Dendritic cells transfected with lentiviral vector-encoding hTERT peptide augment antitumor T cell response in vitro

Abstract Transfection of dendritic cells (DC) with in vitro transcribed RNA was shown to be a highly efficient method to generate antigen specific T cells, probably due to the induction of a polyclonal T cell response directed against multiple antigens presented on different HLA allels. However, the experimental evidence of this assumption remains to be demonstrated. To accomplish this, we used monocyte derived DC that were electroporated with RNA coding for the CMV pp65 antigen. The induction and expansion of antigen specific CD8+ and CD4+ T cells was assessed using a pannel of peptides derived from this antigen and presented on HLA-A2, -A1, -A11, -A24, -B35 and -B7 in IFN-g ELISPOT, 51Cr-release and proliferation assays. Autologous DC generated from CMV positive healthy donors were pulsed with peptides or transfected with pp65 RNA and utilized as stimulators. Autologous purified CD8+ and CD4+ lymphocytes were used as effector cells. By applying this approach we found that transfection of DC with pp65 RNA induces an expansion of polyclonal CD8+ mediated T cell responses that recognized peptide antigens presented on different HLA molecules. These in vitro generated cytotoxic T cells were able to efficiently lyse DC pulsed with pp65 derived peptides or transfected with the cognate RNA in an antigen specific manner after several in vitro restimulations. Furthermore, this experimental approach allowed the identification of the immunodominace of T cell epitopes presented upon RNA transfection. The HLA-2 directed responses were more pronounced as compared to the HLA-A1, -A11, -A24 or -B35 allels. In contrast, in 7 out of 7 HLA-A2 and HLA-B7 positive donors B7-peptides elicited a stronger T cell response than the A2-peptide, indicating the immunodominance of HLA-B7 epitopes. Interestingly, transfection of DC with pp65 RNA resulted in the induction of CD4+ antigen specific T cells that produced IFN-g and proliferated upon stimulation with transfected DC. In the next set of experiments we analyzed the possible induction of CMV specific T cells that recognize epitopes deduced from different antigens. Co-transfection of DC with a mixture of RNAs coding for the CMV pp65 and IE1 antigens elicited polyclonal T lymphocytes specific for peptides derived from both antigens. More importantly, polyclonal cytotoxic T cells could be elicited in peripheral blood of 2 out of 3 CMV negative donors demonstrating the efficiency of this approach. Our results demonstrate that DC transfected with RNA can elicit polyclonal T cell responses and have implications for the development of immunotherapeutic strategies to target viral or tumor associated antigens.

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Generation of Leukemia-Derived Dendritic Cells (DCleu) as a Basis for Specific Immunotherapy in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS).

Blood ◽

10.1182/blood.v104.11.2547.2547 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2547-2547

Author(s):

Helga Schmetzer ◽

Christoph Schmid ◽

Susanne Kufner ◽

Renate Pelka-Fleischer ◽

Tanja Kroell ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Cell Response ◽

T Cell Response ◽

Aberrant Expression ◽

Immune Phenotype ◽

Cytogenetic Aberrations ◽

Stimulation Of

Abstract The presentation of leukemic antigens can be improved in AML and MDS by in vitro generation of dendritic cells of leukemic origin (DCleu), thereby creating a platform for the generation of leukemia-specific cytotoxic lymphocytes (CTL). To further investigate this approach, we developed a serum-free culture system using MCM-Mimic medium (X-vivo + GM-GSF + IL-4 + FL + IL1β + IL-6 + TNFα + PGE2) for differentiation of malignant myeloid blasts to DCleu. Periperal blood mononuclear cells (PB-MNC) were obtained from 100 AML and 55 MDS-patients. Samples contained a mean of 59%/and 6% of blasts, respectively. After 14 days, cultures contained on average 34% (AML) and 20%(MDS) DC. DC yields were best in monocytic subtypes (AML M4, M5 and CMML). Cytogenetic aberrations of the leukemia had no influence. The leukemic origin of cultured DC was demonstrated using a acombined FISH/immune phenotyping assay (FISH/IPA): In cells showing a characteristic DC morphology and immune phenotype, FISH was used to detect specific cytogenetic aberrations identified in the leukemic population at time of diagnosis. Alternativly, DCleu were identified by detecting coexpression of DC markers and a leukemia-specific immune phenotype (as defined by aberrant expression of lineage markers or a characteristic CD34+/CD117+ phenotype in MDS). However, in most cases not all leukemic blasts in a given sample could be differentiated, since on average 47% of clonal cells did not acquire a DC-like immunophenotype. Vice versa, not all DC identified at the end of the culture period were DCleu. The capacity of DCleu to elicit a specific T-cell response was demonstrated by upregulation of contact-molecules, responsible for DC/T-cell contact, by DC-activated T-cell proliferation and by the capacity of DC-activated T-cells to specifically lyse naive blasts. In 6%/31% of AML/MDS-cases, <10% DC could be generated. Therefore, other DC-culture-assays were compared with respect to DC harvest, to identify the best method for DC-generation in each individual patient. Compared to `MCM-Mimic`, harvest of DC could be improved by `CA-Ionophore-media`(A23187 + Il4) in 4 of 7 cases, whereas in 3 cases, MCM led to higher DC-yields. In conclusion, DCleu can be generated in the majority of patients with AML and MDS. To optimize DC harvest for in vitro and in vivo use, different culture assays should be compared in each individual case. DCleu are able to elicit a specific T-cell response in vitro. Nevertheless, cultures containing DCleu also contain relevant numbers of undifferentiated blasts and DC of non-leukemic origin. These cells may represent an obstacle for the clinical use of DCleu, since they may cause specific anergy or unspecific stimulation of effector T-cells. Improvement of culture conditions for generation of DCleu, and methods to separate DCleu before stimulation of effector cells will be required, before clinical trials are feasible.

