Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal phenotypes and functions and elicit an HIV-specific cytotoxic T-lymphocyte response in vitro

Dendritic cells (DCs) genetically modified to continually express and present antigens may be potent physiologic adjuvants for induction of prophylactic or therapeutic immunity. We have previously shown that an env and nef deleted HIV-1 vector (HIV-1ΔEN) pseudotyped with VSV-G transduced monocyte-derived macrophages as well as CD34+ precursors of DCs. Here we extended these findings with HIV-1ΔEN to highly differentiated human DCs derived in culture from circulating monocytes (DCs). In addition, a new vector derived from HIV-1ΔEN but further deleted in its remaining accessory genes vif, vpr, and vpu(HIV-1ΔEN V3) was also tested. Both vectors efficiently transduced DCs. Transduction of DCs did not significantly alter their viability or their immunophenotype when compared with untransduced DCs. Furthermore, the phagocytic potential of immature DCs, as well as their ability to differentiate into mature DCs capable of stimulating T-cell proliferation, was not affected. Finally, DCs transduced by the HIV-1ΔEN vector were able to elicit a primary antiviral cytotoxic T-cell response in autologous CD8 T cells. These results suggest that HIV-1–based vectors expressing viral antigens may be useful for in vivo active immunization as well as ex vivo priming of cytotoxic T cells for adoptive T-cell therapy.

Download Full-text

Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal phenotypes and functions and elicit an HIV-specific cytotoxic T-lymphocyte response in vitro

Blood ◽

10.1182/blood.v96.4.1327 ◽

2000 ◽

Vol 96 (4) ◽

pp. 1327-1333 ◽

Cited By ~ 105

Author(s):

Andreas Gruber ◽

June Kan-Mitchell ◽

Kelli L. Kuhen ◽

Tetsu Mukai ◽

Flossie Wong-Staal

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Ex Vivo ◽

T Cell Response ◽

Adoptive T Cell Therapy ◽

Cytotoxic T Cell ◽

T Cell Therapy ◽

Hiv 1

Abstract Dendritic cells (DCs) genetically modified to continually express and present antigens may be potent physiologic adjuvants for induction of prophylactic or therapeutic immunity. We have previously shown that an env and nef deleted HIV-1 vector (HIV-1ΔEN) pseudotyped with VSV-G transduced monocyte-derived macrophages as well as CD34+ precursors of DCs. Here we extended these findings with HIV-1ΔEN to highly differentiated human DCs derived in culture from circulating monocytes (DCs). In addition, a new vector derived from HIV-1ΔEN but further deleted in its remaining accessory genes vif, vpr, and vpu(HIV-1ΔEN V3) was also tested. Both vectors efficiently transduced DCs. Transduction of DCs did not significantly alter their viability or their immunophenotype when compared with untransduced DCs. Furthermore, the phagocytic potential of immature DCs, as well as their ability to differentiate into mature DCs capable of stimulating T-cell proliferation, was not affected. Finally, DCs transduced by the HIV-1ΔEN vector were able to elicit a primary antiviral cytotoxic T-cell response in autologous CD8 T cells. These results suggest that HIV-1–based vectors expressing viral antigens may be useful for in vivo active immunization as well as ex vivo priming of cytotoxic T cells for adoptive T-cell therapy.

Download Full-text

IMMU-31. QUANTITATIVE EVALUATION OF ROUTES OF ADMINISTRATION OF IMMUNOTHERAPEUTIC T CELLS TO ORTHOTOPIC GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.461 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii111-ii111

Author(s):

Lan Hoang-Minh ◽

Angelie Rivera-Rodriguez ◽

Fernanda Pohl-Guimarães ◽

Seth Currlin ◽

Christina Von Roemeling ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Ex Vivo ◽

