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2.
Front Immunol ; 13: 850358, 2022.
Article in English | MEDLINE | ID: mdl-35432319

ABSTRACT

Immunotherapy of cancer has made tremendous progress in recent years, as demonstrated by the remarkable clinical responses obtained from adoptive cell transfer (ACT) of patient-derived tumor infiltrating lymphocytes, chimeric antigen receptor (CAR)-modified T cells (CAR-T) and T cell receptor (TCR)-engineered T cells (TCR-T). TCR-T uses specific TCRS optimized for tumor engagement and can recognize epitopes derived from both cell-surface and intracellular targets, including tumor-associated antigens, cancer germline antigens, viral oncoproteins, and tumor-specific neoantigens (neoAgs) that are largely sequestered in the cytoplasm and nucleus of tumor cells. Moreover, as TCRS are naturally developed for sensitive antigen detection, they are able to recognize epitopes at far lower concentrations than required for CAR-T activation. Therefore, TCR-T holds great promise for the treatment of human cancers. In this focused review, we summarize basic, translational, and clinical insights into the challenges and opportunities of TCR-T. We review emerging strategies used in current ACT, point out limitations, and propose possible solutions. We highlight the importance of targeting tumor-specific neoAgs and outline a strategy of combining neoAg vaccines, checkpoint blockade therapy, and adoptive transfer of neoAg-specific TCR-T to produce a truly tumor-specific therapy, which is able to penetrate into solid tumors and resist the immunosuppressive tumor microenvironment. We believe such a combination approach should lead to a significant improvement in cancer immunotherapies, especially for solid tumors, and may provide a general strategy for the eradication of multiple cancers.


Subject(s)
Neoplasms , Receptors, Chimeric Antigen , Epitopes , Humans , Immunotherapy, Adoptive , Neoplasms/therapy , Receptors, Antigen, T-Cell/genetics , Receptors, Chimeric Antigen/genetics , T-Lymphocytes , Tumor Microenvironment
4.
Mol Ther ; 26(11): 2553-2566, 2018 11 07.
Article in English | MEDLINE | ID: mdl-30217730

ABSTRACT

Checkpoint inhibitors and adoptive cell therapy provide promising options for treating solid cancers such as HBV-related HCC, but they have limitations. We tested the potential to combine advantages of each approach, genetically reprogramming T cells specific for viral tumor antigens to overcome exhaustion by down-modulating the co-inhibitory receptor PD-1. We developed a novel lentiviral transduction protocol to achieve preferential targeting of endogenous or TCR-redirected, antigen-specific CD8 T cells for shRNA knockdown of PD-1 and tested functional consequences for antitumor immunity. Antigen-specific and intrahepatic CD8 T cells transduced with lentiviral (LV)-shPD-1 consistently had a marked reduction in PD-1 compared to those transduced with a control lentiviral vector. PD-1 knockdown of human T cells rescued antitumor effector function and promoted killing of hepatoma cells in a 3D microdevice recapitulating the pro-inflammatory PD-L1hi liver microenvironment. However, upon repetitive stimulation, PD-1 knockdown drove T cell senescence and induction of other co-inhibitory pathways. We provide the proof of principle that T cells with endogenous or genetically engineered specificity for HBV-associated HCC viral antigens can be targeted for functional genetic editing. We show that PD-1 knockdown enhances immediate tumor killing but is limited by compensatory engagement of alternative co-inhibitory and senescence program upon repetitive stimulation.


