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.

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 G(D2)-specific chimeric receptors containing either the T-cell receptor zeta 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 zeta 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-gamma and tumor necrosis factor-alpha, 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 2B4zeta-expressing NK cells may be a powerful new tool for adoptive immunotherapy of leukemia and other malignancies.

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

Immunosuppressive CD4+CD25(hi)FoxP3+ T cells (T(reg) 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, gammadelta T cells, central and effector memory CD8+ and CD4+ T cells as well as T cells with regulatory phenotype (T(reg) 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 T(reg) 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, T(reg) 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 T(reg) 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.

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 G(D2) to the signaling domains of human 2B4 and/or TCRzeta 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 TCRzeta. 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+CD25(hi)FoxP3+). 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 gammadelta 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, gammadelta T cells have been suggested to be involved in the initiation and maintenance of adaptive immune responses. Here, we investigated the capacity of human gammadelta T cells to induce expansion of virus-specific T cells to Epstein Barr virus (EBV) antigens. Aminobisphosphonate-stimulated human peripheral blood-derived gammadelta T cells (Vgamma2+Vdelta2+) acquired a dual phenotype characteristic for both APCs and effector memory T cells. Coincubation of activated gammadelta 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 CD3CD8 cytolytic effector memory T cells. Furthermore, gammadelta T APCs efficiently processed and presented endogenous antigen, as demonstrated by the capacity of LMP2a gene-transduced gammadelta T cells to induce expansion of T cells with broad specificity for various LMP2a peptides. The capacity of autologous gammadelta T cells to induce LMP2a-specific autologous cytotoxic T lymphocytes was confirmed in 2 patients with Hodgkin lymphoma. In summary, bisphosphonate-activated human gammadelta 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.

Rhabdomyosarcoma lysis by T cells expressing a human autoantibody-based chimeric receptor targeting the fetal acetylcholine receptor.

Rhabdomyosarcomas are the most frequent malignant soft tissue tumors of childhood; however, because current multimodality treatments fail to improve the poor survival rate of children with metastatic rhabdomyosarcoma, new treatments are required. We previously identified the gamma-subunit of the fetal acetylcholine receptor (fAChR) as a specific cell surface target in rhabdomyosarcoma. Here, we engineered human T lymphocytes to express chimeric receptors composed of the antigen-binding domain of a human anti-fAChR antibody joined to the signaling domain of the human T-cell receptor zeta-chain. The interaction of fAChRzeta-transduced T cells with fAChR-positive rhabdomyosarcoma cell lines, but not with fAChR-negative control cells, induced T-cell activation characterized by strong secretion of IFN-gamma and delayed lysis of tumor cells. Importantly, we found that in six of six rhabdomyosarcoma patients, chemotherapy increased fAChR expression on residual tumor cells in vivo. Our observations suggest that these fully human chimeric fAChRzeta-transduced T cells, which should be well tolerated by the patient, have potential use in vivo both as a primary treatment for rhabdomyosarcoma and as a complementary approach to eradicate residual tumor cells after chemotherapy.

Transmembrane domain II of the Na+/proline transporter PutP of Escherichia coli forms part of a conformationally flexible, cytoplasmic exposed aqueous cavity within the membrane.

The Na+/proline transporter PutP of Escherichia coli is a member of a large family of Na+/substrate symporters. Previous work on PutP suggests an involvement of the region ranging from Asp-55 to Gly-58 in binding of Na+ and/or proline (Pirch, T., Quick, M., Nietschke, M., Langkamp, M., Jung, H. (2002) J. Biol. Chem. 277, 8790-8796). In this study, a complete Cys scanning mutagenesis of transmembrane domain II (TM II) of PutP was performed to further elucidate the role of the TM in the transport process. Strong defects of PutP function were observed upon substitution of Ala-48, Ala-53, Trp-59, and Gly-63 by Cys in addition to the previously characterized residues Asp-55, Ser-57, and Gly-58. However, except for Asp-55 none of these residues proved essential for function. The activity of eight mutants was sensitive to N-ethylmaleimide inhibition with the sensitive positions clustering predominantly on a hydrophilic face in the cytoplasmic half of TM II. The same face was also highly accessible to the bulky sulfhydryl reagent fluorescein 5-maleimide in randomly oriented membrane vesicles, suggesting an unrestricted accessibility of the corresponding amino acid positions via an aqueous pathway. Na+ stimulated the reactivity of Cys toward fluorescein 5-maleimide at two positions while proline inhibited reaction of the sulfhydryl group at nine positions. Taken together, the results demonstrate that TM II of PutP is of particular functional importance. It is proposed that hydrophilic residues in the cytoplasmic half of TM II participate in the formation of an aqueous cavity in the membrane that allows Na+ and/or proline binding to residues located in the middle of the TM (e.g. Asp-55 and Ser-57). In addition, the data indicate that TM II participates in Na+- and proline-induced conformational alterations.