Adoptive T Cell Therapy ◽

Whole Body ◽

T Cell Therapy ◽

Clinical Responses

Abstract SIGNIFICANCE Adoptive T cell therapy (ACT) has emerged as the most effective treatment against advanced malignant melanoma, eliciting remarkable objective clinical responses in up to 75% of patients with refractory metastatic disease, including within the central nervous system. Immunologic surrogate endpoints correlating with treatment outcome have been identified in these patients, with clinical responses being dependent on the migration of transferred T cells to sites of tumor growth. OBJECTIVE We investigated the biodistribution of intravenously or intraventricularly administered T cells in a murine model of glioblastoma at whole body, organ, and cellular levels. METHODS gp100-specific T cells were isolated from the spleens of pmel DsRed transgenic C57BL/6 mice and injected intravenously or intraventricularly, after in vitro expansion and activation, in murine KR158B-Luc-gp100 glioma-bearing mice. To determine transferred T cell spatial distribution, the brain, lymph nodes, heart, lungs, spleen, liver, and kidneys of mice were processed for 3D imaging using light-sheet and multiphoton imaging. ACT T cell quantification in various organs was performed ex vivo using flow cytometry, 2D optical imaging (IVIS), and magnetic particle imaging (MPI) after ferucarbotran nanoparticle transfection of T cells. T cell biodistribution was also assessed in vivo using MPI. RESULTS Following T cell intravenous injection, the spleen, liver, and lungs accounted for more than 90% of transferred T cells; the proportion of DsRed T cells in the brains was found to be very low, hovering below 1%. In contrast, most ACT T cells persisted in the tumor-bearing brains following intraventricular injections. ACT T cells mostly concentrated at the periphery of tumor masses and in proximity to blood vessels. CONCLUSIONS The success of ACT immunotherapy for brain tumors requires optimization of delivery route, dosing regimen, and enhancement of tumor-specific lymphocyte trafficking and effector functions to achieve maximal penetration and persistence at sites of invasive tumor growth.

Download Full-text

Generation of in Vitro Autologous Human Cytotoxic T-Cell Response to E7 and HER-2/Neu Oncogene Products Using Ex-Vivo Peptide Loaded Dendritic Cells

Advances in Experimental Medicine and Biology - Dendritic Cells in Fundamental and Clinical Immunology ◽

10.1007/978-1-4757-9966-8_94 ◽

1997 ◽

pp. 583-589 ◽

Cited By ~ 6

Author(s):

H. Navabi ◽

B. Jasani ◽

M. Adams ◽

A. S. Evans ◽

M. Mason ◽

...

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Cell Response ◽

Ex Vivo ◽

T Cell Response ◽

Cytotoxic T Cell ◽

Her 2 ◽

Neu Oncogene ◽

Oncogene Products

Download Full-text

A combination of anti-CD3 and anti-CD7 ricin A-immunotoxins for the in vivo treatment of acute graft versus host disease

Blood ◽

10.1182/blood.v95.12.3693 ◽

2000 ◽

Vol 95 (12) ◽

pp. 3693-3701 ◽

Cited By ~ 26

Author(s):

Ypke V. J. M. van Oosterhout ◽

Liesbeth van Emst ◽

Anton V. M. B. Schattenberg ◽

Wil J. M. Tax ◽

Dirk J. Ruiter ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Graft Versus Host Disease ◽

T Cell Response ◽

Cytotoxic T Cell ◽

Host Disease ◽

Graft Versus Host ◽

Ricin A

Abstract This study evaluated the anti-graft versus host disease (GVHD) potential of a combination of immunotoxins (IT), consisting of a murine CD3 (SPV-T3a) and CD7 (WT1) monoclonal antibody both conjugated to deglycosylated ricin A. In vitro efficacy data demonstrated that these IT act synergistically, resulting in an approximately 99% elimination of activated T cells at 10−8 mol/L (about 1.8 μg/mL). Because most natural killer (NK) cells are CD7+, NK activity was inhibited as well. Apart from the killing mediated by ricin A, binding of SPV-T3a by itself impaired in vitro cytotoxic T-cell cytotoxicity. Flow cytometric analysis revealed that this was due to both modulation of the CD3/T-cell receptor complex and activation-induced cell death. These results warranted evaluation of the IT combination in patients with refractory acute GVHD in an ongoing pilot study. So far, 4 patients have been treated with 3 to 4 infusions of 2 or 4 mg/m2 IT combination, administered intravenously at 48-hour intervals. The T1/2 was 6.7 hours, and peak serum levels ranged from 258 to 3210 ng/mL. Drug-associated side effects were restricted to limited edema, fever, and a modest rise of creatine kinase levels. One patient developed low-titer antibodies against ricin A. Infusions were associated with an immediate drop of circulating T cells, followed by a more gradual but continuing elimination of T/NK cells. One patient mounted an extensive CD8 T-cell response directly after treatment, not accompanied with aggravating GVHD. Two patients showed nearly complete remission of GVHD, despite unresponsiveness to the extensive pretreatment. These findings justify further investigation of the IT combination for treatment of diseases mediated by T cells.