Subject(s)
Carcinoma, Hepatocellular/therapy , Hepatitis B, Chronic/therapy , Liver Neoplasms/therapy , Programmed Cell Death 1 Receptor/immunology , Receptors, Antigen, T-Cell/therapeutic use , Antigens, Neoplasm/immunology , Antigens, Neoplasm/therapeutic use , Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/virology , Genetic Vectors/genetics , Hepatitis B virus/immunology , Hepatitis B virus/pathogenicity , Hepatitis B, Chronic/immunology , Hepatitis B, Chronic/pathology , Hepatitis B, Chronic/virology , Humans , Immunotherapy, Adoptive/methods , Lentivirus/genetics , Liver/immunology , Liver/metabolism , Liver Neoplasms/immunology , Liver Neoplasms/pathology , Liver Neoplasms/virology , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/therapeutic use , Receptors, Antigen, T-Cell/immunology , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology
5.
Clin Cancer Res ; 23(20): 6178-6189, 2017 Oct 15.
Article in English | MEDLINE | ID: mdl-28710313

ABSTRACT

Purpose: Carcinoembryonic antigen (CEA) is a candidate target for cellular immunotherapy of pancreatic cancer. In this study, we have characterized the antigen-specific function of autologous cytotoxic T lymphocytes (CTL) specific for the HLA-A2-restricted peptide, pCEA691-699, isolated from the peripheral T-cell repertoire of pancreatic cancer patients and sought to determine if ex vivo PD-L1 and TIM-3 blockade could enhance CTL function.Experimental Design: CD8+ T-cell lines were generated from peripheral blood mononuclear cells of 18 HLA-A2+ patients with pancreatic cancer and from 15 healthy controls. In vitro peptide-specific responses were evaluated by flow cytometry after staining for intracellular cytokine production and carboxy fluorescein succinimydyl ester cytotoxicity assays using pancreatic cancer cell lines as targets.Results: Cytokine-secreting functional CEA691-specific CTL lines were successfully generated from 10 of 18 pancreatic cancer patients, with two CTL lines able to recognize and kill both CEA691 peptide-loaded T2 cells and CEA+ HLA-A2+ pancreatic cancer cell lines. In the presence of ex vivo PD-L1 blockade, functional CEA691-specific CD8+ T-cell responses, including IFNγ secretion and proliferation, were enhanced, and this effect was more pronounced on Ag-specific T cells isolated from tumor draining lymph nodes.Conclusions: These data demonstrate that CEA691-specific CTL can be readily expanded from the self-restricted T-cell repertoire of pancreatic cancer patients and that their function can be enhanced by PD-L1 blockade. Clin Cancer Res; 23(20); 6178-89. ©2017 AACR.


Subject(s)
B7-H1 Antigen/metabolism , Carcinoembryonic Antigen/immunology , Programmed Cell Death 1 Receptor/metabolism , Signal Transduction , T-Cell Antigen Receptor Specificity/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Adult , Aged , Aged, 80 and over , Antineoplastic Agents, Immunological/pharmacology , Biomarkers, Tumor , Cell Line, Tumor , Cytokines/metabolism , Cytotoxicity, Immunologic/drug effects , Cytotoxicity, Immunologic/immunology , Female , Hepatitis A Virus Cellular Receptor 2/antagonists & inhibitors , Hepatitis A Virus Cellular Receptor 2/metabolism , Humans , Immunophenotyping , Male , Middle Aged , Neoplasm Staging , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/mortality , Pancreatic Neoplasms/pathology , T-Lymphocyte Subsets/drug effects , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism
6.
Oncotarget ; 7(16): 21199-221, 2016 Apr 19.
Article in English | MEDLINE | ID: mdl-27028870

ABSTRACT

Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/ß T-cell receptors (TCRα/ß). However, potential mispairing of introduced TCRα/ß-chains with endogenous ß/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vß-fragment to the TCR Cß-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/ß-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vß-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vß. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/ß-positive T-cells.


