Dendritic cells matured with recombinant human sperm associated antigen 9 (rhSPAG9) induce CD4+, CD8+ T cells and activate NK cells: a potential candidate molecule for immunotherapy in cervical cancer

Abstract Background Dendritic cell (DC)-based immunotherapy is capable of activating the immune system and in particular tumor-specific cytotoxic T lymphocytes (CTLs) to eradicate the tumor. However, major limitations are the availability of autologous tumor cells as antigenic source and the selection of antigen that may have potential to activate both CD4+ and CD8+ T cells in immune-specific manner. Recently, we reported the expression of sperm associated antigen 9 (SPAG9) that is associated with various types of malignancies including cervical cancer. We examined the recombinant human SPAG9 (rhSPAG9) as an antigenic source for generating efficient DCs to stimulate CD4+ and CD8+ T cell responses for future DCs-based vaccine trials in cervical cancer patients. Methods Human monocytes derived DCs were pulsed with different concentrations (250 ng/ml to 1000 ng/ml) of recombinant human SPAG9 (rhSPAG9) and evaluated for their phenotypic and functional ability. The efficacy of DCs primed with 750 ng/ml of rhSPAG9 (SPDCs) was compared with DCs primed with autologous tumor lysates (TLDCs), to induce CD4+, CD8+ T cells and activating NK cells. In addition, we investigated the effect of the chemotherapeutic drug cisplatin on phenotypic and functional potential of SPDCs. Results Phenotypic and functional characterization of DCs pulsed with 750 ng/ml rhSPAG9 was found to be optimal and effective for priming DCs. SPDCs were also capable of stimulating allogeneic T cells similar to TLDCs. SPDCs showed a statistically insignificant increase in the expression of maturation marker CD83 and migration towards CCL19 and CCL21 compared with TLDCs (CD83; P = 0.4; migration; P = 0.2). In contrast, although TLDCs showed better proliferation and secretion of Th1 cytokines (IL12p40, IL12p70 and IFNγ) compared to SPDCs, this difference was not statistically significant (IL12p40, P = 0.06). Further we also observed that clinical dose of cisplatin (200 µM) treated SPDCs were able to stimulate the proliferation of cytotoxic T lymphocytes without increasing the FOXP3+ Tregs in autologous co-cultures. Conclusions In summary, in order to overcome the limitation of the availability of autologous tumor cells as antigenic sources, our present strategy provides an insight to consider rhSPAG9 as a strong immunogen for DC-based immunotherapy for cervical cancer trials and warrants further studies. This is the first report to suggest that rhSPAG9 is an effective antigen for pulsing DCs that are capable of eliciting a potent Th1 response which, in turn, may help in decreasing the tumor burden when used along with a cisplatin based combinatorial regimen for therapeutic intervention.

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Dendritic cells matured with recombinant human sperm associated antigen 9 (rhSPAG9) induce CD4+, CD8+ T cells and activate NK cells: A potential candidate molecule for immunotherapy in cervical cancer

10.21203/rs.3.rs-46780/v1 ◽

2020 ◽

Author(s):

Hemavathi Dhandapani ◽

Hascitha Jayakumar ◽

Abirami Seetharaman ◽

Selvaluxmy Ganeshrajah ◽

Shirley Sunder Singh ◽

...

Keyword(s):

