scholarly journals 714 Selective expansion of antigen-specific CD8 T cells with engineered antigen presenting exosome

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A743-A743
Author(s):  
Tomoyoshi Yamano ◽  
Xiabing Lyu ◽  
Rikinari Hanayama
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Cd8 T Cells ◽  
Costimulatory Molecule ◽  
Animal Experiments ◽  
Surface Proteins ◽  
Hek293 Cells ◽  
Target Cells ◽  
Single Chain ◽  
Antigen Presenting

BackgroundExosomes are vesicular granules of about 100 nm and are secreted by many types of cells. Exosomes contain various proteins, lipids, and RNAs that are transported to target cells which induce functional and physiological changes. Exosomes are promising nano-vesicles for clinical application, owing to their high biocompatibility, low immunogenicity, and high drug delivery efficacy. Recent studies have demonstrated that exosomes from tumor cells or antigen presenting cells (APCs) regulate immune responses. Tumor derived exosomes express PD-L1 on their surface and suppress tumor immunity systemically. On the other hand, mature dendritic cells derived exosomes exert immune activation, and tumor immunotherapy using DCs exosome has been developed. However, few studies have been found to exert a significant effect on cancer treatment, may be because of low expression of costimulatory molecules and lack of cytokines on DCs derived exosomes.MethodsIt has been demonstrated that GFP can be conveyed into exosomes by conjugating GFP with tetraspanins, exosome-specific surface proteins. First, we generated a tetraspanin fusion protein with a single-chain MHCI trimer (scMHCI). IL-2 is inserted on the second extracellular loop of CD81, allowing robust and functional expression of IL-2 on the exosome. We collected exosomes from HEK293 cells culture, which stably express scMHCI-CD81-IL2 and CD80-MFGE8, and used as Antigen-presenting exosome(AP-Exo).ResultsAP-Exo expresses high expression of MHCI-peptide complex, costimulatory molecule, and cytokine, activating cognate CD8 T cells as dendritic cells do. AP-Exo selectively delivered co-stimulation and IL-2 to antigen-specific CD8 T cells, resulting in a massive expansion of antigen-specific CD8 T cells without severe adverse effects in mice. AP-Exo can expand endogenous tumor-specific CD8 T cells and induce the potent anti-tumor effect.ConclusionsOur strategy for building engineered exosomes that work like APCs might develop novel methods for cancer immunotherapy.Ethics ApprovalAll mice were housed in a specific pathogen-free facility, and all animal experiments were performed according to a protocol approved by Kanazawa University, Kanazawa, Japan.

scholarly journals Generation of Human CD8 T Regulatory Cells by CD40 Ligand–activated Plasmacytoid Dendritic Cells

2002 ◽  
Vol 195 (6) ◽  
pp. 695-704 ◽  
Author(s):  
Michel Gilliet ◽  
Yong-Jun Liu
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Transforming Growth Factor ◽  
Cd8 T Cell ◽  
Cd40 Ligand ◽  
Plasmacytoid Dendritic Cells ◽  
Target Cells ◽  
Ifn Γ

Although CD8 T cell–mediated immunosuppression has been a well-known phenomenon during the last three decades, the nature of primary CD8 T suppressor cells and the mechanism underlying their generation remain enigmatic. We demonstrated that naive CD8 T cells primed with allogeneic CD40 ligand–activated plasmacytoid dendritic cells (DC)2 differentiated into CD8 T cells that displayed poor secondary proliferative and cytolytic responses. By contrast, naive CD8 T cells primed with allogeneic CD40 ligand–activated monocyte-derived DCs (DC1) differentiated into CD8 T cells, which proliferated to secondary stimulation and killed allogeneic target cells. Unlike DC1-primed CD8 T cells that produced large amounts of interferon (IFN)-γ upon restimulation, DC2-primed CD8 T cells produced significant amounts of interleukin (IL)-10, low IFN-γ, and no IL-4, IL-5, nor transforming growth factor (TGF)-β. The addition of anti–IL-10–neutralizing monoclonal antibodies during DC2 and CD8 T cell coculture, completely blocked the generation of IL-10–producing anergic CD8 T cells. IL-10–producing CD8 T cells strongly inhibit the allospecific proliferation of naive CD8 T cells to monocytes, and mature and immature DCs. This inhibition was mediated by IL-10, but not by TGF-β. IL-10–producing CD8 T cells could inhibit the bystander proliferation of naive CD8 T cells, provided that they were restimulated nearby to produce IL-10. IL-10–producing CD8 T cells could not inhibit the proliferation of DC1-preactivated effector T cells. This study demonstrates that IL-10–producing CD8 T cells are regulatory T cells, which provides a cellular basis for the phenomenon of CD8 T cell–mediated immunosuppression and suggests a role for plasmacytoid DC2 in immunological tolerance.