Subject(s)
CD3 Complex/immunology , Leukemia, T-Cell/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes/immunology , Adoptive Transfer , Animals , Biomarkers, Tumor , CD3 Complex/genetics , Cell Membrane , Cell Proliferation , Humans , Immunotherapy, Adoptive , Leukemia, T-Cell/genetics , Leukemia, T-Cell/pathology , Mice , Receptors, Antigen, T-Cell, alpha-beta/genetics , Signal Transduction , T-Lymphocytes/metabolism , Tumor Cells, Cultured
7.
Haematologica ; 101(4): 482-90, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26802053

ABSTRACT

Due to the lack of specificity for tumor antigens, allogeneic T-cell therapy is associated with graft-versus-host disease. Enhancing the anti-tumor specificity while reducing the graft-versus-host disease risk of allogeneic T cells has remained a research focus. In this study, we demonstrate that the introduction of 'dominant' T-cell receptors into primary murine T cells can suppress the expression of endogenous T-cell receptors in a large proportion of the gene-modified T cells. Adoptive transfer of allogeneic T cells expressing a 'dominant' T-cell receptor significantly reduced the graft-versus-host toxicity in recipient mice. Using two bone marrow transplant models, enhanced anti-tumor activity was observed in the presence of reduced graft-versus-host disease. However, although transfer of T-cell receptor gene-modified allogeneic T cells resulted in the elimination of antigen-positive tumor cells and improved the survival of treated mice, it was associated with accumulation of T cells expressing endogenous T-cell receptors and the development of delayed graft-versus-host disease. The in-vivo deletion of the engineered T cells, mediated by endogenous mouse mammary tumor virus MTV8 and MTV9, abolished graft-versus-host disease while retaining significant anti-tumor activity of adoptively transferred T cells. Together, this study shows that the in-vitro selection of allogeneic T cells expressing high levels of a 'dominant' T-cell receptor can lower acute graft-versus-host disease and enhance anti-tumor activity of adoptive cell therapy, while the in-vivo outgrowth of T cells expressing endogenous T-cell receptors remains a risk factor for the delayed onset of graft-versus-host disease.


Subject(s)
Graft vs Host Disease/prevention & control , Immunotherapy, Adoptive/methods , Mammary Tumor Virus, Mouse/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/transplantation , Animals , Bone Marrow Transplantation/methods , Cell Line, Tumor , Female , Gene Expression , Genes, Dominant , Genetic Vectors/immunology , Graft vs Host Disease/genetics , Graft vs Host Disease/immunology , Graft vs Host Disease/pathology , Humans , Lymphocyte Depletion/methods , Mammary Tumor Virus, Mouse/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred DBA , Receptors, Antigen, T-Cell/genetics , Survival Analysis , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Transgenes , Transplantation, Homologous , Whole-Body Irradiation
8.
J Hepatol ; 62(2): 486-91, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25308176

ABSTRACT

HBV-DNA integration frequently occurs in HBV-related hepatocellular carcinoma (HCC), but whether HBV antigens are expressed in HCC cells and can be targeted by immune therapeutic strategies remains controversial. Here, we first characterized HBV antigen expression in HCC metastases, occurring in a patient who had undergone liver transplantation for HBV-related HCC. We then deployed for the first time in HCC autologous T cells, genetically modified to express an HBsAg specific T cell receptor, as therapy against chemoresistant extrahepatic metastases. We confirmed that HBV antigens were expressed in HCC metastases (but not in the donor liver) and demonstrated that tumour cells were recognized in vivo by lymphocytes, engineered to express an HBV-specific T cell receptor (TCR). Gene-modified T cells survived, expanded and mediated a reduction in HBsAg levels without exacerbation of liver inflammation or other toxicity. Whilst clinical efficacy was not established in this subject with end-stage metastatic disease, we confirm the feasibility of providing autologous TCR-redirected therapy against HCC and advocate this strategy as a novel therapeutic opportunity in hepatitis B-associated malignancies.


