ALBERT

Description: Traditionally, the focus of anti-tumor immunity has been on CD8+ CTL responses. It is now realized that CD4+ T cells play a relevant role in protective anti-tumor responses. HLA Class II molecules are expressed on AML blasts, predicting that AML cells may stimulate CD4+ T cells. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we developed a novel method of generating multiple autologous AML reactive T cell lines by competitive limiting dilution (LD) AML MNC culture. Most autologous AML reactive T cell lines generated from this culture were CD4+. These CD4+ T cell lines with high IFN-gamma secretion in response to autologous AML cells showed low to moderate specific lysis of autologous AML cells in 4-hour 51Cr release assays. However, co-culture assays demonstrated that these CD4+ AML reactive T cell lines exerted intense cytotoxicity toward autologous AML cells, depleting more than 95% of autologous AML line cells in 2 days, and more than 99% in 7 days, with no or weak toxicity to allogeneic AML cells and cell lines, HL60, NB4, U937 KG1a and ak LCL cell line. Anti-HLA class II, Dr, Dp, Dq mAb (67±16% inhibition) and anti-HLA-Dr mAb (69±11% inhibition) but not anti-HLA class I mAb significantly inhibited IFN-gamma secretion of six CD4+ T cell lines stimulated by autologous AML cells confirmed the HLA class II restriction of reactivity of these CD4+ T cell lines. Flow cytometry analysis showed that these CD4+ T cell lines induced significant Annexin-V expression on autologous AML cells. The induction of Annexin-V+ on AML cells by CD4+ T cell lines correlated significantly with IFN-gamma secretion in response to autologous AML cells (n=11; r=0.84). This result suggested that the most important mechanism of autologous AML cell elimination by CD4+ AML reactive T cells generated from LD-AML-MNC cultures was apoptosis induction related to IFN-gamma secretion.

Description: 2035 Poster Board II-12 Although hematologic remissions can be achieved in the majority of patients with adult acute lymphocytic leukemia (ALL) by chemotherapy, long term survival is only 30–40%. The inability of the immune system to recognize and eliminate residual malignant leukemia cells may be an important mechanism contributing to relapse. In this study, we investigated the possibility of using pre-B-ALL cells as antigen presenting cells in an in vitro culture to induce autologous ALL reactive T cells. After 7 day culture of Pre-B-ALL peripheral blood mononuclear cells (CD19+ 93±4%) in 96 well culture plates in culture medium supplemented with a cytokine combination of IL-2/IL-3/IL-4/IL-7/GM-CSF, CD80, CD86, CD83, CD54, HLA-Dr and CD40 expression was analyzed. Significant enhancement of CD80, CD86, CD83 and CD40 on ALL cells was observed (n=8, P≦0.001-0.02). Addition of lipopolysaccharide (LPS) to the cytokine combination further increased CD80, CD86 or CD40 expression by 3 of 8 ALL samples above the baseline enhancement by cytokines (Figure), while CD40 ligand (CD40L) enhanced expression of the co-stimulatory molecules in 4 of 8 cases. Autologous T cells remaining in the culture after day 7 were then expanded with high dose IL-2 and autologous ALL reactive T cell lines were identified by IFN-g release by T cells in response to autologous ALL cells. Autologous ALL reactive T cells were generated from 5 of 8 pre-B ALL samples studied. The data from 3 experiments demonstrated that although the cytokine combination plus LPS or CD40L could effectively induce ALL cell expression of co-stimulatory molecules, the presence of CD40L and LPS in the culture induced significantly greater activation of autologous ALL-reactive T cells than did the cytokine combination plus LPS alone, as assessed by average IFN-g release of 24-48 culture wells (P≦0.004). ALL-reactive T cell lines selected by high IFN-g release response showed effective elimination of autologous ALL cells in a 2 day co-culture assay with an E:T ratio of 1:1 by flow cytometry analysis. Average residual CD19+ cells was 2.0±1.4% for highly reactive T cell lines (n=17) vs 16.5±25.9% for the less reactive control T cell lines (n=9) (P

