Tr and Tc entered the cell cycle and expressed cyclin E and cyclin A at 24 and 48 hrs of culture. However, p27 was downregulated only in Tc and not in Tr and hyperphosphorylation of Rb, which is the hallmark of cell cycle progression, was detected only in the Tc and not in the Tr population. At 72 hrs of culture, expression of cyclin E and cyclin A was dramatically diminished in Tr whereas it remained unchanged in Tc. More strikingly, expression of p27 in Tr was increased to levels higher than background. Since Tr do not produce IL-2, we examined whether addition of exogenous IL-2 would downregulate p27 and rescue Tr from defective cell cycle progression, similarly to its effect on anergic cells. Addition of exogenous IL-2 resulted in decrease of p27, sustained increase of cyclin E and cyclin A and cell cycle progression. Besides inhibiting cell cycle progression, p27 also promotes apoptosis. Therefore, we examined whether Tr had a higher susceptibility to apoptosis. As determined by Annexin V staining, Tr had a high degree of apoptosis only at 72 hrs of culture, when p27 expression was highly upregulated. Exogenous IL-2 reversed both p27 upregulation and apoptosis. Addition of IL-2 to Tr, also resulted in sustained IL-2-receptor-mediated activation of Erk1/2 at levels equivalent to those of Tc. Thus Tr cells share many biochemical and molecular characteristics of anergy, including defective TCR/CD3-plus-CD28-mediated activation of Ras and MAP kinases, increased expression of p27, defective cell cycle progression and high susceptibility to apoptosis. Moreover, these results suggest that TCR/CD3-mediated and IL-2 receptor-mediated signals converge at the level of MAP kinases to determine the fate of Tr cells towards expansion or cell cycle arrest and subsequent apoptosis.

Description: Purpose: Determine the current pattern of use of non-ablative and reduced intensity conditioning regimens (NST) with volunteer unrelated donor progenitor cell transplantation (URD-T) in patients with malignant diseases, and identify potential prognostic factors for transplant outcomes. Patients and Methods: The National Marrow Donor Program (NMDP) database was queried to identify adult patients (greater than 18 years old) who had undergone an URD-T from 01/01/94 until 05/31/01, had a malignant disorder and received a conditioning regimen which fulfilled one of the following criteria: 500 cGy or less of total body irradiation, 9 mg/kg or less of total busulfan dose; 140 mg/m2 or less total melphalan dose and included a purine analog (fludarabine, cladribine, or pentostatin). A total of 293 patients were available for analysis of which 38% had acute leukemia or MDS, 18% had CML or another myeloproliferative disorder (MPD), 33% had CLL or lymphoma and 11% had a plasma cell disorder (PCD). The following transplant outcomes were analyzed: acute and chronic graft versus host disease (GVHD), non-relapse mortality (NRM), overall survival (OS) and event free survival (EFS). Results: The number of URD-T performed as NST have increased for all disease categories. From 1995 to 1999, 24 transplants were performed for acute leukemia (AML, ALL and MDS); 13 for MPD; 25 for lymphoproliferative disorders (CLL and lymphoma) and 1 for PCD. In the year 2000 alone these increased to 56 transplants for acute leukemia, 53 for CLL or lymphoma, 30 for MPD and 20 for PCD. When compared to 7015 NMDP recipients of ablative allografts performed during the same time period, recipients of NST were older (52 vs. 32 years), were more likely to have undergone transplant in a disease phase other than CR1, CR2 or first Chronic Phase (79% vs. 51%) and were more commonly treated for NHL and AML. NST patients had received a prior transplant in 63% of cases, and 35% had a Karnofsky performance score (KPS) of 〈 90 prior to transplant. The incidence of Grade II-IV acute GVHD for the whole group was 39% ± 5%. The 100 day and 1 year NRM rates were 20% ± 4% and 31% ± 5% respectively. Currently 83 patients are alive at a median of 985 days (range = 99–1825) with 77 patients free of progression. The 2 year OS and EFS rates are 33% ± 5% and 31% ± 5% respectively. On multivariate analysis favorable prognostic factors for OS were better HLA match, blood versus marrow stem cells and pre-transplant KPS ≥ 90%. Hazard Ratio 95% Confidence Intervals HLA Matching Match 1 Potential Match 4.701 3.097–7.136 Mismatch 2.128 1.509–3.001 Karnofsky Performance Scale 〈 90 1 ≥ 90 0.607 0.443–0.834 Stem Cell Source Peripheral Blood 1 Bone Marrow 1.602 1.151–2.229 Conclusions: URD-T with NST regimens are being performed more frequently, particularly in older patients. Although feasible and effective in a small fraction of patients, the procedure is still associated with significant morbidity and mortality. Factors relevant for outcomes after conventional myeloablative allografting (HLA match and performance status) remain important for outcomes after NST.

