Human immunodeficiency virus gag and pol-specific CD8 T cells in perinatal HIV infection.

BACKGROUND: Binding of fluorochrome-conjugated MHC class I tetramers is a powerful means to detect antigen-specific CD8 T lymphocytes. In human immunodeficiency virus (HIV) infection, cellular immune response is essential in curtailing HIV disease progression but gaps persist in our understanding of HIV-specific cells during the disease course. In this study, we evaluated tetramer binding HIV-specific CD8 T cells in HIV-infected children. METHODS: Fluorescently labeled tetramers for HIV gag and pol were utilized to quantify antigen-specific cells by flow cytometry using a whole blood labeling method in a cohort of 19 HLA-A2+ HIV-infected children (age range 1 month to 17 years). RESULTS: Fourteen children had detectable gag (median 0.4%) and pol (median 0.1%) binding CD8 T cells, three children had gag binding cells only, and two had neither. Numbers of gag and pol binding cells correlated with each other and each correlated independently with total CD8 T cells and total CD4 T cells. CONCLUSIONS: HIV gag and pol-specific CD8 T cells are maintained during the chronic phase of HIV infection in children and CD4 lymphocytes appear to be important for sustaining their levels.     

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry. This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor. While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents. Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection. Mononuclear cells are purified from peripheral blood by Ficoll underlay. CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads. Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2. The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25. After labeling, the cells can be directly analyzed by flow cytometry. The tetramer positive cells typically form a distinct population among the expanded CD4+ cells. Tetramer positive cells are usually CD25+ and often CD4 high. Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer. Multiple variations of this basic assay are possible. Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays. Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies. Open in a separate windowClick here to view.^{(85M, flv)}     

加载HCMV抗原肽的HLA-A*0201单体及其四聚体制备和鉴定

细胞毒T淋巴细胞(CTL)在控制病原体感染以及抗肿瘤过程中发挥重要作用,因而特异性CTL的检测相当重要;而过去检测CTL的方法都是间接的,最近发展起来的四聚体技术则是直接检测抗原特异性CTL的有效而特异的方法,成为目前研究T细胞免疫应答的关键技术。报道一种简化的四聚体制备程序,利用该程序成功制备加载人巨细胞病毒(HCMV)抗原肽的HLA-A2四聚体,具有特异性结合CTL活性。HLA-A*0201重链基因是通过RT-PCR方法从HLA-A2⁺供者白细胞中克隆,进而以PCR方法构建在羧基端融合生物素化酶BirA底物肽(BSP)的HLA-A*0201(A2)重链胞外区原核表达载体, A2重组蛋白在大肠杆菌中得到高表达,主要以包涵体形式存在。加载抗原肽的可溶性A2单体是A2胞外区在轻链β2微球蛋白和HLA-A2限制性HCMV pp65_495-503抗原肽(NLVPMVATV,NLV)存在时通过稀释法复性获得,以BirA对其进行生物素化,然后以阴离子交换树脂纯化,得到的纯化A2-NLV单体与Streptavidin_PE按4:08比例混合形成四聚体,结合程度在85％以上,流式细胞仪分析显示该四聚体具有与HLA-A2+供者的特异性CTL结合活性。总之,这种简化的四聚体制备程序,不仅有利于该技术的推广,为特异性T细胞免疫研究建立必要的技术平台,而且A2-NLV四聚体在临床监测CMV特异性CTL水平等方面也有应用价值。     

Stability screening of arrays of major histocompatibility complexes on combinatorially encoded flow cytometry beads

The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.     

H-2Db四聚体的制备及其在检测LCMV特异性CD8+ T细胞中的应用

MHC四聚体技术是研究抗原特异性淋巴细胞应答的关键技术之一。为研究H-2Db基因型(如C57BL/6)小鼠的特异性CD8+ T细胞免疫应答, 需要建立H-2Db四聚体制备技术平台。首先以RT-PCR方法克隆H-2Db重链基因的cDNA, 进而构建H-2Db胞外域与生物素化酶BirA底物肽(BSP)融合蛋白的表达载体, 并在大肠杆菌中获得表达。在LCMV GP33-41抗原肽(KAVYNFATC, KAV)和人β2-微球蛋白存在时, 通过稀释法复性获得H-2Db/KAV单体。该单体经生物素化并纯化后与PE-链亲和素按4: 1的比例混合, 即形成四聚体。通过流式细胞术检测经KAV肽免疫的C57BL/6小鼠体内的LCMV特异性CD8+ T细胞的频率, 结果表明在外周血、引流淋巴结和脾脏中均可检测到一定频率的LCMV特异性CD8+ T细胞, 其中以对外周血标本染色的效果最佳。成功建立了小鼠H-2Db四聚体制备技术平台, 为监测及分析基于H-2Db基因型小鼠的实验性免疫治疗创造了条件。     

