Xiaocai Yan

Depletion of CD25⁺ T cells from hematopoietic stem cell grafts increases posttransplantation vaccine-induced immunity to neuroblastoma.

A multifaceted immunotherapeutic strategy that includes hematopoietic stem cell (HSC) transplantation, T-cell adoptive transfer, and tumor vaccination can effectively eliminate established neuroblastoma tumors in mice. In vivo depletion of CD4⁺ T cells in HSC transplantation recipients results in increased antitumor immunity when adoptively transferred T cells are presensitized, but development of T-cell memory is severely compromised. Because increased percentages of regulatory T (Treg) cells are seen in HSC transplantation recipients, here we hypothesized that the inhibitory effect of CD4⁺ T cells is primarily because of the presence of expanded Treg cells. Remarkably, adoptive transfer of presensitized CD25-depleted T cells increased tumor vaccine efficacy. The enhanced antitumor effect achieved by ex vivo depletion of CD25⁺ Treg cells was similar to that achieved by in vivo depletion of all CD4⁺ T cells. Depletion of CD25⁺ Treg cells resulted in elevated frequencies of tumor-reactive CD8 and CD4⁺ T cells and increased CD8-to-Treg cell ratios inside tumor masses. All mice given presensitized CD25-depleted T cells survived a tumor rechallenge, indicating the development of long-term CD8⁺ T-cell memory to tumor antigens. These observations should aid in the future design of immunotherapeutic approaches that promote the generation of both acute and long-term antitumor immunity.

Murine CD8 lymphocyte expansion in vitro by artificial antigen-presenting cells expressing CD137L (4-1BBL) is superior to CD28, and CD137L expressed on neuroblastoma expands CD8 tumour-reactive effector cells in vivo.

The ability to expand tumour‐infiltrating lymphocytes in vitro has been greatly enhanced by the use of antigen‐independent mechanisms of immune cell costimulation. We have produced human, using the K562 cell line, and murine, using YAC‐1 cells, artificial antigen presenting cells (aAPC) and demonstrate that these cell types stimulate murine lymphocyte populations in distinct ways. Using aAPC that have been transfected with CD137L (4‐1BBL) and CD32 (FcRγII), as a means to bind anti‐CD3 and anti‐CD28 antibody, we found that CD4 cells preferentially expanded in vitro with K562 aAPC, while CD8 cells expanded with both K562 and YAC‐1 aAPC. Co‐stimulation mediated by CD137L on aAPC was superior to that mediated by anti‐CD28 antibody. This was seen in both long and short‐term expansion assays, and by the rapid induction of a CD8+ DX5+ population. DX5 serves, under these in vitro conditions, as a general marker for lymphocyte activation. In vivo, the superiority of CD137L was demonstrated by the induction of T helper 1 effectors seen in freshly isolated splenocytes from mice immunized with CD137L‐expressing neuroblastoma tumour vaccines. The ability to stimulate a strong CD8 CTL response in vivo correlated with the induction of a DX5+ cell population in splenocytes with a memory‐effector phenotype. The presence of this unique DX5+ cell population, phenotypically distinct with regards to CD69 and CD62L expression from DX5+ cells induced by aAPC in vitro, may be associated with the ability of CD137L to induce strong anti‐tumour immunity.

Dual expression of CD80 and CD86 produces a tumor vaccine superior to single expression of either molecule.

A murine model for neuroblastoma, Neuro-2a (N2a), was used to establish a model tumor vaccine. An aggressive subclone of N2a and the less aggressive parental line were transfected with CD80, CD86, or both molecules and stable lines were established. The less aggressive N2a expressing either CD80 or CD86 induced anti-tumor immunity. In contrast, dual expression of CD80 and CD86 was required to initiate a protective anti-tumor immune response against the aggressive subclone. Control of tumor growth was dependent on CD8+ lymphocytes that infiltrated dual-expressing (CD80 and CD86) lesions. These tumor-infiltrating lymphocytes (TIL) exhibited a non-classical mechanism of tumor cell lysis that may require both the up-regulation of cell surface molecules on the tumor and the subsequent lytic activity normally associated with CD8+ TIL. Although Fas was up-regulated by the tumor in the presence of IFN-gamma, N2a and transfected N2a cell lines were not sensitive to Fas-mediated lysis.

Expression of Macrophage Migration Inhibitory Factor by Neuroblastoma Leads to the Inhibition of Antitumor T Cell Reactivity In Vivo

Neuroblastomas and many other solid tumors produce high amounts of macrophage migration inhibitory factor (MIF), which appears to play a role in tumor progression. We found that MIF expression in neuroblastoma inhibits T cell proliferation in vitro, raising the possibility that MIF promotes tumorigenesis, in part, by suppressing antitumor immunity. To examine whether tumor-derived MIF leads to suppression of T cell immunity in vivo, we generated MIF-deficient neuroblastoma cell lines using short hairpin small interfering RNAs (siRNA). The MIF knockdown (MIFKD) AGN2a neuroblastoma cells were more effectively rejected in immune-competent mice than control siRNA-transduced or wild-type AGN2a. However, the increased rejection of MIFKD AGN2a was not observed in T cell-depleted mice. MIFKD tumors had increased infiltration of CD8+ and CD4+ T cells, as well as increased numbers of macrophages, dendritic cells, and B cells. Immunization with MIFKD AGN2a cells significantly increased protection against tumor challenge as compared with immunization with wild-type AGN2a, and the increased protection correlated with elevated frequencies of tumor-reactive CD8+ T cells in the lymphoid tissue of treated animals. Increased numbers of infiltrating tumor-reactive CD8+ T cells were also observed at the site of tumor vaccination. In vitro, treatment of AGN2a-derived culture supernatants with neutralizing MIF-specific Ab failed to reverse T cell suppressive activity, suggesting that MIF is not directly responsible for the immune suppression in vivo. This supports a model whereby MIF expression in neuroblastoma initiates a pathway that leads to the suppression of T cell immunity in vivo.

