Sibylle Pscherer

2B4 (CD244) signaling by recombinant antigen-specific chimeric receptors costimulates natural killer cell activation to leukemia and neuroblastoma cells.

Purpose: Novel natural killer (NK) cell–directed strategies in cancer immunotherapy aim at specifically modulating the balance between NK cell receptor signals toward tumor-specific activation. The signaling lymphocyte activation molecule–related receptor 2B4 (CD244) is an important regulator of NK cell activation. We investigated whether 2B4-enhanced activation signals can redirect the cytolytic function of human NK cells to NK cell–resistant and autologous leukemia and tumor targets. Experimental Design: In vitro–stimulated NK cells from healthy donors and pediatric leukemia patients were gene modified with CD19 or GD2-specific chimeric receptors containing either the T-cell receptor ζ or 2B4 endodomain alone or combined. Results: Chimeric 2B4 signaling alone failed to induce interleukin-2 receptor up-regulation and cytokine secretion but triggered a specific degranulation response. Integration of the 2B4 endodomain into T-cell receptor ζ chimeric receptors significantly enhanced all aspects of the NK cell activation response to antigen-expressing leukemia or neuroblastoma cells, including CD25 up-regulation, secretion of IFN-γ and tumor necrosis factor-α, release of cytolytic granules, and growth inhibition, and overcame NK cell resistance of autologous leukemia cells while maintaining antigen specificity. Conclusion: These data indicate that the 2B4 receptor has a potent costimulatory effect in NK cells. Antigen-specific 2B4ζ-expressing NK cells may be a powerful new tool for adoptive immunotherapy of leukemia and other malignancies.

Human γδ T cells as mediators of chimaeric‐receptor redirected anti‐tumour immunity

Human peripheral blood γδ T cells (Vγ9+ Vδ2+) can be selectively expanded in vivo by the systemic administration of aminobisphosphonates without prior antigen priming. To assess the potential of human γδ T cells to serve as effector cells of specific anti‐tumour immunity, we expanded peripheral blood‐derived γδ T cells and transduced them with recombinant retrovirus encoding GD2‐ or CD19‐specific chimaeric receptors. Flow cytometric analysis of T cells from four individual donors cultured in the presence of zoledronate at day 14 of culture showed selective enrichment of the γδ T cell population (Vγ9+ Vδ2+ CD3+ CD4− CD8−) to 73–96% of total CD3+ T cells. Retroviral gene transfer resulted in chimaeric receptor surface expression in 73 ± 12% of the population. Transduced γδ T cells efficiently recognized antigen‐expressing tumour cell targets, as demonstrated by target‐specific upregulation of CD69 and secretion of interferon‐α. Moreover, transduced γδ T cells efficiently and specifically lysed the antigen‐expressing tumour targets. They could be efficiently expanded in vitro and maintained in culture for prolonged periods. Zoledronate‐activated human γδ T cells expressing chimaeric receptors may thus serve as potent and specific anti‐tumour effector cells. Their responsiveness to stimulation with aminobisphosphonates may enable the selective re‐expansion of adoptively transferred T cells in vivo, permitting long lasting anti‐tumour immune control.

A high proportion of bone marrow T cells with regulatory phenotype (CD4+CD25hiFoxP3+) in Ewing sarcoma patients is associated with metastatic disease