Construction of bioactive chimeric MHC class I tetramer by expression and purification of human-murine chimeric MHC heavy chain and beta(2)m as a fusion protein in Escherichia coli

Major histocompatibility (MHC) class I tetramers are used in the quantitative analysis of epitope peptide-specific CD8+ T-cells. An MHC class I tetramer was composed of 4 MHC class I complexes and a fluorescently labeled streptavidin (SA) molecule. Each MHC class I complex consists of an MHC heavy chain, a beta(2)-microglobulin (beta(2)m) molecule and a synthetic epitope peptide. In most previous studies, an MHC class I complex was formed in the refolding buffer with an expressed MHC heavy chain molecule and beta(2)m, respectively. This procedure inevitably resulted in the disadvantages of forming unwanted multimers and self-refolding products, and the purification of each kind of monomer was time-consuming. In the present study, the genes of a human/murine chimeric MHC heavy chain (HLA-A2 alpha1, HLA-A2 alpha2 and MHC-H2D alpha3) and beta(2)m were tandem-cloned into plasmid pET17b and expressed as a fusion protein. The recombinant fusion protein was refolded with each of the three HLA-A2 restricted peptides (HBc18-27 FLPSDFFPSI, HBx52-60 HLSLRGLPV, and HBx92-100 VLHKRTLGL) and thus three chimeric MHC class I complexes were obtained. Biotinylation was performed, and its level of efficiency was observed via a band-shift assay in non-reducing polyacrylamide gel electrophoresis (PAGE). Such chimeric MHC class I tetramers showed a sensitive binding activity in monitoring HLA/A2 restrictive cytotoxic T lymphocytes (CTLs) in immunized HLA/A*0201 transgenic mice.     

The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor zeta chain.

Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by interaction of both the T cell receptor and its CD8 coreceptor with peptide-major histocompatibility complex (pMHC) class I molecules. We examine the relative roles of these receptors in the activation of human CTL using mutations in MHC class I designed to diminish or abrogate the CD8/pMHC interaction. We use surface plasmon resonance to determine that point mutation of the alpha3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting cells expressing HLA A2 which does not bind to CD8 fail to activate CTL at any peptide concentration. Comparison of CTL activation by targets expressing HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show that the CD8/pMHC interaction enhances sensitivity to antigen. We determine that the biochemical basis for coreceptor dependence is the activation of the 23-kDa phosphoform of the CD3zeta chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. These reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to phenotype antigen-specific CD8+ lymphocytes.     

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU₃₀/10⁶ cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.     

Intracellular CD154 expression reflects antigen-specific CD8+ t cells but shows less sensitivity than intracellular cytokine and MHC tetramer staining

A recent report showed that analysis of CD154 expression in the presence of the secretion inhibitor Brefeldin A (Bref A) could be used to assess the entire repertoire of antigen-specific CD4(+) T helper cells. However, the capacity of intracellular CD154 expression to identify antigen-specific CD8(+) T cells has yet to be investigated. In this study, we compared the ability of intracellular CD154 expression to assess antigen-specific CD8(+) T cells with that of accepted standard assays, namely intracellular cytokine IFN-gamma staining (ICS) and MHC class I tetramer staining. The detection of intracellular CD154 molecules in the presence of Bref A reflected the kinetic trend of antigen-specific CD8+ T cell number, but unfortunately showed less sensitivity than ICS and tetramer staining. However, ICS levels peaked and saturated 8 h after antigenic stimulation in the presence of Bref A and then declined, whereas intracellular CD154 expression peaked by 8 h and maintained the saturated level up to 24 h post-stimulation. Moreover, intracellular CD154 expression in antigen-specific CD8+ T cells developed in the absence of CD4(+) T cells changed little, whereas the number of IFN-gamma-producing CD8(+) T cells decreased abruptly. These results suggest that intracellular CD154 could aid the assessment of antigen-specific CD8(+) T cells, but does not have as much ability to identify heterogeneous CD4(+) T helper cells. Therefore, the combined analytical techniques of ICS and tetramer staining together with intracellular CD154 assays may be able to provide useful information on the accurate phenotype and functionality of antigen-specific CD8(+) T cells.     

