Sufang Li

Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity.

Natural killer (NK) cells are important mediators of anti-tumor immunity and are active against several hematologic malignancies, including multiple myeloma (MM). Umbilical cord blood (CB) is a promising source of allogeneic NK cells but large scale ex vivo expansion is required for generation of clinically relevant CB-derived NK (CB-NK) cell doses. Here we describe a novel strategy for expanding NK cells from cryopreserved CB units using artificial antigen presenting feeder cells (aAPC) in a gas permeable culture system. After 14 days, mean fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for NK cells (CD56+/CD3−) and less than 1% CD3+ cells. Though surface expression of some cytotoxicity receptors was decreased, aAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded with IL-2 alone with respect to various inhibitory receptors, NKG2C and CD94 and maintained strong expression of transcription factors Eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses with and demonstrated cytotoxicity against various MM targets. Finally, aAPC-expanded CB-NK cells showed significant in vivo activity against MM in a xenogenic mouse model. Our findings introduce a clinically applicable strategy for the generation of highly functional CB-NK cells which can be used to eradicate MM.

Cord blood natural killer cells exhibit impaired lytic immunological synapse formation that is reversed with IL-2 exvivo expansion.

Peripheral blood natural killer (NK) cell therapy for acute myeloid leukemia has shown promise in clinical trials after allogeneic stem cell transplantation. Cord blood (CB) is another potentially rich source of NK cells for adoptive immune therapy after stem cell transplantation. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. However, despite expressing normal surface activating and inhibitory NK receptors, CB-derived NK cells have poor cytolytic activity. In this study, we investigate the cellular mechanism and demonstrate that unmanipulated CB-NK cells exhibit an impaired ability to form F-actin immunologic synapses with target leukemia cells compared with peripheral blood-derived NK cells. In addition, there was reduced recruitment of the activating receptor CD2, integrin leukocyte function-associated antigen-1, and the cytolytic molecule perforin to the CB-NK synapse site. Exvivo interleukin (IL)-2 expansion of CB-NK cells enhanced lytic synapse formation including CD2 and leukocyte function-associated antigen-1 polarization and activity. Furthermore, the acquired antileukemic function of IL-2-expanded CB-NK cells was validated using a nonobese diabetic severe combined immunodeficient IL-2 receptor common γ-chain null mouse model. We believe our results provide important mechanistic insights for the potential use of IL-2-expanded CB-derived NK cells for adoptive immune therapy in leukemia.

A prospective study of pulmonary function in Hodgkin’s lymphoma patients

BACKGROUND To prospectively study changes in lung function in Hodgkins lymphoma (HL) patients and to explore predictors for these changes over time. METHODS In all, 52 patients with HL receiving bleomycin-based chemotherapy with (n = 23) or without (n = 29) mediastinal radiotherapy were enrolled. Pretreatment pulmonary function tests were carried out. These were repeated at 1 month, 6 months, and 1 year after therapy. RESULTS With chemotherapy alone, the median %DLCO declined significantly at 1 month but returned to baseline by 6 months. The median %DLCO did not further decrease with radiotherapy, but remained persistently reduced at 1 year. In patients who received radiotherapy, having >33% of lung volume receive 20 Gy (V20) and a mean lung dose (MLD) of >13 Gy significantly predicted for persistently reduced %DLCO at 6 months (P = 0.035). Smoking significantly predicted for a persistently reduced %DLCO at 1 year (P = 0.036). On multivariable analysis, significant predictors for decline in %DLCO at 1 year were higher baseline %DLCO (P = 0.01), higher MLD (P = 0.02), and a smoking history (P = 0.02). CONCLUSIONS Several factors contribute to decline in %DLCO in HL patients who received bleomycin-based computed tomography. The identification of threshold radiation dosimetric parameters for reduced lung function may provide guidance in the radiation planning of these patients.

