Eckart Schott, Nicolas Bertho, Qing Ge, +1, Madelon M. Maurice, and Hidde L. Ploegh
October 8, 2002
99 (21) 13735-13740
Crosslinking of the T cell receptor has been proposed to be a prerequisite for T cell activation. Although the evidence supports this notion for CD4 T cells, the situation for CD8 T cells is less clear. Soluble class I monomers have been used to determine activation requirements in vitro with contradictory results. The possibility of transfer of peptide from soluble class I molecules onto class I molecules present on the surface of CD8 T cells, with ensuing presentation to other CD8 T cells, has been widely ignored. We show that monomeric and tetrameric class I molecules as well as free peptide can stimulate naïve CD8 T cells in vitro. We generate and characterize CD8 T cells that express the OT-I T cell receptor (for K b/SIINFEKL) yet lack K b and D b molecules, and show that their activation requirements differ from their class I positive counterparts when stimulated with soluble K b molecules. By eliminating the confounding effect of peptide transfer, we unmask the true activation requirements for naïve CD8 T cells and show that multivalent engagement of T cell receptors, as well as costimulation, is required for optimal stimulation.
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T cells are activated through their T cell receptor (TCR) when it combines with an MHC–peptide complex on an antigen-presenting cell (APC). Interaction of MHC products with the coreceptors CD4 and CD8 provides essential additional signals. Crosslinking of TCRs using αCD3 monoclonal antibodies results in activation of T cells, an observation that led to the hypothesis that T cell activation requires dimerization of the TCR. Numerous studies have addressed the minimal requirements for T cell activation and the extent of crosslinking involved. Using MHC molecules that had been immobilized or multimerized in solution, kinetic and light-scattering experiments have yielded divergent outcomes. Kinetic data support a TCR dimerization model for both class I and class II restricted TCRs, but data to the contrary have likewise been published. The results from cellular assays that explore TCR–MHC interactions support TCR oligomerization for CD4 T cells, but experiments with CD8 T cells yield a less consistent picture. Monomeric class I molecules could induce calcium flux of primed CD8 T cells, suggesting that coligation of a single TCR and a CD8 molecule suffices for activation. However, data from Daniels and Jameson obtained for primary OT-I CD8 T cells specific for K b/SIINFEKL contradict this result and imply that activation by a monomeric MHC–peptide complex is not sufficient for CD8 T cell activation when using calcium flux as a readout. Oligomerization was also required for activation of two T cell clones expressing different TCRs, although monomers were sufficient to trigger association of TCR with CD8/lck. Monomeric K b molecules loaded with agonist peptide for the OT-I or the 2C TCR induce association of TCR, CD3ɛ, and CD8, as assessed by fluorescence resonance energy transfer. In summary, published data suggest that TCR crosslinking is required for CD4 T cells. Data on the same issue are equivocal for CD8 T cells.
MHC-I tetramers have been used widely to stain antigen-specific T cells. Tetramers can also activate T cells in vitro, allowing differentiation of direct effects of the TCR–MHC interaction from those mediated by additional molecules present on APCs.
Peptide is bound to K b noncovalently, albeit with a binding constant that suggests stable binding [the affinity of SIINFEKL to K b is about 10−8 M]. CD8 T cells can present peptide to each other and become target cells for adjoining CD8 T cells, referred to as “fratricide”. There is no published evidence that documents this possibility for naïve CD8 T cells. On the contrary, reports on the stimulatory capacity of tetramers or transfected K b molecules have found no evidence for direct activation by peptide or have largely ignored this issue. Also, the question of how much peptide is released from soluble class I molecules and whether that amount is sufficient to induce activation has not been answered. Here we use monomers and tetramers loaded with agonist peptides, null peptides, or a mixture of both to investigate the quantity and valency of peptide-loaded K b molecules required for activation of CD8 T cells. We find that soluble K b molecules in tetrameric and monomeric form, as well as free peptide, activate naïve CD8 T cells without need for costimulation. We then generate K b-negative CD8 T cells to investigate the effects of soluble K b molecules in the absence of confounding effects of free peptide by eliminating the possibility of peptide binding to class I molecules on CD8 T cells. We find that class I negative naïve CD8 T cells require multivalent engagement of the TCR as well as costimulation for optimal activation.
Naïve CD8 T cells were isolated from spleens and lymph nodes of OT-I transgenic mice. OT-I is a transgenic TCR (Vα2/Vβ5) specific for an ovalbumin-derived peptide (SIINFEKL) restricted by H-2K b. To obtain naïve OT-I CD8 T cells, cell suspensions from lymph nodes and spleens were depleted for B, NK, myeloid, erythroid, and CD4 T cells by magnetic separation using the CD8 isolation kit from Miltenyi Biotec (Auburn, CA). The purity of isolated CD8 T cells was determined by FACS after staining with αCD8-FITC antibody (PharMingen).
Class I negative CD8 T cells were generated by adoptive transfer of 1.3 × 10 7 bone marrow cells from OT-I K b D b−/− animals into irradiated RAG−/− hosts (The Jackson Laboratory). Eight weeks after the transfer, lymph nodes and spleens were harvested, and CD8 T cells were enriched by depletion of K b-positive cells derived from the RAG−/− host by using biotinylated K b antibody (PharMingen) followed by streptavidin-coated magnetic beads (Miltenyi Biotec) and purification on Macs columns (Miltenyi Biotec). In a second step, CD8 T cells were enriched as described above. Purified cells were subjected to complement-mediated lysis after incubation with K b antibody (PharMingen) to eliminate residual recipient-derived K b-positive cells.