Immunosuppressive CD4+CD25hiFoxP3+ T cells (Treg cells) have been found at increased densities within the tumor microenvironment in many malignancies and interfere with protective antitumor immune responses. Osseous Ewing sarcomas (ESs) are thought to derive from a bone marrow (BM) mesenchymal cell of origin, and microscopic marrow involvement defines a subpopulation of patients at a high risk of relapse. We hypothesized that BM‐resident T cells may contribute to a permissive milieu for immune escape of ESs. Using 6‐color‐flow cytometry, we investigated the pattern of immune cell subset distribution including NK cells, γδ T cells, central and effector memory CD8+ and CD4+ T cells as well as T cells with regulatory phenotype (Treg cells) in BM obtained at diagnosis from 45 primary or relapsed ES patients treated within standardized protocols. Although patients at relapse had an inverted CD4:CD8 T‐cell ratio, neither CD8+ effector/memory T‐cell subsets nor Treg cells significantly differed from patients at diagnosis. No significant associations of innate and effector/memory T‐cell subpopulations with known risk factors were found, including age, gender, tumor site, primary metastases and histological tumor response. By contrast, Treg cells were found at significantly higher frequencies in patients with primary metastatic disease compared with localized ESs (5.0 vs. 3.3%, p = 0.01). Thus, increased BM Treg cells in patients with metastasized ES may reflect an immune escape mechanism that contributes to the development of metastatic disease. Immunotherapeutic strategies will have to adequately consider the regulatory milieu within areas of Ewing tumor‐immune interactions.

NK cells are dysfunctional in human chronic myelogenous leukemia before and on imatinib treatment and in BCR–ABL-positive mice

Although BCR–ABL+ stem cells in chronic myeloid leukemia (CML) resist elimination by targeted pharmacotherapy in most patients, immunological graft-versus-leukemia effects can cure the disease. Besides cytotoxic T cells, natural killer (NK) cells may have a role in immune control of CML. Here, we explored the functionality of NK cells in CML patients and in a transgenic inducible BCR–ABL mouse model. Compared with controls, NK-cell proportions among lymphocytes were decreased at diagnosis of CML and did not recover during imatinib-induced remission for 10–34 months. Functional experiments revealed limited in vitro expansion of NK cells from CML patients and a reduced degranulation response to K562 target cells both at diagnosis and during imatinib therapy. Consistent with the results in human CML, relative numbers of NK1.1+ NK cells were reduced following induction of BCR–ABL expression in mice, and the defects persisted after BCR–ABL reversion. Moreover, target-induced degranulation by expanded BCR–ABL+ NK cells was compromised. We conclude that CML is associated with quantitative and functional defects within the NK-cell compartment, which is reproduced by induced BCR–ABL expression in mice. Further work will aim at identifying the mechanisms of NK-cell deficiency in CML and at developing strategies to exploit NK cells for immunotherapy.

Target antigen expression on a professional antigen-presenting cell induces superior proliferative antitumor T-cell responses via chimeric T-cell receptors

Human T cells expressing tumor antigen-specific chimeric receptors fail to sustain their growth and activation in vivo, which greatly reduces their therapeutic value. The defective proliferative response to tumor cells in vitro can partly be overcome by concomitant CD28 costimulatory signaling. We investigated whether T-cell activation via chimeric receptors (chRec) can be further improved by ligand expression on antigen-presenting cells of B-cell origin. We generated Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) expressing a CD19-specific chRec. These CTLs are provided with native receptor stimulation by autologous EBV-transformed B-lymphoblastoid cell lines (LCLs) but exclusively with chRec (CD19-specific) stimulation by allogeneic, human leukocyte antigen (HLA)-mismatched CD19+ LCLs. CD19ζ-transduced EBV-specific CTLs specifically lysed both allogeneic EBV targets and CD19+ tumor cells through the chRec in a major histocompatibility complex-independent manner, while maintaining their ability to recognize autologous EBV targets through the native T-cell receptor. The transduced CTLs failed to proliferate in response to CD19+ tumor targets even in the presence of CD28 costimulatory signaling. By contrast, CD19 expressed on HLA-mismatched LCL-induced T-cell activation and long-term proliferation that essentially duplicated the result from native receptor stimulation with autologous LCLs, suggesting that a deficit of costimulatory molecules on target cells in addition to CD28 is indeed responsible for inadequate chRec-mediated T-cell function. Hence, effective tumor immunotherapy may be favored if engagement of the chRec on modified T cells is complemented by interaction with multiple costimulator molecules. The use of T cells with native specificity for EBV may be one means of attaining this objective.