Subject(s)
Carcinoma, Hepatocellular/secondary , Hepatitis B Surface Antigens/metabolism , Immunotherapy/methods , Liver Neoplasms/pathology , Receptors, Antigen, T-Cell/therapeutic use , Tacrolimus/therapeutic use , Aged , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/therapy , Fatal Outcome , Follow-Up Studies , Humans , Immunosuppressive Agents/therapeutic use , Liver Neoplasms/immunology , Liver Neoplasms/therapy , Male , Neoplasm Metastasis
9.
J Immunol ; 194(3): 1080-9, 2015 Feb 01.
Article in English | MEDLINE | ID: mdl-25539815

ABSTRACT

Ag receptors used for cancer immunotherapy are often directed against tumor-associated Ags also expressed in normal tissues. Targeting of such Ags can result in unwanted autoimmune attack of normal tissues or induction of tolerance in therapeutic T cells. We used a murine model to study the phenotype and function of T cells redirected against the murine double minute protein 2 (MDM2), a tumor-associated Ag that shows low expression in many normal tissues. Transfer of MDM2-TCR-engineered T cells into bone marrow chimeric mice revealed that Ag recognition in hematopoietic tissues maintained T cell function, whereas presentation of MDM2 in nonhematopoietic tissues caused reduced effector function. TCR-engineered CD8(+) T cells underwent rapid turnover, downmodulated CD8 expression, and lost cytotoxic function. We found that MDM2-TCR-engineered CD4(+) T cells provided help and restored cytotoxic function of CD8(+) T cells bearing the same TCR. Although the introduction of the CD8 coreceptor enhanced the ability of CD4(+) T cells to recognize MDM2 in vitro, the improved self-antigen recognition abolished their ability to provide helper function in vivo. The data indicate that the same class I-restricted TCR responsible for Ag recognition and tolerance induction in CD8(+) T cells can, in the absence of the CD8 coreceptor, elicit CD4 T cell help and partially reverse tolerance. Thus MHC class I-restricted CD4(+) T cells may enhance the efficacy of therapeutic TCR-engineered CD8(+) T cells and can be readily generated with the same TCR.


Subject(s)
Antigens, Neoplasm/immunology , Autoantigens/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Immune Tolerance , Receptors, Antigen, T-Cell/genetics , Adoptive Transfer , Animals , Cell Communication , Cytotoxicity, Immunologic , Gene Expression , Immunophenotyping , Mice , Mice, Transgenic , Phenotype , Proto-Oncogene Proteins c-mdm2/genetics , Receptors, Antigen, T-Cell/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transduction, Genetic
10.
Cancer Res ; 74(20): 5711-22, 2014 Oct 15.
Article in English | MEDLINE | ID: mdl-25261236

ABSTRACT

Immune escape is a fundamental trait of cancer. Dendritic cells (DC) that interact with T cells represent a crucial site for the development of tolerance to tumor antigens, but there remains incomplete knowledge about how DC-tolerizing signals evolve during tumorigenesis. In this study, we show that DCs isolated from patients with metastatic or locally advanced breast cancer express high levels of the adiponectin receptors AdipoR1 and AdipoR2, which are sufficient to blunt antitumor immunity. Mechanistic investigations of ligand-receptor interactions on DCs revealed novel signaling pathways for each receptor. AdipoR1 stimulated IL10 production by activating the AMPK and MAPKp38 pathways, whereas AdipoR2 modified inflammatory processes by activating the COX-2 and PPARγ pathways. Stimulation of these pathways was sufficient to block activation of NF-κB in DC, thereby attenuating their ability to stimulate antigen-specific T-cell responses. Together, our findings reveal novel insights into how DC-tolerizing signals evolve in cancer to promote immune escape. Furthermore, by defining a critical role for adiponectin signaling in this process, our work suggests new and broadly applicable strategies for immunometabolic therapy in patients with cancer.