Cells were incubated at 10 5 cells/well in 96-well plates at 37°C for 20 h in the presence or absence of 10 μg/ml of αCD28 antibody (PharMingen) and mIL-2 (Roche Diagnostics) in the concentrations indicated. For control experiments, cells were activated by 5 μg/ml platebound αCD3 antibody (PharMingen) or 10 ng/ml phorbol myristate acetate and 700 ng/ml of ionomycin (Sigma).
DAP-3/K b is a fibroblast cell line transfected with K b. Cells were irradiated before incubation with peptide for 3 h. Cells were then washed three times before addition of CD8 T cells.
All experiments were performed in duplicates and are depicted as mean ± standard deviation. All figures represent data from at least two independent experiments.
K b monomers were prepared as described, by using the peptides SIINFEKL (agonist) and SSYSYSSL (polyS, null peptide). Peptides were synthesized on an Advanced ChemTech peptide synthesizer and analyzed for purity by mass spectrometry. Peptides used directly in activation assays were further purified by rpHPLC. Refolded K b/β2m–peptide complexes were purified by FPLC on a SuperDex 200 column (Amersham Pharmacia). To obtain K b tetramers, monomers were biotinylated by using recombinant BirA enzyme and purified by FPLC. K b molecules were mixed in a molar ratio of 4:1 with streptavidin or phycoerythrin (PE)-conjugated streptavidin (Molecular Probes). To obtain mixed tetramers, biotinylated monomers loaded with agonist peptide or null peptide were mixed in ratios of 3:1, 2:2, or 1:3 before addition of streptavidin. The tetramerized molecules were resolved as a single peak on a Superdex 200A column. Purity and absence of lower-order oligomers were confirmed by native gel electrophoresis. To prevent adsorption of monomers to the tissue culture plates that might allow polyvalent interaction with responsive T cells, all dilutions of monomers and tetramers were done in media (RPMI 1640) containing 10% FCS before addition to responsive T cells. To assess the degree of peptide release from purified monomers, freshly prepared monomers were diluted in full media to the maximal concentration used in activation assays and concentrated on a centricon concentrator (molecular mass cutoff 10,000 Da; Millipore). The flow-through fraction was used directly and in serial dilutions in activation assays.
Increasing concentrations of unlabeled monomers or tetramers loaded with either agonist or null peptides were mixed with a fixed suboptimal concentration (10−9 M) of agonist-loaded PE-labeled tetramers. CD8 T cells were plated in 96-well plates at 1 × 10 5 cells per well on ice before incubation with the mixture described above. After incubation on ice for 1 h, cells were washed twice and analyzed by FACS.
Cells were collected and washed with FACS buffer (PBS/0.5% BSA/0.02% sodium azide). Cells were stained with αCD8-FITC (53-5.8), αCD69-PE (H1.2F3), and αCD44-Cy-Chrome (IM7) antibody (PharMingen) or with PE-labeled tetramers loaded with SIINFEKL or SSYSYSSL. Cells were washed twice in FACS buffer before FACS analysis.
CD8 T cells (8 × 10 6) were stimulated with 10−7 M of K b molecules in tetrameric or monomeric form or with 10−7 M of SIINFEKL peptide, for the times indicated, at 37°C. Cells were harvested by a brief centrifugation at 800 × g and the pellet was lysed in buffer (1% BRIJ 96) containing protease inhibitors (1 mM 7-amino-1-chloro-3-tosylamido-2-heptanone/1 mM PMSF/0.02 mg/ml leupeptin/0.002 mg/ml aprotinin) and phosphatase inhibitors (1 mM orthovanadate/10 mM NaF/10 mM Na-pyrophosphate). Immunoprecipitation was carried out by using αCD3ζ antibody H146 (a kind gift from I. F. Luescher, Ludwig Institute for Cancer Research, Lausanne, Switzerland), in conjunction with fixed protein A-bearing Staphylococcus aureus. Immunoprecipitates were resolved on a 12.5% reducing SDS/PAGE before transfer to a polyvinylidene difluoride membrane (Millipore). Membranes were probed with α-phosphotyrosine (pTyr) antibody 4G10 (Upstate Biotechnology, Lake Placid, NY). Reaction products were visualized by probing with a horseradish peroxidase-conjugated anti-mouse IgG2b antiserum (Southern Biotechnology Associates) followed by enhanced chemiluminescence (NEN). After stripping, blots were reprobed with αCD3ζ antibody H146 followed by horseradish peroxidase-conjugated anti-Armenian hamster IgG (Jackson) to confirm equal loading of lanes.
Naïve CD8 T cells were isolated from OT-I TCR transgenic mice by depletion of B, NK, myeloid, erythroid, and CD4 T cells. The purity of CD8 T cells was >95% (Fig. 1A). More than 99% of CD8 T cells expressed the transgenic TCR, as shown by staining for Vα2 and Vβ5 (Fig. 1
