ALBERT

Description: Background. Diffuse large B-cell lymphoma (DLBCL) is the most frequent subtype of non-Hodgkin lymphoma. High total metabolic tumor volume (TMTV) or total lesion glycolysis (TLG) calculated using 18F-FDG PET/CT images at diagnosis predicts poor prognosis of patients with DLBCL. However, high cost and poor access to the imaging facilities hamper wider use of 18F-FDG PET/CT. Methods. In order to explore a surrogate marker for TMTV/TLG, we evaluated the correlations between the serum levels of soluble interleukin-2 receptor (sIL-2R) and TMTV/TLG in 64 patients with DLBCL, and the results were verified in an independent validation cohort of 86 patients. The study procedures were in accordance with the Helsinki Declaration and institutional ethical guidelines, conducted under the auspices of the institutional ethics committee, and approved by the institutional review boards. Results. In the training cohort(n=64), OS and EFS were significantly lower in patients with TMTV≥150cm3 than in those with less than 150cm3 [5-year OS; 84.0% vs 29.1%, P=0.000194, 5-year EFS; 71.4% vs 28.7%, P=0.000384]. We also found that TLG≥1500cm3 at diagnosis predicted significantly inferior OS and EFS[5-year OS; 84.0% vs 31.7%; P=0.000477, 5-year EFS; 70.2% vs 30.6%, P=0.00107].Pearson's correlation tests demonstrated highly significant positive correlations between sIL-2R and TMTV[R2=0.490; P=0.00004] and between sIL-2R and TLG[R2=0.543; P=0.00000357]. Serum sIL-2R≥1300 U/ml was a strong prognostic factor both for worse OS and EFS[5-year OS; 85.2% vs 25.9%, P=0.000035, 5-year EFS; 72.0% vs 26.8%, P=0.000076].In a univariate analysis, B symptom, LDH, sIL-2R, TMTV and TLG were associated with poor 5-year OS; and B symptom, LDH, PS, sIL-2R, TMTV and TLG were identified as poor prognostic factors for 5-year EFS. We performed multivariate analysis that included sIL-2R and all factors in NCCN-IPI; age, LDH, clinical stage, ECOG PS and major organ involvement. In this multivariate analysis, age and sIL-2R were independently associated with poor 5-year OS(age; HR, 4.44; 95% CI, 1.05 to 18.7, P=0.0424, sIL-2R; HR, 4.45; 95% CI, 1.04 to 19.1, P=0.0444). Another multivariate analysis that included TMTV and all factors for NCCN-IPI demonstrated that TMTV was an only independent prognostic factor for 5-year OS(HR, 3.87; 95% CI, 1.08 to 13.8; log-rank, P=0.0373).Subgroup analyses included the patients with NCCN-IPI High-Int and High(n=49) demonstrated that the cut off value of TMTV 150cm3 stratified treatment outcomes in this poor prognostic group [5-year OS; 75.0% vs 27.7%, P=0.0355, 5-year EFS; 66.7% vs 29.7%, P=0.0493]. Similar results were obtained using the cut-off value of sIL-2R 1300U/mL [5-year OS; 75.0% vs 25.9%, P=0.0182, 5-year EFS; 58.3% vs 29.7%, P=0.0499]. In the validation cohort(n=86), Kaplan-Meier curves showed that OS and EFS in patients with TMTV≥150cm3 was again lower than in those with TMTV

Description: Abstract 4728 Imatinib mesylate (imatinib) has been widely used clinically for the treatment of CML and Ph-positive ALL patients with tremendous success. However, the effect is limited in advanced stage by rapid development of resistance to imatinib. Tregs play a pivotal role in peripheral tolerance and their impairment results in autoimmune disease and GVHD. Previous studies have revealed that imatinib inhibits the proliferation of several types of immune cells. However, immunomodulatory function of imatinib in CML remains to be elucidated. In the present study, we investigated whether imatinib exerts an immunosuppressive effect on naturally occurring Tregs in T cell responses to CML, especially imatinib-resistant CML. Peripheral blood mononuclear cells were obtained from 5 healthy volunteers with informed consent. CD4+CD25+ and CD4+CD25- T cells were isolated by magnetic cell sorting using human CD4+CD25+ T regulatory cell isolation kit (Miltenyi Biotec). Both CD4+CD25+ and CD4+CD25- T cells, pre-stimulated with anti-CD3 and anti-CD28 monoclonal antibody-coated beads for 2 days, were cultured with increasing doses of imatinib for 3 days and survival of the cells was evaluated by a WST1 tetrazolium assay. Viability decreased at higher concentrations (〉10μM) of imatinib. There was no difference in the sensitivity to imatinib between CD4+CD25+ and CD4+CD25- T cells. To examine