Subject(s)
Breast Neoplasms/immunology , Dendritic Cells/metabolism , Receptors, Adiponectin/metabolism , Tumor Escape , Adiponectin/physiology , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Clonal Anergy , Cyclooxygenase 2/metabolism , Cytotoxicity, Immunologic , Disease Progression , Enzyme Activation , Female , Humans , Interleukin-10/metabolism , MAP Kinase Signaling System , Mice, Inbred C57BL , NF-kappa B/metabolism , Neoplasm Transplantation , PPAR gamma/metabolism , T-Lymphocytes, Cytotoxic/immunology
11.
Oncoimmunology ; 2(1): e22590, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-23483821

ABSTRACT

In this study, we generated human MHC Class I-restricted CD4+ T cells specific for Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two herpesviridae associated with lymphoma, nasopharyngeal carcinoma and medulloblastoma, respectively. Retroviral transfer of virus-specific, HLA-A2-restricted TCR-coding genes generated CD4+ T cells that recognized HLA-A2/peptide multimers and produced cytokines when stimulated with MHC Class II-deficient cells presenting the relevant viral peptides in the context of HLA-A2. Peptide titration revealed that CD4+ T cells had a 10-fold lower avidity than CD8+ T cells expressing the same TCR. The impaired avidity of CD4+ T cells was corrected by simultaneously transferring TCR- and CD8-coding genes. The CD8 co-receptor did not alter the cytokine signature of CD4+ T cells, which remained distinct from that of CD8+ T cells. Using the xenogeneic NOD/SCID mouse model, we demonstrated that human CD4+ T cells expressing a specific TCR and CD8 can confer efficient protection against the growth of tumors expressing the EBV or CMV antigens recognized by the TCR. In summary, we describe a robust approach for generating therapeutic CD4+ T cells capable of providing MHC Class I-restricted immunity against MHC Class II-negative tumors in vivo.

12.
Blood ; 118(13): 3528-37, 2011 Sep 29.
Article in English | MEDLINE | ID: mdl-21750319

ABSTRACT

The function of T-cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR α/ß heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T-cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T-cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognized lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared with T cells modified by TCR only. After tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.


Subject(s)
CD3 Complex/physiology , Genes, T-Cell Receptor/genetics , Genetic Therapy , Animals , CD3 Complex/genetics , CD3 Complex/metabolism , Cells, Cultured , Down-Regulation , Female , Genetic Therapy/methods , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Lymphoma, T-Cell/genetics , Lymphoma, T-Cell/pathology , Lymphoma, T-Cell/therapy , Mice , Mice, Inbred C57BL , Mice, Transgenic , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Treatment Outcome
13.
Blood ; 118(2): 319-29, 2011 Jul 14.
Article in English | MEDLINE | ID: mdl-21606483

ABSTRACT

We have tested whether affinity-matured TCRs that retain peptide specificity improve the ability of primary human CD8(+) T cells to mount antigen-specific responses. We found that TCR affinity correlated with the speed of T-cell responses. High affinity TCR-antigen interactions rapidly initiated T-cell responses, but low affinity TCR/antigen interactions required longer time periods to elicit the same responses. Within the "natural" affinity range, increased TCR-to-antigen affinity correlated with improved ability of T cells to recognize low concentration of antigen. However, affinity-matured TCR with 700-fold enhanced affinity for MHC-to-antigen required 100-fold higher antigen-density to initiate T-cell responses than did wild-type TCR. Using modified peptides to reduce the affinity of TCR-to-antigen interaction, we demonstrate that affinity-matured TCRs are not defective, being superior to wild-type TCR in recognizing low concentration of modified peptides. These data indicate that enhancing TCR affinity can accelerate the speed of T-cell activation and reduce the ability to recognize low density of MHC-to-peptide antigen. We predict that future studies of the human T-cell repertoire will reveal 2 types of low avidity T cells: fast and slow responders, with high-affinity and low-affinity TCR, respectively.


