CD24⁺ Cell Depletion Permits Effective Enrichment of Thymic iNKT Cells While Preserving Their Subset Composition

Invariant NKT (iNKT) cells are a small subset of thymus-generated T cells that produce cytokines to control both innate and adaptive immunity. Because of their very low frequency in the thymus, in-depth characterization of iNKT cells can be facilitated by their enrichment from total thymocytes. Magnetic-activated cell sorting (MACS) of glycolipid antigen-loaded CD1d-tetramer-binding cells is a commonly used method to enrich iNKT cells. Surprisingly, we found that this procedure also dramatically altered the subset composition of enriched iNKT cells. As such, NKT2 lineage cells that express large amounts of the transcription factor promyelocytic leukemia zinc finger were markedly over-represented, while NKT1 lineage cells expressing the transcription factor T-bet were significantly reduced. To overcome this limitation, here, we tested magnetic-activated depletion of CD24⁺ immature thymocytes as an alternative method to enrich iNKT cells. We found that the overall recovery in iNKT cell numbers did not differ between these 2 methods. However, enrichment by CD24⁺ cell depletion preserved the subset composition of iNKT cells in the thymus, and thus permitted accurate and reproducible analysis of thymic iNKT cells in further detail.

Recent advances in pMHC multimer technology / 中国免疫学杂志

To overcome the intrinsic low affinity between peptide bound major histocompatibility complex(pMHC) and T cell receptors (TCRs) on T cell surface,pMHC can be multimerized to enhance its avidity to TCRs.Since 20 years ago pMHC tetramer was first applied in the detection of antigen specific T cells,it has become one of the most important immunoassay tools.Recently,pMHC multimers have been significantly improved over the original tetramer.pMHC multimers with higher valence have been produced in order to enhance detection sensitivity.Additionally,reversible pMHC multimers,which can be released from T cell surface to avoid potential damage to T cells,also have been applied in the isolation of antigen specific T cells.As a class of molecular tools,pMHC multimers play an important role in immunoassays and immunotherapies.A good knowledge and effective utilization of those tools will be greatly helpful in scientific and clinical applications.

Detection of peripheral CD14+CD277+monocyte-macrophage ratio with γδ TCR tetramer and analy-sis of its relationship with treatment outcome / 中华微生物学和免疫学杂志

Objective To investigate the role of peripheral CD14+monocyte-macrophages in the recognition of phosphorylated antigen by γδ T cells and its relationship with treatment outcome. Methods Three kinds of γδ TCR tetramers were used to stain PBMC collected from patients with tuberculosis ( TB) and neonatal umbilical cord blood samples. The proportions of various TB-specific antigen presenting cells (APC) in peripheral blood were analyzed, and their relationships with treatment outcome were assessed based upon clinical data. Results CD14+monocyte-macrophages both in tuberculosis patients′ peripheral blood and neonatal umbilical cord blood were the strongest binding cells to CD277 antibody and γδ TCR tet-ramers. The median (P50) of CD14+monocyte-macrophages reached the highest peak after taking anti-tu-berculosis treatment for about one month and patients′condition was improved obviously during this period. Conclusion This study elucidated that CD14+monocyte-macrophages accounted for the largest proportion of APC when γδ T cells recognized phosphorylated antigens, which provided reference data for further study on the mechanism of γδ T cells restrictively recognizing phosphorylated antigen and their significance in innate and adaptive immunity.

