Increased chemosensitivity via BRCA2-independent DNA damage in DSS1- and PCID2-depleted breast carcinomas.

Breast cancer, the most common malignancy among women, is closely associated with mutations in the tumor suppressor gene BRCA. DSS1, a component of the TRanscription-EXport-2 (TREX-2) complex involved in transcription and mRNA nuclear export, stabilizes BRCA2 expression. DSS1 is also related to poor prognosis in patients with breast cancer owing to the induction of chemoresistance. Recently, BRCA2 was shown to be associated with the TREX-2 component PCID2, which prevents DNA:RNA hybrid R-loop formation and transcription-coupled DNA damage. This study aimed to elucidate the involvement of these TREX-2 components and BRCA2 in the chemosensitivity of breast carcinomas. Our results showed that compared with that in normal breast tissues, DSS1 expression was upregulated in human breast carcinoma, whereas PCID2 expression was comparable between normal and malignant tissues. We then compared patient survival time among groups divided by high or low expressions of DSS1, BRCA2, and PCID2. Increased DSS1 expression was significantly correlated with poor prognosis in recurrence-free survival time, whereas no differences were detected in the high and low BRCA2 and PCID2 expression groups. We performed in vitro analyses, including propidium iodide nuclear staining, single-cell gel electrophoresis, and clonogenic survival assays, using breast carcinoma cell lines. The results confirmed that DSS1 depletion significantly increased chemosensitivity, whereas overexpression conferred chemoresistance to breast cancer cell lines; however, BRCA2 expression did not affect chemosensitivity. Similar to DSS1, PCID2 expression was also inversely correlated with chemosensitivity. These results strongly suggest that DSS1 and PCID2 depletion is closely associated with increased chemosensitivity via BRCA2-independent DNA damage. Together with the finding that DSS1 is not highly expressed in normal breast tissues, these results demonstrate that DSS1 depletion confers a druggable trait and may contribute to the development of novel chemotherapeutic strategies to treat DSS1-depleted breast carcinomas independent of BRCA2 mutations.

Impact of germinal center-associated nuclear protein polymorphisms on breast cancer risk and prognosis in a Japanese population.

BACKGROUND: Germinal center-associated nuclear protein (GANP) is a phosphoprotein involved in mRNA export and regulation of DNA recombination. Although GANP expression in human breast cancer tissue is associated with breast cancer prognosis, the association between the genetic background of GANP and susceptibility and prognosis of breast cancer is unclear. METHODS: We selected 694 breast cancer cases and 1376 age- and menopausal status-matched non-cancer controls from the Hospitable-based Epidemiologic Research Program, conducted at Aichi Cancer Center between 2001 and 2005. We evaluated the impact of two polymorphisms at the GANP locus (rs2839178 and rs11702450) on the susceptibility and prognosis of breast cancer. Reference alleles were defined as the A allele for rs2839178 and G allele for rs11702450. RESULTS: The GG genotype of rs2839178 was statistically significantly associated with breast cancer risk (odds ratio [OR] 0.48, 95% confidence interval [CI] 0.30-0.76, P = 0.002). In prognostic analysis, compared to those with AA genotype of rs2839178, patients with AG or GG genotypes had longer disease-free survival (DFS) (hazard ratio [HR] 0.71, 95% CI 0.49-1.04 and HR 0.42, 95% CI 013-1.42, respectively, P for trend = 0.04). eQTL analysis indicated that association with rs2839178 can be explained by the effect of rs2839173 on expression of GANP/MCM3AP. CONCLUSIONS: The G allele of rs2839178 at the GANP locus was significantly associated with reduced breast cancer risk and longer DFS in breast cancer patients, showing a consistent direction in the association between susceptibility and clinical outcome. GANP is, therefore, important for the occurrence and progression of sporadic breast cancer.

Improving TCR affinity on 293T cells.

