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* Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel; and
Viral Immunology Section, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A cascade of events can influence which peptides are found on the cell surface, including 1) the presence of endogenous and exogenous proteins; 2) the appropriate degradation of these proteins in intracellular compartments; 3) the ability of the degraded peptides to bind in the groove of the particular HLA molecules; and 4) the successful transport of these molecules to the cell surface.
This feature of T cell immune recognition has precluded direct tracking of APCs in vivo, because analysis of antigenic protein distribution cannot determine whether properly processed peptides derived from this molecule are bound to MHC proteins and expressed at the surface of the identified cells.
In the recent years, the advent of the application of tetrameric arrays of class I peptide-MHC complexes has enabled us to detect and study rare populations of Ag-specific CD8+ T cells (4, 5). However, there is a tremendous shortage of reagents to study and visualize class I MHC Ag presentation, the other side of the coin to TCR interactions that are analyzed efficiently by using MHC tetramers. Direct detection of particular peptide-MHC molecule combinations using flow cytometry or immunohistochemistry would allow quantitation of TCR ligands on individual cells, phenotyping of such APCs, and localization of these APCs within normal or pathologic tissues, while confocal immunofluorescence microscopy would permit analysis of the intracellular site(s) of peptide-MHC molecule interaction and trafficking. In situ localization of APCs bearing particular TCR ligands would be especially valuable in characterizing the cell-cell interactions involved in initiation, propagation, and maintenance of T cell immune responses. Multicolor histochemistry could be used to reveal not only the type and location of APCs, but also the phenotype of interacting T cells, including the set of cytokines elicited.
Abs with peptide-specific, MHC-restricted recognition pattern, termed as TCR-like Abs, would therefore serve as an ideal tool to address these issues and become effective reagents to complement the gap between phenotyping T lymphocytes by MHC tetramers and analyzing MHC Ag presentation on target cells, APCs, and lymphoid tissues.
Abs that specifically recognize class I or class II peptide-MHC complexes have already been used in murine systems to study Ag presentation, to localize and quantify APCs displaying a T cell epitope, or as a targeting tool in a mouse model (6, 7, 8, 9, 10, 11, 12, 13, 14, 15). However, only recently, by using large human Ab phage libraries, such unique Abs were generated toward human class I MHC molecules complexed with tumor-associated peptides (16, 17, 18). We now used this strategy to isolate human TCR-like Abs directed toward a human viral epitope and show that such Abs can be used for phenotypic analysis of MHC Ag presentation on APCs by direct visualization and in situ detection of the particular specific peptide-MHC complex.
As a model system, we used the human superhaplotype HLA-A2 complexed with a human T cell lymphotropic virus type I (HTLV-1)-derived Tax11–19 epitope (19).
HTLV-1 was the first human retrovirus identified (20). It causes a variety of diseases, including adult T cell leukemia/lymphoma (21) and a nonneoplasic inflammatory neurological syndrome called HTLV- I-associated myelopathy/tropical virus spastic paraparesis (22). Several other clinical conditions have been linked to HTLV- I infection on the basis of seroepidemiological studies; these include Sjogren’s syndrome, inflammatory arthropathies, polymyositis, and pneumopathies (23). The viral protein TAX seems to play a major role in the pathogenesis of HTLV- I-associated diseases. TAX is known to stimulate the transcription of viral and cellular genes such as the genes coding for IL-2, IL-2R, proto-oncogenes, c-jun and c-fos, several cytokines, and MHC molecules (24). The transforming potential of TAX has been demonstrated in different experimental systems. It has been shown that rodent fibroblastic cell lines expressing TAX form colonies in soft agar and tumors in nude mice (25). Also, TAX transforms primary fibroblasts in cooperation with the Ras protein (26) and immortalizes primary T cells in the presence of IL-2 (27). Transgenic mice carrying the tax gene develop different types of tumors (28). TAX binds directly to DNA, but acts in cooperation with several cellular transcription factors, but the role of these different interactions in the cell transformation mediated by TAX is still unclear (23). HTLV-1-associated myelopathy is a slowly progressive neurologic disease characterized by inflammatory infiltrates in the CNS that consist predominantly of monocytes and CD8+ T cells (19, 29). Systematically, there is an increase in viral load associated with clonal expansion of HTLV-1-reactive CD8+ T cells, which are primarily reactive with the Tax protein. In patients carrying the HLA-A2 allele, the immune response is dominated by CD8+ T cells that recognize the Tax11–19 peptide (19, 29).
