ALBERT

Description: A pilot study was undertaken to assess the safety, activity, and immunogenicity of a polyvalent Wilms tumor gene 1 (WT1) peptide vaccine in patients with acute myeloid leukemia in complete remission but with molecular evidence of WT1 transcript. Patients received 6 vaccinations with 4 WT1 peptides (200 μg each) plus immune adjuvants over 12 weeks. Immune responses were evaluated by delayed-type hypersensitivity, CD4+ T-cell proliferation, CD3+ T-cell interferon-γ release, and WT1 peptide tetramer staining. Of the 9 evaluable patients, 7 completed 6 vaccinations and WT1-specific T-cell responses were noted in 7 of 8 patients. Three patients who were HLA-A0201-positive showed significant increase in interferon-γ–secreting cells and frequency of WT1 tetramer-positive CD8+ T cells. Three patients developed a delayed hypersensitivity reaction after vaccination. Definite related toxicities were minimal. With a mean follow-up of 30 plus or minus 8 months after diagnosis, median disease-free survival has not been reached. These preliminary data suggest that this polyvalent WT1 peptide vaccine can be administered safely to patients with a resulting immune response. Further studies are needed to establish the role of vaccination as viable postremission therapy for acute myeloid leukemia. This study was registered at www.clinicaltrials.gov as #NCT00398138.

Description: Abstract 354 Adoptive cell therapy utilizes unique mechanisms of action to prevent the development of infections in immunocompromised patients and treat chemotherapy resistant malignancies. In adoptive cell therapy, the major effector cells appear to be CD8+ T cells, since they are armed with antigen-specific effector functions, i.e. cytotoxicity and cytokine secretion. However, the roles of antigen-specific CD4+ T cells in T cell immunity are also critical. In immunocompromised patients adoptively transferred with CMV-specific CD8+ T cells, long-term in vivo persistence was achieved only when CMV-specific CD4+ T cells were also present in vivo. Recently, adoptive transfer of a NY-ESO-1 specific CD4+ T cell clone was reported to induce a complete response in a patient with metastatic melanoma. These results suggest that adoptive cell therapy for infectious diseases and cancer can be improved by infusing both antigen-specific CD4+ helper T cells as well as CD8+ CTL. Unfortunately, however, few versatile systems are available for producing large numbers of antigen-specific human CD4+ T cells for the purpose of adoptive therapy. K562 is a human erythroleukemic cell line, which lacks the expression of HLA class I and II, invariant chain (Ii), and HLA-DM, but does express adhesion molecules such as ICAM-1 and LFA-3. Given this unique immunologic phenotype, K562 has served as a useful tool in clinical cancer immunotherapy trials. Previously, we reported the generation of a K562-based artificial APC (aAPC), which expresses HLA-A2, CD80, and CD83. aAPC/A2 can uniquely support the priming and prolonged expansion of large numbers of antigen-specific CD8+ CTL which display a central/effector memory phenotype, possess potent effector function, and can be maintained in vitro without any feeder cells or cloning. aAPC/A2 is equipped with constitutive proteasome and inducible immunoproteasome machinery and can naturally process and present CD8+ T cell peptides via transduced A2 molecules. We have successfully generated clinical grade aAPC/A2 under cGMP conditions and conducted a clinical trial where patient with advanced melanoma are infused with large numbers of MART1-specific CTL generated ex vivo using aAPC/A2, IL-2 and IL-15. Based on our experience with aAPC/A2 and CD8+ T cells, we have generated a series of novel aAPC (aAPC/DR1, DR3, DR4, DR7, DR10, DR11, DR13, and DR15) to stimulate HLA-DR-restricted antigen-specific CD4+ T cells. K562 has been engineered to express HLA-DRα and β chains as a single HLA allele in conjunction with Ii, HLA-DMα and β chains, CD80 and CD83. CD83 delivers a CD80-dependent T cell stimulatory signal that allows T cells to be long-lived. Following the transduction of Ii, CLIP (class II invariant chain-associated peptide) appeared on the cell surface of aAPC. Furthermore, CLIP expression on aAPC was almost completely abrogated by the introduction of HLA-DM. This result is in accordance with previous studies showing that HLA-DM catalyzes the