Download Full-text

Adoptive T Cell Therapy of Human CMV Infection in Immunocompromised Hosts: In Vitro Generation of CMV-Specific T Cells from Naïve Precursors

Blood ◽

10.1182/blood.v112.11.206.206 ◽

2008 ◽

Vol 112 (11) ◽

pp. 206-206 ◽

Cited By ~ 1

Author(s):

Sonja Schmucker ◽

Mario Assenmacher ◽

Jurgen Schmitz ◽

Anne Richter

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Peptide Pool ◽

Adoptive T Cell Therapy ◽

T Cell Therapy ◽

A Cell ◽

Ifn Γ

Abstract Adoptive transfer of virus-specific T cells is a promising therapy for the treatment of infections in immunocompromised patients. Virus-specific T cells can readily be obtained from antigen-experienced, but not naïve donors. In this study we describe a cell culture system for the in vitro generation of CMV-specific T cells from naive T cells derived from CMV-seronegative donors. We isolated naïve T cells by magnetic depletion of non-T cells, CD25+ regulatory T cells, and CD45RO+ effector and memory T cells from peripheral blood mononuclear cells (PBMC) of CMV-seronegative donors. These naïve T cells were co-cultured with autologous mature monocyte-derived DC (MoDC) loaded with a pool of overlapping peptides from the CMV protein pp65. CD3-depleted autologous PBMC were used as feeder cells and CD28 antibody, IL-2, IL-7, and IL-15 were added to the culture. Already only 9–13 days after starting the priming culture, frequencies of 0.0024% and 0.009% pp65495–503/A2-tetramer+ cells among CD8+ T cells were found for 2 HLA-A2+ blood donors. In contrast pp65495–503/A2-tetramer+ T cells were not detectable when naive T cells were cultured with unpulsed MoDC. Tetramers are suitable tools for the identification of antigen-specific T cells but are restricted to single epitopes of mainly CD8+ T cells. To analyze primed CD4+ T cells as well as CD8+ T cells having specificities other than for the peptide pp65495–503, we looked for upregulation of the activation marker CD137 after a second stimulation and found increased frequencies of CD137+ CD4+ T cells as well as CD137+ CD8+ T cells in the pp65-primed cell cultures only when restimulated with the peptide pool of pp65. Because IFN-γ is important for the control of CMV infection, we studied the capability of the in vitro primed pp65-specific CD4+ and CD8+ T cells to produce this cytokine. Restimulation of the T cells with pp65 peptide pool induced IFN-γ secretion in up to 3.9% of the CD8+ T cells and up to 3.8% of the CD4+ T cells in each of six donors tested. No specific IFN-γ production was detected after restimulation with an irrelevant IE-1 peptide pool. As expected the frequency of pp65-specific T cells in the priming cultures is low. For generation of T cell lines, we magnetically enrich pp65- specific T cells according to their IFN-γ secretion using the cytokine secretion assay technology. After further cultivation for 2 weeks the antigen-specificity of the expanded T cells was again evaluated. Only if restimulated with the pp65 peptide pool 56.6% of the CD4+ T cells showed upregulated expression of the activation marker CD154 (CD40L). Cytokine analysis of the cells revealed IFN-γ production in 40.2% of the CD4+ T cells, of which 36% co-expressed IL-2, indicating the functionality of the in vitro primed and expanded T cells. In conclusion, we established a cell culture system for in vitro priming of CMV-specific CD4+ and CD8+ T cells derived from peripheral blood of donors not infected by CMV. This should extend the application of adoptive T cell therapy to patients for whom immune donors are not available.

Download Full-text

Whole-tumor-antigen-pulsed dendritic cells elicit cytotoxic T-cell response against pediatric nasopharyngeal carcinoma in vitro

Medical Oncology ◽

10.1007/s12032-008-9093-8 ◽

2008 ◽

Vol 26 (1) ◽

pp. 78-85 ◽

Cited By ~ 7

Author(s):

Dou Yufeng ◽

Zhang Guocheng ◽

Xu Dongliang ◽

Fu Rong ◽

Cao Yuhong ◽

...

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Nasopharyngeal Carcinoma ◽

Tumor Antigen ◽

Cell Response ◽

T Cell Response ◽

Cytotoxic T Cell

Download Full-text

Antigen sensitivity is a major determinant of CD8+ T-cell polyfunctionality and HIV-suppressive activity

Blood ◽

10.1182/blood-2009-02-206557 ◽

2009 ◽

Vol 113 (25) ◽

pp. 6351-6360 ◽

Cited By ~ 162

Author(s):