Noninvasive Bioluminescent Imaging Demonstrates Long-Term Multilineage Engraftment of Ex Vivo-Expanded CD34-Selected Umbilical Cord Blood Cells†‡

The use of umbilical cord blood (UCB) grafts for hematopoietic stem cell transplantation (HSCT) is a promising technique that permits a degree of human leukocyte antigen mismatch between the graft and the host without the concomitant higher rate of graft‐versus‐host disease that would be observed between an adult marrow graft and a mismatched host. A disadvantage to the use of UCB for HSCT is that immune reconstitution may be significantly delayed because of the low stem cell dose available in the graft. Ex vivo expansion of UCB CD34 cells would provide a greater number of stem cells; however, there are persistent concerns that ex vivo‐expanded CD34 cells may lose pluripotency and the ability to contribute meaningfully to long‐term engraftment. To address this issue, we transduced CD34‐selected UCB cells with a lentiviral construct expressing luciferase, and determined homing and engraftment patterns in vivo by noninvasive bioluminescent imaging in sublethally irradiated NOD/SCID/IL‐2Rγ−/− (NSG) mice. Graft contribution to multilineage commitment was also confirmed by analysis of primary and secondary transplants by flow cytometry and immunohistochemistry. Our results demonstrate that, other than a mild delay at the onset of engraftment, there were no significant differences in lineage repopulation or in long‐term or secondary engraftment between culture‐expanded and unexpanded UCB CD34‐selected cells. The results suggest that multipotent stem cells can be expanded ex vivo and can contribute meaningfully to long‐term hematopoietic engraftment. STEM CELLS 2009;27:1932–1940

De novo T-lymphocyte responses against baculovirus-derived recombinant influenzavirus hemagglutinin generated by a naive umbilical cord blood model of dendritic cell vaccination

Cancer patients and recipients of hematopoietic stem cell transplantation exhibit a negligible response to influenza vaccine. Toward the goal of addressing this issue, we developed an in vitro model of dendritic cell (DC) immunotherapy utilizing DCs generated from naïve umbilical cord blood (UCB). UCB DCs were loaded with purified rHA protein and used to stimulate autologous T-lymphocytes. Upon recall with HA-loaded autologous DC, a 4-10-fold increase in the number of IFN-gamma producing T-lymphocytes was observed in comparison to T-cells stimulated with control DCs. Antigen-specific T-cell functionality was determined by (51)Cr lytic assay. Using a peptide library of predicted HA binding epitopes, we mapped an HA-specific, DR15-restricted CD4 T-cell epitope and observed tetramer positive cells. This model demonstrates that HA-specific immune responses might possibly be generated in a de novo fashion and suggests that dendritic cell immunotherapy for the prevention of influenza in populations of immunosuppressed individuals could be feasible.

Deficient T H-1 responses from TNF-a-matured and α-CD40-matured dendritic cells

The ultimate success of dendritic cell (DC) vaccination for the active immunotherapy of neoplasia is thought to be dependent on a very large number of variables, including DC generation protocol, loading methodology, dose, route of administration, and maturation method. Although the use of a maturation cocktail comprising interleukin (IL)-1β, tumor necrosis factor-α, IL-6, and prostaglandin E2 (ITIP) has recently appeared in the literature, much of the data in the basic and clinical literature have been generated using DCs matured with the single inflammatory cytokine TNF-α. Here, we demonstrate that DCs matured with TNF-α alone or in combination with CD40 agonism are highly deficient, both physiologically and functionally, in comparison with DCs matured with IL-1β, TNF-α, IL-6, and prostaglandin E2. Empirically, the data suggest that DCs matured with these agents are deficient in the induction of type 1 T-helper responses. We further speculate that DCs matured by these methods might be suboptimal for the priming of naive responses.

Transmissible cytotoxicity of multiple myeloma cells by cord blood-derived NK cells is mediated by vesicle trafficking

Natural killer cells (NK) are important effectors of anti-tumor immunity, activated either by the downregulation of HLA-I molecules on tumor cells and/or the interaction of NK-activating receptors with ligands that are overexpressed on target cells upon tumor transformation (including NKG2D and NKP30). NK kill target cells by the vesicular delivery of cytolytic molecules such as Granzyme-B and Granulysin activating different cell death pathways, which can be Caspase-3 dependent or Caspase-3 independent. Multiple myeloma (MM) remains an incurable neoplastic plasma-cell disorder. However, we previously reported the encouraging observation that cord blood-derived NK (CB-NK), a new source of NK, showed anti-tumor activity in an in vivo murine model of MM and confirmed a correlation between high levels of NKG2D expression by MM cells and increased efficacy of CB-NK in reducing tumor burden. We aimed to characterize the mechanism of CB-NK-mediated cytotoxicity against MM cells. We show a Caspase-3- and Granzyme-B-independent cell death, and we reveal a mechanism of transmissible cell death between cells, which involves lipid–protein vesicle transfer from CB-NK to MM cells. These vesicles are secondarily transferred from recipient MM cells to neighboring MM cells amplifying the initial CB-NK cytotoxicity achieved. This indirect cytotoxicity involves the transfer of NKG2D and NKP30 and leads to lysosomal cell death and decreased levels of reactive oxygen species in MM cells. These findings suggest a novel and unique mechanism of CB-NK cytotoxicity against MM cells and highlight the importance of lipids and lipid transfer in this process. Further, these data provide a rationale for the development of CB-NK-based cellular therapies in the treatment of MM.