Description: Abstract 2560 The mammalian orphan receptor tyrosine kinase-1 (ROR1) is expressed in a wide-variety of tissues during early embryonic development. By the late stages of embryogenesis the expression of this developmentally important protein is greatly diminished. Although not expressed in the tissues of post-partum animals, the ROR1 protein is expressed on neoplastic cells in chronic lymphocytic leukemia (CLL), some B-cell malignancies, and a variety of different carcinomas. We examined for expression of ROR1 in primary acute myeloid leukemia (AML) cells harvested from marrow aspirates and their normal counterparts by whole transcriptome paired-end RNA sequencing and by flow-cytometric analyses. These studies revealed selective expression of ROR1 in 62 (35%) of 179 AML samples examined. Many of these samples were found to have cells that co-expressed ROR1 and CD34, suggesting that ROR1 was present on the self-renewing leukemia stem-cell population, which resides in the marrow niche and potentially accounts for resistance to many cytotoxic drugs used in therapy. We investigated the activity of a chimeric anti-ROR1 mAb found effective in clearing CLL cells (UC99961) on AML expansion, growth, and renewal in a leukemia-stem-cell supportive niche assay. Mouse marrow cells lines SL/SL and M2–10B4 (transfected to produce hSCF,hIL3 and hIL3, hG-CSF respectively) were mixed 1:1 after mitomycin-C treatment, and used as a SLM2 stromal monolayer. CD34+ cells were selected from ROR1-positive (n=6) or negative (n=4) AML primary samples. As a normal control, CD34+ cells from cord blood (CB) were used (CB, n=3). In some experiments CD34+ cells were transfected with a GLP-lentivirus prior to co-culture. At the initiation of the co-culture, 10–50 μg/ml of the chimeric anti-ROR-1 mAb (UC99961) or control hIgG were added to the cultures. Two weeks after co-culture initiation, both stromal attached and floating cells were collected and their survival investigated by colony forming assay in methylcellulose. The UC99961 mAb was not cytotoxic to CB or ROR1-negative AML samples. In contrast, the UC99961 mAb provided a dose-dependent inhibition of colony formation for all ROR-1-positive AML samples examined. These results demonstrate the in vitro anti-leukemic specificity of this anti-ROR1 mAb in down-regulating AML stem and progenitor cell populations, without effecting normal CD34+ stem cells. To analyze the effect of ROR1 ligation on AML stem cell populations exclusively, AML self-renewal assays (2-ry colonies) were performed. In these studies, ROR1–positive AML samples were divided based on their response to mAb treatment. Half of the samples (n=3; 50%) demonstrated statistically significant (up to 90%) dose-dependent decreases in colony formation. However, another half was non-responsive and no correlation was found between ROR1 expression on leukemia CD34+ cells and response to anti-ROR1 mAb treatment in the self-renewal assays. Again UC99961 mAb treatment did not negatively impact CD34+ cells from CB or ROR1-negative AML, confirming the specificity and selective toxicity of the mAb for ROR1+ AML stem cells. These studies reveal selective expression of ROR1 on leukemia-stem-cells of large subset of AML patients. Furthermore, this study demonstrates that an anti-ROR1 mAb (UC99961) can inhibit survival and self-renewal in LSC supportive niche assays. Targeted ROR1 inhibition may represent a vital component of therapeutic strategies aimed at eradicating therapeutically recalcitrant malignant stem cells in AML and potentially other refractory cancer-stem-cell-driven malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Naturally occurring cytotoxic T cells directed against various leukemia associated antigens (LAA) expressed by acute myeloid leukemia (AML) cells have been described. However, these LAA-specific T cells are rare and obviously unable to initiate effective anti-leukemia responses. The challenge is how to investigate, select, activate and expand the rare LAA-specific T cells from the vast population of blood cells in patients with AML for immunotherapy. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we have developed a novel method of generating multiple autologous AML reactive T cell lines by limiting dilution AMLDC (LD-AMLDC) culture. The principle of LD-AMLDC is based on the assumption that autologous AML-reactive T cells or precursors are randomly distributed in the AML PBMC suspension, and that each one has an equal opportunity to respond to AML cells in the 96-well plates under optimized culture condition. By culturing AML PBMC (〉90% blasts) in culture medium supplemented with GM-CSF/IL4/IL2/IL7/IL12 to induce AML DC differentiation and activate in situ autologous T cells, highly reactive anti-AML T cell lines (both CD4+ and CD8+ lines) were selected and expanded from LD-AMLDC culture using the appropriate numbers of AML PBMC in each culture well by the criterion of release of IFN-gamma in response to autologous AML blasts. By maximum likelihood solution, the estimated average frequency of AML reactive T cells or precursors is 6±3/1,000,000 AML PBMC (n=8). Strong intracellular IFN-gamma release of T cell lines obtained in LD-AMLDC was demonstrated by flow cytometry analysis after stimulation by autologous AML cells but not autologous B-lymphoblastoid cell line (LCL) (Figure). Effective specific lysis (up to 70% at E:T=20:1) of autologous AML cells but not autologous LCL or allogeneic AML cells by these T cell lines was observed. Two PR1 specific T cell lines were obtained by screening 39 AML reactive HLA-A2+ CD8+ T cell lines generated from 5 LD-AMLDC cultures, suggesting that other unidentified CD4 or CD8 lines with strong autologous AML responses may be reactive to known or unknown LAAs. These results encourage continued efforts to induce, activate and select T cells lines with high autologous AML reactivity using LD-AMLDC culture and to expand multi-LAA reactive T cell lines acquired from limiting dilution AML-DC culture for AML immunotherapy. Figure Figure