In this study, we describe the isolation of a large panel of human recombinant Abs with Ag-specific, MHC-restricted specificity of T cells binding with high affinity to HLA-A2 complexes that display the HTLV-1-derived Tax11–19 peptide. We demonstrate the experimental utility of such human recombinant Abs in direct phenotypic analysis of the specific class I MHC complex on the surface of APCs and virus-infected cells, including visualization and enumeration of the number of complexes on APCs after naturally occurring active intracellular processing of the Ag as well as in situ detection of the specific peptide-MHC complex on the surface of APCs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Single-chain MHC (scMHC)/peptide complexes were produced by in vitro refolding of inclusion bodies produced in Escherichia coli, as described (30). Briefly, a single-chain 
2-microglobulin (2m)-HLA/A2 (scMHC) construct, in which the 2m and HLA-A2 genes are connected to each other by a flexible peptide linker, was engineered to contain the BirA recognition sequence for site-specific biotinylation at the C terminus (scMHC-BirA). In vitro refolding was performed in the presence of a 5–10 molar excess of the antigenic peptides, as described. Correctly folded MHC/peptide complexes were isolated and purified by anion exchange Q-Sepharose chromatography (Pharmacia, Peapack, NJ), followed by site-specific biotinylation using the BirA enzyme (Avidity, Denver, CO), as previously described (4). The homogeneity and purity of the scMHC-peptide complexes were analyzed by various biochemical means, including SDS-PAGE, size exclusion chromatography, and ELISA, as previously described (30).
Selection of phage Abs on biotinylated complexes
Selection of phage Abs on biotinylated complexes was preformed, as described recently (17, 18). Briefly, a large human Fab library containing 3.7 x 1010 different Fab clones (31) was used for the selection. Phages (1013) were first preincubated with streptavidin-coated paramagnetic beads (200 µl; Dynal, Oslo, Norway) to deplete the streptavidin binders. The remaining phages were subsequently used for panning with decreasing amounts of biotinylated scMHC-peptide complexes. The streptavidin-depleted library was incubated in solution with soluble biotinylated scHLA-A2/Tax complexes (500 nM for the first round, and 100 nM for the following rounds) for 30 min at room temperature (RT).
Streptavidin-coated magnetic beads (200 µl for the first round of selection, and 100 µl for the second and third rounds) were added to the mixture and incubated for 10–15 min at RT. The beads were washed extensively 12 times with PBS/Tween 0.1%, and additional two washes were with PBS. Bound phages were eluted with triethylamine (100 mM, 5 min at RT), followed by neutralization with Tris-HCl (1 M, pH 7.4), and used to infect E. coli TG1 cells (OD = 0.5) for 30 min at 37°C.
The diversity of the selected Abs was determined by DNA fingerprinting using a restriction endonuclease (BstNI), which is a frequent cutter of Ab V gene sequences. The Fab DNA of different clones was PCR amplified using the primers pUC-reverse (5'-AGCGGATAACAATTTCACACAGG-3') and fd-tet-seq24 (5'-TTTGTCGTCTTTCCAGACGTTAGT-3'), followed by digestion with BstNI (NEB, Beverly, MA) (2 h, 60°C) and analysis on agarose gel electrophoresis.
Expression and purification of soluble recombinant Fab Abs
Fab Abs were expressed and purified, as described recently. TG1 or BL21 cells were grown to OD600 = 0.8–1.0 and induced to express the recombinant Fab Ab by the addition of 1 mM isopropyl -D-thiogalactoside (IPTG) for 3–4 h at 30°C. Periplasmic content was released using the B-PER solution (Pierce, Rockford, IL), which was applied onto a prewashed TALON column (Clontech, Palo Alto, CA). Bound Fabs were eluted using 0.5 ml of 100 mM imidazole in PBS. The eluted Fabs were dialyzed twice against PBS (overnight, 4°C) to remove residual imidazole.
ELISA with phage clones and purified Fab Abs
The binding specificities of individual phage clones and soluble Fab were determined by ELISA using biotinylated scMHC-peptide complexes. ELISA plates (Falcon) were coated overnight with BSA-biotin (1 µg/well). After having been washed, the plates were incubated (1 h, RT) with streptavidin (1 µg/well), washed extensively, and further incubated (1 h, RT) with 0.5 µg of MHC/peptide complexes. The plates were blocked for 30 min at RT with PBS/2% skim milk and subsequently were incubated for 1 h at RT with phage clones (
109 phages/well) or various concentrations of soluble purified Fab. After having been washed, the plates were incubated with HRP-conjugated/anti-human Fab Ab (for soluble Fabs) or HRP-conjugated anti-M13 phage (for phage-displayed Fabs). Detection was performed using tetramethylbenzidine reagent (Sigma-Aldrich, St. Louis, MO). The HLA-A2-restricted peptides used for specificity studies of the Fab phage clones or purified Fab Abs are as follows.