Neuroblastoma cells transiently transfected to simultaneously express the co-stimulatory molecules CD54, CD80, CD86, and CD137L generate antitumor immunity in mice

The goal of this study was to show that nonviral gene transfection technology can be used to genetically modify neuroblastoma cells with immune stimulatory molecules, and that the modified cells can generate an antitumor immune response. The authors found that an electroporation-based gene transfection method, nucleofection, could be used to modify mouse AGN2a (an aggressive variant of Neuro-2a) neuroblastoma cells to simultaneously express as many as four different immune stimulatory molecules encoded by separate plasmid vectors. Within 18 hours after nucleofection, greater than 60% of the cells typically expressed the transfected gene products, and the percentages of cells expressing the products often exceeded 96%. High levels of plasmid in cell nuclei immediately after nucleofection documented instantaneous availability of gene vectors to the transcriptional machinery. AGN2a cells nucleofected to express the co-stimulatory molecules CD80 and CD86 expressed higher levels of these molecules than cells that had been permanently transfected with these same plasmid vectors, and the nucleofected cells were as effective as the permanently transfected cells at inducing an antitumor response in vivo in a tumor prevention model. AGN2a cells nucleofected with four separate plasmid vectors encoding CD54, CD80, CD86, and CD137L induced a T-cell immune response in vitro and served as a potent tumor vaccine in the tumor prevention model. These data show that transient transfection using a nonviral based method, nucleofection, can be used to rapidly generate novel cell-based tumor vaccines.

Induction of a VLA-2 (CD49b)-Expressing Effector T Cell Population by a Cell-Based Neuroblastoma Vaccine Expressing CD137L

In malignancies where no universally expressed dominant Ag exists, the use of tumor cell-based vaccines has been proposed. We have modified a mouse neuroblastoma cell line to express either CD80 (B7.1), CD137L (4-1BBL), or both receptors on the tumor cell surface. Vaccines expressing both induce a strong T cell response that is unique in that among responding CD8 T cells, a T effector memory cell (TEM) response arises in which a large number of the TEM express the α-chain of VLA-2, CD49b. We demonstrate using both in vitro and in vivo assays that the CD49b+ CD8 T cell population is a far more potent antitumor effector cell population than nonfractionated CD8 or CD49b− CD8 T cells and that CD49b on vaccine-induced CD8 T cells mediates invasion of a collagen matrix. In in vivo rechallenge studies, CD49b+ T cells no longer expanded, indicating that CD49b TEM expansion is restricted to the initial response to vaccine. To demonstrate a mechanistic link between the expression of costimulatory molecules on the vaccine and CD49b on responding T cells, we stimulated naive T cells in vitro with artificial APC expressing different combinations of anti-CD3, anti-CD28, and CD137L. Although some mRNA encoding CD49b was induced by combining anti-CD3 with anti-CD28 or CD137L, the highest level was induced when all three signals were present. This indicates that CD49b expression results from additive costimulation and that the level of CD49b message serves as an indicator of the effectiveness of T cell activation by a cell-based vaccine.

Nucleofection of Mouse Neuroblastoma Cells with Plasmid DNA Vectors Can Be Used To Generate Novel Cell-Based Tumor Vaccines

analog, and the PADRE helper epitope were emulsified in Montanide adjuvant and tested for safety and immunogenicity in HLA-A2+ NSCLC (stage IIb/IIIa) and colon (stage III) cancer patients with no detectable disease following standard treatment. Patients in this Phase I trial received 6 vaccine treatments every 3 wk, each at a dose of 5 mg peptide (0.5 mg/epitope). CTL responses in the peripheral blood were measured for all vaccine epitopes using a validated interferon-gamma ELISPOT assay following a single in vitro stimulation with epitope. Based on 13 patients who have completed treatment so far, the EP-2101 multi-epitope vaccine appears to be safe and welltolerated. Vaccine-induced CTL responses were observed in 12 of the 13 patients, with 8 of the patients generating responses to

Tumor-derived macrophage migration inhibitory factor (MIF) inhibits T lymphocyte activation

5 epitopes in the vaccine. Seven of the patients induced CTLs to

Annexin-V promotes anti-tumor immunity and inhibits neuroblastoma growth in vivo

3 wild-type TAA epitopes, either to the natural wild-type epitopes in EP-2101 or to the corresponding wild-type epitopes of vaccine analogs. Overall, 8 of the 9 CTL epitopes in EP2101 (all 7 analogs and 1 wild-type) were immunogenic and 6 of the 7 analog epitopes induced wild-type cross-reactive CTLs in at least one patient. A Th cell response against PADRE was also detected following vaccination in 6 of 10 patients tested so far. The breadth of CTL responses against multiple native and analog CTL epitopes induced by EP-2101 indicates that this vaccine may provide effective immunotherapy in patients with diverse types of cancer expressing common TAAs.