Description: High-dose therapy and autologous stem cell transplant (SCT) is an option for patients with AML most commonly performed in first complete remission (CR1) or CR2. Stem cell (SC) collection in CR1 typically follows consolidation. SC collection in CR2 is limited by the need to achieve a second remission prior to harvest, and the use of additional induction therapy that may result in marrow toxicity and SC depletion. In addition, a SC product collected in CR2 might be more likely to have leukemia cell contamination that could contribute to subsequent relapse. Prophylactic collection of SC from patients in CR1 who are not imminently going on to SCT may therefore be reasonable and theoretically could improve outcome of SCT in CR2. However, many patients never relapse, and for those who do, alternative options, and the need to achieve a 2nd CR, further limit the chance that these cells will be used. The morbidity and cost of SC collection, and the need for prolonged storage, call into question the practice of routine prophylactic SC harvest. To determine the utility of SC collection in CR1, we identified 67 patients with AML who had autologous SCs collected between 1995 and 2002. Charts were reviewed to assess whether the collection was prophylactic or for immediate use and we reviewed the timing and outcomes of transplant. 61/67 patients had SCs collected in CR1, 5 in CR2, 1 in CR3. 22 had collection for imminent therapeutic use and 45 for potential future use. Among the 22 patients whose cells were collected for planned SCT, cells were collected in CR1 in 17 cases and CR2 in 5 cases. 11 (50%) remain in CR a median of 58 mo (range 4–103) after SCT, and 11 died a median of 11 mo (4 – 28) after SCT. Causes of death were relapse (n=8), transplant related mortality (TRM) (n=1), TRM after allogeneic SCT (n=1), and unrelated causes (n=1). Of 17 patients transplanted in CR1, 8 (47%) remain in CR. Of the patients whose cells were both collected and used in CR2, 3/5 remain in remission 8, 53, and 103 mo after SCT and 2 died of relapse 10 and 24 mo after SCT. 5/45 patients whose stem cells were collected prophylactically in CR1 used these cells for SCT in CR2 a median of 16 months (15 – 28) after collection. 2/5 of these patients remain in CR 21 mo and 51 mo and 3 had died of relapsed disease 9–12 mo after SCT. Stem cells remain unused in 40 of these patients and 21 (53%) remain in CR a median of 39 mo (7–98 ) after collection. 15/21 remain in CR without further therapy. 3 patients are currently alive receiving therapy for relapse and therefore may use cells in the future. 6 patients are in CR after allogeneic SCT in CR1 (2), CR2 (3) or untreated relapse (1). 16/40 will never use stored cells. 12 died of disease a median of 10 mo (4–22) after prophylactic SC collection and 4 died from complications of allogeneic SCT in CR1 (3) or CR2 (1). In summary, of the 45 patients with SCs harvested prophylactically in AML CR1, 5/21 (23%) who required further therapy went on to use these cells with 2/5 long-term survivors. Another 24 have done well with initial therapy but remain at risk for relapse and may use SCs in the future. In our experience, prophylactic autologous SC harvest and storage from patients with AML in CR1 remains a reasonable option. Additionally, SCT for AML in CR has resulted in frequent long-term remission at our institution.