2B4 (CD244) signaling via chimeric receptors costimulates tumor-antigen specific proliferation and in vitro expansion of human T cells

Regulatory NK cell receptors can contribute to antigen-specific adaptive immune responses by modulating T cell receptor (TCR)-induced T cell activation. We investigated the potential of the NK cell receptor 2B4 (CD244) to enhance tumor antigen-induced activation of human T cells. 2B4 is a member of the CD2 receptor subfamily with both activating and inhibitory functions in NK cells. In T cells, its expression is positively associated with the acquisition of a cytolytic effector memory phenotype. Recombinant chimeric receptors that link extracellular single-chain Fv fragments specific for the tumor-associated surface antigens CD19 and GD2 to the signaling domains of human 2B4 and/or TCRζ were expressed in non-specifically activated peripheral blood T cells by retroviral gene transfer. While 2B4 signaling alone failed to induce T cell effector functions or proliferation, it significantly augmented the antigen-specific activation responses induced by TCRζ. 2B4 costimulation did not affect the predominant effector memory phenotype of expanding T cells, nor did it increase the proportion of T cells with regulatory phenotype (CD4+CD25hiFoxP3+). These data support a costimulatory role for 2B4 in human T cell subpopulations. As an amplifier of TCR-mediated signals, 2B4 may provide a powerful new tool for immunotherapy of cancer, promoting sustained activation and proliferation of gene-modified antitumor T cells.

Activated Human γδ T Cells as Stimulators of Specific CD8+ T-cell Responses to Subdominant Epstein Barr Virus Epitopes : Potential for Immunotherapy of Cancer

The efficacy of current cancer vaccines is limited by the functional heterogeneity and poor availability and expansion of professional antigen-presenting cells (APCs). Besides their potent innate effector properties, γδ T cells have been suggested to be involved in the initiation and maintenance of adaptive immune responses. Here, we investigated the capacity of human γδ T cells to induce expansion of virus-specific T cells to Epstein Barr virus (EBV) antigens. Aminobisphosphonate-stimulated human peripheral blood-derived γδ T cells (Vγ2+Vδ2+) acquired a dual phenotype characteristic for both APCs and effector memory T cells. Coincubation of activated γδ T cells pulsed with human leukocyte antigen-restricted epitopes of either the highly stimulatory EBV lytic cycle antigen Bam H1 Z fragment leftward open reading frame or the tumor-associated latent EBV antigen latent membrane protein 2a (LMP2a) with autologous peripheral blood lymphocytes induced selective expansion of peptide-specific, fully functional CD3+CD8+ cytolytic effector memory T cells. Furthermore, γδ T APCs efficiently processed and presented endogenous antigen, as demonstrated by the capacity of LMP2a gene-transduced γδ T cells to induce expansion of T cells with broad specificity for various LMP2a peptides. The capacity of autologous γδ T cells to induce LMP2a-specific autologous cytotoxic T lymphocytes was confirmed in 2 patients with Hodgkin lymphoma. In summary, bisphosphonate-activated human γδ T cells stimulate expansion of cytotoxic effector T cells specific for both subdominant and dominant viral epitopes and thus show promise as a novel source of efficient APCs for immunotherapy of viral and malignant disease.

Gene-Engineered Varicella-Zoster Virus–Reactive CD4+ Cytotoxic T Cells Exert Tumor-Specific Effector Function