whether imatinib exerts an inhibitory effect on the proliferation of Tregs, freshly isolated CD4+CD25+ and CD4+CD25- T cells were cultured with varying concentrations of imatinib together with CD3 and CD28 stimulation for 3 days. The proliferation of these cells was inhibited by higher concentrations of imatinib in a similar fashion. Expression of FoxP3 in CD4+CD25+ Tregs was inhibited with 10μM imatinib by apporoximately 70% as shown in Figure 1. Because Treg has been shown to suppress immunity against cancer, a similar inhibitory effect is expected to occur in CML. This prompts us to examine immune responses to CML. For this purpose, we utilized a cell line (TM) established by us from a patient with CML in blastic crisis and another imatinib-resistant cell line (TM-G) generated by culturing TM with increasing concentrations of imatinib. CFSE-labeled CD4+CD25- and CD8+ responder cells were cultured for 5 days with irradiated autologous CD3- antigen presenting cells which were loaded with cell-lysates from either TM or TM-G in the presence or absence of Tregs which were pre-incubated with increasing concentrations of imatinib for 2 days. Proliferation of both CD4+CD25- and CD8+ responder cells were inhibited by Tregs which were not exposed to imatinib. On the other hand, Tregs which were exposed to imatinib had reduced inhibitory effects on the proliferation of responder cells depending on the concentration of imatinib as shown in Figure 2. These results demonstrate the immunomodulatory functions of imtinib in CML and imply that the use of this drug is a potent inhibitor of Tregs in cancer immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2101 Background and Purpose: Recent success associated with adoptive transfer of antitumor T cells in lymphodepleted patients suggests the potential of adoptive immunotherapy to have a significant clinical impact. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we previously reported a culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL) from HLA-A2-positive melanoma patients by using K562-based artificial antigen-presenting cells (aAPCs). In the present study, we have applied the culture system to HLA-A24, one of the most common HLA class I antigen in the Asian population, and examined whether HLA-A2402-restricted WT1-specific CTL can be generated using aAPCs. Methods: HLA-A2402–positive peripheral blood mononuclear cells (PBMC) were obtained from healthy donors (n=4) and cancer patients (n=10). To establish antigen-specific T cells, CD8+ T cells were purified by positive selection using a magnetic beads method (Miltenyi Biotec). aAPCs were pulsed with an HLA-A2402 restricted, modified 9-mer WT1 immunodominant peptide (CYTWNQMNL). aAPCs were then irradiated with 200 Gy and added to purified CD8+ T cells at a ratio of 1:20 in 96-well plates in RPMI1640 supplemented with 10% human AB serum. Between stimulations, IL-2 (10 U/ml) and IL-15 (10ng/ml) (both from Peprotech) were added to the cultures. Results: Flow cytometry (FACS) analysis confirmed that aAPCs stably expressed transduced HLA class I, CD80 and CD83 molecules. aAPC did not express HLA class II molecules, CD40, CD154, or CD86. Following 3–4 rounds of weekly stimulation with peptide-pulsed aAPCs, WT1 peptide-specific CD8+ T cells were evaluated by a tetramer staining. The percentage of tetramer-positive cells was 0.082±0.0075% before stimulation. It increased to 0.865±0.528% (10.5 fold increase) and 1.89±1.12% (23.0 fold increase) following the third and the forth stimulation, respectively. There was no marked difference in magnitude of increase between healthy donors and cancer patients. However, when CD8+ T cells from patients vaccinated with WT1-peptide pulsed dendritic cells were stimulated with WT1-peptide-pulsed aAPCs, the percentage of tetramer-positive cells was significantly higher (11.72±6.46%) following the third stimulation. CD8+ T cells stimulated with WT1-peptide-pulsed aAPCs were negative for both A0201/WT1 and A2402/CMV tetramers, confirming HLA restriction and antigen specificity. FACS analysis revealed that WT1-specific CTL expanded with WT1-peptide-pulsed aAPCs expressed a memory phenotype. Furthermore, these CTL demonstrated cytotoxicity against CIR-A2402 target cells pulsed with a WT1 peptide in an LDH release assay. Unpulsed or HIV peptide-pulsed target cells were not lysed. Conclusions: These results demonstrated that HLA-A24-restricted WT1 specific CTLs with a memory phenotype can be generated ex vivo using peptide-pulsed gene-engineered aAPCs within a short period of time for clinical use. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3948 Poster Board III-884 Serum soluble interleukin-2 receptor (sIL2R) is known to be elevated in patients with adult T-cell leukemia, non-Hodgkin lymphoma and Hodgkin lymphoma. Some studies reported the clinical usefulness of pre-treatment sIL2R value as a prognostic factor associated with survival in various NHL. However it has not been adequately examined about the significance of pre-treatment sIL2R value in patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL), especially in rituximab era. We retrospectively analyzed the clinical significance of pre-treatment sIL2R value in 90 patients diagnosed as having DLBCL and treated in our hospital between 2004 and 2008. And we also compared them to patients with follicular lymphoma (FL) and to patinets with lymphadenopathy other than lymphoma. Median age of all 90 patients with DLBCL was 68 years (range, 18 to 88). Thirty-one (34%) patients were less than 60 years. Male/female was 53/37. Thirty-three (37%) patients had stage I or II diseases. Sixty-five (72%) patients were in 0/1 of ECOG performance status (PS). LDH value was elevated above normal range in 55 (61%) patients. Sixteen (18%) patients had 2 or more extranodal diseases. According to International Prognostic Index (IPI), 48 patients and 42 patients were classified as low/low-intermediate risk groups and high-intermediate/high risk groups, respectively. Seventy-nine of 90 patients were treated with R-CHOP or R-CHOP-like chemotherapies with or without radiotherapy. Two-year overall survival rate was 68% in all 90 patients with DLBCL. In all 90 patients with DLBCL, pre-treatment sIL2R value was 4258+/-6850 U/ml (mean+/-SD) which was significantly higher than that in 42 patients with lymphadenopathy other than lymphoma (870+/-892, p

Description: Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells offer a promising platform for off-the-shelf immunotherapy against cancer. A unique benefit of iPSC-derived immune effector cells is the possibility to perform multiple precision editing steps at the single cell level to achieve a homogenous effector cell population tailored to target a desired cancer type and equipped with selected functional properties. These functional edits are superimposed on the innate reactivity of NK cells to stress ligands and MHC downregulation (missing self). The ability of NK cells to sense missing self is based on a functional calibration to self MHC during a process termed NK cell education, the latter being critically dependent on signaling through inhibitory receptors, including CD94/NKG2A and killer cell immunoglobulin-like receptors (KIR). Whereas the process of NK cell differentiation into mature effector cells from iPSCs has been well characterized, the role of natural variation in inhibitory receptor expression and NK cell education remains poorly defined in iNK cells. We used mass cytometry to map the receptor repertoire in series of iNK cell lines and genetic edits thereof during differentiation and in vitro expansion (Figure 1A and B). Similar to peripheral blood NK cells, the receptor repertoire was diversified but genetically hardwired showing consistent patterns within each iNK cell line but with slight variation between genetically distinct lines. NKG2A was the dominantly expressed inhibitory receptor ranging from 13% to 87% with the highest expression in multi-edited iNK cell lines engineered to express a chimeric antigen receptor against CD19, a high affinity, non-cleavable FcγRIIIa receptor (CD16) and a recombinant IL15 signaling complex (CAR19-iNK cells). KIR expression was generally low in all tested iNK cell lines but increased gradually during culture and was further increased by genetic silencing of NKG2A receptors. Interestingly, silencing of NKG2A lead to increased levels of the activating receptor NKG2C. We monitored degranulation by iNK cell variants against K562 engineered to express varying levels of HLA-E as well as CD19+ Nalm-6 cells. Genetic silencing of ß2microglobulin (ß2m), associated with reduced levels of