Detection of antigen-specific CD14+ monocytes in blood of tuberculosis patients by using CD4+ T-cell receptor tetramer-based flow cytometric analysis and cell climbing slice assay / 中国感染与化疗杂志

Objective To evaluate the utility of CD4+ TCR tetramers‐based flow cytometric analysis and cell climbing slice assay in detecting antigen‐specific CD14+ monocytes in the blood of tuberculosis (TB) patients .Methods CD4+ TCR tetramers were used to detect tetramer‐positive CD14+ monocytes in the peripheral blood (PBL ) samples of inpatients with advanced pulmonary TB (PTB) by flow cytometric analysis .The PBL samples obtained from non‐TB patients and umbilical cords were used as controls .These tetramers were also used to examine tetramer‐bound CD14+ monocytes and Mycobacterium tuberculosis (MTB) antigen‐specific and tetramer‐bound cells by cell climbing slice in situ staining .Results The median percentage of tetramer‐bound CD14+ monocytes in PBL samples from PTB patients ,non‐TB patients and umbilical cords were 1 .32% , 0 .50% and 0 .26% respectively by using CD4+ Vα21‐J39/Vβ29‐D1‐J2 tetramer , while the medians were 1 .05% , 0 .49% and 0 .19% respectively by using CD4+ Vα21‐J39/Vβ29‐D2‐J2 tetramer . The percentage of tetramer‐bound CD14+ monocytes in PTB patients group was significantly higher than the other two control groups .In cell climbing slice in situ staining ,tetramer‐bound CD14+ monocytes ,and MTB antigen‐specific and tetramer‐bound cells were positive in PTB tissue compared with negative in control tissues . Conclusions CD4+ TCR tetramers‐based flow cytometric analysis and cell climbing slice assay could be used to sensitively detect M TB antigen‐specific CD14+ monocytes in the blood of TB patients ,and more accurately evaluate the changing profile and clinical significance of these cells in TB patients .

Detection of Foreign Antigen-specific CD4+Foxp3+ Regulatory T Cells by MHC Class II Tetramer and Intracellular CD154 Staining

The unrestricted population of CD4+Foxp3+ regulatory T (Treg) cells, which have been known to control the expression of autoimmune diseases and protective immunity to inflammatory reactions, has led to greater appreciation of functional plasticity. Detecting and/or isolating Ag-specific CD4+Foxp3+ Tregs at the single cell level are required to study their function and plasticity. In this study, we established and compared both MHC class II tetramer and intracellular CD154 staining, in order to detect CD4+Foxp3+ Treg specific for foreign Ag in acute and chronic infections with lymphocytic choriomeningitis virus (LCMV). Our results revealed that MHC class II tetramer staining showed a lower detection rate of LCMV GP66-77-specific CD4+ T cells because most of MHC class II tetramers were unbound and unstable when combined staining was performed with intracellular cytokines. In contrast, intracellular CD154 staining was revealed to be easier and simple for detecting LCMV GP66-77-specific CD4+ T cells, compared to MHC class II tetramer staining. Subsequently, we employed intracellular CD154 staining to detect LCMV GP66-77-specific CD4+Foxp3+ Tregs using Foxp3GFP knock-in mouse, and found that LCMV GP66-77-specific CD4+Foxp3+ Tregs and polyclonal CD4+Foxp3+ Tregs showed differential expansion in mice infected with LCMV Arms or Cl13 at acute (8 and 13 days pi) and chronic phases (35 days pi). Therefore, our results provide insight into the valuable use of intracellular CD154 staining to detect and characterize foreign Ag-specific CD4+Foxp3+ Treg in various models.

The preliminary study of CD4+ Vα9-J27/Vβ29-D1-J2 tetramers in detecting Mycobacterium tuberculosis infections / 中华微生物学和免疫学杂志