removal of CLIP from DR thus enabling exogenous peptides to bind to empty DR molecules in late endosomes. In addition to its endogenous pinocytic activity, aAPC was made capable of Fcγ receptor-mediated endocytosis by transduction of CD64. Comparison of naïve aAPC and CD64-transduced aAPC confirmed that Fcγ receptor-mediated endocytosis is more efficient than pinocytosis to take up soluble protein and process and present DR-restricted peptides to CD4+ T cells. Using these standardized and renewable aAPC, we determined novel viral protein-derived DR-restricted CD4+ T cell epitopes and expanded large numbers of viral antigen-specific CD4+ T cells without growing bystander Foxp3+ regulatory T cells. Without any feeder cells or cloning, expansion of CD4+ T cells using aAPC and low dose IL-2 and IL-15 was sustainable up to 150 days. Immunophenotypic analysis using HLA-DR tetramers and specific mAbs revealed that expanded CD4+ T cells were CD45RA−, CD45RO+, CD62L+-, demonstrating a central/effector memory phenotype. Furthermore, intracellular cytokine analysis showed that expanded DR-restricted viral-specific CD4+ T cells secreted IL-2 and IFN-γ but much less IL-4, displaying a Th1-biased phenotype. Taken all together, these results suggest that K562-based aAPC may serve as a translatable platform to generate both antigen-specific CD4+ helper T cells and CD8+ CTL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2086 CD8+ T cells play a primary role in rejecting pathogens and tumors. Studies of CD8 null mice show that CD8 coreceptor is critical for the development of MHC class I-restricted CD8+ T cells in the thymus. However, while these mice possess low numbers of CTL with limited clonality, they are highly avid and contain acute and chronic infections. In humans, CD8 deficiency leads to no or only mild symptoms of immunodeficiency. These results suggest that the CD8 coreceptor is not absolutely necessary for the generation of antigen-specific CTL and that there exists a compensatory mechanism for the loss of CD8 expression. Common γ chain receptor cytokines (e.g. IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) transmit STAT-mediated signaling in T cells. Importantly, it has been demonstrated that crosstalk between TCR and STAT signaling occurs in CD8+ T cells. For example, weak or partial TCR-initiated signaling via the Ras/MAPK pathway can be complemented by IL-2 induced STAT activation. This mechanism enables common γ chain receptor cytokines to modulate optimal and supplement suboptimal TCR signaling. Recently, we reported the generation of K562-based artificial APC (aAPC) expressing HLA-A2 as a single HLA allele in conjunction with CD80 and CD83 (wt A2-aAPC). This aAPC can prime naïve CD8+ T cells ex vivo and generate antigen-specific CD8+ CTL with a central memory~effector memory phenotype. It has been demonstrated that the D227K/T228A mutations of A2 molecules (mut A2) inhibit the CD8/MHC interaction without affecting TCR/pMHC interactions. In this human ex vivo study, using a modified aAPC which expresses mut A2 molecules (mut A2-aAPC), we tested our hypothesis that CD8 coreceptor-independent T cell stimulation in the presence of complementary adaptive cytokines preferentially stimulates high avidity antigen-specific CTL. When freshly isolated A2+ CD8+ T cells were restimulated against recall antigen with mut A2-aAPC, both antigen specificity measured by tetramer positivity and number of expanded antigen-specific CD8+ T cells were significantly lower compared with wt A2-aAPC. Similar results were obtained when naïve CD8+ T cells were primed ex vivo with wt-A2 aAPC in the presence of CD8 coligation and then restimulated with mut-A2 aAPC in the absence of CD8 binding. When naïve CD8+ T cells were initially primed with mut A2-aAPC in the absence of CD8 binding, subsequent restimulation using wt A2-aAPC in the presence of CD8 coligation was not able to induce the proliferation of antigen-specific CD8+ CTL. As expected, when naïve CD8+ T cells were both primed and restimulated with mut A2-aAPC in the complete lack of CD8 coligation, antigen-specific CD8+ CTL did not grow. However, adding IL-21 overcame this deficiency. When CD8-independent T cell priming and restimulation by mut A2-aAPC was supplemented with IL-21, antigen-specific CD8+ CTL expansion with high functional avidity occurred. Lack of CD8 binding to MHC results