Description: CTLA-4 is expressed upon activation of T-cells,and serves as an important negative regulator of their effector function. It is also expressed constitutively on CD4+/CD25+ regulatory T cells (Treg), where its function is not clear. Following allogeneic hematopoietic stem cell transplantation (allo-HCT), CTLA-4 function may be involved in suppression of alloreactive T cells that mediate the graft-versus malignancy effect and GVHD. We have studied the administration of a single dose of Ipilimumab (MDX-010), a fully human monoclonal anti-CTLA-4 antibody, in a dose escalation trial in patients with relapse/progression of malignancy following allo-HCT. Here we report effects of ipilimumab on peripheral CD4+/CD25+ cell levels and FOXP3 mRNA expression in these patients. Seventeen patients with a variety of malignancies were enrolled in this study. Ipilimumab was given intravenously at a dose level of 0.1, 0.33, 0.66, 1, and 3mg/kg. The blood samples were obtained prior and after infusion at day 7, 14 and 30. The immunophynotyping of peripheral blood mononuclear cells (PBMC) was analyzed by flow cytometry. CD4+/CD25+ cells from nine patients at day 0, 7, and five normal donors were separated using a Dynal CD4+/CD25+ Treg kit. FOXP3 mRNA expression on CD4+/CD25+ cells were analyzed by a quantitative RT-PCR. Expression level of FOXP3 was normalized to 18S rRNA. Within CD4+ cell population, the percentage of CD4+/CD25high cells was significantly higher in patients at day 0 (11.6±6.7%, n=17), compared with normal donors (3.8±1.6%, n=12; P