This study presents an efficient method to improve TCR affinity, comprising 1) CDR-directed saturation mutation of TCR cDNA, 2) transient TCR display on CD3-expressing HEK293T (CD3-293T) cells by simple plasmid transfection, 3) staining with HLA-tetramers, and 4) multi-round sorting of cells with CD8-independent tetramer binding on a flow cytometer. Using these procedures, we successfully identified mutant TCRs with enhanced binding from an HLA-A*24:02-restricted, human telomerase reverse transcriptase (hTERT)-specific TCR. Two such clones, 2A7A and 2D162, harboring mutations in CDR1 and CDR2 of TCRß, respectively, were isolated with both showing sequential four amino acid substitutions. When expressed on CD3-293T cells along with wild-type TCRα, the TCR molecules of these mutants as well as their combinatory mutation, bound to HLA-A24/hTERT-tetramers more strongly than the wild-type TCRs, without binding to control tetramers. Besides, in order to facilitate a functional study of TCR, we established an artificial T cell line, designated as CD8I-J2, which expresses a human CD8 and IFN-γ producing cassette by modifying Jurkat-derived J.RT3-T3.5 cells. CD8I-J2 cells expressing wild-type or affinity-enhanced hTERT-specific TCRs were analyzed for their recognition of serially diluted cognate peptide on HLA-A*24:02-transduced T2 cells. CD8I-J2 cells expressing each mutant TCR recognized the hTERT peptide at lower concentrations than wild-type TCR. The hierarchy of peptide recognition is concordant with tetramer binding on CD3-293T cells and none of these mutant TCRs were cross-reactive with irrelevant peptides reported to be present on HLA-A*24:02 molecules as far as tested. These methods might thus be useful for obtaining high affinity mutants from other TCRs of interest.

Generation of TCR-Expressing Innate Lymphoid-like Helper Cells that Induce Cytotoxic T Cell-Mediated Anti-leukemic Cell Response.

CD4+ T helper (Th) cell activation is essential for inducing cytotoxic T lymphocyte (CTL) responses against malignancy. We reprogrammed a Th clone specific for chronic myelogenous leukemia (CML)-derived b3a2 peptide to pluripotency and re-differentiated the cells into original TCR-expressing T-lineage cells (iPS-T cells) with gene expression patterns resembling those of group 1 innate lymphoid cells. CD4 gene transduction into iPS-T cells enhanced b3a2 peptide-specific responses via b3a2 peptide-specific TCR. iPS-T cells upregulated CD40 ligand (CD40L) expression in response to interleukin-2 and interleukin-15. In the presence of Wilms tumor 1 (WT1) peptide, antigen-specific dendritic cells (DCs) conditioned by CD4-modified CD40Lhigh iPS-T cells stimulated WT1-specific CTL priming, which eliminated WT1 peptide-expressing CML cells in vitro and in vivo. Thus, CD4 modification of CD40Lhigh iPS-T cells generates innate lymphoid helper-like cells inducing bcr-abl-specific TCR signaling that mediates effectiveanti-leukemic CTL responses via DC maturation, showing potential for adjuvant immunotherapy against leukemia.

BCR-ABL-specific CD4+ T-helper cells promote the priming of antigen-specific cytotoxic T cells via dendritic cells.

The advent of tyrosine kinase inhibitor (TKI) therapy markedly improved the outcome of patients with chronic-phase chronic myeloid leukemia (CML). However, the poor prognosis of patients with advanced-phase CML and the lifelong dependency on TKIs are remaining challenges; therefore, an effective therapeutic has been sought. The BCR-ABL p210 fusion protein's junction region represents a leukemia-specific neoantigen and is thus an attractive target for antigen-specific T-cell immunotherapy. BCR-ABL p210 fusion-region-specific CD4+ T-helper (Th) cells possess antileukemic potential, but their function remains unclear. In this study, we established a BCR-ABL p210 b3a2 fusion-region-specific CD4+ Th-cell clone (b3a2-specific Th clone) and examined its dendritic cell (DC)-mediated antileukemic potential. The b3a2-specific Th clone recognized the b3a2 peptide in the context of HLA-DRB1*09:01 and exhibited a Th1 profile. Activation of this clone through T-cell antigen receptor stimulation triggered DC maturation, as indicated by upregulated production of CD86 and IL-12p70 by DCs, which depended on CD40 ligation by CD40L expressed on b3a2-specific Th cells. Moreover, in the presence of HLA-A*24:02-restricted Wilms tumor 1 (WT1)235-243 peptide, DCs conditioned by b3a2-specific Th cells efficiently stimulated the primary expansion of WTI-specific cytotoxic T lymphocytes (CTLs). The expanded CTLs were cytotoxic toward WT1235-243-peptide-loaded HLA-A*24:02-positive cell lines and exerted a potent antileukemic effect in vivo. However, the b3a2-specific Th-clone-mediated antileukemic CTL responses were strongly inhibited by both TKIs and interferon-α. Our findings indicate a crucial role of b3a2-specific Th cells in leukemia antigen-specific CTL-mediated immunity and provide an experimental basis for establishing novel CML immunotherapies.

