ALBERT

Description: CD infection is a common complication in immunocompromised patients, including those undergoing HSCT, with reported incidences ranging from 4% to 13%. However, there has been no previous detailed report on the clinical impact of positive CD toxin in patients after allogeneic HSCT with current supportive therapies. Therefore, we retrospectively reviewed the medical records of 422 patients who underwent allogeneic HSCT with a variety of preparative regimens at National Cancer Center Hospital from January 2000 to April 2004. CD toxin in stool samples was measured by the Latex particle agglutination method, and selected patients were examined further by endoscopy. Positive results with CD toxin were observed at least once in 51 patients, at a median time of 40 days (range, −1 to 212 days) following allogeneic HSCT. Most patients had severe watery diarrhea, along with other symptoms including fever, nausea, anorexia and abdominal cramping, which mimics pseudomembrane colitis. Twenty-seven of the 51 patients (53%) underwent endoscopic examination, and macroscopic findings were as follows; normal in 4 (15%), extensive edema in 11 (41%), and mucosal sloughing in 12 (44%), while none had pseudomembrane formation. Histological diagnosis revealed that 26 cases (96%) were compatible with GVHD, including 2 complicated with Cytomegalovirus colitis. Thirty-three patients (65%) were treated with oral vancomycin for a median of 14 days (2 to 46 days), while the remaining 18 were followed conservatively without any specific medication. Forty-four patients were evaluable for follow-up, and, regardless of management, all subsequently became negative for CD toxin. These findings suggest that positive CD toxin in the stool detected during the course of allogeneic HSCT may only reflect nonsignificant colonization or proliferation of CD in the gut, and hence may not necessarily imply clinically relevant pseudomembranous colitis which would require intense medical treatment. Additionally, in patients undergoing allogeneic HSCT, signs and symptoms of intestinal involvement may simply reflect concomitant GVHD per se or a pathology closely related to GVHD.

Description: Abstract 2284 Poster Board II-261 Background: To evaluate the role of autologous and allogeneic SCT in the treatment of PTCLs, Japan Study Group for Cell Therapy and Transplantation conducted a multicenter retrospective survey in Japan and Korea. Methods: After excluding patients with adult T-cell leukemia/lymphoma and NK-cell tumors, patient data were newly collected from 330 patients (222 male and 108 female) with a median age of 49 years (range, 13–71) who underwent SCT between 9/1991 and 12/2008 (196 autologous and 134 allogeneic including 31 patients with previous autograft). Allogeneic SCT (53 BM, 54 PB, 1 BM+PB, 26 CB) was performed using a reduced-intensity conditioning (RIC) in 84 patients (63%). While a pathologic central review will be performed, currently there were 159 (48%) patients with PTCL, not otherwise specified, 63 (19%) with angioimmunoblastic T-cell lymphoma, 47(14%) with anaplastic large cell lymphoma (23 ALK-negative, 14 ALK-positive and 10 unknown), 12 (4%) with enteropathy-associated T-cell lymphoma, and others. The disease status at transplant in the allo-group was significantly worse than that in the auto-group (Table 1). The median number of chemotherapy regimens was 2 (range, 1–7), and the median duration between diagnosis and transplant was 267 days (range, 120-4889 days). Results: The median follow-up for surviving patients was 45 mo (range, 2.3–141 mo). There was no significant difference in overall survival among different groups, including histological subtypes, RIC and myeloablative conditioning in the allo-group and high-dose chemotherapy regimens in the auto-group. Early survival rate after transplant was significantly better for auto-group than allo-group (Wilcoxon P=0.001), but the difference was marginal in the total course (Logrank P=0.06) (Figure). The non-relapse mortality (NRM) in the auto-group was significantly lower than that in the allo-group (P1), cell source (CB/BM+PB), and performance status (PS; 〉1), stage, chemorefractory disease, international prognostic index (IPI; H-I/H risk) and prognostic index for PTCL, unspecified (PIT; group 3/4) at transplant. The risk factors in the allo-group were bulky mass at diagnosis, age (〉50 years), cell source, and PS, stage, IPI and PIT at transplant, while those in the auto-group were age (〉40 years), recurrence after frontline therapy, number of chemotherapy regimens, and stage, chemorefractory disease, IPI and PIT at transplant. Conclusions: Despite a worse disease status at transplant in the allo-group, the overall survival was comparable to that in the auto-group. This supports the notion that early allogeneic SCT is a valuable treatment option for PTCLs, although a large-scale randomized trial to identify a suitable upfront-transplant type for chemosensitive patients with PTCLs is warranted. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2281 Poster Board II-258 Background: A decision analysis using the Markov process is a flexible and convenient analytical method that tracks the clinical events that occur after a certain decision with different probabilities and utilities over time. To address the role of allogeneic hematopoietic cell transplantation (allo-HCT) for acute myeloid leukemia (AML) in CR1, we performed a Markov decision analysis using newly collected clinical data from 2029 patients. Methods: Probabilities and other outcome data were derived from a database of adult AML patients that was constructed from case report files collected for this study. We included patients who were diagnosed with AML other than M3 between 1999 and 2006, aged 16 to 70 years, and who had achieved CR1 after 1 or 2 courses of induction chemotherapy. Using the software package TreeAge Pro 2009, we constructed a Markov decision model that compared 2 strategies: allo-HCT in CR1 (HCT group) and no allo-HCT in CR1 (CTx group). Possible health states considered in each decision included, for the HCT group, 1) no relapse without chronic GVHD, 2) no relapse with chronic GVHD, 3) relapse, and 4) dead, and, for the CTx group, 1) no relapse, 2) relapse, 3) second remission, 4) after salvage allo-HCT, and 5) dead. Quality-of-life (QOL) adjustments were made by incorporating time trade-off utilities that were derived from a questionnaire to 12 physicians who were familiar with the treatment of AML. The cycle length between state transitions was set at 3 months. Results: A total of 2029 patients were eligible for this analysis. The median age was 50 years, and the median follow-up of the surviving patients was 4.10 years. The proportions of patients with favorable, intermediate, unfavorable and unknown cytogenetic risk by SWOG criteria were 20%, 54%, 17% and 9%, respectively. Therapies performed at CR1 were allo-HCT in 494 patients (24%) and chemotherapy in 1535 patients (76%). Among 1076 patients whose HLA typing was performed for allo-HCT in CR1, 431 had HLA-matched or 1-antigen-mismatched related donors. Life expectancy and quality-adjusted life expectancy for the HCT and CTx groups in each risk category are summarized in Table 1. Life expectancy of the HCT group was longer than that of the CTx group (4.96 years vs. 4.44 years). However, quality-adjusted life expectancy of the HCT group was comparable to that of the CTx group (4.06 years vs. 3.98 years) due to a larger reduction of expected life length in the HCT group after QOL adjustment. In a subset analysis of the CTx group, patients with more favorable cytogenetic risk had a longer life expectancy. Whereas allo-HCT in CR1 was associated with a shorter life expectancy in patients with favorable-risk AML, allo-HCT in CR1 was associated with a longer life expectancy in those with intermediate-risk or unfavorable-risk AML. Adjustment for QOL did not change the preferred decision in the intermediate- and unfavorable-risk groups, although the survival advantages for allo-HCT in CR1 were less than those without QOL adjustment. In a subset of patients who had related donors, both life expectancy and quality-adjusted life expectancy were longer in the HCT group. Conclusion: The results of our decision analysis using the Markov process indicated that patients with intermediate- or unfavorable-risk AML have a longer life expectancy and quality-adjusted life expectancy with a decision of allo-HCT in CR1. Our results also showed that a Markov decision analysis that incorporates QOL may be useful as a decision-making tool for patients who might be candidates for allo-HCT. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3370 Poster Board III-258 Although the outcome of patients (pts) with adult T-cell leukemia-lymphoma (ATL) remains poor when they are treated with conventional chemotherapy, we previously showed in a multi-center prospective study that one-third of pts who underwent RIST from a related donor in CR or PR could survive without disease for more than 2 years (Tanosaki R et al., BBMT 2008). In this retrospective study, we reviewed our single-center experience with RIST for ATL pts, focusing on the outcome of those who underwent RIST in non-remission status or who relapsed after RIST. A total of 24 pts underwent RIST from a related donor between 2001 and 2008. The median age was 54 years (range, 44-65). Of the 14 males and 10 females, 19 were acute type and 5 were lymphoma type. Disease status at transplantation was 5 CR, 10 PR, 8 NC and 1 PD. Donors were siblings in 18 and children in 6, including 5 HTLV-1 healthy carriers. HLA in serology was 6/6 in 19 and 5/6 in 5. Stem cell sources were PBSC in 22 and BM in 2. Conditioning regimens were fludarabine (30 mg/m2 iv days -8 to -3) and busulfan (3.2 mg/kg iv days -6 and -5) with (n=8) or without (n=16) rabbit anti-T-lymphocyte globulin (ATG, Fresenius; 2.5 mg/kg iv days -2 and -1). All patients received cyclosporine alone for GVHD prophylaxis. Engraftment was rapid in all 24 pts (neutrophil〉500/uL; median 12 days, range 10 -19), with no graft failure. There were 3 non-relapse mortalities; respiratory failure from bronchiolitis obliterans at 21 months (mos), interstitial pneumonitis at 47 mos, and pneumococcal sepsis at 38 mos. Notably, 10 of the 19 pts who were non-CR at RIST survived without disease progression to a median of 53 mos (range, 20 to 85). All of these pts were acute type, and had circulating ATL cells in the peripheral blood (PB) immediately before RIST (average 33% of WBC, range 5-73). Circulating ATL cells decreased to below 5% within 1 mo in 8 pts. A total of 12 pts relapsed within 16 mos; 7 (58%) within 3 mos, and 11 (92%) within 12 mos. Two patients who had relapsed after RIST showed a significant but transient response to the withdrawal of immunosuppression (CR 1, PR 1). Donor lymphocyte infusion was performed in 6 pts without significant benefits. Seven pts who relapsed at a single site, which was confirmed by CT scan or FDG-PET, were treated with local irradiation alone, and 3 whose HTLV-1 proviral load in PB had become negative at relapse survived to 48, 64 and 77 mos; 1 pt required 2 courses of irradiation because of immediate relapse at the margin of the preceding radiation field, and another pt underwent surgical resection of a residual mass since a biopsy revealed a viable lesion at the irradiated site. The 5-year overall and progression-free survival of all pts were 52% (95% CI, 38-66%) and 37% (95% CI, 22-52%), respectively, at a median follow-up of 59 mos (range, 12 to 85) in surviving pts. HTLV-1 proviral load in PB was examined using real-time PCR for tax in 208 samples from 21 evaluable pts, and it became negative at least once in 15 pts (71%), including 1 pt whose donor was an HTVL-1 carrier; proviral load remained negative in 7 pts at a median follow-up of 32 mos (range, 3 to 84). Since HTLV-1 tax is a promising target molecule for identifying the immunological mechanism, HLA-restricted tax-specific CTLs were examined in HLA-A2- and/or A24-positive pts using tax tetramers by taking blood samples periodically after informed consent was obtained from each pt. A total of 80 samples in 13 pts were analyzed. The number of tax tetramer-positive (tax+) cells did not change significantly up to at least 1 year after RIST, while the clinical responses and decrease/disappearance of HTLV-1 proviral load were observed within 3 mos in most cases. An increase in tax+ cells was observed after 1 year in 2 pts who had achieved CR. In conclusion, about half of the acute-type ATL pts with a significant involvement of ATL cells in PB at RIST could survive for a long time in our cohort. ATL pts who relapsed at a single site after RIST still have a chance to be cured with local treatment using irradiation alone or surgical resection with the aid of HTLV-1 proviral load as a marker for minimum residual disease. Since most ATL pts had already become resistant to chemotherapy and the intensity of conditioning was reduced, potent GV-ATL and GV-HTLV-1 effects might have played a key role in disease control. However, tax-specific CTL kinetics did not correlate with either clinical responses or the HTLV-1 proviral load, which suggested that other molecules may be immunologically targeted. Our results might contribute to the establishment of the cure-oriented treatment strategy for ATL pts. Disclosures: No relevant conflicts of interest to declare.