In Vitro Generation of Cytotoxic T Lymphocytes against MAGE A1 and MAGE A3 Derived From Healthy Donors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4079-4079
Author(s):  
Lei Bao ◽  
Mindy M Stamer ◽  
Kimberly Dunham ◽  
Deepa Kolaseri Krishnadas ◽  
Kenneth G Lucas
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Lymphocytes ◽  
Cell Therapy ◽  
Cytotoxic T Lymphocytes ◽  
Cd8 T Cells ◽  
Target Cells ◽  
Ifn Gamma ◽  
Healthy Donors

Abstract Abstract 4079 Poster Board III-1014 MAGE A1 and MAGE A3 are cancer testis antigens that are expressed on a number of malignant tumor cells, but not by normal cells, except for male germ cells which lack HLA expression. Therefore, MAGE cytotoxic T lymphocytes are strictly tumor-specific. Adoptive transfer of antigen specific cytotoxic T lymphocytes (CTL) provides immediate graft-versus tumor effects while minimizing risk for graft-versus-host disease. The aim of the current study was to find ideal conditions for expansion of CTL targeting tumor-associated antigens from peripheral blood mononuclear cells (PBMCs) of healthy donors to be used in allogenic cell therapy. In this study we investigated the ability to generate MAGE A1 and MAGE A3 specific cytotoxic T cells using autologous dendritic cells (DC) loaded with MAGE A1 and MAGE A3 overlapping peptides. CTL lines specific for MAGE A1 and MAGE A3 were established by stimulating CD8 T cells from healthy donors with autologous dendritic cells loaded with MAGE A1 or MAGE A3 overlapping pooled peptides in round-bottomed, 96-well plates. CD8+ T cells were restimulated with the same ratio of peptide pulsed DC on days 7 and 14 in the presence of IL-2 (50 U/ml), IL-7 and IL-15 (5 ng/ml). These microcultures were screened 10 days after the third stimulation for their capacity to produce interferon-gamma (IFN-gamma) when stimulated with autologous EBV-transformed B lymphocytes (BLCL) transduced with lentivirus(LV) encoding MAGE A1 or MAGE A3 and autologous BLCL transduced with LV encoding GFP. MAGE A1 and MAGE-A3 specific IFN-gamma producing cells were rapidly expanded in OKT3 and IL2. The specificity of the rapidly expanded MAGE A1 and MAGE A3 specific T cells was confirmed by IFN-gamma production as measured by intracellular cytokine staining and ELISA as well as antigen specific cytotoxicity by a standard 51chromium (51Cr) release assay. We successfully generated MAGE A1 and MAGE A3 specific CTL lines from healthy donors using this method. Specific CTL lines showed cytotoxicity in vitro not only to target cells pulsed with MAGE A1 or MAGE A3 peptides but also to target cells transduced with LV-MAGE A1 or LV-MAGE A3. Specific cytolytic activity was accompanied by IFN-gamma secretion. These data indicate that tumor antigen specific CTL can be expanded using overlapping peptides regardless of an individual's HLA specificity. The ability to generate tumor specific CTL from donors of various HLA backgrounds provide a rationale for utilizing MAGE A1 and MAGE A3 overlapping peptides for expansion of antigen specific T cells for adoptive T-cell therapy against MAGE A1 or MAGE A3 expressing tumors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Direct CD4 Help Provision following Interaction of Memory CD4 and CD8 T Cells with Distinct Antigen-Presenting Dendritic Cells

