OBJECTIVE: To screen anti-tumor drugs that improve antigen processing and presentation in acute myeloid leukemia (AML) cells. METHODS: A TCR-like or TCR mimic antibody that can specifically recognize HLA-A*0201:WT1_126-134 ( RMFPNAPYL) complex (hereafter referred to as HLA-A2:WT1) was synthesized to evaluate the function of antigen processing and presentation machinery (APM) in AML cells. AML cell line THP1 was incubated with increasing concentrations of IFN-γ, hypomethylating agents (HMA), immunomodulatory drugs (IMiD), proteasome inhibitors (PI) and γ-secretase inhibitors (GSI), followed by measuring of HLA-ABC, HLA-A2 and HLA-A2:WT1 levels by flow cytometry at consecutive time points. RESULTS: The TCR-like antibody we generated only binds to HLA-A*0201⁺WT1⁺ cells, indicating the specificity of the antibody. HLA-A2:WT1 level of THP-1 cells detected with the TCR-like antibody was increased significantly after co-incubation with IFN-γ, showing that the HLA-A2:WT1 TCR like antibody could evaluate the function of APM. Among the anti-tumor agents screened in this study, GSI (LY-411575) and HMA (decitabine and azacitidine) could significantly increase the HLA-A2:WT1 level. The IMiD lenalidomide and pomalidomide could aslo upregulate the expression of HLA-A2:WT1 complex under certain concentrations of the drugs and incubation time. As proteasome inhibitors, carfilzomib could significantly decreased the expression of HLA-A2:WT1, while bortezomib had no significant effect on HLA-A2:WT1 expression. CONCLUSION HLA-A2:WT1 TCR-like antibody can effectively reflect the APM function. Some of the anti-tumor drugs can affect the APM function and immunogenicity of tumor cells.
Humans ; Leukemia, Myeloid, Acute/immunology* ; Antineoplastic Agents/pharmacology* ; Antigen Presentation/drug effects* ; HLA-A2 Antigen/immunology* ; Receptors, Antigen, T-Cell/immunology* ; Cell Line, Tumor ; Interferon-gamma

Cytokine-activated T cells (CATs) can be easily expanded and are widely applied to cancer immunotherapy. However, the good efficacy of CATs is rarely reported in clinical applications because CATs have no or very low antigen specificity. The low-efficacy problem can be resolved using T cell antigen receptor-engineered CAT (TCR-CAT). Herein, we demonstrate that NY-ESO-1 HLA-A*02:01-specific high-affinity TCR (HAT)-transduced CATs can specifically kill cancer cells with good efficacy. With low micromolar range dissociation equilibrium constants, HAT-transduced CATs showed good specificity with no off-target killing. Furthermore, the high-affinity TCR-CATs delivered significantly better activation and cytotoxicity than the equivalent TCR-engineered T cells (TCR-Ts) in terms of interferon-γ and granzyme B production and in vitro cancer cell killing ability. TCR-CAT may be a very good alternative to the expensive TCR-T, which is considered an effective personalized cyto-immunotherapy.
Cell Line, Tumor ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; Genetic Engineering ; HLA-A2 Antigen ; metabolism ; Humans ; Immunotherapy, Adoptive ; methods ; Lymphocyte Activation ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes ; immunology

OBJECTIVETo find and identify HLA-A*0201 restricted cytotoxic T lymphocyte (CTL) epitopes from epidermal growth factor pathway substrate number 8 (Eps8) for specific immunotherapy based on Eps8-derived epitopes in clinic.

METHODSOnline biological softwares involved C-proteasomal cleavage, MHC class I binding affinity and TAP transport efficiency were used for prediction of HLA-A*0201 restricted epitopes from Eps8. Then, T2-binding assays and peptide/MHC complex stability tests were used to further verify the predicted epitopes. Specific secretion of IFN-γ from human CTL was assayed using the IFN-γ ELISPOT kit, and cytolytic activity was measured by a 4-h lactate dehydrogenase (LDH) release assay. Finally, the functional effects in vivo were measured in HLA-A*0201/Kb transgenic (Tg) mice.

RESULTSFour natural epitopes were designed through online biological softwares. Of the four epitopes selected, p360-368 was found to have the high binding affinity to HLA-A*0201, while p101-109 and p276-284 showed moderate affinities. DC50 of peptide/MHC complexes of the natural epitopes mentioned were all longer than 8 h. In functional assays with human PBMNC in vitro and in HLA-A*0201/Kb transgenic mice in vivo, CTLs primed by each epitope (p101-109, p276-284 and p360-368) secreted IFN-γ and were toxic to cancer cells from a variety of tissue types in an HLA-A*0201-restricted and Eps8-specific manner.

CONCLUSIONNatural epitopes (p101-109, p276-284 and p360-368) may be the HLA-A*0201 restricted epitope derived from Eps8.