Description: To generate highly AML reactive autologous CTL, the optimal protocol of growth factor combination and sequence of administration were screened and compared in an AML-DC culture system aimed to induce AML cell DC differentiation, activate and expand naïve T cells against AML; and simultaneously activate and expand possible rare existing AML reactive memory T cells. Primary AML peripheral blood or marrow cells containing more than 90% AML blasts were cultured in 96 well plates. Every single well was regarded and treated as an independent culture. Various growth factor combinations of GM-CSF, IL2, IL-3, IL-4, IL7, IL12, CD40L, TNF-α, anti-CTLA-4 mAb were compared for efficiency of AML cell DC differentiation induction and autologous T cell activation in 7 day culture. Significantly enhanced CD80/CD86 expression and total cell number (include both AML-DC and T cells, see attached figure) in the culture was observed when GM-CSF/IL-4 combined with IL-12. The T cells in each well were than expanded with high dose IL-2 (6000 u/ml) and CTL activity against autologous AML were examined by both 51Cr release assay and culture supernatant IFN-γ concentration assay. Multiple wells of AML cell culture from the same patient with the same cell number and culture condition exhibited substantial functional variation demonstrated by MHC-restricted autologous AML cell killing (3 - 70% specific lysis) and IFN-γ secretion (25 - 2994 pg/ml), indicating that the T cells randomly put in the independent wells vary greatly in efficiency of AML antigen recognition and activation. IL-12 alone exerted significant enhanced IFN-γ secretion in 4 of 5 patients. IL-12 combined with GM-CSF/IL-2/IL-4/IL-7 enhanced IFN-γ secretion compared with IL-12 alone in 3 of 5 patients. CTLA-4 blockade further significantly enhanced T cell activity in 5 of 5 patients. Highly active CTL lines were selected from high IFN-γ secretion wells and rapidly expanded using OKT-3/CD28 coated, mitomycin-c treated autologous AML cells. After expansion, the highly active lines still maintained high autologous AML cell killing compared to the low active lines. A therapeutic quantity of AML reactive autologous T cells (20.2±30.1)x109, (n=5) can be obtained after 6 weeks of culture from (6.4±2.9)x107 AML PBMC. This study demonstrated AML cells cultured in IL-12 or IL-12 combined with GM-CSF/IL-2/IL-4/IL-7/anti-CTLA4 mAb activated the potential of the patient immune system and may maximize the possibility of AML reactive CTL generation for adoptive immunotherapy. Figure Figure

Description: Autologous T lymphocytes can be employed for immune therapy of patients with acute myeloid leukemia (AML). In an earlier study (Biol Blood Marrow Transplant2002; 8:557), we found that AML cells can be used as antigen presenting cells to activate and expand T cells in culture with IL-2 and monoclonal antibody (mAb) to CD3. In this study, we found that a substantial proportion of the expanded T cells expressed CD33 (34 ± 16%; range: 8 – 58%; N=11) and CD13 (26 ± 5%; range: 22 – 33%), but not CD14. It is unlikely that the myeloid markers were absorbed onto the activated T cells, since the same expression pattern was observed in cultures with purified T cells from normal donors. A literature review disclosed two reports that also observed co-expression of CD33 on activated T cells from both AML patients and normal controls (Schmidt-Wolf et al, Br J Haematol.1995; 90:512; Nakamura et al, letter in Blood, 1994; 83:1442). We measured the functional capacity of the activated T cells in a 4-hr Cr-51 release assay. Pre-incubation of the T cells with anti-CD33 mAb and/or anti-CD13 mAb did not change the cytotoxicity against AML cells. Also, killing of AML cells was not affected by the depletion of CD33+ or CD13+ activated T cells using magnetic beads. The activated T cells migrated in a chemotactic response to a peptide analog (CTCE-0214) of Stromal cell derived factor-1 (SDF-1) when measured in a trans-well assay. When activated T cells were pre-incubated with anti-CD33 mAb, enhanced chemotaxis was observed in response to CTCE-0214 (39.9 ± 0.5% vs 26.9 ± 0.3%, p 〈 0.001). However, incubation with anti-CD13 mAb did not change the proportion of chemotactic cells. The results of this study confirmed that myeloid antigens can be induced on activated T cells from patients with AML and that there are differences in the mobility of CD33+ T cells in the presence of a chemotactic molecule. Further study of the biological significance of CD33 expression on activated T cells in AML and other diseases is warranted.