Subject(s)
Histocompatibility Antigens/immunology , Major Histocompatibility Complex/immunology , Peptides/immunology , Receptors, Antigen, T-Cell/metabolism , T-Cell Antigen Receptor Specificity/physiology , T-Lymphocytes/metabolism , Cells, Cultured , HLA-A Antigens/metabolism , HLA-A2 Antigen , HeLa Cells , Histocompatibility Antigens/metabolism , Humans , Jurkat Cells , Kinetics , Peptides/metabolism , Protein Binding/physiology , Receptors, Antigen, T-Cell/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Time Factors
14.
Cancer Immunol Immunother ; 60(9): 1243-55, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21553146

ABSTRACT

The cancer testis antigen Preferentially Expressed Antigen of Melanoma (PRAME) is overexpressed in many solid tumours and haematological malignancies whilst showing minimal expression in normal tissues and is therefore a promising target for immunotherapy. HLA-A0201-restricted peptide epitopes from PRAME have previously been identified as potential immunogens to drive antigen-specific autologous CTL responses, capable of lysing PRAME expressing tumour cells. CTL lines, from 13 normal donors and 10 melanoma patients, all of whom were HLA-A0201 positive, were generated against the PRAME peptide epitope PRA(100-108). Specific killing activity against PRA(100-108) peptide-pulsed targets was weak compared with CTL lines directed against known immunodominant peptides. Moreover, limiting dilution cloning from selected PRAME-specific CTL lines resulted in the generation of a clone of only low to intermediate avidity. Addition of the demethylating agent 5-aza-2'-Deoxycytidine (DAC) increased PRAME expression in 7 out of 11 malignant cell lines including several B lineage leukaemia lines and also increased class I expression. Pre-treatment of target cells was associated with increased sensitivity to antigen-specific killing by the low avidity CTL. When CTL, as well as of the target cells, were treated, the antigen-specific killing was further augmented. Interestingly, one HLA-A0201-negative DAC-treated line (RAJI) showed increased sensitivity to killing by clones despite a failure of expression of PRAME or HLA-A0201. Together these data point to a general increased augmentation of cancer immunogenocity by DAC involving both antigen-specific and non-specific mechanisms.


Subject(s)
Antigens, Neoplasm/immunology , Azacitidine/analogs & derivatives , Immunotherapy, Adoptive/methods , Neoplasms/immunology , Neoplasms/therapy , T-Lymphocytes, Cytotoxic/immunology , Antibody Affinity , Antigens, Neoplasm/metabolism , Antimetabolites, Antineoplastic/pharmacology , Azacitidine/pharmacology , Cell Line, Tumor , Cytotoxicity, Immunologic/immunology , Decitabine , HL-60 Cells , HLA-A2 Antigen/immunology , Humans , K562 Cells , Transfection
15.
Blood ; 117(25): 6813-24, 2011 Jun 23.
Article in English | MEDLINE | ID: mdl-21447831

ABSTRACT

Recently, vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However, it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here, we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted, WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery, T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells, whereas T cells specific for a non-self-viral antigen retained a CD44(low) naive phenotype. Only the WT1-specific T cells, but not the virus-specific T cells, displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells, the animals did not develop autoimmunity, and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor-associated self-antigen may drive differentiation of functionally competent memory T cells.


Subject(s)
T-Lymphocytes/cytology , T-Lymphocytes/immunology , Thymus Gland/immunology , WT1 Proteins/immunology , Animals , Cell Line , Gene Expression , Gene Transfer Techniques , Humans , Immunologic Memory , Mice , Mice, Inbred C57BL , Mice, Transgenic , Stem Cells/immunology , Stem Cells/metabolism , Vaccination , WT1 Proteins/genetics , Wilms Tumor/immunology
16.
Trans R Soc Trop Med Hyg ; 105(2): 86-94, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21168891

ABSTRACT

Fine needle aspirates from Burkitt's lymphoma and other tumours transferred directly into ThinPrep® PreservCyt® (Cytyc UK Ltd, Crawley, UK) buffered alcohol fixative retain their cellular and viral antigens and nucleic acids for many months at ambient temperatures. Despite the presence of blood and debris, cells dried onto slides from droplets and post-fixed in formalin, or sections of paraffin-embedded cell blocks from formalin post-fixed pellets, prove adequate for morphology, immunocytochemistry, in-situ hybridization and molecular biological analyses. Where there is lack of expertise in making thin smears or hospitals lack pathology laboratories and services, PreservCyt® provides an excellent medium for transport elsewhere for diagnosis and research.