T cells with grafted specificities for surface antigens provide an avenue for rapidly producing immune effector cells with tumor specificity. However, the function of chimeric receptor (chRec) gene-modified T cells is limited by lack of T-cell expansion and persistence. We propose to use varicella zoster virus (VZV)-reactive T cells as host for the chRec because these cells can be expanded both in vitro and in vivo by stimulation of their native receptor during endogenous reexposure to the virus or by administration of VZV vaccine. We obtained human T cells reactive with VZV from the peripheral blood of seropositive donors by stimulation with VZV lysate and evaluated their characteristics after genetic modification with two tumor-specific model chRecs. Cultures dominated by cytolytic CD4(+) T cells (VZV-CTL) could be expanded and maintained in vitro. Gene-modified VZV-CTL recognized and lysed tumor targets in a MHC-independent manner while maintaining functional, MHC-restricted interaction with VZV antigen through their native receptor. Thus, chRec-transduced VZV-CTL may provide a source of potent tumor-reactive cells for adoptive immunotherapy of cancer. The availability of a safe and effective VZV vaccine provides the option of repeated in vivo stimulation to maintain high T-cell numbers until the tumor is eliminated.

Activated human γδ T cells induce peptide-specific CD8+ T-cell responses to tumor-associated self-antigens

Specific cellular immunotherapy of cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated self-antigens. Here, we investigated the capacity of human γδ T cells to induce expansion of CD8+ T cells specific for peptides derived from the weakly immunogenic tumor-associated self-antigens PRAME and STEAP1. Coincubation of aminobisphosphonate-stimulated human peripheral blood-derived γδ T cells (Vγ9+Vδ2+), loaded with HLA-A*02-restricted epitopes of PRAME, with autologous peripheral blood CD8+ T cells stimulated the expansion of peptide-specific cytolytic effector memory T cells. Moreover, peptide-loaded γδ T cells efficiently primed antigen-naive CD45RA+ CD8+ T cells against PRAME peptides. Direct comparisons with mature DCs revealed equal potency of γδ T cells and DCs in inducing primary T-cell responses and peptide-specific T-cell activation and expansion. Antigen presentation by γδ T-APCs was not able to overcome the limited capacity of peptide-specific T cells to interact with targets expressing full-length antigen. Importantly, T cells with regulatory phenotype (CD4+CD25hiFoxP3+) were lower in cocultures with γδ T cells compared to DCs. In summary, bisphosphonate-activated γδ T cells permit generation of CTLs specific for weakly immunogenic tumor-associated epitopes. Exploiting this strategy for effective immunotherapy of cancer requires strategies that enhance the avidity of CTL responses to allow for efficient targeting of cancer.

CD28 co-stimulation via tumour-specific chimaeric receptors induces an incomplete activation response in Epstein–Barr virus-specific effector memory T cells

Expression of tumour antigen‐specific chimaeric receptors in T lymphocytes can redirect their effector functions towards tumour cells. Integration of the signalling domains of the co‐stimulatory molecule CD28 into chRec enhances antigen‐specific proliferation of polyclonal human T cell populations. While CD28 plays an essential role in the priming of naive CD4+ T cells, its contribution to effector memory T cell responses is controversial. We compared the function of the chRec with and without the CD28 co‐stimulatory domain, expressing it in peripheral blood T cells or Epstein–Barr virus (EBV)‐specific T cell lines. The chimaeric T cell receptors contain an extracellular single‐chain antibody domain, to give specificity against the tumour ganglioside antigen GD2. The transduced cytotoxic T lymphocytes (CTL) maintained their specificity for autologous EBV targets and their capacity to proliferate after stimulation with EBV‐infected B cells. Intracellular cytokine staining demonstrated efficient and comparable antigen‐specific interferon (IFN)‐γ secretion by CTL following engagement of both the native and the chimaeric receptor, independent of chimaeric CD28 signalling. Furthermore, tumour targets were lysed in an antigen‐specific manner by both chRec. However, while antigen engagement by CD28ζ chRec efficiently induced expansion of polyclonal peripheral blood lymphocytes in an antigen‐dependent manner, CD28 signalling did not induce proliferation of EBV–CTL in response to antigen‐expressing tumour cells. Thus, the co‐stimulatory requirement for the efficient activation response of antigen‐specific memory cells cannot be mimicked simply by combining CD28 and ζ signalling. The full potential of this highly cytolytic T cell population for adoptive immunotherapy of cancer requires further exploration of their co‐stimulatory requirements.