in partial TCR signaling because of the absence of CD8/Lck recruitment to the proximity of MHC/TCR. While the responses of IL-21R- Jurkat and its Lck-null mutant, J.CaM1.6, to IL-21 were minimal, both showed robust IL-21 responses when stably transduced with IL-21R. Intracellular staining revealed that IL-21 induced robust phosphorylation of STAT3 but not STAT1 upon stimulation in both IL-21R-transduced Jurkat and J.CaM1.6. When IL-21R-transduced Lck-null J.CaM1.6 cells were stimulated in the presence of IL-21, T cell responses were completely abrogated by STAT3 inhibition. In contrast, the MAPK inhibitor only partially blocked the T cell responses. The combination of suboptimal doses of the STAT3 inhibitor and the MAPK inhibitor completely nullified the T cell responses, indicating an additive effect of STAT3 and MAPK inhibition. These results suggest that STAT3 but not STAT1 is critically involved in IL-21 signaling that rescues the defective T cell responses caused by the lack of Lck. Taken all together, this study suggests that CD8 ligation is critical for the expansion of post-thymic peripheral antigen-specific CTL in humans. However, STAT3-mediated IL-21 signaling can complement partial TCR signaling caused by the lack of CD8 association and expand CTL with high functional avidity. Since high functional avidity CTL are optimal effectors for the clearance of pathogens and tumors in vivo, our findings may be important for generating high avidity CTL ex vivo for effective adoptive therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4284 While adoptive T cell therapy is a promising treatment modality for cancer, the optimal approach to generate T cell grafts ex vivo is currently unknown. CD4+ T cells help generate effective immune responses by sustaining CD8+ T cell proliferation, preventing exhaustion, and establishing long-lived functional memory. Incorporation of CD4+ T cell help to expand CD8+ T cells may provide a novel strategy to generate CTL grafts for adoptive therapy. In mouse models, common γ-chain receptor cytokines and CD40/CD40L can mediate CD4+ T cell help. However, CD4+ T cell help in humans has yet to be fully defined. We therefore developed an in vitro model for human CD4+ T cell help, which utilizes a novel artificial APC, aAPC/mOKT3. K562-based aAPC/mOKT3 expresses a membranous form of anti-CD3 mAb, CD54, CD58, CD80, and CD83 and stimulates CD3+ T cells regardless of HLA haplotype or antigen specificity. Using aAPC/mOKT3, we stimulated CD8+ T cells in the presence or absence of CD4+ T cells and found that CD8+ T cells expanded better when coincubated with CD4+ T cells, suggesting the presence of CD4+ T cell help. Coculture experiments using transwell plates suggested that the observed CD4+ T cell help of CD8+ T cell expansion involved both soluble factors and cell-cell contact. To identify molecules mediating the observed CD4+ T cell help, supernatants of CD4+/CD8+ T cell mixed and separate cultures were measured for a panel of soluble factors. IL-2 and IL-21 were detected at lower levels in mixed cultures, consistent with more consumption or less production of these cytokines. Blockade of either IL-2 or IL-21 in CD4+/CD8+ T cell mixed cultures resulted in a reduction of CD8+ T cell expansion, indicating that, for both cytokines, more consumption rather than less production occurred and that IL-2 and IL-21 may serve as mediators of CD4+ T cell help. However, the addition of IL-21 to CD8+ T cells stimulated with aAPC/mOKT3 in the presence of IL-2 did not improve CD8+ T cell expansion, suggesting that IL-2 plus IL-21 cannot solely replace CD4+ T cell help. We found that the presence of CD4+ T cells upregulated the expression of IL-21R on CD8+ T cells. When we introduced IL-21R on CD8+ T cells and stimulated with aAPC/mOKT3 in the presence of IL-2 and IL-21, CD8+ T cell proliferation was restored. These results suggest that CD4+ T cells help CD8+ T cells proliferate ex vivo by secreting both IL-2/IL-21 and upregulating IL-21R. When peripheral CD3+ T cells from normal donors were stimulated with aAPC/mOKT3, the number of both CD4+ and CD8+ T cells increased. However, in contrast to other pan T cell expansion systems, aAPC/mOKT3 preferentially expanded CD8+ T cells. No obvious skewing in the Vβ usage of both CD4+ and CD8+ T cell populations was revealed by TCR Vβ repertoire analysis, supporting “unbiased” T