HBsAg-specific CD8+ T cells as an indispensable trigger to induce murine hepatocellular carcinoma.

Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) is mediated by an inappropriate attack by HBV-specific T cells in patients. However, this immunopathogenic process has not been clarified because of the lack of a suitable animal model. Here, we used immunocompetent Fah-/- mice as the recipients in the adoptive transfer of HBsAg+ hepatocytes from HBs-Tg mice to replace the recipient hepatocytes (HBs-HepR). HBs-HepR mice exhibited persistent HBsAg expression with chronic hepatitis and eventually developed HCC with a prevalence of 100%. HBsAg-specific CD8+ T cells were generated and specifically and continuously induced hepatocyte apoptosis with progressive chronic inflammation, and the depletion of CD8+ T cells or their deficiency prevented HCC, which could then be reproduced by the transfer of HBsAg-specific CD8+ T cells. In summary, our results demonstrated that CD8+ T cells plays a critical role in HBsAg-driven inflammtion and HCC tumorigenesis.

Correlation analysis of serum IL-7 with evaluation of cellular immunity in chronic hepatitis B / 中华微生物学和免疫学杂志

Objective:To explore the possible correlation between serum detection of IL-7, IL-21, HBV-specific cytotoxic T lymphocytes (CTLs), HBV DNA, and the expression of CD127 on the T lymphocytes, and discuss the effect of IL-7 to cellular immune response in patients with chronic hepatitis B (CHB).Methods:Five hundred and sixty serum samples were collected from patients with CHB in Beijing Friendship Hospital from September 2017 to March 2020. The serum IL-7 and IL-21 were detected by enzyme-linked immunosorbent assay (ELISA), and HBV-specific CTLs and the expression of CD127 on the T lymphocytes were determined by flow cytometry. While HBV DNA were tested using quantitative real-time PCR (qRT-PCR). Subjects were divided into groups A, B, and C, according to the IL-7 levels (low: IL-7<20 pg/ml, medium: 20 pg/ml≤IL-7<30 pg/ml, and high: IL-7≥30 pg/ml).Results:The average concentration of serum IL-7 in patients with CHB was significantly lower than that of healthy controls ( P<0.01), and the difference among three groups was statistically significant ( P<0.01). Meanwhile, levels of IL-21, percentages of HBV-specific CTL, and the expression of CD127 on the CD8 + T lymphocytes showed an upward trend among groups, and there were significant differences among three groups ( P<0.01) with a positive correlation between each two variables ( P<0.01). However, HBV DNA showed a downward trend in group A, B and C, and the difference of the three groups were statistically significant ( P<0.01), which were negatively correlated with other variables ( P<0.01). Multiple linear regression analysis showed that HBV-specific CTL was an independent influencing factor for HBV DNA ( P<0.01), and IL-7, the expression of CD127 on the CD8 + T lymphocytes and IL-21 had an independent effect on HBV-specific CTL ( P<0.05). Conclusions:IL-7 could regulate HBV-specific immune response, and might be used as an effective cellular immune indicator to evaluate the cellular immune status of patients with chronic hepatitis B.

HBVsvp-Pulsed Dendritic Cell Immunotherapy Induces Th1 Polarization and Hepatitis B Virus-Specific Cytotoxic T Lymphocytes Production.

BACKGROUND: In chronic hepatitis B virus (CHB) patients, both dendritic cells (DCs) and T cells are functionally impaired and consequently the HBV-specific cellular immune responses are downregulated. The present study aims to investigate whether monocyte-derived DC (MoDCs)-pulsed-HBV subviral particles (HBVsvp) can polarize Th1 cells to induce HBV-specific cytotoxic T-lymphocytes (CTL) responses in CHB patients. METHODS AND MATERIALS: To this end, the human hepatoma HepG2.2.15 cell line was used to produce HBVsvp as a culturing system, and HBVsvp were concentrated for highly virus titer using the polyethylene glycol protocol. Peripheral blood mononuclear cells (PBMCs), collected from CHB patients and healthy donors, were differentiated into MoDCs and T cells. PBMCs-derived MoDCs were first pulsed with HBVsvp and then cultured with PBMCs-derived T cells. MoDCs and/or T subsets cells were identified for phenotypic activation by FACS analysis. The cytokine secretion of IL-4, IL-12, and IFN-γ in the culture supernatants was detected. RESULTS: The MoDCs were restored for their activation upon pulsing with HBVsvp in vitro, as identified by significantly overexpression of both CD86 and HLA-DR, and overproduction of IL-4 and IL-12. Furthermore, MoDCs-pulsed-HBVsvp induced Th1 frequencies and activated HBV-specific CTL to produce significantly highest amount of IFN-γ. Enhanced HBV-specific CTL led to strong cytolytic capacity against HepG2.2.15. CONCLUSION: Overall, our data suggest that in vitro activation of MoDCs with HBVsvp overcomes the functionally impaired DCs and T cells in CHB patients offering a promising tool for therapeutic or vaccine-based approaches against HBV.