2010 ◽  
Vol 185 (2) ◽  
pp. 1028-1036 ◽  
Author(s):  
Marie-Ghislaine de Goër de Herve ◽  
Bamory Dembele ◽  
Mélissa Vallée ◽  
Florence Herr ◽  
Anne Cariou ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Cd8 T Cells ◽  
Cd4 Help ◽  
Antigen Presenting

scholarly journals Fcγ Receptor Type I (CD64)-Mediated Impairment of the Capacity of Dendritic Cells to Activate Specific CD8 T Cells by IgG-opsonized Friend Virus

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 145 ◽  
Author(s):  
Zoltán Bánki ◽  
Roland Werner ◽  
Lydia Riepler ◽  
Annika Rössler ◽  
Brigitte Müllauer ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Cd8 T Cells ◽  
Cytotoxic T Cell ◽  
Type I ◽  
Friend Virus ◽  
Fcγ Receptor ◽  
Fcγ Receptors ◽  
Viral Antigens ◽  
Antigen Presenting

Dendritic cells (DCs) express Fcγ receptors (FcγRs) for the binding immune complexes (ICs) consisting of IgG and antigens (Ags). IC–FcγR interactions have been demonstrated to enhance activation and antigen-presenting functions of DCs. Utilizing Friend virus (FV), an oncogenic mouse retrovirus, we investigated the effect of IgG-opsonization of retroviral particles on the infection of DCs and the subsequent presentation of viral antigens by DCs to virus-specific CD8 T cells. We found that opsonization by virus-specific non-neutralizing IgG abrogated DC infection and as a consequence significantly reduced the capacity of DCs to activate virus-specific CD8 T cells. Effects of IgG-opsonization were mediated by the high-affinity FcγR type I, CD64, expressed on DCs. Our results suggest that different opsonization patterns on the retroviral surface modulate infection and antigen-presenting functions of DCs, whereby, in contrast to complement, IgG reduces the capacity of DCs to activate cytotoxic T cell (CTL) responses.

scholarly journals Expression and functional significance of an additional ligand for CTLA-4

1993 ◽  
Vol 90 (23) ◽  
pp. 11054-11058 ◽  
Author(s):  
D J Lenschow ◽  
G H Su ◽  
L A Zuckerman ◽  
N Nabavi ◽  
C L Jellis ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Costimulatory Molecule ◽  
T Cell Proliferation ◽  
Antigen Presenting

Effective T-cell activation requires antigen/major histocompatibility complex engagement by the T-cell receptor complex in concert with one or more costimulatory molecules. Recent studies have suggested that the B7 molecule, expressed on most antigen presenting cells, functions as a costimulatory molecule through its interaction with CD28 on T cells. Blocking the CD28/B7 interaction with CTLA4Ig inhibits T-cell activation in vitro and induces unresponsiveness. We demonstrate that another molecule(s), termed B7-2, is expressed constitutively on dendritic cells, is differentially regulated on B cells, and costimulates naive T cells responding to alloantigen. B7-2 is up-regulated by lipopolysaccharide in < 6 hr and is maximally expressed on the majority of B cells by 24 hr. In contrast, B7 is detected only on a subset of activated B cells late (48 hr) after stimulation. In addition, Con A directly induces B7-2 but not B7 expression on B cells. Finally, although both anti-B7 monoclonal antibodies and CTLA4Ig blocked T-cell proliferation to antigen-expressing B7 transfectants, only CTLA4Ig had any significant inhibitory effect on T-cell proliferation to antigens expressed on natural antigen presenting cells, such as dendritic cells. Thus, B7 is not the only costimulatory molecule capable of initiating T-cell responses since a second ligand, B7-2, can provide a necessary second signal for T-cell activation.