Highly functional T-cell receptor repertoires are abundant in stem memory T cells and highly shared among individuals.

To expand our knowledge of the ontogeny of the T-cell receptor (TCR) repertoire of antigen-specific T-cell subsets, we combined next-generation deep sequencing and single-cell multiplex clonotype analysis to evaluate the diversity and frequency of paired TCRs, their functions and whether clonotypic TCRs are shared among different individuals. Using an HLA-A*02-restricted cytomegalovirus (CMV) pp65-derived immunogenic peptide, we found that the more dominant pp65-specific TCR clonotypes in the blood of healthy donors have higher binding affinities for the CMV peptide and arise from clonotypes that are highly shared among individuals. Interestingly, these highly shared HLA-A*02-restricted CMV-specific TCRs were detected in a CMV-seronegative individual as well as in HLA-A*02-negative donors albeit at lower frequency. More intriguingly, these shared TCR clonotypes were abundant in the stem memory T-cell subset, and TCR diversity of the stem memory T-cell repertoire was significantly lower than in the central memory and effector memory T-cell repertoires. These results suggest that the stem memory T-cell subset may serve as a reservoir of highly shared and highly functional memory T-cells.

Systemic DC Activation Modulates the Tumor Microenvironment and Shapes the Long-Lived Tumor-Specific Memory Mediated by CD8+ T Cells.

Strategies to reprogram the tumor microenvironment are being explored to improve cancer immunotherapy. In one approach, we have targeted dendritic cells (DC) to improve their function with adjuvant vector cells (aAVC) that are engineered from NKT ligand-loaded CD1d(+) allogeneic cells transfected with tumor antigen mRNAs. Here, we report the finding that this approach also programs local immune responses by establishing tertiary lymphoid structures (TLS), which include expanded antigen-specific CD8(+) T-cell clones, mobilized DCs, and normalized tumor vasculature. aAVC therapy also expanded specific Vß-expressing antitumor T-cell clones, leading to the formation of long-term memory T cells. When combined with PD-1 blockade, aAVC infusion triggered regression of poorly immunogenic tumor cells that did not respond to PD-1 blockade alone, as well as expansion of antigen-specific CD8(+) T-cell clones in the tumor. The findings of this study help to inform a next-generation platform for the generation of efficacious cancer vaccines. Cancer Res; 76(13); 3756-66. ©2016 AACR.

A Functionally Superior Second-Generation Vector Expressing an Aurora Kinase-A-Specific T-Cell Receptor for Anti-Leukaemia Adoptive Immunotherapy.