Production of fluorescent tetramerized Fabs
The genes encoding to the L and H chain of Fab T3F2 were cloned separately into a T7-promotor pET-based expression vector. The L chain gene was engineered to contain the BirA recognition sequence for site-specific biotinylation at the C terminus (T3F2 light-BirA). These constructs were expressed separately in E. coli BL21 cells and upon induction with IPTG, intracellular inclusion bodies that contain large amounts of the recombinant protein accumulated. Inclusion bodies of both chains were purified, solubilized, reduced, and subsequently refolded at a 1:1 ratio in a redox-shuffling buffer system containing 0.1 M Tris, 0.5 M arginine, and 0.09 mM oxidized glutathione, pH 8.0. Correctly folded Fab was then isolated and purified by anion exchange MonoQ chromatography (Pharmacia). The Fab peak fractions were concentrated using Centricon-30 (Amicon, Beverly, MA) to 1 mg/ml, and the buffer was exchanged to Tris-HCl (10 mM, pH 8.0). Biotinylation was performed using the BirA enzyme (Avidity), as previously described. Excess biotin was removed from biotinylated Fabs using a G-25 desalting column. PE-labeled streptavidin (Jackson ImmunoResearch, West Grove, PA) was added at a molar ratio of 1:4 to produce fluorescent tetramers of the biotinylated Fab.
Flow cytometry
The B cell line RMAS-HHD, which is transfected with a single-chain 2m-HLA-A2 gene, the EBV-transformed HLA-A2+ JY cells, mature human HLA-A2+ dendritic cells, and the HLA-A2- B cell line APD-70 were used to determine the reactivity of the recombinant Fab Abs with cell surface-expressed HLA-A2/peptide complexes. Peptide pulsing was performed as indicated: 106 cells were washed twice with serum-free RPMI and incubated overnight at 26°C or 37°C, respectively, in medium containing 1–50 µM of the peptide. The RMAS-HHD cells were subsequently incubated at 37°C for 2–3 h to stabilize cell surface expression of MHC-peptide complexes.
Altenatively, 20 x 106 JY or APD cells were transfected with 20 µg of the eukaryotic expression vector pcDNA 3.1 (Invitrogen, San Diego, CA) containing cDNA encoding the TAX protein (pcTAX). The cDNA was a kind gift of M. Yutsudo (Osaka University, Japan) and T. Oka (Okayama University, Okayama, Japan). Twelve to 24 h after transfection, cells were incubated for 60–90 min at 4°C with recombinant Fab Abs (20 µg/ml) in 100 µl. After three washes, the cells were incubated with 1 µg anti-human Fab (Jackson ImmunoResearch). After another three washes, the cells were resuspended in ice-cold PBS. All subsequent washes and incubations were performed under ice-cold conditions, as already described for peptide-loaded cells. The cells were analyzed by a FACStar flow cytometer (BD Biosciences, San Jose, CA), and the results were analyzed with the WinMDI program (J. Trotter, http://facs.scripps.edu/).
Competition binding assays
Flexible ELISA plates were coated with BSA biotin, and scMHC-peptide complexes (1 µg in 100 µl) were immobilized, as already described. The binding of soluble purified Fab was performed by competitive binding analysis, which examined the ability of purified Fab to inhibit the binding of 125I-labeled Fab to the specific immobilized scMHC-peptide complex. The recombinant Fab Ab were labeled with 125I using the Bolton-Hunter reagent. The labeled Fab was added to the wells as a tracer (3–5 x 105 cpm/well) in the presence of increasing concentrations of the unlabeled Fab as a competitor. The binding assays were performed at RT for 1 h in PBS. The plates were washed (five times) with PBS, and the bound radioactivity was determined using a gamma counter. The apparent affinity of the Fab was determined by extrapolating the concentration of competitor necessary to achieve 50% inhibition of 125I-labeled Fab binding to the immobilized scMHC-peptide complex. Nonspecific binding was determined by adding a 20- to 40-fold excess of unlabeled Fab.
Immunohistochemistry
JY or APD cells were transfected with the pcTAX vector, as described above. After 24 h, the cells were incubated with 20 µg/ml HRP-labeled T3F2 Fab tetramer for 1 h on ice in RMPI containing 10% FCS. The cell suspension was applied onto glass slides precoated with 0.1% poly(L-lysine) (Sigma-Aldrich), as described (32). Cells were then incubated for 1 h at RT. Slides were washed three times with PBS, and incubated with a diaminobenzidine+ solution (DAKO, Carpenteria, CA) for 1 min, followed by washing with PBS to remove excess of staining reagent.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immune response in HTLV-1-infected patients carrying the HLA-A2 allele is primarily directed to the Tax11–19 by clonal expansion of HTLV-1-reactive CD8+ T cells.
Recombinant peptide-HLA-A2 complexes that present the Tax11–19 HTLV-1-derived peptide were generated using a scMHC construct that was described previously (30). In this construct, the extracellular domains of HLA-A2 are connected into a single-chain molecule with 2m using a 15-aa flexible linker. The scMHC-peptide complexes were produced by in vitro refolding of inclusion bodies in the presence of the HTLV-1-derived Tax11–19 peptide. The refolded scHLA-A2/Tax complexes were found to be very pure, homogenous, and monomeric by SDS-PAGE and size exclusion chromatography analyses (data not shown). Recombinant scMHC-peptide complexes generated by this strategy had been previously characterized in detail for their biochemical, biophysical, and biological properties, and were found to be correctly folded and functional (30, 33).