Construction and Pre-Clinical Evaluation of An Anti-CD19 Chimeric Antigen Receptor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4623-4623 ◽  
Author(s):  
James Kochenderfer ◽  
Steven Feldman ◽  
Yangbing Zhao ◽  
Hui Xu ◽  
Mary Black ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Costimulatory Molecule ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Target Cells ◽  
Single Chain ◽  
Cell Counts

Abstract Adoptive T cell immunotherapy can cause regression of established malignancy. One promising approach is to transfer genes encoding chimeric antigen receptors (CARs) that specifically recognize tumor-associated antigens to T cells before the T cells are adoptively transferred to patients. We have constructed a CAR that consists of an anti-CD19 single chain variable region (scFv) that is coupled to a portion of the CD28 costimulatory molecule and the signaling component of the CD3-zeta chain. CD19 is a promising target for immunotherapy because most malignant B cell express CD19, but the only normal cells that express CD19 are B cells, B cell precursors, and perhaps follicular dendritic cells. We have demonstrated that gamma-retroviruses encoding the anti-CD19 CAR can be used to transduce human T cells and that these transduced T cells specifically recognize CD19+ targets. To transduce T cells, we stimulated T cells with the anti-CD3 monoclonal antibody OKT3 on day 0 then conducted sequential retroviral transductions on day 2 and on day 3. Transductions were performed by spin-loading retroviruses onto RetroNectin (Takara) coated culture plates followed by overnight incubation of the OKT3- stimulated T cells on the plates. Forty-five to sixty-seven percent of T cells expressed the anti-CD19 CAR as measured by flow cytometry 7–8 days after transduction (n=8). Anti-CD19-CAR-transduced CD8+ and CD4+ T cells produced IFNg and IL-2 specifically in response to stimulation with CD19+ target cells. The transduced T cells specifically killed primary chronic lymphocytic leukemia (CLL) cells. T cells from CLL patients that were either untreated or previously treated with fludarabine plus rituximab could be transduced and induced to proliferate sufficiently to provide enough cells for clinical adoptive T cell transfer. In addition, we adapted this protocol for use in CLL patients with very high peripheral blood leukemia cell counts by depleting CD19+ cells using magnetic bead sorting prior to OKT3 stimulation. In preparation for a clinical trial that will enroll patients with advanced B cell malignancies, we have generated a producer cell clone that produces GALV (Gibbon ape leukemia virus)-enveloped gamma-retroviruses encoding the anti-CD19 CAR, and we have produced sufficient retroviral supernatant for the proposed clinical trial under good manufacturing practice (GMP) conditions.

Efficient Generation of HLA-A24-Restricted WT1-Specific Cytotoxic T Lymphocytes Using Gene-Engineered Artificial Antigen-Presenting Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2101-2101
Author(s):  
Masahiro Ogasawara ◽  
Tomohiro Yamakawa ◽  
Yuki Katsura ◽  
Kanako Shima ◽  
Toshihiro Matsukawa ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Hla Class I ◽  
Fold Increase ◽  
Target Cells ◽  
Facs Analysis ◽  
The Third ◽  
Healthy Donors ◽  
Artificial Antigen ◽  
Antigen Presenting

Abstract Abstract 2101 Background and Purpose: Recent success associated with adoptive transfer of antitumor T cells in lymphodepleted patients suggests the potential of adoptive immunotherapy to have a significant clinical impact. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we previously reported a culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL) from HLA-A2-positive melanoma patients by using K562-based artificial antigen-presenting cells (aAPCs). In the present study, we have applied the culture system to HLA-A24, one of the most common HLA class I antigen in the Asian population, and examined whether HLA-A2402-restricted WT1-specific CTL can be generated using aAPCs. Methods: HLA-A2402–positive peripheral blood mononuclear cells (PBMC) were obtained from healthy donors (n=4) and cancer patients (n=10). To establish antigen-specific T cells, CD8+ T cells were purified by positive selection using a magnetic beads method (Miltenyi Biotec). aAPCs were pulsed with an HLA-A2402 restricted, modified 9-mer WT1 immunodominant peptide (CYTWNQMNL). aAPCs were then irradiated with 200 Gy and added to purified CD8+ T cells at a ratio of 1:20 in 96-well plates in RPMI1640 supplemented with 10% human AB serum. Between stimulations, IL-2 (10 U/ml) and IL-15 (10ng/ml) (both from Peprotech) were added to the cultures. Results: Flow cytometry (FACS) analysis confirmed that aAPCs stably expressed transduced HLA class I, CD80 and CD83 molecules. aAPC did not express HLA class II molecules, CD40, CD154, or CD86. Following 3–4 rounds of weekly stimulation with peptide-pulsed aAPCs, WT1 peptide-specific CD8+ T cells were evaluated by a tetramer staining. The percentage of tetramer-positive cells was 0.082±0.0075% before stimulation. It increased to 0.865±0.528% (10.5 fold increase) and 1.89±1.12% (23.0 fold increase) following the third and the forth stimulation, respectively. There was no marked difference in magnitude of increase between healthy donors and cancer patients. However, when CD8+ T cells from patients vaccinated with WT1-peptide pulsed dendritic cells were stimulated with WT1-peptide-pulsed aAPCs, the percentage of tetramer-positive cells was significantly higher (11.72±6.46%) following the third stimulation. CD8+ T cells stimulated with WT1-peptide-pulsed aAPCs were negative for both A0201/WT1 and A2402/CMV tetramers, confirming HLA restriction and antigen specificity. FACS analysis revealed that WT1-specific CTL expanded with WT1-peptide-pulsed aAPCs expressed a memory phenotype. Furthermore, these CTL demonstrated cytotoxicity against CIR-A2402 target cells pulsed with a WT1 peptide in an LDH release assay. Unpulsed or HIV peptide-pulsed target cells were not lysed. Conclusions: These results demonstrated that HLA-A24-restricted WT1 specific CTLs with a memory phenotype can be generated ex vivo using peptide-pulsed gene-engineered aAPCs within a short period of time for clinical use. Disclosures: No relevant conflicts of interest to declare.