In-vivo gp100-specific Cytotoxic CD8+ T Cell Killing Assay.

Cytotoxic CD8+ T lymphocytes (CTLs) represent a crucial component of the adaptive immune system and play a prominent role in the anti-tumor immune responses of both mice and humans. Cytotoxic CD8+ T cells are responsible for the lysis of cells expressing peptides associated with MHC class I molecules and derived from infection with a pathogen or from mutated antigens. In order to quantify in vivo this antigen-specific CD8+ T cell killing activity, we use the in vivo killing assay (IVKA). Here, we describe the protocol for the lysis of cells expressing a CD8+ T cell melanoma epitope of the hgp10025-33 protein (KVPRNQDWL). C57BL/6 recipient mice, receive first target cells, prepared from naive congenic (CD45.1) C57BL/6 spleen cells pulsed with the hgp10025-33 peptide and labeled with CFSE and of non-pulsed control cells labeled with Brilliant violet. One day later, the spleen cells of recipient mice are isolated and analyzed by FACS to measure the amount of CFSE cells and Brillant Violet (BV) cells. The percentage of lysis is calculated by the difference between CFSE versus BV. Measuring the ability of antigen-specific CD8+ T cells to lyse their antigen in vivo is very important to evaluate the adaptive cytotoxic response induced against a pathogen or a tumor antigen.

Phase I clinical trial of cell division associated 1 (CDCA1) peptide vaccination for castration resistant prostate cancer.

Cell division associated 1 (CDCA1) was screened as an oncogene that is overexpressed on several cancers, including prostate cancer. A highly immunogenic HLA-A*2402-restricted epitope peptide corresponding to part of the CDCA1 protein was also identified. A phase I clinical trial was conducted for patients with castration resistant prostate cancer (CRPC) using a CDCA1 peptide vaccination. Twelve patients having HLA-A*2402 with CRPC after failure of docetaxel chemotherapy were enrolled. They received subcutaneous administration of the CDCA1 peptide as an emulsion with Montanide ISA51VG once a week in a dose-escalation manner (doses of 1.0 or 3.0 mg/body, six patients received each dose). The primary endpoint was safety, and the secondary endpoints were the immunological and clinical responses. Vaccination with CDCA1 peptide was well tolerated without any serious adverse events. Peptide-specific cytotoxic T lymphocyte (CTL) responses using ELISPOT assay and dextramer assay were observed in three patients receiving the 1.0 mg dose and five patients receiving the 3.0 mg dose. The median overall survival time was 11.0 months and specific CTL reacting to CDCA1 peptide were recognized in long-surviving patients. CDCA1-derived peptide vaccine treatment was tolerable and might effectively induce peptide-specific CTLs for CRPC patients. This novel peptide vaccine therapy for CRPC appears promising. (ClinicalTrials.gov number, NCT01225471).

Streptamer technology allows accurate and specific detection of CMV-specific HLA-A*02 CD8+ T cells by flow cytometry.