Description: Since allogeneic hematopoietic stem cell transplantation (HSCT) is reported to have a high mortality rate for advanced lymphoma patients, less toxic technique has been expected. We evaluated the safety and preliminary efficacy of RIST in patients with advanced NHL on the basis of the WHO classification. We retrospectively reviewed the medical records of 52 patients with NHL who underwent RIST from February 2000 to April 2004 at National Cancer Center Hospital. The median age of the patients at the time of RIST was 50 years (range, 26 to 67 years). Eighteen patients had indolent B-cell lymphoma (follicular 16, lymphoplasmacytic 1, chronic lymphocytic leukemia 1), 11 had aggressive B-cell lymphoma (diffuse large B cell 8, mediastinal large B cell 2, mantle cell 1), and 23 had T-cell and NK-cell lymphoma (adult T-cell leukemia/lymphoma 12, peripheral T-cell, unspecified 8, extranodal NK/T-cell 1, enteropathy-type T-cell 1, anaplastic large cell 1). The median number of prior chemotherapy regimens was 3 (range, 1–8), and 17 (33%) patients had experienced autologous HSCT before RIST. Thirty (58%) patients had progressive/refractory disease at the time of transplantation. The RIST regimens were fludarabine- (n=47) and cladribine-based (n=5), of whom 9 patients received additional anti-thymocyte globulin. Stem cell sources were related peripheral blood (n=36), unrelated bone marrow (n=8), and unrelated cord blood (n=8). GVHD prophylaxis was cyclosporin alone (n=36) or cyclosporin and short-term methotrexate (n=16). The median duration of follow-up after transplantation was 309 days (range, 2 to 1492 days). Except for one patient who died early on day 2, all patients showed sustained engraftment. Acute GVHD gradeII–IV developed in 32 patients (62%), gradeIII–IV developed in 12 (23%) and chronic GVHD developed in 29 (56%). The day-100 TRM rate was 6%, including sepsis (n=2) and liver failure (n=1). The estimated one-year overall-survival and estimated progression-free survival rates were 75% and 63%, respectively, for all patients; 94% and 94% for indolent B cell, 76% and 58% for T or NK cell and 42% and 21% for aggressive B cell, as shown in Figures 1 and 2. (P = 0.0037 and, 0.0004 by log-rank test). In our analysis, GVHD did not clearly affect the clinical response or survival. Sustained engraftment and the low TRM rate indicate that RIST is feasible in patients with relapsed or refractory NHL. Although this is a preliminary study with a heterogeneous population and a short follow-up, the results suggest that RIST might be a promising procedure in relapsed or refractory NHL, especially in indolent B-, T- and NK-NHL. Further large-scale prospective studies are warranted.

Description: [Introduction] The effectiveness of allogeneic hematopoietic stem cell transplantation (allo-HSCT) against autoimmune disease has been suggested by studies in animal models and patients who had undergone transplantation for hematological disorders. However, its significant treatment-related mortality (TRM) has limited its application to life-threatening cases. Reduced-intensity stem cell transplantation (RIST) is a rapidly expanding treatment strategy and diminishes TRM by decreasing conditioning regimen toxicity compared to conventional myeloablative transplants. The benefit of RIST for autoimmune disease still remains unclear. [Purpose] The purpose of this study is to analyze outcomes of patients with autoimmune diseases treated with RIST for coexisting hematological diseases. [Patients and Methods] Between April 2001 and March 2004, 19 patients (median age, 53 y; range, 20-70) underwent RIST from allogeneic donor. All of the patients were considered inappropriate for conventional allo-HSCT due to age 〉 50 years and/or organ dysfunction (generally attributable to autoimmune disease-related). Stem cell sources were unrelated CB (n=10), unmanipulated related PB (n=7) and unmanipulated unrelated BM (n=2). The preparative regimens were Flu/Mel-based (n=11), Flu/Bu-based (n=7), and Flu/CY (n=1). The primary disease were NHL (n=7), MDS (n=4), AML (n=3), HD (n=2), ATL (n=2), and SAA (n=1). Concurrent autoimmune diseases were ulcerative colitis (n=4), psoriasis (n=3), rheumatoid arthritis (n=2), interstitial pneumonitis (n=2), atopic dermatitis (n=1), ITP (n=1), CIDP (n=1), polychondritis (n=1), SLE (n=1), and MCTD (n=1). Eleven out of 19 patients were active in autoimmune disease, and seven needed immunosuppressive therapy. [Results] Median follow up period is 174 days (range, 33–987). All patients achieved neutrophile engraftment (median 22 days; range 12–42) and sustained 100% donor chimerism without DLI. 13 patients (68%) developed grade II-IV acute GVHD, and six (31%) developed chronic GVHD. Nine patients (47%) who were treated with steroid, died of transplant-related toxicity; sepsis (n=7), GI bleeding (n=1), and multiple organ failure (n=1). One patient died of leukemia relapse. As of August 2004, ten patients are alive at median follow-up of 402 days, and the actuarial overall survival rate is 51 % at 2 years. Regarding the clinical response for autoimmune disease, no patients experienced flares of the disease: complete remission is observed in seven patients (70%) and they become therapy-free. In the other three patients (30%), disease remains stable. [Conclusion] Our results suggest that a strategy that incorporates RIST for autoimmune disease may be worth considering for further intense evaluation. It may have a considerable graft-versus-autoimmunity (GVA) effect due to the recipient lymphoablation and reconstitution of donor T cells. Organ failure due to autoimmune disease, impaired immune response, and GVHD may contribute to high TRM. Management of regimen-related toxicity and the control of GVHD will be the focus of future investigation.

