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

2004 ◽  
Vol 79 (4) ◽  
pp. 377-383 ◽  
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

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

2008 ◽  
Vol 26 (1) ◽  
pp. 78-85 ◽  
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

CD8alpha+ dendritic cells enhance the antigen-specific CD4+ T-cell response and accelerate development of collagen-induced arthritis

2007 ◽  
Vol 111 (2) ◽  
pp. 76-83 ◽  
Author(s):  
Young Ok Jung ◽  
So-Youn Min ◽  
Mi-La Cho ◽  
Min-Jung Park ◽  
Ju-Youn Jeon ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Cell Response ◽  
T Cell Response ◽  
Cd4 T Cell ◽  
Collagen Induced Arthritis ◽  
Accelerate Development

Transfection of Dendritic Cells with In Vitro Transcribed RNA Induces Polyclonal CMV-Specific CD8+ and CD4+ Mediated T Cell Responses.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1354-1354
Author(s):  
Annkristin Heine ◽  
Tobias Holderried ◽  
Frank Grünebach ◽  
Silke Appel ◽  
Markus M. Weck ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Response ◽  
Cytotoxic T Cells ◽  
T Cell Responses ◽  
T Cell Response ◽  
Cell Responses ◽  
Rna Transfection

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.

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

Blood ◽  
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.

Targeting Myeloma-Associated Macrophage Inhibits Multiple Myeloma Progression By Enhancing Anti-Tumor Cytotoxic CD4+ T Cell Response

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 481-481
Author(s):  
Wang Qiang ◽  
Yong Lu ◽  
Rong Li ◽  
Zhen Cai ◽  
Jian Hou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Cell Response ◽  
Myeloma Cell ◽  
T Cell Response ◽  
Cd4 T Cell ◽  
Antibody Treatment ◽  
Stimulating Factor

Abstract Background: Multiple myeloma (MM) is an incurable plasma cell cancer characterized by tumor cell accumulation and expansion in the bone marrow (BM). One of the major problems is that MM BM microenvironment is a tumor promoting and immune suppressive milieu. We previously discovered that macrophages (MФs), in particular myeloma-associated MФs (mMФs), heavily infiltrated into MM BM and mediated chemoresistance in MM. Colony-stimulating factor 1 receptor (CSF1R), also known as macrophage colony-stimulating factor receptor (M-CSFR), plays an important role as regulator of the development, morphology, survival, and functions of tissue macrophages as well as tumor-associated macrophages (TAMs). CSF1R blockade by inhibitors and antibodies has been shown promising to treat different tumors, such as glioma, pancreatic cancer, and diffuse-type giant cell tumor. We here assessed the impact of CSF1R blockade by CSF1R antibodies from Imclone (Lilly) on human and murine mMФs, MM growth in vivo, and anti-myeloma immune response in the BM microenvironment as well as chemotherapy in MM. Methods: MФ infiltration was determined in the bone marrow (BM) patients with MGUS (n=6), MM (n=6), and compared to healthy donors (HD, n=6). CSF1R signaling blockade was assessed in the monocytes differentiation with M-CSF in the presence of CSF1R antibody or IgG control. CSF1R antibody impact on MФs growth/viability/proliferation was measured by trypan blue exclusion analysis, MTS, and Ki67 flow cytometry analysis. Tumor burden of 5TGM-1-bearing mice with CSF1R blockade treatment was determined by in vivo bioluminescent imaging, ELISA of IgG2b in mouse serum, and flow cytometry analysis of CD138+ myeloma cell infiltration in mouse BM. The effect of CSF1R antibody treatment on mMФ depletion and M1/M2 polarization was determined by flow cytometry and real-time PCR. Impact of CSF1R antibody treatment on cytotoxic CD8+ and CD4+ T cell immune response was measured by intracellular granzyme B flow cytometry and granzyme B ELISPOT. Effector cell-mediated MM cytotoxicity in the presence of mMФs with or without CSF1R treatment was measured by CD138/Annexin V flow cytometry. Survival rate of MM-bearing mice with CSF1R antibody and chemotherapy was evaluated using Kaplan-Meier estimates and log-rank tests using GraphPad Prism 5 software. Results: MΦ accumulation in BM was associated with myeloma development. Blocking CSF1R by humanized and murine CSF1R monoclonal antibodies (CS4 and CS7) not only inhibited monocyte survival and differentiation but also suppressed human and mouse mMΦ survival and development in vitro. Further, Targeting of MФs by either CS7 antibody or DT-mediated MФ killing in LysmCre X Csf1rLsL-DTR C57BL/6 mouse had marked inhibitory effects on established myeloma progression. CSF1R blockade by CS7 treatment reprogrammed the tumor microenvironment toward to an anti-tumor phenotype. CSF1R antibody treatment reduced myeloma cell load in mouse BM, however the anti-MM activity by CSF1R antibody was abolished in immunodeficient Rag-/- mice. Strikingly we found CSF1R blockade mediated anti-MM effect mainly based on CD4+ T cell response by CD8 and CD4 neutralizing antibody. Correspondingly cytotoxic anti-MM CD4+ T cell response enhanced by CSF1R antibody treatment was confirmed by ex vivo ELISPOT and in vitro cytotoxicity assay. Additionally, CSF1R antibody treatment promoted MM drug sensitivity. Conclusion: Our data demonstrated thatMФs play an important role in MM growth and development. Targeting mMФs by CSF1R blockade achieves anti-MM activity by enhancing cytotoxic CD4+ T cell response and promotes chemotherapy on MM, therefore suggesting therapeutic strategies based on interfering with myeloma-macrophage interactions. Disclosures No relevant conflicts of interest to declare.