cell expansion by aAPC/mOKT3. Moreover, HLA-restricted antigen-specific CD8+ CTL with high functional avidity could be generated from CD3+ T cells initially expanded for 4 weeks using aAPC/mOKT3. Using aAPC/mOKT3, tumor-infiltrating lymphocytes (TIL) were successfully expanded without adding soluble mAb or allogeneic feeder cells. As in peripheral T cell cultures, CD8+ T cells predominantly expanded in all cultures, including those that initially contained a minimal percentage of CD8+ T cells. Importantly, Foxp3+ Treg cells did not proliferate. Expanded T cells highly expressed CD27 and CD28, which are associated with T cell survival and persistence in vivo. They also secreted high levels of IFN-γ and IL-2, lower amounts of IL-4, and no IL-10. These results demonstrate that the aAPC/mOKT3-based system can expand functional CD8+ TIL in the presence of autologous CD4+ T cells. In conclusion, we have determined that CD4+ T cell-dependent CD8+ T cell expansion required both soluble factors secreted by and cell contact with CD4+ T cells. Among the soluble factors secreted by CD4+ T cells, IL-2 and IL-21 were necessary. Furthermore, upregulation of IL-21R on CD8+ T cells by CD4+ T cells was critical for an optimized response to IL-21. Thus, in humans, CD4+ T cells help CD8+ T cells proliferate by secreting IL-2/IL-21 and upregulating IL-21R. Our aAPC enabled expansion of CD8+ TIL in the presence of CD4+ T cell help without using soluble mAb or allogeneic feeder cells. Taken together, these results demonstrate the indispensable role of CD4+ T cell help on expanding CD8+ T cells and suggest a novel strategy to generate anti-tumor T cells ex vivo for adoptive therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 999 Human CD1d-restricted invariant natural killer T (iNKT) cells are a unique subset of innate T-cells that carry an invariant T-cell receptor (TCR) Vα24 chain paired with a TCR Vβ11 chain. They constitutively express a variety of activation and memory markers and possess the capacity to rapidly produce a variety of cytokines including IFN-γ and IL-4 upon TCR engagement. As an immune response launches, iNKT cells can induce innate immune responses and serve as a bridge between innate and adaptive immunity. In addition, it has also been shown that iNKT cells themselves have anti-tumor and anti-infectious effects in mice. However, to translate these findings and develop iNKT cell-mediated immunotherapy, iNKT cells must recognize target cells while sparing normal cells. Autoreactivity is primarily dictated by 1) the specificity and avidity of the TCR on iNKT cells; 2) the expression level of CD1d on target cells; and 3) the density and stability of endogenous ligand(s) presented by CD1d on target cells. In this study, we sought to determine the TCR Vβ11 CDR3 sequence motifs that distinguish high avidity and low avidity iNKT cells. We developed an artificial antigen-presenting cell (aAPC) to expand iNKT cells by transducing K562 with CD1d, CD80, and CD83. Using this CD1d+aAPC loaded with or without α-galactosylceramide (αGC), we expanded CD1d-restricted iNKT cells by stimulating primary CD3+ T cells expressing canonical iNKT TCR Vα24 chain. Expanded iNKT cells were stained with αGC-loaded CD1d tetramers and were found to express TCR Vβ11 chain in conjunction with transgenic canonical TCR Vα24 chain. Sequence analysis of cloned Vβ11 CDR3 revealed that the clonality of iNKT cells generated using unloaded aAPC was significantly lower than that of iNKT cells generated using loaded aAPC. Surprisingly, when cotransfected with canonical TCR Vα24 chain, some TCR Vβ11 chains isolated from iNKT cells generated using unloaded aAPC were stained with unloaded CD1d tetramers produced in HEK293 cells. This result suggests that these reconstituted iNKT TCR recognized endogenous ligand(s) derived from HEK293 cells in the context of CD1d. A comprehensive analysis of the structural avidity demonstrated that these TCR Vβ11 chains reconstituted TCR with significantly higher structural avidity than those cloned from NKT cells generated using loaded aAPC. However, this significant difference was only observed when the structural avidity was measured using unloaded tetramers but not αGC-loaded tetramers. A univariate analysis found that structural avidity was significantly higher when 1) Vβ11 CDR3 used J2-5; 2) Vβ11 CDR3 consisted of exactly 23 amino