Description: Background: Within the concept of reduced-intensity stem cell transplantation (RIST), there is a wide range of differences in regimens utilized, in terms of toxicities and antileukemia effects, and only a little information is available on the clinical impact of chimerism status in patients conditioned with a busulfan-containing regimen. To examine this point, we reviewed the pattern of lineage-specific chimerism to correlate with subsequent clinical outcomes. Patients and Methods: We retrospectively reviewed the data of 117 patients (median age, 52 years: range, 29–68) who had various hematological malignancies and underwent busulfan-containing RIST with related blood stem cells (n=81), related marrow (n=4) or unrelated marrow (n=32), between January 2000 and December 2006. The conditioning regimen consisted of busulfan (8 mg/kg) and fludarabine (180 mg/m2, n=64) or cladribine (0.66 mg/kg, n=53), with or without 2–4 Gy TBI (n=26) or anti-thymocyte globulin (5–10 mg/kg: n=31). Prophylaxis for GVHD consisted of cyclosporin or tacrolimus, with or without methotrexate. Chimerism was evaluated with peripheral blood samples taken on days 30, 60 and 90 after transplantation by PCR-based amplification of polymorphic short tandem repeat regions. Results: The median follow-up of surviving patients was 857 days (50–2535). Percent donor-chimerism was significantly higher in granulocytes than T-cell fraction throughout the entire course, and the mean values were, respectively, 96% vs 83%, 98% vs 89% and 98% vs 91% at days 30, 60 and 90 after RIST. At day 30, the numbers of patients with T-cell chimerism 〉90%, 60–90% and

Description: Previous studies on the tetramer-based quantification of CMV-specific CTL have shown that the reconstitution of CMV-specific CTL to levels greater than 10/μL may protect against CMV reactivation. To evaluate the usefulness of CMV-specific tetramer for monitoring the number of CMV-specific CTL, assays with CMVpp65 495–503 tetramer were performed in 34 patients with HLA-A02 while CMVpp65 328–336 tetramer was used in 47 HLA-A24 patients who received non-T-cell-depleted SCT from a serologically full-matched donor after a myeloablative or nonmyeloablative regimen without ATG. Patients were assessed after recovery from CMV reactivation. Although the average number of CTL detected in patients with HLA-A02 [23.5/μL (1.33%/lymphocytes)] was significantly higher than that in patients with HLA-A24 [0.48/μL (0.02%)], the CMV reactivation rate was similar (67.6% and 62.5% in HLA-A02 and A24, respectively). This result demonstrates that these assays are of limited value since the number of CTL detected varies among different HLA-restricted epitopes. To further evaluate whether there is any relationship between CMV-specific CTL and CMV reactivation, the number of CTL in HLA-A02 patients was assessed in detail. The average number of CTLs in 11 patients without CMV reactivation, 23 with CMV reactivation, 13 with a peak CMV antigenemia of 〉10/50000, and 3 who developed CMV disease was 12.3/μL (0.85%), 29.3/μL (1.35%), 13.8/μL (0.8%) and 16.3/μL (1.33%), respectively. No significant correlation was observed between the number of CTL and CMV reactivation after the reconstitution of CMV immunity. Since CMV reactivation usually occurs within 100 days after SCT, tetramer was assessed biweekly until day 100 in 13 HLA-A02 patients. In those who had CMV reactivation, simultaneous intracellular IFN-γ staining was performed with the same peptide used for tetramer. CMV reactivation was observed in 10 patients between day 23 and day 56 (median, day 34); among them, 5 had a peak antigenemia of 〉10/50000, and required GCV therapy, and 3 developed CMV colitis. The average number of CTL at CMV reactivation was 5.67 (0.08–22.65) /μL in 10 patients who had reactivation, with 3 showing 〉10/μL, while this was 1.08 (0–1.98) /μL at day 30 in those who did not. Two of the 3 patients who developed CMV colitis had 〉10/μL CTL at the time of disease onset, while among 8 who did not require GCV therapy, only 1 and 2 patients recovered CTL 〉10/μL at day 30 and day 60, respectively. The number of intracellular IFN-γ-secreting cells among those with CMV colitis was 18.2/μL (1.8%) at the time of disease onset, and this increased to 47.3/μL (3.5%) after recovery from CMV disease. These results suggest that tetramer-based monitoring of CTL is of limited value in predicting CMV reactivation compared to intracellular IFN-γ assay that assesses the functional properties of CTL.