BACKGROUND: Multimer technology is widely used to screen antigen-specific immune recovery after allogeneic hematopoietic stem cell transplantation (allo-HSCT) as it enables identification, enumeration, phenotypic characterization and isolation of virus-specific T-cells. Novel approaches of multimerization might improve on classical tetramer staining; however, their use as standard monitoring technique to quantify antigen-specific cells has not been validated yet. We have compared two of these available multimeric complexes: pentamer and streptamer to select the best strategy for the incorporation into clinical monitoring practice. METHODS: CMVpp65495-503 -specific HLA-A*02:01 CD8+ T lymphocytes (CTLA *02:01 -CMVpp65495-503 ) were examined with pentamer and streptamer in peripheral blood cells of 77 healthy volunteers. Quantitative and qualitative analyses were performed to compare the precision and repeatability, sensitivity and accuracy and specificity of both technologies by flow cytometry. RESULTS: Standard deviation for both techniques was less than 0.05 showing that they are repetitive and precise. Both techniques significantly correlated at high frequencies (rSpearman = 0.9422; P < 0.0001) but it was lost at lower levels (<1%) of CTLA *02:01 -CMVpp65495-503 (rSpearman = 0.3351; P = 0.1376). Streptamer is more accurate for the detection of CTLA *02:01 -CMVpp65495-503 providing significantly closer values to the theoretical ones (P < 0.0001) as pentamer binds unspecifically to a notable proportion of non-CMV-specific CD8+ T-cells. CONCLUSION: Our results suggest that streptamer multimer provides precise, accurate and specific results to detect CTLA *02:01 -CMVpp65495-503 by flow cytometry. Streptamer multimer can be used not only for the monitoring of early CTLA *02:01 -CMVpp65495-503 reconstitution in immunosuppressed patients following allo-HSCT but also, in conjunction with its reversibility role, for the isolation of CTLA *02:01 -CMVpp65495-503 for its future use in adoptive immunotherapy. © 2016 International Clinical Cytometry Society.

Development of oral CTL vaccine using a CTP-integrated Sabin 1 poliovirus-based vector system.

We developed a CTL vaccine vector by modification of the RPS-Vax system, a mucosal vaccine vector derived from a poliovirus Sabin 1 strain, and generated an oral CTL vaccine against HIV-1. A DNA fragment encoding a cytoplasmic transduction peptide (CTP) was integrated into the RPS-Vax system to generate RPS-CTP, a CTL vaccine vector. An HIV-1 p24 cDNA fragment was introduced into the RPS-CTP vector system and a recombinant poliovirus (rec-PV) named vRPS-CTP/p24 was produced. vRPS-CTP/p24 was genetically stable and efficiently induced Th1 immunity and p24-specific CTLs in immunized poliovirus receptor-transgenic (PVR-Tg) mice. In challenge experiments, PVR-Tg mice that were pre-immunized orally with vRPS-CTP/p24 were resistant to challenge with a lethal dose of p24-expressing recombinant vaccinia virus (rMVA-p24). These results suggested that the RPS-CTP vector system had potential for developing oral CTL vaccines against infectious diseases.

HCV-specific CD8+ cell detection at week 12 of chronic hepatitis C treatment with PEG-interferon-α2b/ribavirin correlates with infection resolution.

Lower than 2-log viral-load (VL) decrease at week 12 (w12) of chronic hepatitis C (CHC) treatment with Peg-interferon/ribavirin has 100% negative predictive value (PV) of sustained virologic response (SVR), and this could be related with absence of HCV-specific cytotoxic T lymphocyte (CTL) response. In this study, percentage of cases with SVR, according to peripheral HCV-specific cytotoxic response at w12, was analysed (Group-1: detection(+), Group-2: detection(-)). SVR was higher in group-1 (93%) than in group-2 (47%) (p=0.003). An increase on HCV-specific CTL frequency between baseline and w12 and higher specific reactivity were observed in group-1 (p=0.011 and p=0.025). HCV-specific CTL detection at w12 correlated with level of VL decrease (p=0.016, r=0.389), and among HCV genotype-1 patients with either early or delayed virologic response (EDVR), 100% positive PV of SVR was observed. In summary, HCV-specific CTL detection at w12 of Peg-interferon/ribavirin treatment correlates with SVR and in EDVR genotype-1 cases predicts SVR.

The abrogation of TCR-independent interactions with human serum ensures a selective capture of therapeutic virus-specific CD8+ T-cells by multimer technology in Adoptive Immunotherapy.

Multimers are complexes of recombinant MHC-class I molecules conjugated with antigenic immunodominant peptides and labeled with fluorescent molecules or magnetic microbeads that allow the quantification and selection of virus-specific cytotoxic T-cell subpopulations. Specific T-cell receptors recognize the immunodominant peptides and bind to the multimers. Although these complexes are only recognized by CD8(+) T cells with specific T-cell receptors for the particular antigen, it has been observed that multimers can also bind non-specifically to CD8- cells, such as B-cells and monocytes. Using PBMCs from CMV-seropositive healthy donors, we analyze the tendency of Pentamer and Streptamer multimers towards non-specific interactions and describe a method to avoid this unwanted event. We find that a notable proportion of multimer-positive cells are likely to represent cross-contamination by cells lacking a TCR specific for pp65. In addition, we demonstrate that this unspecific interaction can be overcome by the pre-incubation of multimer-stained PBMCs with human AB serum, without altering their capacity to bind specifically to the CD8(+) T cell population of interest. In conclusion, in this study we characterize a novel method to abrogate TCR-independent interactions of multimers to ensure a pure and safe therapeutic product for Adoptive Immunotherapy.