Subject(s)
Biopsy, Needle/methods , Burkitt Lymphoma/pathology , Adolescent , Burkitt Lymphoma/epidemiology , Child , Child, Preschool , Female , Humans , Immunohistochemistry , Infant , Malawi/epidemiology , Male , Paraffin Embedding , Sensitivity and Specificity , Socioeconomic Factors , Specimen Handling
17.
J Hepatol ; 55(1): 103-10, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21145860

ABSTRACT

BACKGROUND & AIMS: Virus-specific T cells capable of controlling HBV and eliminating hepatocellular carcinoma (HCC) expressing HBV antigens are deleted or dysfunctional in patients with chronic HBV or HBV-related HCC. The goal of this study was to determine if T cell receptor (TCR) gene transfer can reconstitute HBV-specific T cell immunity in lymphocytes of chronic HBV patients and investigate whether HCC cells with natural HBV-DNA integration can be recognized by genetically modified T cells. METHODS: We used vector-mediated gene transfer to introduce HLA-A2-restricted, HBV-specific TCRs into T cells of chronic HBV as well as HBV-related HCC patients. RESULTS: The introduced TCRs were expressed on the cell surface, evidenced by Vß and pentamer staining. TCR transduced T cells produced IFN-γ, TNF-α, IL-2, and lysed HBV infected hepatocyte-like cell lines. Furthermore, HCC cell lines with natural HBV-DNA integration could be recognized by HBV-specific TCR-re-directed T cells. CONCLUSIONS: TCR re-directed HBV-specific T cells generated from PBMC of chronic HBV and HBV-related HCC patients were multifunctional and capable of recognizing HBV-infected cells and HCC tumor cells expressing viral antigens from naturally integrated HBV DNA. These genetically modified T cells could be used to reconstitute virus-specific T cell immunity in chronic HBV patients and target tumors in HBV-related HCC.


Subject(s)
Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/virology , Hepatitis B virus/immunology , Liver Neoplasms/immunology , Liver Neoplasms/virology , T-Lymphocytes/immunology , T-Lymphocytes/virology , Cell Line , Cell Line, Tumor , Gene Expression , Genetic Engineering , Genetic Vectors , HLA-A2 Antigen/metabolism , Hepatitis B Surface Antigens/genetics , Hepatitis B Surface Antigens/immunology , Hepatitis B, Chronic/immunology , Hepatitis B, Chronic/virology , Humans , Immunity, Cellular , Receptors, Antigen, T-Cell/genetics , Transduction, Genetic
18.
Haematologica ; 95(1): 126-34, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19679884

ABSTRACT

BACKGROUND: The Wilms' tumor antigen (WT1) is an attractive target for immunotherapy of leukemia. In the past, we isolated and characterized the specificity and function of a WT1-specific T-cell receptor. The goal of this translational study was to develop a safe and efficient WT1-T-cell receptor retroviral vector for an adoptive immunotherapy trial with engineered T cells. DESIGN AND METHODS: We generated a panel of retroviral constructs containing unmodified or codon-optimized WT1-T-cell receptor alpha and beta genes, linked via internal ribosome entry sites or 2A sequences, with or without an additional inter-chain disulfide bond in the T-cell receptor constant domains. These constructs were functionally analyzed in vitro, and the best one was tested in an autologous primary leukemia model in vivo. RESULTS: We identified a WT1-T-cell receptor construct that showed optimal tetramer staining, antigen-specific cytokine production and killing activity when introduced into primary human T cells. Fresh CD34(+) cells purified from a patient with leukemia were engrafted into NOD/SCID mice, followed by adoptive immunotherapy with patient's autologous T cells transduced with the WT1-T-cell receptor. This therapeutic treatment evidently decreased leukemia engraftment in mice and resulted in a substantial improvement of leukemia-free survival. CONCLUSIONS: This is the first report that patient's T cells, engineered to express the WT1-T-cell receptor, can eliminate autologous leukemia progenitor cells in an in vivo model. This study provides a firm basis for the planned WT1-T-cell receptor gene therapy trial in leukemia patients.