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

2017 ◽  
Vol 91 (6) ◽  
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.

scholarly journals Broadly directed SARS-CoV-2-specific CD4+ T cell response includes frequently detected peptide specificities within the membrane and nucleoprotein in patients with acute and resolved COVID-19

2021 ◽  
Vol 17 (9) ◽  
pp. e1009842
Author(s):  
Janna Heide ◽  
Sophia Schulte ◽  
Matin Kohsar ◽  
Thomas Theo Brehm ◽  
Marissa Herrmann ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Response ◽  
Ex Vivo ◽  
Intracellular Cytokine Staining ◽  
T Cell Responses ◽  
T Cell Response ◽  
Cd4 T Cell ◽  
Cell Responses ◽  
Single Peptide

The aim of this study was to define the breadth and specificity of dominant SARS-CoV-2-specific T cell epitopes using a comprehensive set of 135 overlapping 15-mer peptides covering the SARS-CoV-2 envelope (E), membrane (M) and nucleoprotein (N) in a cohort of 34 individuals with acute (n = 10) and resolved (n = 24) COVID-19. Following short-term virus-specific in vitro cultivation, the single peptide-specific CD4+ T cell response of each patient was screened using enzyme linked immuno spot assay (ELISpot) and confirmed by single-peptide intracellular cytokine staining (ICS) for interferon-γ (IFN-γ) production. 97% (n = 33) of patients elicited one or more N, M or E-specific CD4+ T cell responses and each patient targeted on average 21.7 (range 0–79) peptide specificities. Overall, we identified 10 N, M or E-specific peptides that showed a response frequency of more than 36% and five of them showed high binding affinity to multiple HLA class II binders in subsequent in vitro HLA binding assays. Three peptides elicited CD4+ T cell responses in more than 55% of all patients, namely Mem_P30 (aa146-160), Mem_P36 (aa176-190), both located within the M protein, and Ncl_P18 (aa86-100) located within the N protein. These peptides were further defined in terms of length and HLA restriction. Based on this epitope and restriction data we developed a novel DRB*11 tetramer (Mem_aa145-164) and examined the ex vivo phenotype of SARS-CoV-2-specific CD4+ T cells in one patient. This detailed characterization of single T cell peptide responses demonstrates that SARS-CoV-2 infection universally primes a broad T cell response directed against multiple specificities located within the N, M and E structural protein.

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

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.

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

2011 ◽  
Vol 79 (11) ◽  
pp. 4493-4502 ◽  
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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