Immunophenotyping at the time of diagnosis distinguishes two groups of nasopharyngeal carcinoma patients: implications for adoptive immunotherapy.

BACKGROUND: Adoptive immunotherapy with EBV-specific CTLs (EBV-CTL) has been used to treat EBV-associated nasopharyngeal carcinoma (NPC) but only a fraction of the patients shows noticeable clinical response. PATIENTS AND METHODS: Sixty-seven newly diagnosed NPC patients from 2005 to 2007 and 21 healthy donors were collected. Immunological parameters and immune function of PBMCs and EBV-CTL were analyzed by flow cytometer analysis (FACS) and 5¹Cr releasing experiment; Molecular characteristics on NPC tumor cells were investigated by immunochemical staining and statistic analysis. RESULTS: NPC patients can be classified into two groups based on the percentage of CD3+ T cells in peripheral blood before accepted any treatment, (>52.6%, mean-2SE from healthy controls, NPC Group 1; <52.6%, NPC Group 2). The patients in Group 2 showed a significant decrease of CD3+CD8+ T-cells, CD3+CD4+ T-cells and CD3+CD45RO+ memory T cells, and increase of CD3⁻CD16+ NK cells compared to Group 1 patients and healthy controls (P<0.001). EBV-specific T cell responses, were weaker in this group of patients and their tumor cells expressed lower levels of the EBV encoded latent membrane protein (LMP)-1 and HLA class II protein compared with the patients of NPC Group 1 (P<0.05). CONCLUSION: These findings demonstrate that NPC patients could be distinguished on the basis of their immune status which will affect the efficacy of EBV-CTL immunotherapy.

Construction and primary application of Sh-2K~d-HBc tetramer / 中国免疫学杂志

Objective:To prepare and test tetrameric sH-2K~d-HBc complex for the further measurement of the specific CTL response.Methods:PE labled streptavidin with 4 biotinylated binding sites can bind to 4 biotinylated monomer to form the corresponding tetramer.Mice were immunized via different methods of genetic immunization by use of the construted pcDNA3-C plasmid to get the specific CTLs.Then our prepared tetramer was applied to stain the specific CTLs by the analysis of flow cytometry.Results:We applied our prepared tetramer to stain the cells from the experimental groups and control group.The results showed the tetramer was able to discriminate the frequencies of specific CTL induced by the three immunol methods(0.24%,0.26%,0.36% vs 0.07%,P≤0.05).This demonstrated that the prepared tetramer could bind its targets specifically and efficiently.The three immunol methods induced different levels of immune responses.Compared with the traditional muscle injection,gene gun induced weaker humoral immune response and stronger cellular immune response,and hydrodynamic injection induced the strongest humoral and cellular immune responses.Conclusion:Have successfully constructed the sH-2K~d-HBc tetramer.The techniques and methods can be used for preparation of tetramers of other types of MHCⅠ molecules.

Study on DC loaded with freeze-thrawing antigen from breast cancer cells to the specific cytotoxicity of CTL in vitro / 中国免疫学杂志

Objective:To study the influence of the DCs from mononuclear cells in axillary draining lymph nodes of patients with breast cancer to the cytotoxicity of the antigenic specific CTLs stimulated by freeze-thrawing antigen from breast cancer cells in vitro.Methods:The mononuclear cells were isolated from axillary draining lymph nodes,and were cultured with cytokines to induce DCs and tumor draining lymph node cells(TDLNCs) respectively.DCs stimulated by the auto-breast cancer freeze-thrawing antigen(DCs-Ag).TDLNCs were co-cultured with DCs-Ag to derive tumor antigenic specific CTLs(DC-Ag-TDLNC).Then the cytotoxicty of specific CTLs to auto-breast cancer cells and MCF-7 cells was detected.Results:The cytotoxicity of DC-Ag-TDLNC,DC-TDLNC and TDLNC to the auto-breast cancer cells was 67.64%、31.25% and 26.36% respectively,P0.05.Conclusion:Typical DCs can be induced from the mononuclear cells from axillary draining lymph nodes after stimulated with cytokines(rhGM-CSF,rhIL-4 and TNF-?).The DCs possess stronger antigen presentation and can obviously increase the killing activity of tumor antigen specific CTLs to auto-breast cancer cells.