Subject(s)
Antigens, Neoplasm/immunology , Blast Crisis/immunology , Genetic Engineering/methods , Leukemia/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/therapeutic use , T-Lymphocytes/pathology , Wilms Tumor/pathology , Adult , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Blast Crisis/genetics , Blast Crisis/therapy , Genetic Therapy/methods , Genetic Vectors/biosynthesis , Genetic Vectors/chemistry , Hepatitis B Virus, Woodchuck/genetics , Humans , Jurkat Cells , Leukemia/genetics , Leukemia/therapy , Mice , Mice, Inbred NOD , Mice, SCID , Receptors, Antigen, T-Cell/physiology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transplantation, Autologous/immunology , Wilms Tumor/immunology , Wilms Tumor/therapy
19.
Proc Natl Acad Sci U S A ; 106(45): 19078-83, 2009 Nov 10.
Article in English | MEDLINE | ID: mdl-19884493

ABSTRACT

Regulatory T cells (Tregs) can suppress a wide range of immune cells, making them an ideal candidate for the treatment of autoimmunity. The potential clinical translation of targeted therapy with antigen-specific Tregs is hampered by the difficulties of isolating rare specificities from the natural polyclonal T cell repertoire. Moreover, the initiating antigen is often unknown in autoimmune disease. Here we tested the ability of antigen-specific Tregs generated by retroviral gene transfer to ameliorate arthritis through linked suppression and therefore without cognate recognition of the disease-initiating antigen. We explored two distinct strategies: T cell receptor (TCR) gene transfer into purified CD4+CD25+ T cells was used to redirect the specificity of naturally occurring Tregs; and co-transfer of FoxP3 and TCR genes served to convert conventional CD4(+) T cells into antigen-specific regulators. Following adoptive transfer into recipient mice, the gene-modified T cells engrafted efficiently and retained TCR and FoxP3 expression. Using an established arthritis model, we demonstrate antigen-driven accumulation of the gene modified T cells at the site of joint inflammation, which resulted in a local reduction in the number of inflammatory Th17 cells and a significant decrease in arthritic bone destruction. Together, we describe a robust strategy to rapidly generate antigen-specific regulatory T cells capable of highly targeted inhibition of tissue damage in the absence of systemic immune suppression. This opens the possibility to target Tregs to tissue-specific antigens for the treatment of autoimmune tissue damage without the knowledge of the disease-causing autoantigens recognized by pathogenic T cells.


Subject(s)
Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/therapy , Immunotherapy, Adoptive/methods , T-Lymphocytes, Regulatory/immunology , Adoptive Transfer , Animals , Autoantigens/immunology , Flow Cytometry , Gene Transfer Techniques , Genetic Vectors/genetics , Mice , Mice, Inbred C57BL , Retroviridae , T-Cell Antigen Receptor Specificity/genetics
20.
J Clin Invest ; 118(11): 3619-28, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18846251

ABSTRACT

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.


Subject(s)
CD4 Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , Genes, T-Cell Receptor , T-Lymphocytes, Regulatory/immunology , Transplantation Tolerance/immunology , Animals , CD4 Antigens/genetics , CD4 Antigens/metabolism , CD4-Positive T-Lymphocytes/metabolism , Crosses, Genetic , Gene Transfer Techniques , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class II/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Retroviridae/genetics , Transduction, Genetic , Transplantation Tolerance/genetics , Transplantation, Homologous
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