Cervical Cancer ◽

T Cells ◽

T Lymphocytes ◽

Nk Cells ◽

Cytotoxic T Lymphocytes ◽

Cd8 T Cells ◽

Functional Characterization ◽

Potential Candidate ◽

Autologous Tumour ◽

Antigenic Source

Abstract Background Dendritic cell (DC)-based immunotherapy is capable of activating the immune system, and in particular tumour-specific cytotoxic T lymphocytes (CTLs) to eradicate the tumour. However, major limitations are the availability of autologous tumour cells as antigenic source and the selection of antigen that may have potential to activate both CD8 + and CD4 + T cells in immune-specific manner. Recently, we reported the expression of sperm associated antigen 9 (SPAG9) that is associated with various types of malignancies including cervical cancer. We examined the recombinant human SPAG9 (rhSPAG9) as an antigenic source for generating efficient DCs to stimulate CD4 + and CD8 + T cell responses for future DCs-based vaccine trials in cervical cancer patients. Methods Human monocytes derived DCs were pulsed with different concentrations (250 ng/ml to 1000 ng/ml) of recombinant human SPAG9 (rhSPAG9) and evaluated for their phenotypic and functional ability. Subsequently, the efficacy of DCs primed with 750 ng/ml of rhSPAG9 (SPDCs) was compared with DCs primed with autologous tumour lysate (TLDCs), to induce CD4+, CD8 + T cells and activating NK cells. In addition, we investigated the effect of the chemotherapeutic drug cisplatin on phenotypic and functional potential of SPDCs. Results Phenotypic and functional characterization of DCs pulsed with 750 ng/ml rhSPAG9 was found to be optimal and effective for priming DCs. SPDCs were also capable of stimulating allogeneic CD4 + and CD8 + T cells similar to TLDCs. SPDCs showed a statistically insignificant increase in the expression of maturation marker CD83 and migration towards CCL19 and CCL21 compared with TLDCs (CD83 p = 0.4; for migration p = 0.2). In contrast, TLDCs showed better proliferation and secretion of Th1 cytokines (IL12p40, IL12p70 and IFNγ) compared to SPDCs, but this was also not statistically significant (IL12p40, p = 0.06). We also found that clinical dose of cisplatin (200 µM) treated SPDCs were able to stimulate the proliferation of cytotoxic T lymphocytes without increasing the FOXP3+Tregs in autologous co-cultures. Conclusions This is the first report to suggest that rhSPAG9 is an effective antigen for pulsing DCs that are capable of eliciting a potent Th1 response which, in turn, may help in decreasing the tumour burden when used along with a cisplatin based combinatorial regimen for therapeutic intervention. This strategy provides an insight to consider rhSPAG9 as a strong immunogen for DC-based immunotherapy for cervical cancer.

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Frequency analysis of tumor-reactive cytotoxic T lymphocytes in peripheral blood of a melanoma patient vaccinated with autologous tumor cells

Cancer Immunology Immunotherapy ◽

10.1007/s002620050099 ◽

1994 ◽

Vol 39 (2) ◽

pp. 93-99 ◽

Cited By ~ 1

Author(s):

Wolfgang Herr ◽

Thomas W�lfel ◽

Michael Heike ◽

Karl-Hermann Meyer zum B�schenfelde ◽

Alexander Knuth

Keyword(s):

T Lymphocytes ◽

Tumor Cells ◽

Cytotoxic T Lymphocytes ◽

Frequency Analysis ◽

Peripheral Blood ◽

Melanoma Patient ◽

Autologous Tumor

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Frequency analysis of tumor-reactive cytotoxic T lymphocytes in peripheral blood of a melanoma patient vaccinated with autologous tumor cells

Cancer Immunology Immunotherapy ◽

10.1007/bf01525314 ◽

1994 ◽

Vol 39 (2) ◽

pp. 93-99 ◽

Cited By ~ 21

Author(s):

Wolfgang Herr ◽

Thomas W�lfel ◽

Michael Heike ◽

Karl-Hermann Meyer zum B�schenfelde ◽

Alexander Knuth

Keyword(s):

T Lymphocytes ◽

Tumor Cells ◽

Cytotoxic T Lymphocytes ◽

Frequency Analysis ◽

Peripheral Blood ◽

Melanoma Patient ◽

Autologous Tumor

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Generation of tumor-specific cytotoxic T-lymphocytes from peripheral blood of colorectal cancer patients for adoptive immunotherapy.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.3056 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 3056-3056

Author(s):

Marco Bregni ◽

Serena Del Bue ◽

Andrea Galli ◽

Alberto della Valle ◽

Pasquale Ferrante ◽

...