Aurora Kinase A is a cancer-associated protein normally involved in the regulation of mitosis. Being over-expressed in a range of cancers, it is a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor sequences cognisant of HLA-A*0201-restricted Aurora Kinase A antigen has previously been shown to transfer specific immunoreactivity against the target peptide in a Human Lymphocyte Antigen-restricted manner. While T cell receptor gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumour-reactive lymphocytes from a patient's own cells, one hurdle is potential mispairing and competition between exogenous and endogenous T cell receptor chains. We have used a retroviral vector design bearing a short-interfering RNA that downregulates endogenous T cell receptor chains, without affecting expression of the transgenic T cell receptor sequences. The T cell receptor expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the T cell receptor alpha and beta chains, further enhancing formation of the desired T cell receptor. Via a simple, modular cloning method, we have cloned the alpha and beta chains of the anti-Aurora Kinase A-reactive T cell receptor into this 'siTCR' vector. We then compared the activity of this vector against the original, 'conventional' vector across a panel of assays. T cell receptors expressed from the siTCR-vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed T cell receptors was superior using the siTCR design, and this was achieved at lower vector copy numbers. Maintaining T cell receptor efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of T cell receptor gene-transfer will be crucial for clinical applications of this technology.

Epstein-Barr virus-associated leukemic lymphoma after allogeneic stem cell transplantation.

Leukemic Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative diseases (PTLD) following allogeneic hematopoietic stem cell transplantation are extremely rare. We can successfully treat an EBV-associated leukemic lymphoma patient with rituximab, cidofovir, and donor lymphocyte infusion (DLI). In the present case, EBV-specific T cells that were present in the peripheral blood before rituximab administration treatment rapidly increased after DLI in association with a decrease in the EBV-DNA load.

Development of Engineered T Cells Expressing a Chimeric CD16-CD3ζ Receptor to Improve the Clinical Efficacy of Mogamulizumab Therapy Against Adult T-Cell Leukemia.

PURPOSE: Mogamulizumab (Mog), a humanized anti-CC chemokine receptor 4 (CCR4) mAb that mediates antibody-dependent cellular cytotoxicity (ADCC) using FcγR IIIa (CD16)-expressing effector cells, has recently been approved for treatment of CCR4-positive adult T-cell leukemia (ATL) in Japan. However, Mog failure has sometimes been observed in patients who have accompanying chemotherapy-associated lymphocytopenia. In this study, we examined whether adoptive transfer of artificial ADCC effector cells combined with Mog would overcome this drawback. EXPERIMENTAL DESIGN: We lentivirally gene-modified peripheral blood T cells from healthy volunteers and ATL patients expressing the affinity-increased chimeric CD16-CD3ζ receptor (cCD16ζ-T cells). Subsequently, we examined the ADCC effect mediated by those cCD16ζ-T cells in the presence of Mog against ATL tumor cells both in vitro and in vivo RESULTS: cCD16ζ-T cells derived from healthy donors killed in vitro Mog-opsonized ATL cell line cells (n = 7) and primary ATL cells (n = 4) depending on both the number of effector cells and the dose of the antibody. cCD16ζ-T cells generated from ATL patients (n = 3) also exerted cytocidal activity in vitro against Mog-opsonized autologous ATL cells. Using both intravenously disseminated model (n = 5) and subcutaneously inoculated model (n = 4), coadministration of Mog and human cCD16ζ-T cells successfully suppressed tumor growth in xenografted immunodeficient mice, and significantly prolonged their survival (P < 0.01 and P = 0.02, respectively). CONCLUSIONS: These data strongly suggest clinical feasibility of the novel combined adoptive immunotherapy using cCD16ζ-T cells and Mog for treatment of aggressive ATL, particularly in patients who are ineligible for allogeneic hematopoietic stem cell transplantation. Clin Cancer Res; 22(17); 4405-16. ©2016 AACR.

Cellular Adjuvant Properties, Direct Cytotoxicity of Re-differentiated Vα24 Invariant NKT-like Cells from Human Induced Pluripotent Stem Cells.

Vα24 invariant natural killer T (iNKT) cells are a subset of T lymphocytes implicated in the regulation of broad immune responses. They recognize lipid antigens presented by CD1d on antigen-presenting cells and induce both innate and adaptive immune responses, which enhance effective immunity against cancer. Conversely, reduced iNKT cell numbers and function have been observed in many patients with cancer. To recover these numbers, we reprogrammed human iNKT cells to pluripotency and then re-differentiated them into regenerated iNKT cells in vitro through an IL-7/IL-15-based optimized cytokine combination. The re-differentiated iNKT cells showed proliferation and IFN-γ production in response to α-galactosylceramide, induced dendritic cell maturation and downstream activation of both cytotoxic T lymphocytes and NK cells, and exhibited NKG2D- and DNAM-1-mediated NK cell-like cytotoxicity against cancer cell lines. The immunological features of re-differentiated iNKT cells and their unlimited availability from induced pluripotent stem cells offer a potentially effective immunotherapy against cancer.