Secretion of bioactive interleukin-1β by dendritic cells is modulated by interaction with antigen specific T cells

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3809-3815 ◽  
Author(s):  
Stefania Gardella ◽  
Cristina Andrei ◽  
Sara Costigliolo ◽  
Lucia Olcese ◽  
M. Raffaella Zocchi ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Lymphocytes ◽  
Cd8 T Cells ◽  
Interleukin 1Β ◽  
T Lymphocyte Subsets ◽  
Cd8 T Lymphocyte ◽  
Human Dendritic Cells ◽  
Antigen Presenting

The role of interleukin-1β (IL-1β) as a regulator of the immune response, although extensively investigated, is still debated. We then studied the expression of IL-1β by human dendritic cells (DCs), the professional antigen presenting cells, and its modulation during immune reactions in vitro. Our results show that, on maturation or tetanus toxoid presentation to specific CD4+ CD40L+T lymphocytes, DCs begin to accumulate IL-1β precursor (pro–IL-1β) but do not secrete bioactive IL-1β. In contrast, interaction with alloreactive T cells results in both stimulation of pro–IL-1β synthesis and secretion of processed isoforms of the cytokine, that display biologic activity. Both CD4+ and CD8+ subsets of allospecific T lymphocytes are required: CD4+ T cells drive the synthesis of pro–IL-1β through CD40 engagement but have no effects on pro–IL-1β processing; CD8+ T cells, unable to induce synthesis of pro–IL-1β per se, are responsible for the generation of mature IL-1β by pro–IL-1β–producing DCs. Interleukin-1β–converting enzyme (ICE) inhibitors do not prevent the recovery of IL-1β bioactivity after allorecognition, indicating that allospecific CD8+ T cells may induce the release of bioactive IL-1β via mechanism(s) other than ICE activation. Altogether, these findings suggest that CD4+ and CD8+ T-lymphocyte subsets have distinct roles in the induction of IL-1β secretion by DCs and support the hypothesis that IL-1β plays a role in cell-mediated immune responses.

Human CD1c+ dendritic cells secrete high levels of IL-12 and potently prime cytotoxic T-cell responses

Blood ◽  
2013 ◽  
Vol 122 (6) ◽  
pp. 932-942 ◽  
Author(s):  
Giulia Nizzoli ◽  
Jana Krietsch ◽  
Anja Weick ◽  
Svenja Steinfelder ◽  
Federica Facciotti ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cytotoxic T Cell ◽  
Cell Responses ◽  
Key Points ◽  
Cytotoxic Molecules ◽  
Cytotoxic T Cell Responses ◽  
Antigen Presenting

Key Points CD1c+ DC but not BDCA-3+ DC or other antigen-presenting cells secrete high amounts of bioactive IL-12. CD1c+ DC efficiently cross-present antigens, prime CD8+ T cells, and induce the highest levels of cytotoxic molecules.

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