Phenotypic changes in CD8+ peripheral blood lymphocytes in cats infected with feline immunodeficiency virus

It is well documented that several cell surface molecules of T lymphocytes are altered by immune activation. We previously reported that feline immunodeficiency virus (FIV) infection induces a reduction in CD8beta chain expression of peripheral blood lymphocytes (PBLs) in cats. In this study, we performed three-color flow-cytometric analysis for activation-associated cell surface molecules (CD2, CD11a, CD45RA-like and major histocompatibility complex antigen class II (MHC II)) and light scatters (cellular size and complexity) to examine whether phenotypic changes also occurred in CD4(+) PBLs in addition to CD8(+) PBLs, of five FIV-infected cats and one uninfected cat. It was shown that (i) CD8alpha(+) PBLs, but not CD4(+) PBLs, had a distinct subpopulation with increased CD11a expression accompanying a reduced CD8beta chain and increased intracellular granules (ii) CD8alpha(+) PBLs, but not CD4(+) PBLs, expressed CD45RA-like antigen with diverse expression levels and (iii) MHC II expression was greater in CD8alpha(+) PBLs than CD4(+) PBLs, and the CD8beta chain reduction was correlated with the MHC II decrease within CD8alpha(+) PBLs. These results suggest that FIV infection induces phenotypically heterogeneous subpopulations in CD8(+) PBLs, including activated phenotypes, rather than in CD4(+) PBLs.     

One-pot, mix-and-read peptide-MHC tetramers

Background

Cytotoxic T Lymphocytes (CTL) recognize complexes of peptide ligands and Major Histocompatibility Complex (MHC) class I molecules presented at the surface of Antigen Presenting Cells (APC). Detection and isolation of CTL''s are of importance for research on CTL immunity, and development of vaccines and adoptive immune therapy. Peptide-MHC tetramers have become important reagents for detection and enumeration of specific CTL''s. Conventional peptide-MHC-tetramer production involves recombinant MHC production, in vitro refolding, biotinylation and tetramerization; each step followed by various biochemical steps such as chromatographic purification, concentration etc. Such cumbersome production protocols have limited dissemination and restricted availability of peptide-MHC tetramers effectively precluding large-scale screening strategies involving many different peptide-MHC tetramers.

Methodology/Principal Findings

We have developed an approach whereby any given tetramer specificity can be produced within 2 days with very limited effort and hands-on time. The strategy is based on the isolation of correctly oxidized, in vivo biotinylated recombinant MHC I heavy chain (HC). Such biotinylated MHC I HC molecules can be refolded in vitro, tetramerized with streptavidin, and used for specific T cell staining-all in a one-pot reaction without any intervening purification steps.

Conclusions/Significance

We have developed an efficient “one-pot, mix-and-read” strategy for peptide-MHC tetramer generation, and demonstrated specific T cell straining comparable to a commercially available MHC-tetramer. Here, seven peptide-MHC tetramers representing four different human MHC (HLA) class I proteins have been generated. The technique should be readily extendable to any binding peptide and pre-biotinylated MHC (at this time we have over 40 different pre-biotinylated HLA proteins). It is simple, robust, and versatile technique with a very broad application potential as it can be adapted both to small- and large-scale production of one or many different peptide-MHC tetramers for T cell isolation, or epitope screening.     

Influence of calcium ions in the flow cytometric analysis of human CD8-positive cells.

BACKGROUND: The CD8 co-receptor is an important marker used to identify various lymphocyte subsets. A significant decrease in CD8alpha staining intensity was observed in the presence of divalent cation chelators. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers were treated with calcium chelators, stained with different anti-human CD8 mAbs, and analyzed by flow cytometry. RESULTS: Calcium chelators caused a dose-dependent decrease in fluorescence intensity, using specific anti-human CD8alpha mAbs. This phenomenon was not due to CD8 internalization and could be reversed by the addition of calcium ions. In contrast, calcium depletion increased staining intensity with one anti-CD8beta mAb. CONCLUSIONS: Divalent cation chelators are used as cell anti-clumping agents in MACS or FACS applications. Researchers should be aware that such treatment could lead to the almost complete loss of fluorescence with selected anti-human CD8alpha mAbs. Since CD8 staining is used in conjunction with tetramer staining to identify antigen-specific cytotoxic human T cells, the effect of calcium depletion should be taken into account in experimental design.     