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Human Cytomegalovirus (HCMV)-Specific CD4+ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

Journal of Virology ◽

10.1128/jvi.02128-16 ◽

2017 ◽

Vol 91 (6) ◽

Cited By ~ 28

Author(s):

Sarah E. Jackson ◽

George X. Sedikides ◽

Gavin M. Mason ◽

Georgina Okecha ◽

Mark R. Wills

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Human Cytomegalovirus ◽

Immune Cells ◽

Cell Response ◽

T Cell Response ◽

Cell Responses ◽

Infected Cells

ABSTRACT Human cytomegalovirus (HCMV) infection and periodic reactivation are generally well controlled by the HCMV-specific T cell response in healthy people. While the CD8+ T cell response to HCMV has been extensively studied, the HCMV-specific CD4+ T cell effector response is not as well understood, especially in the context of direct interactions with HCMV-infected cells. We screened the gamma interferon (IFN-γ) and interleukin-10 (IL-10) responses to 6 HCMV peptide pools (pp65, pp71, IE1, IE2, gB, and US3, selected because they were the peptides most frequently responded to in our previous studies) in 84 donors aged 23 to 74 years. The HCMV-specific CD4+ T cell response to pp65, IE1, IE2, and gB was predominantly Th1 biased, with neither the loss nor the accumulation of these responses occurring with increasing age. A larger proportion of donors produced an IL-10 response to pp71 and US3, but the IFN-γ response was still dominant. CD4+ T cells specific to the HCMV proteins studied were predominantly effector memory cells and produced both cytotoxic (CD107a expression) and cytokine (macrophage inflammatory protein 1β secretion) effector responses. Importantly, when we measured the CD4+ T cell response to cytomegalovirus (CMV)-infected dendritic cells in vitro, we observed that the CD4+ T cells produced a range of cytotoxic and secretory effector functions, despite the presence of CMV-encoded immune evasion molecules. CD4+ T cell responses to HCMV-infected dendritic cells were sufficient to control the dissemination of virus in an in vitro assay. Together, the results show that HCMV-specific CD4+ T cell responses, even those from elderly individuals, are highly functional and are directly antiviral. IMPORTANCE Human cytomegalovirus (HCMV) infection is carried for a lifetime and in healthy people is kept under control by the immune system. HCMV has evolved many mechanisms to evade the immune response, possibly explaining why the virus is never eliminated during the host's lifetime. The dysfunction of immune cells associated with the long-term carriage of HCMV has been linked with poor responses to new pathogens and vaccines when people are older. In this study, we investigated the response of a subset of immune cells (CD4+ T cells) to HCMV proteins in healthy donors of all ages, and we demonstrate that the functionality of CD4+ T cells is maintained. We also show that CD4+ T cells produce effector functions in response to HCMV-infected cells and can prevent virus spread. Our work demonstrates that these HCMV-specific immune cells retain many important functions and help to prevent deleterious HCMV disease in healthy older people.

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Lentiviral gp34/OX40L Gene Transfer into Dendritic Cells Facilitates Alloreactive CD4 + T-Cell Response In Vitro

International Journal of Hematology ◽

10.1532/ijh97.03120 ◽

2004 ◽

Vol 79 (4) ◽

pp. 377-383 ◽

Cited By ~ 8

Author(s):

Masayuki Kobayashi ◽

Akifumi Takaori-Kondo ◽

Keiko Fukunaga ◽

Hiroyuki Miyoshi ◽

Takashi Uchiyama

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Gene Transfer ◽

Cell Response ◽

T Cell Response ◽

Cd4 T Cell

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Abstract 152: Oxidized Low-Density Lipoprotein Immune Complexes Generate an Inflammatory Immune Response by Signaling Through Multiple Receptors on Dendritic Cells