Keyword(s):

T Lymphocytes ◽

Tumor Cell ◽

Tumor Cells ◽

Cytotoxic T Lymphocytes ◽

Peripheral Blood ◽

Tumor Cell Line ◽

Interleukin 4 ◽

Autologous Tumor ◽

The Third ◽

Ifn Γ

3056 Background: Adoptive T-cell transfer (ACT) using autologous TIL, grown ex vivo and then infused into the cancer patient after lymphoablative chemotherapy, has emerged as an effective treatment for patients with metastatic melanoma. However, this approach has been hampered by the difficulty of isolating TILs from tumors other than melanoma, and of amplifying a sufficient quantity of autologous tumor-reactive T cells. So we decided to adopt a recently described procedure for generating in vitro large numbers of anti-tumor HLA-restricted CTLs, by stimulating patient’s CD8-enriched peripheral blood mononuclear cells (PBMCs) with DCs pulsed with apoptotic solid tumor cells (TCs) as a source of tumor antigens. Methods: 61 patients affected by CRC were enrolled. Tumor biopsies were obtained at surgery, together with 100 ml of heparinized peripheral blood. Tumors were dissociated to a single-cell suspension and cultured in order to obtain tumor cell line from each patient. Dendritic cells (DCs) were generated from previously separated PBMCs, using a magnetic positive selection of CD14+ monocytes, cultured in presence of Interleukin-4 and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Anti-tumor cytotoxic T lymphocytes (CTLs) were elicited using DCs as antigen-presenting cells, autologous apoptotic tumor cells as source of antigens and T CD-8 lymphocytes enriched effectors, with weekly stimulation. To evaluate the cytotoxic activity of CTLs, interferon-γ (IFN-γ) secretion was assessed by ELISPOT. Results: Tumor cell lines and DCs were obtained from 19 out of 61 patients. ELISPOT was performed so far for 6 patients: strong IFN-γ secretion was detected at the third, fourth and fifth stimulations for one patient and at the second for another patient, whereas for three patients a weak secretion was detected during the second and the third stimulations. CTLs from one patient did not react to the stimulations. Conclusions: Generation of CTLs suitable for ACT immunotherapy is feasible from peripheral blood in patients with CRC.

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Human Papillomavirus Type 16 (HPV-16) Virus-Like Particle L1-Specific CD8+ Cytotoxic T Lymphocytes (CTLs) Are Equally Effective as E7-Specific CD8+ CTLs in Killing Autologous HPV-16-Positive Tumor Cells in Cervical Cancer Patients: Implications for L1 Dendritic Cell-Based Therapeutic Vaccines

Journal of Virology ◽

10.1128/jvi.02443-08 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6779-6789 ◽

Cited By ~ 27

Author(s):

Stefania Bellone ◽

Karim El-Sahwi ◽

Emiliano Cocco ◽

Francesca Casagrande ◽

Marilisa Cargnelutti ◽

...

Keyword(s):

Cervical Cancer ◽

T Lymphocytes ◽

Cancer Patients ◽

Tumor Cells ◽

Copy Number ◽

Autologous Tumor ◽

Hpv 16 ◽

Expression Levels ◽

E6 And E7

ABSTRACT Papillomavirus-like particles (VLPs) based on L1 capsid protein represent a promising prophylactic vaccine against human papillomavirus (HPV) infections. However, cell-mediated immune responses against this antigen are believed to be of limited therapeutic value in established HPV-infected cervical lesions and, for this reason, have not been intensively investigated in cervical cancer patients. In this study we analyzed and quantified by real-time PCR (RT-PCR) the RNA expression levels of E6, E7, and L1 genes in flash-frozen HPV-16 cervical carcinomas. In addition, the kinetics of expression of E6, E7, and L1 in HPV-16-infected primary cell lines established as long-term cultures in vitro was also evaluated at RNA and protein levels. Finally, in order to evaluate the therapeutic potential of L1-specific CD4+ and CD8+ T lymphocytes responses in cervical cancer patients, L1 VLP-loaded dendritic cells (DCs) were used to stimulate peripheral blood lymphocytes from cervical cancer patients and such responses were compared to those elicited by the E7 oncoprotein. We show that 22 of 22 (100%) flash-frozen cervical biopsy samples collected from HPV-16-positive cervical cancer patients harbor L1, in addition to E6 and E7 RNA, as detected by RT-PCR. E7 RNA copy number (mean, 176.2) was significantly higher in HPV-16-positive cervical cancers compared to the E6 RNA copy number (mean, 47.3) and the L1 copy number (mean, 58.3) (P < 0.0001 and P < 0.001, respectively). However, no significant differences in expression levels between E6 and L1 were found. Kinetic studies of E6, E7, and L1 RNA and protein expression levels in primary tumors showed a sharp reduction in L1 expression after multiple in vitro passages compared to E6 and E7. Autologous DCs pulsed with HPV-16 VLPs or recombinant full-length E7 elicited strong type 1 L1- and E7-specific responses in CD4+ and CD8+ T cells from cervical cancer patients. Importantly, L1 VLP-specific CD8+ T lymphocytes expressed strong cytolytic activity against autologous tumor cells and were as effective as E7-specific cytotoxic T lymphocytes in lysing naturally HPV-16-infected autologous tumor cells. Taken together, these data demonstrate a consistent expression of L1 in primary cervical tumors and the possibility of inducing effective L1/tumor-specific CD4+ and CD8+ T-lymphocyte responses in patients harboring HPV-infected cervical cancer. These results may have important implications for the treatment of patients harboring established HPV-infected lesions with L1 VLPs or combined E7/L1 DC-based vaccinations.