Generation of mouse pluripotent stem cell-derived proliferating myeloid cells as an unlimited source of functional antigen-presenting cells.

The use of dendritic cells (DC) to prime tumor-associated antigen-specific T-cell responses provides a promising approach to cancer immunotherapy. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can differentiate into functional DCs, thus providing an unlimited source of DCs. However, the previously established methods of generating practical volumes of DCs from pluripotent stem cells (PSC) require a large number of PSCs at the start of the differentiation culture. In this study, we generated mouse proliferating myeloid cells (pMC) as a source of antigen-presenting cells (APC) using lentivirus-mediated transduction of the c-Myc gene into mouse PSC-derived myeloid cells. The pMCs could propagate almost indefinitely in a cytokine-dependent manner, while retaining their potential to differentiate into functional APCs. After treatment with IL4 plus GM-CSF, the pMCs showed impaired proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex (MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposure to maturation stimuli induced the production of TNFα and IL12p70, and enhanced the expression of MHC-II, CD40, and CD86, which is thus suggestive of typical DC maturation. Similar to bone marrow-derived DCs, they stimulated a primary mixed lymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2K(b)-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection in mice against challenge with OVA-expressing melanoma. Overall, myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may be used to address issues associated with the preparation of DCs.

Increased T-cell responses to Epstein-Barr virus with high viral load in patients with Epstein-Barr virus-positive diffuse large B-cell lymphoma.

The immunological status of patients with Epstein-Barr virus-positive diffuse large B-cell lymphoma (EBV+ DLBCL) without obvious immunodeficiency has not been elucidated. A multicenter prospective study was conducted to assess pretreatment T-cell responses to EBV, EBV-DNA load and anti-EBV antibody in these patients. The proliferative and interferon (IFN)-γ-producing capacity of T-cells in response to autologous B-lymphoblastoid cell lines was determined using carboxyfluorescein diacetate succinimidyl ester (CFSE)-based assay. Frequencies of EBV-specific CD4+ T-cells in patients with EBV+ DLBCL (n = 13) were significantly higher than in healthy controls (HCs) (n = 16) after both ex vivo and in vitro stimulation. Frequencies of EBV-specific CD8+ T-cells in patients with EBV+ DLBCL tended to be higher than in HCs after in vitro stimulation. Patients with EBV+ DLBCL also showed increased immunoglobulin G (IgG) responses to lytic EBV-encoded antigens. Pretreatment plasma EBV-DNA level was significantly higher in patients with EBV+ DLBCL than in patients with EBV- DLBCL or HCs. In conclusion, EBV-specific T-cells showed increased reactivity, accompanied by higher levels of plasma virus DNA in patients with EBV+ DLBCL.

Role of latent membrane protein 1 in chronic active Epstein-Barr virus infection-derived T/NK-cell proliferation.

Epstein-Barr virus (EBV) predominantly infects B cells and causes B-cell lymphomas, such as Burkitt lymphoma and Hodgkin lymphoma. However, it also infects other types of cells, including T and natural killer (NK) cells, and causes disorders, such as chronic active EBV infection (CAEBV) and T/NK-cell lymphoma. The CAEBV is a lymphoproliferative disease with poor prognosis, where EBV-positive T or NK cells grow rapidly, although the molecular mechanisms that cause the cell expansion still remain to be elucidated. EBV-encoded latent membrane protein 1 (LMP1) is an oncogene that can transform some cell types, such as B cells and mouse fibroblasts, and thus may stimulate cell proliferation in CAEBV. Here, we examined the effect of LMP1 on EBV-negative cells using the cells conditionally expressing LMP1, and on CAEBV-derived EBV-positive cells by inhibiting the function of LMP1 using a dominant negative form of LMP1. We demonstrated that LMP1 was responsible for the increased cell proliferation in the cell lines derived from CAEBV, while LMP1 did not give any proliferative advantage to the EBV-negative cell line.