For selection of TCR-like Abs, we used a large Ab phage library, consisting of a repertoire of 3.7 x 1010 human recombinant Fab (31). Due to the selection strategy (described in Materials and Methods), we first depleted the library of streptavidin binders and subsequently applied the library to panning in solution on soluble recombinant scHLA-A2-peptide complexes containing the Tax11–19 peptide. A 1300-fold enrichment in phage titer was observed after three rounds of panning (Table I). The specificity of the selected phage Abs was determined by a differential ELISA analysis on streptavidin-coated wells incubated with biotinylated scMHC HLA-A2 complexes containing either the Tax11–19 peptide or control complexes containing other HLA-A2-restricted peptides. Phage clones analyzed after the third round of selection exhibited two types of binding patterns toward the scHLA-A2-peptide complex: one class of Abs consisted of pan-MHC binders that cannot differentiate between the various MHC-peptide complexes; the second type consisted of Abs that bound the MHC-peptide complex in a peptide-specific manner. The ELISA screen revealed that 78 of 90 randomly selected clones screened (87%) from the third round of panning appeared to be binding to HLA-A2/peptide complex.
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Using E. coli BL21 or TG1 cells, we produced soluble Fab from three phage clones (analyzed above; Fig. 1) that exhibited the most specific binding pattern to the HTLV-1-derived HLA-A2-Tax complexes.
These Fab Abs that are tagged with a hexahistidine tag fused to the CH1 domain were purified by metal affinity chromatography from the periplasmic fraction. SDS-PAGE analysis of the affinity-purified material revealed homogenous, pure Fab Abs with the expected m.w. (Fig. 2A). Approximately 2–4 mg of pure material could be obtained from 1 L of bacterial shake flask culture. For further manipulations; i.e., to increase the avidity of the monomeric Fab, we also produced the Fabs by in vitro refolding; the Fd and L chains were subcloned into a T7-promotor pET-based expression vector, and upon induction with IPTG, large amounts of recombinant protein accumulated as intracellular inclusion bodies (Fig. 2B). Upon in vitro redox-shuffling refolding, purified monomeric Fab was obtained in high yields (4–6 mg of purified Fab was obtained from two 1-L shake flask cultures, each expressing the Fab Fd or L chain domain) (Fig. 2C).
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25–30 nM for the T3F2 Ab. Similar results were observed for the T3E3 Ab (data not shown). Detection of complexes on pulsed APCs
To demonstrate that the isolated Fab can bind the specific MHC-peptide complex not only in the recombinant soluble form, but also in the native form, as expressed on the cell surface, we used murine TAP2-deficient RMA-S cells transfected with the human HLA-A2 gene in a single-chain format (34) (HLA-A2.1/Db-2m single chain, RMA-S-HHD cells). The HTLV-1-derived Tax peptide and control peptides were loaded on RMA-S-HHD cells, and the ability of the selected Fab Abs to bind to peptide-loaded cells was monitored by flow cytometry. Peptide-induced MHC stabilization of the TAP2 mutant RMA-S-HHD cells was demonstrated by the reactivity of mAbs w6/32 (HLA conformation dependent) and BB7.2 (HLA-A2 specific) with peptide-loaded, but not unloaded cells (data not shown). Fabs T3E3 and T3F2 reacted only with Tax-loaded RMA-S-HHD cells, but not with cells loaded with the gp100-derived G9–154 peptide (Fig. 5, A and B). Similar results were observed in FACS analysis using 10 other HLA-A2-restricted peptides (data not shown).
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Because the density of a particular peptide-HLA complex on cells is expected to be low compared with peptide-pulsed APCs, we increased the avidity of Fab T3F2 by making Fab tetramers, which are directly tagged with a fluorescent probe. This approach was used previously to increase the binding avidity of peptide-MHC complexes to the TCR or to increase the sensitivity of recombinant Ab molecules (35). Another advantage of using fluorescently labeled tetramers is that only a single staining step is required, whereas monomeric unlabeled Fabs require a fluorescently labeled secondary Ab. We thus used our Fab tetramers, which were generated with fluorescently labeled streptavidin, to measure the expression of the HTLV-1-derived Tax-MHC complexes on the surface of peptide-pulsed APCs. As shown in Fig. 6, the intensity of the binding as measured by flow cytometry with peptide-pulsed RMAS-HHD (Fig. 6A), JY cells (Fig. 6B), and human dendritic cells (Fig. 6C) was dramatically increased by two logs compared with the staining intensity with the T3F2 Fab monomer.