Description: Background: In a mouse model, it has been shown that inflammatory cytokines play a primary role in the development of acute graft-versus-host disease (GVHD). Here, we evaluated whether the pre-engraftment CRP value, which is used as a surrogate marker of inflammation, could predict post-transplant complications including GVHD. Methods: The medical records of 224 adult patients (median age, 47 years; range, 18–68 y), who underwent conventional (CST, n=105) or reduced-intensity (RIST, n=119) allogeneic stem cell transplantation between January 2002 and July 2006 were reviewed retrospectively. Their diagnosis included AML (n=94), ALL (n=23), NHL (n=62), MDS (n=27) and others (n=18). Stem cell sources included bone marrow (n=108), peripheral blood stem cells (n=98) and cord blood cells (n=18). Patients were categorized according to the maximum CRP value during the pre-engraftment neutropenic period: the “low CRP” group (CRP 〈 15 mg/dL) included 157 patients and the “high CRP” group (CRP≥15 mg/dL) included 67 patients. We assessed the occurrence of acute GVHD, non-relapse mortality (NRM) and overall survival. Results: The incidence of documented infections during neutropenia was higher in the high CRP group (34% vs 17%, P=0.004). The CRP value was significantly lower after RIST than after CST (P=0.017) or after related than after unrelated transplantation (P

Description: Background: Natural killer T (NKT) cells are one of the primary effectors of the innate immune systems, and also have an important role to initiate and regulate adaptive immune responses. Previously, we reported that Vα24+ NKT cells proliferated more efficiently from granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (PBMC) compared with steady-state blood cells. However, optimal culture conditions and characterization of ex vivo expanded NKT cells have not been fully evaluated. Object: We examined several different conditions including cell ratios, mediums, growth factors and incubation schedules to seek an optimal culture system for expansion of NKT cells. Methods: PBMC were collected from donors for hematopoietic stem cell transplantation before and after G-CSF administration, and were cultured in AIM-V medium supplemented with 10% auto-plasma, 100 ng/mL α-galactosylceramide (α-GalCer) and 100 U/mL recombinant human (rh) IL-2. IL-2 alone was repeatedly charged every 3 days to maintain its biological activity. After 12 days culture, we compared the expansion efficacy of Vα24+CD3+ NKT cells derived from PBMC with or without G-CSF. For depletion analysis, we used a magnetic cell sorting (MACS) system with labeling magnetic beads-conjugated monoclonal antibody against CD14, 56, 34, and TCR Vα24 chain. Results: The expansion fold of Vα24+CD3+ NKT cells were significantly higher with G-CSF (669 vs 182 fold, n=20). Among cell populations we tested, the proportion of CD14+CD16+ cells before cultures were associated with the efficacy of Vα24+CD3+ NKT cells expansion, and the proportion of CD34+, Vα24+, CD56+ and CD56+CD161+ cells were not. The magnitude of expansion of Vα24+CD3+ NKT cells was correlated with the percentage of CD14+ cells at the initiation of cultures. Proliferation of Vα24+CD3+ NKT cells was abrogated by the depletion of Vα24+cells, but notCD34+ cells. Depletion of CD56+ T cells induced higher expansion ratio of Vα24+CD3+ NKT, which was abolished when CD56+ and CD56− cells were cultured separately using a 3 μm pored-membrane filter. Furthermore, co-culture of enriched Vα24+ cells and purified CD56+ cells inhibited the proliferation of Vα24+CD3+ NKT cells. It was hypothesized that the repeated IL-2 supplementation resulted in enhancement of CD56+ cells (NK cells) to suppress the proliferation of Vα24+CD3+ NKT cells. We tested different administration schedule of IL-2 as follow: on day 0 only, day 0 & 3, day 0, 3 & 6, day 0, 3, 6 & 9 (that is every 3 days), and we found that Vα24+CD3+ NKT cells expanded most effectively when IL-2 was supplemented on day 0 only. In order to modify the number of CD14+ cells in culture system, we added back CD14+ cells to CD14− cells culture on day 0, 3, 6, 9 or every 3 days, but this did not result in significant enhancement of proliferation of Vα24+CD3+ NKT cells. Conclusions: For efficient ex vivo culture of Vα24+ NKT cells, the presence of Vα24+ NKT cells and CD14+ cells are critical. The NK cells may interfere the interaction between antigen presenting cells (APC) and NKT cells by hindering a function of antigen presentation or yielding direct cytotoxicity against APC. These findings are helpful to develop an efficient expansion system of NKT cells in feature adaptive immunotherapy.