Gene-modified human α/ß-T cells expressing a chimeric CD16-CD3ζ receptor as adoptively transferable effector cells for anticancer monoclonal antibody therapy.

The central tumoricidal activity of anticancer monoclonal antibodies (mAb) is exerted by FcγR IIIa (CD16)-expressing effector cells in vivo via antibody-dependent cell-mediated cytotoxicity (ADCC), as observed for natural killer (NK) cells. In practice, chemotherapy-induced leukopenia and exhaustion of NK cells resulting from ADCC often hamper the clinical efficacy of cancer treatment. To circumvent this drawback, we examined in vivo the feasibility of T cells, gene-modified to express a newly generated affinity-matured (158V/V) chimeric CD16-CD3ζ receptor (cCD16ζ-T cells), as a transferable alternative effector for cancer mAb therapy. cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human interleukin-2 (rhIL-2), and they successfully displayed ADCC-mediated tumoricidal activity in vitro. During ADCC, ligation of opsonized cancer cells to the introduced cCD16ζ-T cells stimulated the effector cells to produce proinflammatory cytokines and release toxic granules through the activation of the Nuclear factor of activated T cells (NFAT) pathway after phosphorylation of the CD3ζ chain. In parallel, these stimulated cCD16ζ-T cells transiently proliferated and differentiated into effector memory T cells. In contrast, NK cells activated by rhIL-2 displayed similar ADCC activity, but failed to proliferate. Human cCD16ζ-T cells infused concomitantly with anti-CD20 mAb synergistically inhibited the growth of disseminated Raji cells, a CD20(+) lymphoma cell line, in immunodeficient mice, whereas similarly infused rhIL-2-treated NK cells survived for a shorter time and displayed less effective tumor suppression. Our findings strongly suggest the clinical feasibility of cCD16ζ-T cells as adoptively transferable ADCC effector cells that could potentially enhance the clinical responses mediated by currently available anticancer mAbs.

Adoptive transfer of genetically engineered WT1-specific cytotoxic T lymphocytes does not induce renal injury.

Because WT1 is expressed in leukemia cells, the development of cancer immunotherapy targeting WT1 has been an attractive translational research topic. However, concern of this therapy still remains, since WT1 is abundantly expressed in renal glomerular podocytes. In the present study, we clearly showed that WT1-specific cytotoxic T lymphocytes (CTLs) certainly exerted cytotoxicity against podocytes in vitro; however, they did not damage podocytes in vivo. This might be due to the anatomical localization of podocytes, being structurally separated from circulating CTLs in glomerular capillaries by an exceptionally thick basement membrane.

An HLA-modified ovarian cancer cell line induced CTL responses specific to an epitope derived from claudin-1 presented by HLA-A*24:02 molecules.

In an attempt to induce cytotoxic T lymphocytes (CTLs) that react to ovarian cancer cells, we isolated a CTL clone that specifically recognizes claudin-1 in an HLA-A*24:02-restricted manner. Naïve CD8(+) T lymphocytes were obtained from a healthy adult donor and stimulated twice in vitro with HLA-modified TOV21G cells that were originally derived from an ovarian clear-cell carcinoma line. The TOV21G modification involved RNAi-mediated gene silencing of intrinsic HLA molecules and lentiviral transduction of a synonymously mutated HLA-A*24:02. Then, cDNA library construction using mRNA extracted from the parental TOV21G cells and subsequent expression cloning were conducted. These experiments revealed that a CTL clone obtained from the bulk culture recognized a minimal epitope peptide RYEFGQALF, which was derived from an autoantigen claudin-1 presented by HLA-A*24:02 molecules. This clone exhibited cytolytic activities against three ovarian cancer cell lines and normal bronchial epithelial cells in an HLA-A*24:02-restricted manner. Our data indicate that HLA-modified cancer cells can be used as an artificial antigen-presenting cell to generate antigen-specific CTLs in a manner restricted by an HLA allele of interest.