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These results further demonstrated the ability of these high affinity TCR-like Abs to detect the specific Tax MHC-peptide complex on the surface of APCs.
Detection of complexes formed by active intracellular processing
To examine the ability of the TCR-like Fab Abs to detect HLA-A2/Tax complexes produced by physiological Ag processing, we transfected the HTLV-1 Tax gene into the EBV-transformed B cell HLA-A2-positive Ag-presenting JY cells and into HLA-A2-negative APD cells as controls. Twenty-four hours after transfection, we tested the reactivity of the HLA-A2/Tax-specific Ab T3F2 by flow cytometry. The analysis was done using our sensitive tetrameric Fab T3F2. As shown in Fig. 7A, significant staining above control could be clearly seen only with HLA-A2-positive JY cells transfected with the Tax gene, but not with HLA-A2-negative cells transfected with the Tax gene (Fig. 7B). A control TCR-like Fab Ab specific for the melanoma gp100-derived G9–154 epitope did not react with the Tax-transfected JY cells (Fig. 7A). The peptide-specific, MHC-restricted pattern of reactivity by T3F2 is not due to differences in transfection efficiency or HLA expression of JY and APD cells because the percentage of transfected cells was similar, as determined in control experiments transfecting GFP into these cells (Fig. 7C), and the staining intensity of these cells with w6/32, a pan-MHC mAb, was similar (data not shown). These results indicate that the TCR-like Fab Abs are capable of detecting the specific MHC-peptide complex after active and naturally occurring endogenous intracellular processing. Moreover, we attempted to use Fab T3F2 for detecting HLA-A2/Tax complexes on virus-infected cells. To this end, we used HLA-A2-negative HUT 102 and HLA-A2-positive RSCD4 cells, which are lines of human CD4+ T cells infected with HTLV-1. As shown in Fig. 7D, a significant staining with Fab T3F2 was observed on RSCD4, but not on HUT 102 cells, indicating that the TCR-like Fab is capable of detecting the specific HLA-A2/Tax complex on the surface of virus-infected cells. The staining pattern revealed two subpopulations with a high and low-moderate reactivity with the TCR-like Fab, which may indicate variability in the expression of the specific Tax epitope within subpopulations of RSCD4 HTLV-1-infected cells. Similar variability was observed in staining experiments with an anti-Tax protein Ab (data not shown). A control TCR-like Fab, G2D12, specific for the melanoma gp100-differentiation Ag epitope, G9–154, did not stain RSCD4 cells (Fig. 7D).
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Sensitivity of ligand detection and direct quantitation of surface peptide-MHC complexes on APCs and virus-infected cells
The data that we have presented to date demonstrate the fine and unique specificity of the HLA-A2/Tax-specific Fab Abs as well as their ability to detect naturally processed Tax peptide bound to HLA-A2. To demonstrate that ligand recognition by these Fabs in vitro approaches the sensitivity of T cells to ligand-induced activation through the TCR, we examined the sensitivity of ligand detection. To that end, the staining with Fab T3F2 was tested over a broad range of peptide concentrations. As shown in Fig. 8, A and B, the titration of peptide-pulsed JY with different concentrations of the Tax peptide demonstrated that the staining intensity was dependent on the concentration of the peptide used for pulsing, and that peptide concentration at the low nM range was sufficient to detect binding. The staining intensity of peptide-pulsed JY cells observed with T3F2 was compared with calibration beads that display known fixed numbers of PE sites. This comparison enabled us to determine the number of HLA-A2/Tax complexes displayed on the surface of cells that are pulsed with various concentrations of the Tax peptide (Fig. 8A and Table I). Specific ligand detection sensitivity was observed with as low as 100 complexes per cell (when cells were pulsed with 6 nM peptide) and reached saturation at 1.1–1.2 x 105 complexes per cell at high peptide concentration of 25–50 µM. These results demonstrate that the sensitivity of ligand detection by T3F2 is in the same range of the minimal concentrations of peptides needed to elicit measurable cytokine secretion (IL-2 or IFN-
) from T cell hybridomas or target cell lysis by CD8+ CTL lines (36, 37).
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3 x 104 complexes. As demonstrated in Fig. 7D, Fab T3F2 recognized two subpopulations of HTLV-1-infected RSCD4 cells with high and moderate reactivity. The highly reactive cells express on their surface 3 x 104 HLA-A2/Tax complexes, while the cell population with low to moderate staining intensity expresses several hundred sites. These results clearly demonstrate the power of such TCR-like Abs to provide useful and easily obtained direct quantitative data on specific peptide-MHC complex expression on each cell in a population and the high level of occupancy of MHC class I molecules attained by determinants derived from proteins produced upon transfection with an intact whole gene or directly on virus-infected cells.