ObjectiveTo investigate the specificity of CD4+ Vα9-J27/Vβ29-D1-J2 tetramer in detecting Mycobacterium tuberculosis(MTB) infections.MethodsThe above TCR tetramer by using biotinylated monomers expressed and purified from constructed stable Drosophila Schneider 2 cell( S2 cell) lines was prepared.The PE-labled TCR tetramer was used to costain with S2 cell lines expressing MTB prptide/HLA-DR complexes on the cell membrane,and also was used to detect tetramer-bound CD14+ monocytes and macrophages in the peripheral blood mononuclear cells (PBMC) of pulmonary tuberculosis (PTB) patients and three control groups by flow cytometric analysis.And the FITC-labled tetramer was used to examine tetramer-bound CD14+ monocytes and macrophages,and MTB antigen-specific and tetramer-bound cells by in situ staining.ResultsThe TCR tetramer was well binding with S2 cell lines expressing C14/HLA-DR *1504 on the cell membrane.By flow cytometric analysis,the percentage of tetramer-bound CD14+ monocytes and macrophages in PTB patients group was higher than the other three control groups( P＜0.001 ).By in situ staining,tetramer-bound CD14+ monocytes and macrophages,and MTB antigen-specific and tetramer-bound cells were positive in PTB tissue and negative in control pneumonia tissue.ConclusionThe spcificity of TCR tetramer in monitoring MTB infections by flow cytometric analysis and in situ staining could be seen,which laid a laboratory foundation in the diagnosis and immune mechanism research of TB by using TCR tetramers.

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.

Analysis of the frequency and function of antigen specific CTL in different courses' patients with condyloma acuminata / 中华微生物学和免疫学杂志

HPV in remission CA,so the disease can be cured.

Identification of predicted epitopes of HLA-A*0201-restricted cytotoxic T lymphocytes derived from human papillomavirus type 11 E7 antigen / 中华微生物学和免疫学杂志

Objective To screen and identify the predicted epitopes of synthesized HLA-A*0201restricted CTL derived from HPVll E7 antigen.Methods Five HPVll E7 CTL epitope peptides and terramers consisting of HLA-A*0201 were selected by way of computer and synthesized by Sanquin company,including HPVllE7 7-15(TLKDIVLDL),15-23(LQPPDPVGL),47-55(PLTQHYQIL),81-89(DLLLGTLNI)and 82-90(LLLGTLNIV).These peptides binding to human peripheral blood-derived DCs were tested for their ability to activate T cells isolated from peripheral blood lymphocytes of HLA-A*0201 healthy individuals.the number of specific tetramer+CD8+T cells by flow cytometry,the level of the section of IFN-γ by ELISA,and the ability of the CTL to kill the target cells were observed.Results The immature DCs could be fully activated by all the five HPV11 E7 peptides.Peptide-loaded mature DCs were able to stimulate the epitope-specific T cells responses in vitro.An increased frequency(P<0.05)of T ceils specific for the E7 7-15 epitope compared to other epitopes of HPV11E7.The epitope-specific CTL of E7 7-15 induced by the activated DCs specifically killed HPV11E7 expressing 293 cell line,and in a ratio of 50:1,the specific cytolytic activity was the strongest than the others(P<0.05).Conclusion DCs loaded with HPV11 E7 7-15(TLKDIVLDL)peptide can induce highly effective and specific ectogenic processed epitopespecific CTL responses in vitro.This peptide may be the candidate for development of CTL based vaccine in the treatment of HPV infeetions.

Mucosal CD8+ T Cell-Mediated Immunity of Vaccinized Animals to Genital Infection of Herpes Simplex Virus Type 1

In the present study, we directly evaluated mucosal CD8+ T cell-mediated immunity using ex vivo cytotoxic T lymphocyte (CTL) assay and MHC class I tetramer staining method in iliac lymph node (LN) and vaginal tracts of mice immunized mucosally with several prime-boost protocols after genital infection of herpes simplex virus type 1 (HSV-1). Ex vivo CTL activity in iliac LN of infected mice was evaluated at 3-day post-infection without in vitro 5-day stimulation. Iliac LN of mice immunized with recombinant viral vaccine-priming and DNA vaccine-boosting protocol showed more potent CTL activity than those of other groups. Such ex vivo CTL activity was consistent with mucosal gB498-505 (SSIEFARL)-specific CD8+ T cell number of vaginal tract determined by MHC class I (H-2b) tetramer containing immunodominant peptide. Furthermore, the number of mucosal SSIEFARL-specific CD8+ T cells recruited into infected genital tracts appeared to decide the protective outcome against genital infection of virulent HSV-1. These results support that mucosal CD8+ T cells are principal mediators for the protection against genital infection of HSV-1.