Description: AML cell line 2006-5 was established by serial culture of an AML patients’ PBMC (M5). Compared to primary AML cells, this line expresses CD13, CD33, CD15, CD4, CD86, CD54 but lost CD34, CD64, CD123 and HLA-Dr. A group of CD13−/CD33− cells was detected consisting of 0.2% of total cells, but no significant CD34, CD90 or CD123 expression cells could be detected by FACS. After depleting of CD13+/CD33+ cells by magnetic beads, the CD13−/CD33− cells were enriched to 95%. Significant CD34+, CD90+ or CD123+ cells (0.2–0.5%) appeared in the CD13−/CD33− enriched fraction of AML cells. Leukemia colony forming (LCF) cell assay analyzed at day 14 demonstrated CD34+, CD90+ or CD123+ cells selected by magnetic beads formed typical compact large colonies of more than 1000 cells representing 40–60% of total colonies (Fig left panel). The CD13−/CD33− enriched fraction formed medium colonies with a dense center surrounded by a halo of migrating cells (80–90% of total colonies) (Fig right). Untreated 2006-5 cells formed small diffuse colonies (70–80%). Re-plating of compact colonies selected from CD34+, CD90+ or CD123+ cell LCF assays formed the above 3 type of colonies in one culture dish. Although a daughter line can be generated from a single colony formed by CD13-CD33− cells, no compact colonies could be observed in re-plating experiment. After 14 days of culture, immature erythroid cell clusters were observed in the area surrounding compact colonies suggesting that the progenitors of the colony forming leukemia cells were at the GEMM stage. Small colonies of untreated cells gradually became apoptotic and necrotic after 10 days. These results support the theory that AML is organized as a hierarchy similar to the normal hematopoietic system. CD34+, CD90+ or CD123+ cells with very low frequency of 1–2 in 1×105 2006-5 line cells demonstrated high proliferation and differentiation potential, and may represent leukemia stem cells. CD13−/CD33− cells with lower proliferation rate may belong to more differentiated leukemia progenitors without self renew potential. If additional experiments support the notion that cells forming compact colonies are leukemia stem cells, they may serve as a useful resource to test the biology of leukemia and its therapy. Figure Figure Figure Figure

Description: Many numerical methods have been developed for in-plane vibration of orthotropic rectangular plates with various boundary conditions; however, the exact results for such structures with elastic boundary conditions are very scarce. Therefore, the object of this paper is to present an accurate solution for free in-plane vibration of orthotropic rectangular plates with various boundary conditions by the method of reverberation ray matrix (MRRM) and improved golden section search (IGSS) algorithm. The boundary condition studied in this paper is defined as that a set of opposite edges is with one kind of simply supported boundary conditions, while the other set is with any kind of classical and general elastic boundary conditions or their combination. Its accuracy, reliability, and efficiency are verified by some numerical examples where the results are compared with other exact solutions in the published literature and the FEA results based on the ABAQUS software. Finally, some new accurate results for free in-plane vibration of orthotropic rectangular plates with elastic boundary conditions are examined and further can be treated as the reference data for other approximate methods or accurate solutions.

Description: The present work is concerned with dynamic characteristics of beam-stiffened rectangular plate by an improved Fourier series method (IFSM), including mobility characteristics, structural intensity, and transient response. The artificial coupling spring technology is introduced to establish the clamped or elastic connections at the interface between the plate and beams. According to IFSM, the displacement field of the plate and the stiffening beams are expressed as a combination of the Fourier cosine series and its auxiliary functions. Then, the Rayleigh–Ritz method is applied to solve the unknown Fourier coefficients, which determines the dynamic characteristics of the coupled structure. The Newmark method is adopted to obtain the transient response of the coupled structure, where the Rayleigh damping is taken into consideration. The rapid convergence of the current method is shown, and good agreement between the predicted results and FEM results is also revealed. On this basis, the effects of the factors related to the stiffening beam (including the length, orientations, and arrangement spacing of beams) and elastic parameters, as well as damping coefficients on the dynamic characteristics of the stiffened plate are investigated.