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.152 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Jillian P Rhoads ◽

Ashley J Wilhelm ◽

Amy S Major

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Immune Complexes ◽

Cell Response ◽

Oxidized Ldl ◽

T Cell Response ◽

Critical Function ◽

Activation Markers ◽

Multiple Receptors

Antibodies to oxidized LDL (oxLDL) and resulting immune complexes (ICs) are a prominent feature of atherosclerosis and diseases associated with increased atherosclerosis including Type 2-diabetes and rheumatoid arthritis. Although levels of oxLDL-ICs correlate with disease severity and associate with pro-inflammatory activation of macrophages in vitro , it is currently unclear whether these ICs are simply biomarkers or play an active role in disease pathogenesis. One possible mechanism by which oxLDL-ICs may regulate inflammation in atherosclerosis is by interacting with Fc gamma receptors (FcgRs) expressed on the surface of antigen presenting cells, such as dendritic cells (DCs). Not surprisingly, signaling through FcgRs is tightly regulated and dependent on their relative cell surface density. In addition, FcgR signaling has been shown to be linked to Toll like receptor-4 (TLR-4), a pattern recognition receptor. We hypothesize that oxLDL-ICs exacerbate atherosclerosis via signaling through FcgRs and TLR4 on DCs resulting in a pro-inflammatory T cell response. To test this hypothesis, bone marrow derived DCs (BMDCs) were treated with in vitro generated oxLDL ICs. Interestingly, BMDCs treated with oxLDL-ICs had increased expression of the activation markers MHC-II and CD40 and produced greater levels of the Th17 polarizing cytokines IL-1beta and IL-23 compared to cells treated with oxLDL alone. Secretion of pro-inflammatory cytokines was significantly decreased by pre-treatment of BMDCs with a TLR4 inhibitor and by blocking FcgR signaling. This suggests that responses to oxLDL-ICs involve both TLR-4 and FcgRs. Furthermore, incubation of OT-II T cells with BMDCs treated with oxLDL-IC prior to incubation with ovalbumin peptide displayed increased pro-inflammatory cytokine secretion compared to incubation with oxLDL alone. In conclusion, our studies provide new evidence that oxLDL-ICs signal through multiple receptors on DCs resulting in increased inflammatory potential. Because of their critical function in shaping the T cell response, we believe that DC signaling via FcgRs and oxLDL-ICs represents an important link between innate and adaptive immunity in atherosclerosis.

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Immunization with Apoptotic Phagocytes Containing Histoplasma capsulatum Activates Functional CD8+T Cells To Protect against Histoplasmosis

Infection and Immunity ◽

10.1128/iai.05350-11 ◽

2011 ◽

Vol 79 (11) ◽

pp. 4493-4502 ◽

Cited By ~ 13

Author(s):

Shih-Hung Hsieh ◽

Jr-Shiuan Lin ◽

Juin-Hua Huang ◽

Shang-Yang Wu ◽

Ching-Liang Chu ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Response ◽

Cell Activation ◽

Histoplasma Capsulatum ◽

T Cell Response ◽

Content Type

ABSTRACTWe have previously revealed the protective role of CD8+T cells in host defense againstHistoplasma capsulatumin animals with CD4+T cell deficiency and demonstrated that sensitized CD8+T cells are restimulatedin vitroby dendritic cells that have ingested apoptotic macrophage-associatedHistoplasmaantigen. Here we show that immunization with apoptotic phagocytes containing heat-killedHistoplasmaefficiently activated functional CD8+T cells whose contribution was equal to that of CD4+T cells in protection againstHistoplasmachallenge. Inhibition of macrophage apoptosis due to inducible nitric oxide synthase (iNOS) deficiency or by caspase inhibitor treatment dampened the CD8+T cell but not the CD4+T cell response to pulmonaryHistoplasmainfection. In mice subcutaneously immunized with viableHistoplasmayeasts whose CD8+T cells are protective againstHistoplasmachallenge, there was heavy granulocyte and macrophage infiltration and the infiltrating cells became apoptotic. In mice subcutaneously immunized with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled apoptotic macrophages containing heat-killedHistoplasma, the CFSE-labeled macrophage material was found to localize within dendritic cells in the draining lymph node. Moreover, depleting dendritic cells in immunized CD11c-DTR mice significantly reduced CD8+T cell activation. Taken together, our results revealed that phagocyte apoptosis in theHistoplasma-infected host is associated with CD8+T cell activation and that immunization with apoptotic phagocytes containing heat-killedHistoplasmaefficiently evokes a protective CD8+T cell response. These results suggest that employing apoptotic phagocytes as antigen donor cells is a viable approach for the development of efficacious vaccines to elicit strong CD8+T cell as well as CD4+T cell responses toHistoplasmainfection.

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