Description: Relapse remains the major limitation to successful autologous stem cell transplantation for Hodgkin’s lymphoma (HL) and is associated with limited long-term survival. There is now strong evidence that allogeneic donor cells can induce a direct and potent graft vs tumor reaction in patients with HL, ie, a Graft vs HL (GvHL) effect. Based on these findings, we evaluated the outcomes for 17 patients with incurable HL, all having relapsed after high dose therapy with autologous PBSC rescue, treated with non-myeloablative conditioning with allogeneic stem cell transplant (NMT). Patients were conditioned with fludarabine/cytoxan +/− Campath followed by allogeneic peripheral blood stem cells mobilized with G-CSF (12 from HLA-identical sibling donors, 3 from matched unrelated donors, 2 from single antigen mis-matched unrelated donors). Median recipient age was 37 (range 12–51). Median time from prior autotransplantation to relapse was 4.8 months (range 1–15m); median time from autotransplantation to NMT was 12m (range 5–53m), identifying this cohort as an extremely poor risk group. Conditioning in all patients included fludarabine 90–120 mg/m2/d combined with either ‘low dose’ cytoxan (900 mg/m2/d over 3 d) in 11 patients with sibling donors; or with ‘high dose cytoxan (3–4 gm/m2) in 6 pts with unrelated donors. 4 pts with unrelated donors, receiving high dose cytoxan with fludara also received 100 mg Campath 1-H divided over 5 days from D-9 to D-5. Post transplant GvHD prophylaxis consisted of cyclosporine (CSA) alone in 7 pts, CSA/methotrexate in 4 pts, and CSA/mycophenolate mofetil in 6 pts. Median MNC content of the graft was 7.5 x 108/kg (range 2.4–14); median CD34+ content was 4.1 x 106/kg (range .9–15.4). The median survival post allotransplant for all patients was 21.5 months (range 1–48.5m) and EFS was 8.5 months (range 1–41.5m) and was not influenced by graft source (sibling vs URD) or conditioning regimen. The 100 day TRM for all patients was 12%. The incidence of grade II-IV acute GvHD in 15 evaluable patients was 6/15 (40%). The incidence of chronic GvHD was 4/15 (27%) and was not correlated to a lower relapse rate among affected patients. Two patients were inevaluable for chronic GvHD and relapse secondary to early TRM. Of the 15 patients evaluable for response, 7 achieved CR (46%), 6 PR (40%), and 2 SD for an overall response rate of 86% in this chemoresistant cohort. Despite a high response rate, death from progressive disease occurred in the majority of patients. These data suggest a GVHL response can be induced, contributing to a response rate beyond what one would expect from salvage chemotherapy alone in this resistant cohort. However, the durability of response remains a limiting factor. Further studies designed to combine cytoreduction with allogeneic cellular therapy to enhance GVHL activity are warranted to improve outcomes after NMT for patients with poor-risk Hodgkin’s Lymphoma.

Description: CD4+CD25+ T regulatory (Treg) cells have been shown to critically regulate self and more recently allograft tolerance in mice. Studies of human Treg have been hindered by the presence of CD25-dim conventional T cells that copurify during Treg isolation. We compared adult and cord blood sources for Treg, with the hypothesis that cord blood should lack significant numbers of memory cells or environmentally reactive T cells. We found that cord blood was a superior source for Treg isolation compared to adult blood. CD4+CD25+ cells were readily purified, and generated cell lines that consistently exhibited potent suppressor activity, with 〉95% suppression of allogeneic MLR (29/30 donors). The cells could markedly suppress an allo-MLR at a 1/16-1/32 suppressor/responder cell ratio. The cultured Treg cells blocked cytokine accumulation in MLR, with a less robust inhibition of chemokine production. Cultured Treg uniformly expressed CD25, CD62L, CCR7, CD27, and intracellular CTLA4. Upon re-stimulation with anti-CD3/CD28 beads, the cultured Treg produced minimal cytokines (IL2, IFN-gamma, and IL10), and preferentially expressed TGF-beta latency associated protein on the cell surface, while conventional CD4+CD25− derived cell lines did not. Cytokine production however, could be largely restored by stimulation with PMA/ionomycin. Abundant FoxP3 mRNA was detected in fresh, cultured, and anti-CD3/CD28 bead restimulated CD4+CD25+ cells (approximately 2–4 fold less than cyclophillin A). Low amounts of FoxP3 mRNA were present in fresh CD25− cells, and they increased 32 fold on culture. However, FoxP3 protein, as assessed by western blot, was specifically expressed in the CD25+ derived cell lines, and was not detected in the CD25− derived cell lines. Restimulation with anti-CD3/CD28 beads led to increased expression of FoxP3 protein in CD25+ derived cell lines, but not in CD25− derived cell lines. Cord blood derived cultured suppressor cells were not cytolytic in chromium release assays, and mediated suppression of alloreactivity that was cell contact dependent and predominantly independent of IL10 and TGF-beta. Antibodies to GITR, GITR-L, OX40, OX40-L, CTLA4, and PD1 did not significantly affect suppression of the culture activated Treg cells. These results demonstrate virtually pure populations of potent suppressor cells can be cultured from cord blood, and these cell lines form an ideal model system for the evaluation of suppressor cell biology and mechanisms of action.