HLA-class I and HLA-E, led to dampened global functional responses in iNK cells, suggesting a positive impact of education during iNK cell differentiation and expansion (Figure 1C). Subset stratification revealed that NKG2A+ iNK cells showed superior functionality compared to NKG2A- iNK cells across all iNK cell lines tested, albeit less striking in CAR19-iNK cells that showed the highest overall natural cytotoxicity (Figure 1D). Knockdown of NKG2A led to a general reduction in functional capacity of NK92 cells (Figure 1E-F) and CAR19-iNK cells (Figure 1H), supporting a critical role for NKG2A-driven education in iNK cells. Given the superior functionality of NKG2A+ iNK cells, we next addressed whether this advantage was countered by expression of the check point ligand HLA-E during target cell interactions. Although we noted a slight inhibitory impact on natural cytotoxicity in NK cells isolated and expanded from peripheral blood (PB-NK) against K562 cells expressing physiological levels of HLA-E, this effect was completely overridden in iNK cells and did not interfere with NKG2A+ CAR-iNK cell recognition of HLA-E expressing CD19+ target cells (Figure 1G-H). Indeed, NKG2A+ CAR19-iNK showed superior degranulation against HLA-E expressing CD19+ Nalm-6 targets compared to CRISPR-edited NKG2A-/- CAR19-iNK cells (Figure 1I). Our results shed light on the regulatory gene circuits and cellular programs that determine functional potential in iPSC-derived NK cells products. Specifically, our results point to a crucial role for NKG2A-driven acquisition of a mature effector cell phenotype in combination with functional education through cognate ligands. Importantly, iNK cell education is operational during iNK cell differentiation and expansion without interfering with recognition of tumor targets expressing HLA-E. Figure 1 Disclosures Cichocki: Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding. Mahmood:Fate Therapeutics, Inc: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Chu:Fate Therapeutics, Inc: Current Employment. Groff:Fate Therapeutics, Inc: Current Employment. Denholtz:Fate Therapeutics, Inc: Current Employment. Miller:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; GT Biopharma: Consultancy, Patents & Royalties, Research Funding. Lee:Fate Therapeutics, Inc.: Current Employment. Kaufman:Fate Therapeutics: Consultancy. Goodridge:Fate Therapeutics, Inc: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Malmberg:Fate Therapeutics: Consultancy, Patents & Royalties; Vycellix: Membership on an entity's Board of Directors or advisory committees.

Description: Neutrophils have been suggested to play a critical role in terminal differentiation of NK cells. Whether this is a direct effect or a consequence of global immune changes with effects on NK cell homeostasis remains unknown. Here, we used high-resolution flow- and mass cytometry to examine NK cell repertoires in 64 patients with neutropenia and 27 healthy age- and gender-matched donors. A subgroup of patients with chronic neutropenia showed severely disrupted NK cell homeostasis manifested as increased frequencies of CD56bright NK cells and a lack of mature CD56dim NK cells. These immature NK cell repertoires were characterized by expression of proliferation/exhaustion markers Ki-67, Tim-3 and TIGIT and displayed blunted tumor target cell responses. Systems-level immune mapping revealed that the changes in immunophenotypes were confined to NK cells, leaving T cell differentiation intact. RNA sequencing of NK cells from these patients showed upregulation of a network of genes, including TNFSF9, CENPF, MKI67 and TOP2A, associated with apoptosis and the cell cycle, different from conventional CD56bright signatures. Profiling of 249 plasma proteins showed a coordinated enrichment of pathways related to apoptosis and cell turnover, which correlated with immature NK cell repertoires. Notably, most of these patients exhibited severe-grade neutropenia, suggesting that the profoundly altered NK cell homeostasis was connected to the severity of their underlying etiology. Hence, although our data suggests that neutrophils are dispensable for NK cell development and differentiation, some patients displayed a specific gap in the NK repertoire, associated with poor cytotoxic function and more severe disease manifestations.