Jorge R. Almeida ◽

Delphine Sauce ◽

David A. Price ◽

Laura Papagno ◽

So Youn Shin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Ex Vivo ◽

Cd8 T Cell ◽

Basic Knowledge ◽

Suppressive Activity ◽

Antiviral Efficacy ◽

Hiv 1

Abstract CD8+ T cells are major players in the immune response against HIV. However, recent failures in the development of T cell–based vaccines against HIV-1 have emphasized the need to reassess our basic knowledge of T cell–mediated efficacy. CD8+ T cells from HIV-1–infected patients with slow disease progression exhibit potent polyfunctionality and HIV-suppressive activity, yet the factors that unify these properties are incompletely understood. We performed a detailed study of the interplay between T-cell functional attributes using a bank of HIV-specific CD8+ T-cell clones isolated in vitro; this approach enabled us to overcome inherent difficulties related to the in vivo heterogeneity of T-cell populations and address the underlying determinants that synthesize the qualities required for antiviral efficacy. Conclusions were supported by ex vivo analysis of HIV-specific CD8+ T cells from infected donors. We report that attributes of CD8+ T-cell efficacy against HIV are linked at the level of antigen sensitivity. Highly sensitive CD8+ T cells display polyfunctional profiles and potent HIV-suppressive activity. These data provide new insights into the mechanisms underlying CD8+ T-cell efficacy against HIV, and indicate that vaccine strategies should focus on the induction of HIV-specific T cells with high levels of antigen sensitivity to elicit potent antiviral efficacy.

Download Full-text

Role of viral infectivity in the induction of influenza virus-specific cytotoxic T cells.

Journal of Experimental Medicine ◽

10.1084/jem.147.4.1236 ◽

1978 ◽

Vol 147 (4) ◽

pp. 1236-1252 ◽

Cited By ~ 76

Author(s):

T J Braciale ◽

K L Yap

Keyword(s):

T Cells ◽

Influenza Virus ◽

T Cell ◽

Cell Response ◽

Infectious Virus ◽

Cytotoxic T Cells ◽

T Cell Response ◽

Cytotoxic T Cell

This report examines the requirement for infectious virus in the induction of influenza virus-specific cytotoxic T cells. Infectious influenza virus was found to be highly efficient at generating both primary and secondary cytotoxic T-cell response in vivo. Inactivated influenza virus however, failed to stimulate a detectable cytotoxic T-cell response in vivo even at immunizing doses 10(5)-10(6)-fold higher than the minimum stimulatory dose of infectious virus. Likewise inactivated virus failed to sensitize target cells for T cell-mediated lysis in vitro but could stimulate a specific cytotoxic response from primed cells in vitro. Possible requirements for the induction of virus-specific cytotoxic T-cell responses are discussed in light of these observations and those of other investigators.

Download Full-text

Expansion of HIV-specific CD4+ and CD8+ T cells by dendritic cells transfected with mRNA encoding cytoplasm- or lysosome-targeted Nef

Blood ◽

10.1182/blood-2005-04-1513 ◽

2006 ◽

Vol 107 (5) ◽

pp. 1963-1969 ◽

Cited By ~ 44

Author(s):

Daniel G. Kavanagh ◽

Daniel E. Kaufmann ◽

Sherzana Sunderji ◽

Nicole Frahm ◽

Sylvie Le Gall ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Cd8 T Cells ◽

Mononuclear Cells ◽

Ex Vivo ◽

Cytotoxic T Cells ◽

T Cell Response ◽

Peripheral Blood Mononuclear ◽

T Cell Lines

Transfection with synthetic mRNA is a safe and efficient method of delivering antigens to dendritic cells for immunotherapy. Targeting antigens to the lysosome can sometimes enhance the CD4+ T-cell response. We transfected antigen-presenting cells (APCs) with mRNA encoding Gag-p24 and cytoplasmic, lysosomal, and secreted forms of Nef. Antigen-specific cytotoxic T cells were able to lyse the majority of transfected targets, indicating that transfection was efficient. Transfection of APCs with a Nef construct bearing lysosomal targeting signals produced rapid and prolonged antigen presentation to CD4+ and CD8+ T cells. Polyclonal CD4+ and CD8+ T-cell lines recognizing multiple distinct epitopes were expanded by coculture of transfected dendritic cells with peripheral blood mononuclear cells from viremic and aviremic HIV-infected subjects. Importantly, lysosome-targeted antigen drove a significantly greater expansion of Nef-specific CD4+ T cells than cytoplasmic antigen. The frequency of recognition of CD8 but not CD4 epitopes by mRNA-expanded T cells was inversely proportional to sequence entropy and was similar to ex vivo responses from a large chronic cohort. Thus human dendritic cells transfected with mRNA encoding lysosome-targeted HIV antigen can expand a broad, polyclonal repertoire of antiviral T cells, offering a promising approach to HIV immunotherapy.

Download Full-text