Development of a novel redirected T-cell-based adoptive immunotherapy targeting human telomerase reverse transcriptase for adult T-cell leukemia.

Although adult T-cell leukemia (ATL) has a poor prognosis, successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) in some cases suggests that a cellular immune-mediated strategy can be effective. So far, however, no effective target for anti-ATL immunotherapy has been defined. Here we demonstrated for the first time that human telomerase reverse transcriptase (hTERT) is a promising therapeutic target for ATL, and we developed a novel redirected T-cell-based immunotherapy targeting hTERT. hTERT messenger RNA was produced abundantly in ATL tumor cells but not in steady-state normal cells. Rearranged human leukocyte antigen-A*24:02 (HLA-A*24:02) -restricted and hTERT461-469 nonameric peptide-specific T-cell receptor (TCR) α/ß genes were cloned from our previously established cytotoxic T lymphocyte clone (K3-1) and inserted into a novel retroviral TCR expression vector encoding small interfering RNAs for endogenous TCR genes in redirected T cells (hTERT-siTCR vector). Consequently, allogeneic or autologous gene-modified CD8(+) T cells prepared using the hTERT-siTCR vector successfully killed ATL tumor cells, but not normal cells including steady-state hematopoietic progenitors, in an HLA-A*24:02-restricted manner both in vitro and in vivo. Our experimental observations support the development of a novel hTERT-targeting redirected T-cell-based adoptive immunotherapy for ATL patients, especially those for whom suitable allo-HSCT donors are lacking.

Co-introduced functional CCR2 potentiates in vivo anti-lung cancer functionality mediated by T cells double gene-modified to express WT1-specific T-cell receptor.

BACKGROUND AND PURPOSE: Although gene-modification of T cells to express tumor-related antigen-specific T-cell receptor (TCR) or chimeric antigen receptor (CAR) has clinically proved promise, there still remains room to improve the clinical efficacy of re-directed T-cell based antitumor adoptive therapy. In order to achieve more objective clinical responses using ex vivo-expanded tumor-responsive T cells, the infused T cells need to show adequate localized infiltration into the tumor. METHODOLOGY/PRINCIPAL FINDINGS: Human lung cancer cells variously express a tumor antigen, Wilms' Tumor gene product 1 (WT1), and an inflammatory chemokine, CCL2. However, CCR2, the relevant receptor for CCL2, is rarely expressed on activated T-lymphocytes. A HLA-A2402(+) human lung cancer cell line, LK79, which expresses high amounts of both CCL2 and WT1 mRNA, was employed as a target. Normal CD8(+) T cells were retrovirally gene-modified to express both CCR2 and HLA-A*2402-restricted and WT1(235-243) nonapeptide-specific TCR as an effector. Anti-tumor functionality mediated by these effector cells against LK79 cells was assessed both in vitro and in vivo. Finally the impact of CCL2 on WT1 epitope-responsive TCR signaling mediated by the effector cells was studied. Introduced CCR2 was functionally validated using gene-modified Jurkat cells and human CD3(+) T cells both in vitro and in vivo. Double gene-modified CD3(+) T cells successfully demonstrated both CCL2-tropic tumor trafficking and cytocidal reactivity against LK79 cells in vitro and in vivo. CCL2 augmented the WT1 epitope-responsive TCR signaling shown by relevant luciferase production in double gene-modified Jurkat/MA cells to express luciferase and WT1-specific TCR, and CCL2 also dose-dependently augmented WT1 epitope-responsive IFN-γ production and CD107a expression mediated by these double gene-modified CD3(+) T cells. CONCLUSION/SIGNIFICANCE: Introduction of the CCL2/CCR2 axis successfully potentiated in vivo anti-lung cancer reactivity mediated by CD8(+) T cells double gene-modified to express WT1-specific TCR and CCR2 not only via CCL2-tropic tumor trafficking, but also CCL2-enhanced WT1-responsiveness.