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At present, there are no direct means available for the enumeration and phenotyping of individual cells bearing physiological levels of peptide-MHC complexes in mixed cell populations. The TCR-like Fabs are ideally suited to conduct such analyses. Prospective uses of such molecules will be in the analysis of tumor samples or study of Ag presentation in lymphoid tissues within a heterogeneous cell population. To simulate the situation of a heterogeneous population of cells in which only a small fraction might express the specific peptide-MHC complex, we mixed Tax-transfected and control-nontransfected JY cells at various ratios and analyzed the reactivity of T3F2 by flow cytometry. As shown in Fig. 8C, staining with T3F2 allows accurate identification of the admixed Tax-transfected JY cells that express on their surface HLA-A2/Tax complexes, generated by active processing, using a simple one-color flow cytometry analysis. Using various ratios of mixtures between transfected and nontransfected cells, we show that T3F2 was able to detect as low as 1% Tax-transfected JY cells within a background population of 99% nontransfected cells (Fig. 8, C and D), resulting in the ability to detect 0.5% of positive cells (calculated from a maximal 61.2% transfection efficiency of JY cells; see Fig. 8D).
These results demonstrate the ease with which the isolated TCR-like Fab can reveal a cell subpopulation bearing specific endogenously generated peptide-MHC complexes.
In situ detection of cells bearing specific peptide-MHC complexes formed after active intracellular processing
Another major potential use for the TCR-like Fab Abs is the direct in situ visualization of ligand-bearing cells within undisturbed tissues using immunohistological methods. As a first step to assess this potential, we determined whether T3F2 can be used for such studies by testing its ability to detect in situ HLA-A2/Tax complexes on JY cells following naturally occurring active intracellular processing. Tax-transfected JY cells were subjected to a single-step immunohistochemistry staining with an HRP-labeled T3F2 Fab Ab. As shown in Fig. 9, these experiments showed a strong positive staining of the Tax-transfected JY cells (Fig. 9, A and B), but not of control-nontransfected JY cells (Fig. 9C). A control TCR-like Fab, G2D12, specific for the melanoma gp100 G9–154 epitope, did not exhibit any significant immune reactivity on Tax-transfected JY cells (Fig. 9D). Further evidence for the specific, MHC-restricted reactivity of Fab T3F2 in these in situ immunohistochemistry experiments is provided by the lack of reactivity with Tax-transfected (Fig. 9E) and nontransfected (Fig. 9F) APD cells that are HLA-A2 negative (HLA-A2-/HLA-A1+). These data show the ability of the T3F2 TCR-like Fab Ab to detect specific peptide-MHC complexes in situ on cells and potentially in tissue sections after naturally occurring active intracellular processing. To our knowledge, this is the first demonstration of in situ detection of Ag presentation in a human system.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These Abs exhibit an exquisite, very specific, and special binding pattern: they can bind in a peptide-specific manner only to HLA-A2/Tax complexes; hence, these are recombinant Abs with T cell Ag receptor-like specificity. In contrast to the inherent low affinity of TCRs, these molecules display the high affinity binding characteristics of Abs, while retaining TCR specificity. We demonstrate the utility of these Abs as a novel tool to characterize phenotypically MHC Ag presentation of a particular class I peptide-MHC complex on the surface of APCs after naturally occurring active intracellular processing of the Ag as well as detection of the particular HLA-A2/peptide complex on virus-infected cells.
Our findings demonstrate the critical importance and utility of these molecules in establishing new tools for studying Ag presentation in health and disease. We present some crucial features of these Abs, including: their ability to bind the native HLA-A2/Tax complex as displayed on the cell surface of APCs and virus-infected cells; the ability to quantitate the number of a particular peptide-MHC complex on the surface of APCs after active intracellular processing; and last but not least, the ability to detect in situ by immunohistochemistry methods the expression of a particular peptide-MHC complex on the surface of intact APCs after active naturally occurring intracellular processing of the Ag.
An important feature of the TCR-like Fab Abs isolated in this study is their capacity to detect TCR ligands at cell surface densities near the threshold limit for T cell signaling. Studies from other laboratories using a TCR-like mAb directed to a murine H-2Kb class I MHC in complex with the OVA peptide SIINFEKL indicated that the lower limit of sensitivity of flow cytometry detection is in the range of 100–500 ligands/cell using single-step or sandwich-staining techniques (12). Our data presented in this work for a human HLA-A2/peptide ligand are in good agreement with these numbers; thus, the HLA-A2/Tax-specific TCR-like Fab Ab was able to detect in a reproducible manner as low as 100 sites/cell. These numbers agree with several estimates of the threshold number of peptide-MHC complexes required to elicit nuclear responses from T cells, such as cytokine secretion (38, 39), but are 10-fold greater than what may be required for CTL-mediatedcell lysis (40, 41). Using flow cytometry, it was possible to use the T3F2 Fab Ab to detect specific ligand on cells pulsed with peptide concentrations similar to those required to activate T cell hybridoma or CTL lines to cytokine secretion and within a few fold of the minimal concentration able to sensitize target cells for lysis in a short-term assay (12). These data indicate that when applied to dissociated cell populations using flow cytometry, the detection of ligand with T3F2 and other TCR-like Fabs with similar affinity approaches the sensitivity of T cells, and hence that these molecules are suitable reagents for evaluating antigenic complex expression at low, but physiologically relevant levels. In our study, the detection sensitivity of specific ligand was observed with as low as 100 complexes per cell (when cells were pulsed with 6 nM peptide). Although this is a high degree of sensitivity, it may not be sufficient enough for expression of very few peptide-MHC complexes in which robust target cell lysis by CTL is brought about with peptide concentrations in the picomolar range.