Adaptor Proteins, Signal Transducing ; immunology ; Animals ; Epitopes, T-Lymphocyte ; metabolism ; HLA-A2 Antigen ; metabolism ; Humans ; Mice ; Mice, Transgenic ; T-Lymphocytes, Cytotoxic

OBJECTIVETo construct vitiligo-specific HLA-A*0201-peptide tetramers and to apply the constructed tetramers in detection of vitiligo-specific cytotoxic T lymphocytes (CTL).

METHODSProteins HLA-A0201*-BSP and β2M were obtained by effective prokaryotic expression. The purified proteins were refolded with vitiligo antigen peptides MelanA 26-35, gp100 209-217, and tyrosinase 1-9, respectively to form HLA-A*0201-peptide complex. The complex was biotinylated by BirA enzyme and purified by gel-filtration chromatography. The tetramers were generated by mixing the complex with phycoerythrin (PE)-streptavidin at a ratio of 4∶1 and identified by Dot-blot assay. The capacity of tetramer to detect vitiligo-specific CTL was analyzed by flow cytometry.

RESULTSThe biotinylation of vitiligo-specific HLA-A*0201-peptide tetramers were successfully performed by Dot-blot. Flow cytometry analysis indicated that the tetramer effectively bound to specific CTL from peripheral blood of patients with vitiligo.

CONCLUSIONThree kinds of biotinylated vitiligo-specific HLA-A*0201-peptide tetramers have been constructed successfully. The tetramer can detect antigen specific CTL from patients with vitiligo.

Biotinylation ; Flow Cytometry ; HLA-A2 Antigen ; Humans ; Peptides ; T-Lymphocytes, Cytotoxic ; cytology ; Vitiligo ; diagnosis ; immunology

Cytotoxic T cells (CTLs) play a key role in the control of Hepatitis B virus (HBV) infection and viral clearance. However, most of identified CTL epitopes are derived from HBV of genotypes A and D, and few have been defined in virus of genotypes B and C which are more prevalent in Asia. As HBV core protein (HBc) is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool covering HBc to screen and identify specific CTL epitopes. An unconventional HLA-A2-restricted epitope HBc141-149 was discovered and structurally characterized by crystallization analysis. The immunogenicity and anti-HBV activity were further determined in HBV and HLA-A2 transgenic mice. Finally, we show that mutations in HBc141-149 epitope are associated with viral parameters and disease progression in HBV infected patients. Our data therefore provide insights into the structure characteristics of this unconventional epitope binding to MHC-I molecules, as well as epitope specific CTL activity that orchestrate T cell response and immune evasion in HBV infected patients.
Adult ; Amino Acid Sequence ; Animals ; Binding Sites ; Epitopes ; chemistry ; immunology ; metabolism ; Female ; Genotype ; HEK293 Cells ; HLA-A2 Antigen ; metabolism ; Hepatitis B Core Antigens ; chemistry ; immunology ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; Humans ; Hydrogen Bonding ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Middle Aged ; Molecular Dynamics Simulation ; Mutation ; Protein Binding ; Protein Structure, Tertiary ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism

This report aims to investigate the dynamical changes of HBcAg18-27 epitope specific cytotoxic T lymphocytes(CTL), alanine aminotransferase (ALT), HBV DNA and HBsAg in peripheral blood of acute hepatitis B patients, and to explore the roles of HBcAg18-27-specific CTLs in virus clearance and liver injury. Acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients were divided into two groups according to results of HLA-A0201. Patients with positive HLA-A0201 were classified into HBcAg-specific CTL group and those with negative HLA-A0201 were referred as control group. The specific CTLs were stained with HLA-A0201 limited HBcAg18-27 epitope MHC-Pentamer and the frequencies of CTLs, T, B, NK and NKT cells were detected by flow cytometry (FCM). The serum ALT, HBV DNA and HBsAg were examined using speed analysis, quantitative PCR and abbott chemiluminescent technology. The frequencies of HBcAg18-27-specific CTLs in AHB patients were higher in the early three weeks as compared to the late three weeks. The apex time of HBV-specific CTL frequencies lagged behind those of HBV DNA, HBsAg and ALT. The loss of HBsAg in patients with high frequencies of HBV-specific CTL was earlier than that in patients with low frequencies (t = 2.018, P value is less than 0.05). In the second week the peak frequencies of CD3+CD8+ cells overlapped with that of HBcAg18-27-specific CTLs and with a positive correlation between (r = 0.420, P value is less than 0.05). During the early stages of AHB, the frequencies of NK and NKT cells were found significantly lower than that of control group and CHB group and the levels were back to normal after recovery. Moreover, a negative correlation existed between the frequencies of NK cells and the dynamic changes of HBcAg18-27-specific CTLs (r = -0.435, P value is less than 0.01) in AHB group. The frequencies of HBcAg18-27-specific CTLs were significantly higher as compared to CHB group in the first three weeks (z = -3.258, -4.04, and -3.259, P value is less than 0.01). The early loss of HBsAg was closely related to the high frequencies of HBcAg18-27 specific CTLs in AHB patients. HBcAg-specific CTL frequencies in peripheral blood could be used to predict clinical outcome after HBV infection. The frequencies of CD8+ T cells can reflect the changes of frequencies of HBcAg-specific CTL during acute HBV infection.
Adolescent ; Adult ; Case-Control Studies ; Female ; HLA-A2 Antigen ; immunology ; Hepatitis B ; immunology ; Hepatitis B Core Antigens ; blood ; immunology ; Humans ; Killer Cells, Natural ; immunology ; Lymphocyte Count ; Male ; Middle Aged ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Young Adult