Soluble major histocompatibility complex-peptide octamers with impaired CD8 binding selectively induce Fas-dependent apoptosis

Fluorescence-labeled soluble major histocompatibility complex class I-peptide "tetramers" constitute a powerful tool to detect and isolate antigen-specific CD8(+) T cells by flow cytometry. Conventional "tetramers" are prepared by refolding of heavy and light chains with a specific peptide, enzymatic biotinylation at an added C-terminal biotinylation sequence, and "tetramerization" by reaction with phycoerythrin- or allophycocyanin-labeled avidin derivatives. We show here that such preparations are heterogeneous and describe a new procedure that allows the preparation of homogeneous tetra- or octameric major histocompatibility complex-peptide complexes. These compounds were tested on T1 cytotoxic T lymphocytes (CTLs), which recognize the Plasmodium berghei circumsporzoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on Lys(259) in the context of H-2K(d). We report that mutation of the CD8 binding site of K(d) greatly impairs the binding of tetrameric but not octameric or multimeric K(d)-PbCS(ABA) complexes to CTLs. This mutation abolishes the ability of the octamer to elicit significant phosphorylation of CD3, intracellular calcium mobilization, and CTL degranulation. Remarkably, however, this octamer efficiently activates CTLs for Fas (CD95)-dependent apoptosis.     

Flow cytometric analysis of macaque whole blood for antigen-specific intracellular cytokine production by T lymphocytes

We report here the standardized conditions for stimulation of macaque whole blood samples with various protein or peptide antigens, and production of significant intracellular levels of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in CD4+ as well as CD8+ T lymphocytes. We observed significantly higher levels of TNF-alpha compared with IFN-gamma in both CD4+ and CD8+ T lymphocytes from all the macaque whole blood samples stimulated with staphylococcal enterotoxin B (SEB) as an antigen. Similarly, when whole blood samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine were stimulated with either the peptide cocktail or recombinant gp160, we observed production of significant levels of TNF-alpha by both CD4+ and CD8+ T lymphocytes. These results strongly support the utility of the whole blood cytokine flow cytometry methodology for determining antigen-specific immune responses of macaques in vaccine studies.     

Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two-dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8(+) immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h.     

Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity

There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.     

An MHC interaction site maps to the amino-terminal half of the T cell receptor alpha chain variable domain.

We have used cloned T cell receptor (TCR) genes from closely related CD4 T cell lines to probe the interaction of the TCR with several specific major histocompatibility complex (MHC) class II ligands. Complementarity determining region 3 (CDR3) equivalents of both alpha and beta TCR chains are required for antigen-MHC recognition. Our data provide novel information about the rotational orientation of TCR-MHC contacts in that exchange of the amino terminal portion of the TCR alpha chain containing the putative CDR1 and CDR2 regions results in both gain and loss of MHC class II specificity by the resulting receptor. These two TCRs differ primarily in recognition of polymorphisms in the second hypervariable region of the MHC class II alpha chain. These results document the involvement of CDR1 and/or CDR2 of the TCR alpha chain in MHC recognition and suggest a rotational orientation of this TCR to its MHC ligand.     

Tetramer-guided analysis of TCR beta-chain usage reveals a large repertoire of melan-A-specific CD8+ T cells in melanoma patients

The assessment of the TCR repertoire expressed by tumor-specific CD8+ T lymphocytes has been hampered to date by the difficulty of targeting the analysis to lymphocytes directed against a single epitope. In the present study we have used fluorescent A2/Melan-A tetramers in conjunction with anti-CD8 and anti-TCR beta-chain variable (BV) mAbs to analyze by flow cytometry the BV segment usage by Melan-A-specific CD8+ T cells in tumor-infiltrated lymph nodes (TILN) and tumor-infiltrating lymphocytes (TIL) from A2 melanoma patients. Analysis of TILN populations revealed small proportions of A2/Melan-A tetramer+ cells expressing many different BV together with over-representation of A2/Melan-A tetramer+ cells expressing certain BVs. The BV usage by A2/Melan-A tetramer+ lymphocytes in TIL was more restricted than that in TILN. Moreover, the predominant BV segments were quite distinct in populations derived from different patients. A2/Melan-A tetramer+ cells expressing the dominant BVs found in TILN could also be found in the corresponding peptide-stimulated autologous PBMC, although A2/Melan-A tetramer+ lymphocytes expressing additional BVs were also identified. Together, these results suggest that a large and diverse repertoire of Melan-A-specific T cells using different BV TCR segments is available in A2 melanoma patients.