A novel triple purge strategy for eliminating chronic myelogenous leukemia (CML) cells from autografts.

Imatinib-refractory chronic myelogenous leukemia (CML) patients can experience long-term disease-free survival with myeloablative therapy and allogeneic hematopoietic cell transplantation; however, associated complications carry a significant risk of mortality. Transplantation of autologous hematopoietic cells has a reduced risk of complications, but residual tumor cells in the autograft may contribute to relapse. Development of methods for purging tumor cells that do not compromise the engraftment potential of the normal hematopoietic cells in the autograft has been a long-standing goal. Since primitive CML cells differentiate more rapidly in vitro than their normal counterparts and are also preferentially killed by mafosfamide and imatinib, we examined the purging effectiveness on CD34+ CML cells using a strategy that combines a brief exposure to imatinib (0.5–1.0 μM for 72 h) and then mafosfamide (30–90 μg/ml for 30 min) followed by 2 weeks in culture with cytokines (100 ng/ml each of stem cell factor, granulocyte colony-stimulating factor and thrombopoietin). Treatment with 1.0 μM imatinib, 60 μg/ml mafosfamide and 14 days of culture with cytokines eliminated BCR-ABL+ cells from chronic phase CML patient aphereses, while preserving normal progenitors. This novel purging strategy may offer a new approach to improving the effectiveness of autologous transplantation in imatinib-refractory CML patients.

AML-loaded DC generate Th1-type cellular immune responses in vitro.

BACKGROUND The generation of AML-specific T-lymphocyte responses by leukemia-derived DC has been documented by multiple investigators and is being pursued clinically. An obstacle to widespread use of this strategy is that it has not been possible to generate leukemic DC from all patients, and an alternative approach is needed if the majority of leukemia patients are to receive therapeutic vaccination in conjunction with other treatment protocols. METHODS In the present study, we generated DC from CD14-selected monocytes isolated from healthy donor PBPC and loaded them with a total cell lysate from AML patient blasts. RESULTS Immature in vitro-derived DC exhibited robust phagocytic activity, and mature DC demonstrated high expression of CD80, CD83, CD86 and the chemokine receptor CCR7, important for DC migration to local lymph nodes. Mature, Ag-loaded DC were used as APC for leukemia-specific cytotoxic T-lymphocyte (CTL) induction and demonstrated cytotoxic activity against leukemic targets. CTL lysis was Ag-specific, with killing of both allogeneic leukemic blasts and autologous DC loaded with allogeneic AML lysate. HLA-matched controls were not lysed in our system. DISCUSSION These data support further research into the use of this strategy as an alternative approach to leukemia-derived DC vaccination.

Ex vivo B cell depletion using the Eligix B Cell SC system and autologous peripheral blood stem cell transplantation in patients with follicular non-Hodgkin's lymphoma.

Summary:One limitation of ASCT is the potential reinfusion of tumor cells contaminating PBSC. The Eligix B cell SC system consists of high-density microparticles coated with anti-B cell antibodies. To determine if this system eliminates B cells and lymphoma cells from PBSC, immunocytochemistry and PCR of the bcl-2/IgH rearrangement were performed, and correlated with patient outcome after ASCT. Eligible patients (n=29) had relapsed or transformed follicular NHL with bone marrow involvement <20%, and all lymph nodes <5 cm. PBSCs were mobilized with cyclophosphamide/G-CSF (n=21), and patients were conditioned with cyclophosphamide, carmustine and etoposide.Using immunocytochemistry on PBSC, the median number of CD20+ cells pre-purge was 310/106 (range 0–16692) and post-purge was 0.75/106; the median log B cell depletion was 2.7 (range 1.4–3.9). B cell depletion correlated with PFS after ASCT (P=0.06). Of 17 available samples for PCR, only four had a detectable t(14;18) breakpoint. After purging, all four remained PCR+; two had a 1–3 log depletion of lymphoma cells. At median follow-up of 18 months, 10 patients, including five infused with PCR-negative PBSC, have had disease progression. The paucity of PCR-informative patients, possibly related to in vivo rituximab therapy, limited the utility of minimal residual disease as a surrogate marker of clinical outcome.