OBJECTIVETo investigate the clinical safety and effects of auto-dendritic cells pulsed with HLA-A201-binding peptides prostate-specific antigen (PSA) , prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP) in the treatment of hormone-refractory metastatic prostate cancer (HRPC).

METHODSSixteen HRPC patients with positive HLA-A201 were enrolled and their monocytes isolated and induced into dendritic cells with the combination of rhGM-CSF and rhIL4. The patients were inoculated subcutaneously near the inguinal region with auto-DCs pulsed with peptides PSA (KLQCVDLHV) , PSMA (ALDVYNGL L) and PAP (LLHETDSAV) every 2 weeks for 4 times, and the immunological and clinical responses were examined within 1 -2 weeks after the final vaccination.

RESULTSVaccination of dendritic cells was well tolerated and no toxicity was observed. The cytokine levels in the serum such as IL-2, IL-12 and IFN-gamma were significantly increased after the vaccination (P < 0.01). The delayed type hyper- sensitivity (DTH) test was positive in 4 of the patients (4/11), the percentage of antigen-special IFN-gamma+ CD8+ T increased in 5 (5/11), the level of the tumor marker PSA decreased in 6 (6/16) , hydrops abdominis reduced in 1 (1/16), and the size of the cervical lymph node lessened in 1 (1/16). Three patients showed partial remission (PR), 7 stability of the disease (SD), and the other 6 progression of the disease (PD).

CONCLUSIONAuto-DC vaccines loaded with PSA, PSMA and PAP peptides, capable of eliciting specific immune responses in HRPC patients, is a safe and effective option for the treatment of advanced HRPC.

Acid Phosphatase ; Aged ; Antigens, Surface ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Cancer Vaccines ; immunology ; Cytokines ; blood ; Dendritic Cells ; immunology ; Glutamate Carboxypeptidase II ; immunology ; HLA-A2 Antigen ; immunology ; Humans ; Male ; Middle Aged ; Prostate-Specific Antigen ; immunology ; Prostatic Neoplasms ; therapy ; Protein Tyrosine Phosphatases ; immunology ; Treatment Outcome

OBJECTIVESTo identify HLA-restricted epitope of mucoprotein 4 (MUC4) antigen as a tumor associated antigen of pancreatic ductal adenocarcinoma (PDAC), and to validate its natural presentation in PDAC patient peripheral blood.

METHODSTwo epitope prediction databases (SYFPEITHI and ProPred-I) were used to predict HLA-A*0201 restricted MUC4 epitope, T2 cell assay was used to determine the peptide binding affinity with HLA-A*0201 molecule. Dendritic cells (DCs) were induced from the HLA-A* 0201-positive healthy individuals' peripheral blood mononuclear cells (PBMC). Mature DCs were pulsed with synthesized peptides. Autologous CD8(+) T cells from the HLA-A* 0201 healthy donor were stimulated with the peptide-pulsed DCs as CTL. CTL activity was assessed by lactate dehydrogenase release assay and IFN-γ released by enzyme-linked immunospot assay. Pentamer was synthesized for HLA-A* 0201 restricted epitope P1126, then was used to detect specific CTL in PBMC of PDAC patients.

RESULTSFive candidate HLA-A*0201 epitopes were predicted, LLLGVGTFV (P1125) and LLGVGTFVV (P1126) were determined as the two with more HLA-A*0201 affinity. Mature DCs could be induced from PBMCs. CTL induced by peptide P1126 could lyses T2 cells pulsed with peptide P1126 and HCT-116 cells [MUC4(+), HLA-A2(+)]. The number of CTL induced by peptide P1126 which could secret IFN-γ (130.3 ± 6.6) was obviously higher than that in the negative group. By Pentamer assay, P1126-pentamer and CD8 double positive CTL could be detected in PBMC of PDAC patients with MUC4(+) than patients with MUC4(-), but no significant difference of CTL frequency between patients with HLA-A2(+) and with HLA-A2(-) in MUC4(+) PDAC patients.