We have applied this principle in this study to mixtures of the parental JY APCs and its Tax gene-transfected derivative. The latter is actively processing the Ag and displays the relevant Tax11–19 HLA-A2 peptide complex on the surface of the cell, as demonstrated by the ability of T3F2 to stain Tax-transfected, but not control cells. Even when using T3F2 in a single-step staining for flow cytometry, it was possible to readily identify Tax-transfected cells admixed with nontransfected JY cells in as low proportion as 1%. The extreme ease with which precise quantitation of cell surface peptide-MHC complexes can be accomplished using this approach also makes it an invaluable tool for Ag processing and presentation studies. Increasingly, such studies deal with quantitative differences in Ag display resulting from use of distinct forms of an Ag, of various Ag delivery methods, or of cells deficient in some known or suspected component of the processing machinery. Without such reagents, the quantitation of surface TCR ligand levels relies on biochemical isolation of antigenic peptides. This is an expensive and laborious process subject to numerous experimental artifacts and cannot distinguish between intracellular pools of loaded molecules and those on the cell surface accessible to TCRs.
In the data presented in this work, Fab T3F2 was used for quantitation of specific complexes on the cell surface after active intracellular processing by JY APCs transfected with the intact Tax gene or directly on virus-infected cells. This analysis demonstrated that transfection with the Tax gene and active intracellular processing in JY cells led to a display of 1 x 104 complexes per cell. Comparison with total HLA-A2 staining showed that nearly 90% of the HLA-A2 molecules were occupied with a single peptide species (data not shown). These data agree with the work of Porgador et al. (12), who quantified the number of H-2Kb/SIINFEKL complexes on the surface of cells after infection with recombinant vaccinia virus encoding the SIINFEKL sequence in a variety of contexts and with data on the occupancy of class I molecules of infected cells with peptides derived from virally encoded proteins. The later case are estimates that have been obtained by analysis of the extent of stabilization of newly synthesized class I H chain-2m complexes or elution of peptides from expressed class I molecules and reconstruction experiments to determine the peptide concentration in the eluates (42). Direct visualization and quantitation of the number of complexes displayed on the surface of virus-infected cells revealed the ability of TCR-like Fab T3F2 to detect subpopulations of cells with a variable expression of the specific HLA-A2/Tax complex. The biological significance of these subpopulations is currently under investigation.
Immunohistochemical staining with T3F2 permitted the in situ detection of the particular HLA-A2/Tax complex on the surface of intact Tax-transfected JY cells after naturally occurring active intracellular processing of the Ag. HLA background staining with the recombinant Fab was insignificant under these conditions because neither nontransfected cells nor HLA-A2-negative cells transfected with Tax were stained. Our data represent the first in situ visualization of cells bearing specific peptide-MHC class I complexes of a human system and demonstrate, in principle, that this approach can be used to conduct such studies for which alternative methods are not available.
Confocal immunofluorescence microscopy would permit analysis of the intracellular site(s) of peptide-MHC molecule interaction and trafficking. In situ localization of APCs bearing particular peptide-MHC TCR ligands would be especially valuable in characterizing the cell-cell interactions involved in initiation, propagation, and maintenance of T cell immune responses. Multicolor histochemistry could be used to reveal not only the type and location of APCs, but also the phenotype of interacting T cells, including the set of cytokines elicited.