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Induction of Human Papillomavirus-Specific CD4+ and CD8+ Lymphocytes by E7-Pulsed Autologous Dendritic Cells in Patients with Human Papillomavirus Type 16- and 18-Positive Cervical Cancer

Journal of Virology ◽

10.1128/jvi.73.7.5402-5410.1999 ◽

1999 ◽

Vol 73 (7) ◽

pp. 5402-5410 ◽

Cited By ~ 106

Author(s):

Alessandro D. Santin ◽

Paul L. Hermonat ◽

Antonella Ravaggi ◽

Maurizio Chiriva-Internati ◽

Dejin Zhan ◽

...

Keyword(s):

Cervical Cancer ◽

T Cells ◽

Dendritic Cells ◽

Human Papillomavirus ◽

Tumor Cells ◽

Full Length ◽

Autologous Tumor ◽

Hpv 16 ◽

Pulsed Dc ◽

Hpv 18

ABSTRACT Human papillomavirus (HPV) type 16 (HPV 16) and HPV type 18 (HPV 18) are implicated in the induction and progression of the majority of cervical cancers. Since the E6 and E7 oncoproteins of these viruses are expressed in these lesions, such proteins might be potential tumor-specific targets for immunotherapy. In this report, we demonstrate that recombinant, full-length E7-pulsed autologous dendritic cells (DC) can elicit a specific CD8+ cytotoxic T-lymphocyte (CTL) response against autologous tumor target cells in three patients with HPV 16- or HPV 18-positive cervical cancer. E7-specific CTL populations expressed strong cytolytic activity against autologous tumor cells, did not lyse autologous concanavalin A-treated lymphoblasts or autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL), and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. Cytotoxicity against autologous tumor cells could be significantly blocked by anti-HLA class I (W6/32) and anti-CD11a/LFA-1 antibodies. Phenotypically, all CTL populations were CD3+/CD8+, with variable levels of CD56 expression. CTL induced by E7-pulsed DC were also highly cytotoxic against an allogeneic HLA-A2+ HPV 16-positive matched cell line (CaSki). In addition, we show that specific lymphoproliferative responses by autologous CD4+ T cells can also be induced by E7-pulsed autologus DC. E7-specific CD4+ T cells proliferated in response to E7-pulsed LCL but not unpulsed LCL, and this response could be blocked by anti-HLA class II antibody. Finally, with two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, a marked Th1-like bias (as determined by the frequency of gamma interferon- and interleukin 4-expressing cells) was observable for both CD8+ and CD4+ E7-specific lymphocyte populations. Taken together, these data demonstrate that full-length E7-pulsed DC can induce both E7-specific CD4+ T-cell proliferative responses and strong CD8+ CTL responses capable of lysing autologous naturally HPV-infected cancer cells in patients with cervical cancer. These results may have important implications for the treatment of cervical cancer patients with active or adoptive immunotherapy.

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