CONCLUSIONSTumor associated antigen MUC4-derived HLA-A* 0201-restrictive cytotoxic T lymphocyte (CTL) epitope P1126 can induce CTL reaction. The CTL can secret immunologic active material to induce the specific target cells lysis. P1126 epitope can be naturally presented in PBMC of PDAC patients, but its HLA-restriction may not be perfect.

Antigens, Neoplasm ; immunology ; Cells, Cultured ; Dendritic Cells ; immunology ; Epitopes, T-Lymphocyte ; immunology ; HLA-A Antigens ; immunology ; HLA-A2 Antigen ; immunology ; Humans ; Mucin-4 ; immunology ; Pancreatic Neoplasms ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

This study was purposed to investigate the value of combination of pentamer and intracellular IFNgamma staining in the qualitative and quantitative detection of circulating antigen-specific T cells. WT1 expressions in 14 HLA-A*0201+ patients and their matched donors were detected by RT-PCR, and circulating WT1 specific T cells were assayed by HLA-A*0201/WT1 pentamer combined with intracellular IFNgamma+ staining. The results showed that the low level of WT1 expression was found only in 2 cases out of 14 donors, but different levels of WT1 expression could be observed in all leukemic patients. The WT1+CD8+ CTL and WT1+IFNgamma+ cells did not detected in all 14 donors, but WT1+CD8+ CTL cells in 2 patients and WT1+IFNgamma+ cells in 3 patients could be detected before transplantation respectively, there was no significant difference between them, while the WT1+CD8+ CTL cells and WT1+IFNgamma+ cells both could be detected in all 14 patients after transplantation, the positive detection rate after transplantation was obviously higher than that before transplantation. The WT1+CD8+ and WT1+ IFNgamma+ cells could be detected within 30 days after transplantation, but the positive detection rate of WT1+IFNgamma+ cells was higher than that of WT1+CD8+ CTL cells (p=0.014). The median peak value of WT1+CD8+ CTL cells was 0.18% in 14 patients, and the median peak value of WT1+IFNgamma+ cells was 0.83% in 14 patients, the later was significantly higher than former. The median peak time of WT1+CD8+ CTL cells was 75 days after transplantation, while the WT1+IFNgamma+ cells was 105 days after transplantation, there was no significant difference between them. It is concluded that pentamer and intracellular IFNgamma staining may effectively detect circulating WT1 specific T cells in leukemic patients, and the combination of these two methods profit to the exact qualitation and quantitation of circulating antigen-specific T cells.
Adolescent ; Adult ; Child ; Female ; Flow Cytometry ; HLA-A Antigens ; analysis ; HLA-A2 Antigen ; Humans ; Interferon-gamma ; analysis ; Leukemia ; blood ; genetics ; immunology ; Male ; Middle Aged ; Staining and Labeling ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism ; WT1 Proteins ; genetics ; immunology ; metabolism ; Young Adult

This study was aimed to investigate the effect of artificial antigen-presenting cells (aAPCs) on inducing activation and proliferation of specific T-lymphocytes through stimulating biological function of dendritic cells with aAPCs in vitro. The specific antigen of chronic myeloid leukemia CML-28 was screened as objective antigen peptide by using magnetic microbeads as vector; the CML-28 epitope sequence (Vltfaldsv) was obtained by antigen epitope prediction software; this epitope was coupled with MHC molecule and used as first signal molecule, the B7-1 molecule was used as second signal molecule; these 2 molecules simultaneously were loaded onto surface of magnetic microbeads so as to contract aAPC complex. The bone marrow mononuclear cells were derived from HLA-A2(+) healthy bone marrow donors, CD8(+) T lymphocytes were screened and co-cultured with aAPC complex. During culture the 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) was added and proliferation of T-lymphocytes was detected by CPSE and proliferation level of specific T lymphocytes was detected by flow cytometry. The results showed that the proliferation level of CML-28 specific T lymphocytes obviously increased in experimental group, average level was 17.34 +/- 0.65%, while average level in control was 2.25 +/- 0.43%, there was significant difference between them (p < 0.01). It is concluded that the aAPC complex can mimic human APCs in vitro, and stimulate activation and proliferation of CD8(+) specific T lymphocytes.
Adolescent ; Adult ; Antigen Presentation ; immunology ; Antigen-Presenting Cells ; immunology ; metabolism ; Antigens, Neoplasm ; immunology ; Biomimetics ; methods ; CD8-Positive T-Lymphocytes ; immunology ; Female ; HLA-A2 Antigen ; immunology ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; immunology ; Lymphocyte Activation ; Male ; Middle Aged ; Tissue Donors ; Young Adult