Interestingly, we were able to isolate a repertoire of several Abs against the HTLV-1-derived epitope HLA-A2/Tax. Until now, Abs with TCR-like specificity had been generated against murine MHC-peptide complexes using various strategies of immunizations (6, 7, 8, 9, 10, 11, 12, 13, 14, 15). Only recently had we used the same strategy as described in this study to isolate high affinity TCR-like, peptide-specific, MHC-restricted Fab recombinant Abs against tumor-associated Ags, including three epitopes derived from melanoma differentiation Ag gp100 (17) and two epitopes derived from the telomerase catalytic subunit (hTERT) (18). Most strikingly, we were able to select several different TCR-like Abs, whereas all previous experiments were able to isolate only a single Ab clone (7, 12). The fact that 62% of the MHC-peptide-binding Abs had the fine specificity of a TCR-like molecule, i.e., presumably bind to a small portion of the total area of the MHC-peptide complex, is nevertheless surprising, especially because they were selected from a nonimmune repertoire considered not to be biased toward such specificity. The fact that we have been able to isolate from the same phage library recombinant Fabs against a large variety of MHC-peptide complexes containing other cancer-associated or viral HLA-A2-restricted peptides (17, 18) indicates that this behavior is not Tax or peptide related. It is possible that one particular Ab family or Ab V gene segment could have an intrinsic propensity to bind HLA-A2 molecules, and that the high frequency could be explained by a high abundance of such Abs in the nonimmune library. However, the sequences of the selected clones are derived from many different Ab families and germline segments, without any biases visible in the complementarity-determining regions either (Table II). The high frequency and high affinities for some of the Abs isolated in this study suggest that these molecules may be present at a high frequency in the Ab repertoires from the B cell donors of the phage library, but an in vivo role for such Abs remains unclear.
Whatever the reason for this high frequency of Abs to MHC peptides may be, it appears that this phage-based approach can be successfully applied to isolate recombinant Abs with TCR-like specificity to a large variety of MHC-peptide complexes. Thus, it may now be possible to elucidate the role of Ags under various pathological conditions such as cancer, viral infections, and autoimmune disease, not only at the level of the T cell, using MHC tetramers, but also at the level of the APC and diseased cells, using Abs of the type described in this work.
Another interesting application for these TCR-like Fab Abs is for structure-function studies of MHC-peptide-TCR interactions. By mutating particular residues in the specific peptide and testing the influence of these mutations on the binding of the Fab Abs and peptide-specific T cell clones, it may be possible to obtain important data regarding the structure-function relationship and the different nature of the recognition process between the TCR-like Fabs and the native TCR (43). In a recent study, we used TCR-like Abs as molecular sensors to study the influence of anchor position modification in altered peptide ligands (44). Interestingly, we have found that modification of a tumor-derived peptide at an HLA-A2 anchor position can alter the confirmation of the peptide-MHC complex (44).
To improve the sensitivity and targeting capabilities of these TCR-like Ab molecules, two Ab engineering approaches may be used: one increases the affinity of the parental Abs by affinity maturation strategies without altering their TCR-like fine specificity (45), and the second, already used in this study, increases the avidity of these recombinant monovalent molecules by making them multivalent. Combining these strategies may result in improved second-generation Ab molecules that will increase the level of sensitivity and would allow us to use them in more complicated experimental settings of in vitro and in vivo analysis of Ag presentation.
Overall, the experiments presented in this study provide new means for the isolation of useful human recombinant Abs to specific peptide-MHC class I complexes. The data obtained with one such viral epitope-specific recombinant Fab Ab show that such reagents have obvious utility for flow cytometric phenotyping of APCs isolated from various tissues or organs; for visualizing the intracellular localization of peptide-MHC complexes; for quantitating the number of specific complexes formed under various conditions of Ag availability and expression or the function of components of the processing and peptide-loading machinery within cells; and perhaps for tracking APCs in vivo. Abs to peptide-human class I complexes present on tumors (46, 47, 48) could potentially be used to identify patients whose cells express specific complexes before attempting therapeutic vaccination, to follow up vaccinated patients for survival of cells lacking the target Ag, or to target cytotoxic drugs or radionuclides to the tumor. Recently, we have demonstrated the ability of such Abs to block TCR recognition of targets, which suggests that they could also be used to decrease disease by interfering with recognition of normal tissue Ags by autoreactive CD8+ T cells (17, 18), without the risk of Ag administration to an already autoimmune individual and without the loss of function of an entire MHC allele, as would be the case with traditional anti-class I Abs. This wide range of basic and applied uses will encourage further attempts toward the generic isolation and characterization of such molecules. Our approach produces Ab molecules that enable phenotypic analysis of Ag (MHC-peptide) presentation, the other side of the coin to MHC-peptide-TCR interactions. Combining these two new approaches will significantly enhance our ability to understand immune responses in health as well as under various pathological conditions such as cancer, viral infections, and also when applied to class II MHC molecules, autoimmune diseases.
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2 Address correspondence and reprint requests to Dr. Yoram Reiter, Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Room 333, Haifa 32000, Israel. E-mail address: reiter{at}tx.technion.ac.il
3 Abbreviations used in this paper: ER, endoplasmic reticulum; 2m, 2-microglobulin; hTERT, human telomerase reverse transcriptase; HTLV-I, human T cell lymphotropic virus type I; IPTG, isopropyl -D-thiogalactoside; RT, room temperature; sc, single chain.
Received for publication November 26, 2002. Accepted for publication February 19, 2003.
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2 microglobulin (2m) HLA-A2.1 monochain transgenic H-2Db 2m double knockout mice. J. Exp. Med. 185:2043.Related articles in The JI:
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