acids; and 3) Vβ11 CDR3 encoded 3 or more acidic amino acids (asparagic and glutamic acids). A multivariate analysis confirmed that all three variables were independent predictors of higher structural avidity. Furthermore, each variable is sufficient to increase the structural avidity of an iNKT TCR with low structural avidity. Intriguingly, unloaded mouse CD1d tetramers produced in HEK293 cells also stained a human iNKT TCR with high structural avidity reconstituted on both human and mouse T cells. This result suggests that the human iNKT TCR with high structural avidity possessed a cross-species reactivity to mouse CD1d in conjunction with endogenous ligand(s) derived from HEK293 cells. We next studied the autoreactivity of cloned human iNKT TCR with high structural avidity. A human T cell line, Jurkat, reconstituted with high avidity iNKT TCR, was able to recognize cells expressing CD1d endogenously (itself, SUP-T1, and primary human monocytes) and ectopically (K562, C1R, Hela). Furthermore, mouse T cells expressing human iNKT TCR with high structural avidity recognized mouse cell lines, B16, EL4, and 58, all endogenously expressing CD1d. Finally, human primary T cells transduced with the iNKT TCR with high structural avidity were autoreactive, secreting both IFN-γ and IL-4 in response to CD1d+ target cells. Using our CD1d+ aAPC-based system, we successfully isolated human iNKT cells with high structural avidity and identified the TCR Vβ11 CDR3 sequence motifs that dictate the structural avidity of iNKT cells. To develop clinically effective iNKT cell-mediated immunotherapy without unwanted autoimmunity, it will be critically important to define the range of iNKT TCR avidity that enables reactivity to pathologic but not normal cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4292 High-dose chemotherapy with autologous HSCT results in profound immunodeficiency early post transplant. Immune reconstitution is largely derived from the transplanted stem cells and occurs over a period of many months. Sex steroid hormones (testosterone and estrogen) have been shown to inhibit thymic function. Castration and sex steroid inhibition have been reported to stimulate thymic regeneration and production of T-cells and also, improved bone marrow function. We performed a study to test the hypothesis that temporarily blocking production of sex hormones using leuprolide acetate, a gonadotropin hormone-releasing hormone [GnRH] agonist, would improve thymic function and accelerate immune reconstitution following autologous stem cell transplantation. Methods: Adult patients receiving myeloablative autologous peripheral blood progenitor cell transplantation for lymphoma, Hodgkin's disease or multiple myeloma were eligible. Females and males, age 18–50 and 18–65 years, respectively, were included. Patients were required to be in remission or have chemosensitive relapse. Patients could not have received mediastinal radiation, or recent testosterone or estrogen treatment, or post transplant therapy that would affect immune function. Patients were randomized to receive 3 intramuscular injections of leuprolide acetate (Lupron 11.25 mg 3 Month Depot) or placebo approximately 3 months apart to cover the period from the date of transplantation to 8 months post HSCT. The primary endpoint was in vitro antibody and T cell response to vaccination with keyhole limpet haemocyanin (KLH) at 6 months post-HSCT. Immunoglobulins were measured by ELISA and interferon-gamma (IFNγ) by ELISpot. Results: 25 subjects (19 males and 6 females) were enrolled into the study at 3 sites. 13 were randomized to placebo and 12 to leuprolide treatment. As expected, suppression of luteinizing hormone and total testosterone was observed in males in the leuprolide acetate group at all evaluation time points during treatment (P

Description: Magnetic flux quantization is one of the defining properties of a superconductor. We report the observation of half-integer magnetic flux quantization in mesoscopic rings of superconducting β-Bi2Pd thin films. The half-quantum fluxoid manifests itself as a π phase shift in the quantum oscillation of the superconducting critical temperature. This result verifies unconventional superconductivity of β-Bi2Pd and is consistent with a spin-triplet pairing symmetry. Our findings may have implications for flux quantum bits in the context of quantum computing.